Biotechnology Bulletin

Advancing Biological Breeding for Tuber Crops to Ensure National Food Security

XU Jian-fei, SHANG Yi

2024, 40(9): 1-3.

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Application and Risk of RNAi Technology in Potato Insect Pest Management

WANG Ke-ran, YAN Jun-jie, LIU Jian-feng, GAO Yu-lin

2024, 40(9): 4-10. doi:10.13560/j.cnki.biotech.bull.1985.2024-0472

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Insect pest damages to crops is a threat to global food security, farmers urgently need safe and effective crop protection tools to sustainably have yields that meet ever-increasing global demand. RNAi（RNA interference）technology is a molecular biology technique for post-transcriptional regulation of gene silencing. Its principle is based on the binding of small double-stranded RNA molecules composed of 19-25 nucleotide pairs to the target mRNA, which triggers the degradation of the mRNA and thus leads to the silencing of the target gene. RNAi technology has been widely applied in crop pest management, focusing mainly on the prevention and control of Coleoptera, Hemiptera, and Lepidoptera pests targeting potatoes. On December 22, 2023, the world's first RNAi biopesticide was officially approved for commercialization. It is used to control the potato beetle, an internationally recognized important and devastating quarantine pest of potatoes, which is becoming increasingly resistant to pesticides. It is the world's first sprayable RNA biopesticide allowed for commercial use on crops, and it is a landmark green control of potato pests. While products based on RNAi technology are used for agricultural pest control, their resistance, off-target effects and potential risks to environmental safety still need to be considered. This article provides a comprehensive review on the feasibility of applying RNAi technology in potato insect pest management, its applications, and potential risks, aiming to elaborate on the status and prospects of RNAi technology in potato pest management, and provide theoretical references for integrating RNAi technology into the comprehensive management for potato pest management.

CRISPR/Cas9 Editing MeHNL Gene to Generate Cassava Plants with Low Cyanogenic Glycoside

TONG Wei-jing, LUO Shu, LU Xin-lu, SHEN Jian-fu, LU Bai-yi, LI Kai-mian, MA Qiu-xiang, ZHANG Peng

2024, 40(9): 11-19. doi:10.13560/j.cnki.biotech.bull.1985.2024-0520

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【Objective】 Because cassava（Manihot esculenta Crantz）contains potentially toxic cyanogenic glucosides, its food safety is affected, which leads to the increase of processing costs. It is of great significance to cultivate cassava with low cyanogenic glycosides by biotechnology.【Method】 CRISPR/Cas9 technology was used to edit the α-hydroxynitrile lyase gene MeHNL. This gene encodes α-hydroxynitrile lyase that catalyzes the decomposition of cyanogenic glycosides. The editing target is located on its first exon, and 27 positive plants were obtained through Agrobacterium-mediated stable transformation.【Result】Sequencing analysis indicated successful editing in 26 out of 27 transgenic plants, and a high editing efficiency was achieved by 96.3%. The editing types predominantly comprised base insertions and deletions, alongside minor base substitutions and large fragment deletions. HCN colorimetric kit and HPLC analysis confirmed a significant reduction in hydrocyanic acid and cyanogenic glycosides in the mutant lines. Additionally, transgenic plants demonstrated slender leaves compared to non-transgenic counterparts, implicating MeHNL's impact on plant growth and development.【Conclusion】The cassava germplasm with low cyanide is obtained by CRISPR/Cas9 technology, which provides materials for exploring the study of cyanogenic glycoside metabolism on cassava growth and development.

Identification and Bioinformatics Analysis of Gene in the Magnesium Transporter Family in Cassava

TAN Bo-wen, ZHANG Yi, ZHANG Peng, WANG Zhen-yu, MA Qiu-xiang

2024, 40(9): 20-32. doi:10.13560/j.cnki.biotech.bull.1985.2024-0521

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【Objective】Magnesium transporters（MGTs）are vital for magnesium transportation，thus analyzing the basic information, the physiological and chemical characteristics of the MeMGT gene family in cassava serves as a foundational step for understanding the functions of these genes.【Method】In this study, we identified and named 13 putative MeMGTs genes in cassava（Manihot esculenta Crantz）based on the cassava genome database through homologous sequence alignments, designated as MeMGT2;1-MeMGT10;2. We conducted chromosomal localization, conserved motif analysis, and phylogenetic analysis of the MeMGT family genes. Additionally, we predicted the biochemical properties, structure, and function of the corresponding proteins. 【Result】Chromosomal analysis revealed that the 13 MeMGTs genes were distributed across 8 chromosomes, each containing 4 to 13 exons. Tissue-specific expression patterns indicated differential expressions of MeMGTs genes across various developmental stages. MeMGT4;2 was mainly expressed in cassava fibrous root and storage root, which might potentially play a role in storage root development, and MeMGT7 was mainly expressed in cassava leave, vein and stem with function possibly involved in long-distance Mg²⁺ transport. Cis-acting element analysis revealed the presence of numerous photoresponsive and hormone-responsive elements in the promoter regions of MeMGTs genes, suggesting regulation by multiple factors. Phylogenetic analysis of the MGT gene family categorized these genes into five distinct clusters between cassava and selected model species. Protein sequence analysis identified MeMGTs member proteins as hydrophilic proteins with two conserved transmembrane domains and a Gly-Met-Asn（GMN）tripeptide motif, devoid of signal peptides. Phosphorylation analysis indicated the presence of phosphorylation sites, including serine, threonine, and tyrosine sites, which may contribute to the functionality of MeMGTs member proteins.【Conclusion】MeMGTs genes are regulated by various elements including light, hormone, drought and cold responses, might play a crucial role in the growth, development and adversity stress of cassava.

Establishment of CRISPR/Cas9-mediated Highly Efficient Gene Editing System in Microtubers of Potatoes

SONG Qian-na, DUAN Yong-hong, FENG Rui-yun

2024, 40(9): 33-41. doi:10.13560/j.cnki.biotech.bull.1985.2024-0536

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【Objective】 The development of gene editing technology has made it possible to achieve precise molecular breeding in potato. Microtubers are ideal materials for genetic transformation, but its induction and genetic transformation are of genotype-dependence. Thus, establishing an efficient and widely used gene editing technology may provide technical support for precise molecular breeding for potatoes in future. 【Method】Cultivated tetraploid potato variety Qingshu No. 9 and Bingshu No. 6 were used to explore microtuber induction and genetic transformation. Concurrently, the CRISPR/Cas9 gene editing vector was transformed for genome editing. In addition, the screening system was tested in another three potato varieties. 【Result】 The efficient tuberizations of five potato varieties were obtained using stem segment with two leaves on solid media including 10% sucrose and 5 mg/L kinetin under dark condition, while induction effect varied. The optimal ratio of hormones for the regeneration of Qingshu No.9 was 0.5 mg/L 6-benzylaminopurine, 0.2 mg/L indole-3-acetic acid, 0.2 mg/L gibberellin, and 2 mg/L zeatin, under which the regeneration rate, the transformation frequency, genome editing rate was 41.5%, 51.9%, and 82.1%, respectively. Using the above screened the formula of hormones, genetic transformation and regeneration was efficiently achieved in another four potato varieties, and the genome editing rate was 63.2%, 33.3%, and 10% for Bingshu No.6, Desiree, and Jingshu No. 16, respectively. 【Conclusion】We established the highly efficient genetic transformation and regeneration system for five potato varieties, and genome editing can be completed for four potato varieties.

Cloning and Expression Analysis of StDREBs Gene in Solanum tuberosum L.

ZHANG Xiao-mei, ZHOU Nan-ling, ZHANG Sai-hang, WANG Chao, SHEN Yu-long, GUAN Jun-mei, MA Ling

2024, 40(9): 42-50. doi:10.13560/j.cnki.biotech.bull.1985.2024-0509

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【Objective】This study is aimed to investigate the expression characteristics of OsDREB1C homologous genes（StDREBs）in potato（Solanum tuberosum L.）under low nitrogen conditions, evaluating their potential to enhance nitrogen use efficiency in potato, thereby providing a theoretical basis for subsequent gene functional validation and crop improvement. 【Method】The homologous gene of OsDREB1C in potato was obtained by homologous cloning. Four homologous genes of OsDREB1C were found in the potato. Their expressions were analyzed via bioinformatics, subcellular localization and low nitrogen conditions. 【Result】 StDREBs protein in potato contained AP2 conserved domain, and the protein products were located in the nucleus. StDREBs gene was expressed in the roots, stems and leaves, with a higher expression in the roots, followed by in the stems, and the lowest relative expression in the leaves. Under nitrogen deficiency treatment, the expressions of StDREBs gene in different tissues increased with the increasing of treatment time, and the expression of StDREBs gene reached its peak when 1/10N was treated for 18 h, indicating that StDREBs participated in the response to low-nitrogen environment in potato. 【Conclusion】 OsDREB1C homologs（StDREBs）play a key role in potato under low-nitrogen conditions.

Identification and Expression Analysis of AQP Gene Family in Potato

MAN Quan-cai, MENG Zi-nuo, LI Wei, CAI Xin-ru, SU Run-dong, FU Chang-qing, GAO Shun-juan, CUI Jiang-hui

2024, 40(9): 51-63. doi:10.13560/j.cnki.biotech.bull.1985.2024-0439

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【Objective】Aquaporins（AQPs）are crucial in physiological processes such as plant growth and development, water transport and stress response under abiotic stress in plants, and the present study was aimed to reveal the properties and functions of the potato StAQP family. 【Method】The potato's StAQP gene family was identified by genome-wide, and their gene structures, conserved motifs, cis-acting progenitors, chromosomal localization, and gene collinearity were analyzed using bioinformatics, and the gene expression patterns were verified by combining transcriptome data and RT-qPCR. 【Result】The 44 members of the potato StAQP gene family were distributed in five subfamilies: PIP, TIP, NIP, SIP and XIP. The gene structures and conserved motifs of the members in the same subfamily were basically the same, and the most of the members of the potato AQP family were distributed in the plasma membrane; the members of the potato StAQP family contained a large number of cis-acting progenitors related to light and stress response. The results of collinearity analysis showed that there were a large number of homologous gene pairs in AQP family between the potato and Arabidopsis thaliana, Nicotiana attenuata, Solanum lycopersicu and Capsicum annuum. The results of transcriptome and RT-qPCR validation showed that the potato's StAQP family members were highly expressed under drought and salt stress, and that there was a difference in the expressions of different members under different stresses. Differences in the expression of the same genes under different stresses also existed, three of these genes, StAQP7, StAQP28 and StAQP31, were the key genes of the StAQP family in response to abiotic stresses. 【Conclusion】StAQPs genes play an important role in the growth and development of potato and in the response to abiotic stresses.

Identification and Expression Analysis of SAT Gene Family in Potato（Solanum tuberosum L.）

SHEN Peng, GAO Ya-Bin, DING Hong

2024, 40(9): 64-73. doi:10.13560/j.cnki.biotech.bull.1985.2024-0133

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【Objective】 Serine acetyltransferase（SAT）is a critical enzyme in assimilating sulfur into cysteine（Cys）, participating in various biological processes in plants, especially in plant response to abiotic stress. However, analyses on the potato SAT gene family（StSAT）has not yet be reported. This study systematically and comprehensively identified the potato SAT gene family, providing a theoretical basis for further understanding the characteristics of the StSAT gene family and further analysis of their functions in potato resistance to abiotic stress. 【Method】HMM was used to identify the StSAT gene family in potatoes and analyze their chromosomal distribution, gene structure, protein-conserved motifs, and collinearity across species. Additionally, RNA-seq data downloaded from PGSC were used to analyze the expression patterns of StSATs in different tissue parts of doubled-monoploid（DM）potatoes under abiotic stress and exogenous hormone treatments. qPCR（quantitative real-time PCR）was to analyze the relative expressions of StSATs in tetraploid potatoes under NaCl and PEG treatment（0, 1, 3, and 24 h）【Result】Four StSATs were identified in potatoes, and they were distributed across four chromosomes. Based on phylogenetic characteristics, these four StSATs were classified into three subfamilies. According to synteny analysis, there were 4 orthologous SAT genes to Arabidopsis, 4 to tomato（Solanum lycopersicum）, 2 to cabbage（Brassica oleracea）, 1 to rice（Oryza sativa）, and 1 to corn（Zea mays）. Expression analysis showed that the expressions of all four StSATs significantly increased over time under NaCl and PEG treatments compared to 0 h, indicating their likely involvement in the potato's response to salt and osmotic stress.【Conclusion】 StSAT gene family members play an essential role in potato responses to salt and osmotic stress.

Sequence Analysis of MeSDH Protein and Its Relationship with MeH1.2 in Cassava

ZHAO Ping-juan, LIN Chen-yu, WANG Meng-yue, ZHANG Xiu-chun, LI Shu-xia, RUAN Meng-bin

2024, 40(9): 74-81. doi:10.13560/j.cnki.biotech.bull.1985.2024-0441

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【Objective】Sorbitol dehydrogenase（SDH）plays an important role in regulating the conversion between fructose and sorbitol in Roseviaceae plants, and also participates in plant resistance to stress. The study on the function of MeSDH gene may provide a theoretical basis for cultivating cassava germplasm with high yield and resistance to stress. 【Method】MeSDH gene was cloned using ‘SC124’ cDNA as template, and the tissue specificity of MeSDH gene and its response pattern to drought, low temperature, PEG and ABA were analyzed by quantitative real-time PCR（RT-qPCR）. MeH1.2 was used as bait to screen the mixed yeast cDNA library of cassava under drought and low temperature. Yeast two hybridization（Y2H）point-to-point and bimolecular fluorescence complementation（BiFC）experiments were further used to confirm the relationship between target proteins. 【Result】The CDS length of MeSDH gene cloned from ‘SC124’ cDNA is 1 092 bp, encoding 364 amino acids, and the sequence shows no difference with it in the database. MeSDH protein contains catalytic Zn binding site, NADP binding site and structural Zn binding site, belonging to the MDR superfamily. MeSDH protein is localized in the nucleus by transforming tobacco leaves. The expression of MeSDH gene is decreasing in the order of functional leaves, young leaves, fibrous roots and stems. The expression of MeSDH is up-regulated in the cassava leaves treated with drought，low temperature and PEG, and significantly up-regulated in the cassava leaves and roots treated with ABA. Yeast library screening, Y2H point-to-point and BiFC experiments confirm the interaction between MeH1.2 and MeSDH. 【Conclusion】 MeSDH gene is up-regulated in response to various stresses, and probably MeSDH and MeH1.2 work together.

Screening and Expression Analysis of ERF Gene Related to Anthocyanin Synthesis in Colored Potato

WU Juan, WU Xiao-juan, WANG Pei-jie, XIE Rui, NIE Hu-shuai, LI Nan, MA Yan-hong

2024, 40(9): 82-91. doi:10.13560/j.cnki.biotech.bull.1985.2024-0460

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【Objective】Ethylene responsive factor（ERF）is an important transcription factor involved in the regulation of anthocyanin biosynthesis in plants. Screening of StERF genes that may be involved in the regulation of anthocyanin synthesis in potato tubers lays the foundation for the next step of research on the function of anthocyanin-related StERFs genes in colored potato.【Method】Bioinformatics methods were used to analyze their physicochemical properties, subcellular localization, conserved motifs, evolutionary relationships, protein secondary structure, promoter cis-acting elements, and protein interactions, while RT-qPCR was used to analyze the expression patterns of StERFs in potato flesh of different colors.【Result】Seven differentially expressed ERF transcription factors were obtained based on the transcriptome expression profiles of potato tubers of different colors. The seven StERFs are hydrophilic proteins, all of which are predicted to be localized in the nucleus. Motif 1（RWLG）and Motif 2（YRG）are the common conserved motifs in the seven StERFs, which belong to the structural domains of the characteristic sequences of AP2. The promoter sequences of the StERFs genes contain hormone-responsive action elements, as well as cis-action elements in response to defense and stress, low temperature and light, etc. The expressions of the seven StERFs were significantly higher in purple potato flesh than in yellow potato flesh, among which StERF72 and StERF110 were closer to known anthocyanin-associated ERFs（IbERF71 and PyERF3）. The existence of protein interactions between the seven StERFs and 56 transcription factors.【Conclusion】Seven StERFs may be involved in the regulation of anthocyanin synthesis in potato tubers, among which StERF72 and StERF110 can be candidated genes to further validate their functions.

Cloning and Function Analysis of the ScDHNS Gene of Crotonase/Enoyl-CoA Superfamily from a Wild Potato Species

QIAO Yan, YANG Fang, REN Pan-rong, QI Wei-liang, AN Pei-pei, LI Qian, LI Dan, XIAO Jun-fei

2024, 40(9): 92-103. doi:10.13560/j.cnki.biotech.bull.1985.2024-0528

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【Objective】 The DHNS（1,4-dihydroxy-2-naphthoyl-CoA synthase）gene in potatoes is a potentially important in the biosynthesis of glycoalkaloid metabolism in Solanaceae plants. Researching and verifying of the function of the potato DHNS gene can provide a source of genes and materials for the selection of low-glycoalkaloid potato varieties（lines）. 【Method】 The full-length sequence of ScDHNS cDNA was cloned from the wild potato species Solanum chacoense using the RACE technique. This sequence was analyzed for bioinformatics and subcellular localization, and its function was verified by constructing an overexpression vector pBWA（V）HS-DHNS to transform cultivated potato. 【Result】 The read open frame of ScDHNS cDNA sequence is 1 023 bp, encodes 340 amino acids, has a molecular weight of 37.34 kD and an isoelectric point of 8.592, and contains a typical ECH domain. It belongs to the enoyl hydratase/acetyl-CoA superfamily and is present in the genomes of plants such as Brachypodium distachyon and Medicago truncatula with homologous genes, showing gene expansion and contraction events. After the overexpression of the ScDHNS gene, it was found that the expressions of the transformed plants ScDHNS and SGT1 were significantly upregulated, and the expressions were notably higher than those in wild-type（WT）potato plants. Additionally, the total glycoalkaloid content in the corresponding transformed plants was significantly higher than that in WT potato plants, reaching a maximum of 364.3 mg/kg, which is 2.4 times that of the control. Subcellular localization results indicates that ScDHNS is localized in the peroxisome. 【Conclusion】 The ScDHNS gene in potato may regulate the expression of the key gene SGT1, which is involved in glycoalkaloid synthesis. It influences glycoalkaloid synthesis through the β-oxidation and mevalonate pathways. This gene is significantly associated with the subcellular compartmentalization of glycoalkaloids and has considerable application value in the cultivation of potato varieties with reduced glycoalkaloid content.

Identification of Potato G6PDH Gene Family and Its Expression Analysis in Damaged Tubers

SONG Bing-fang, LIU Ning, CHENG Xin-yan, XU Xiao-bin, TIAN Wen-mao, GAO Yue, BI Yang, WANG Yi

2024, 40(9): 104-112. doi:10.13560/j.cnki.biotech.bull.1985.2024-0243

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【Objective】Glucose-6-phosphate dehydrogenase（G6PDH）plays an important role in plant response to abiotic stress. Identification of the G6PDH gene family in potato and analysis of its expression pattern in damaged tubers would provide a reference for the subsequent in-depth study of the role of potato G6PDH genes in damage stress. 【Method】We used bioinformatics methods to identify the G6PDH gene family members of the potato w, and to analyze the chromosomal distribution, protein physicochemical properties, secondary structure, evolutionary relationships, gene structure, conserved motifs, and promoter cis-acting elements of proteins encoded by members of the gene family, as well as the expression pattern in different organs and damaged tubers of potato.【Result】A total of 4 G6PDH family members in potato genome were identified and distributed on 4 chromosomes, named StG6PDH1-StG6PDH4. Based on the subcellular localization and phylogenetic analysis, StG6PDH1, StG6PDH3 and StG6PDH4 were located in the chloroplasts and belonged to the plastidic type, StG6PDH2 was located in the cytoplasm and belonged to the cytoplasmic type. The number of amino acids of potato G6PDH proteins ranged from 511 to 596 aa, molecular weights from 58.48 to 66.65 kD, isoelectric points from 5.83 to 8.57, and instabilities coefficient from 39.79 to 47.53. The secondary structure of protein showed the highest percentage of α-helices and irregular coils and the least of β-turns. In addition, the promoters of StG6PDHs contained a large number of phytohormone, light, and stress response elements. The four StG6PDHs were expressed in the roots, stems, leaves and tubers of potato, and the expression in the leaves was higher than in other organs. The members of StG6PDHs were involved in the responses of potato tubers to injury stress. The expressions of StG6PDH1, StG6PDH2 and StG6PDH3 were upregulated within 36 h after tubers injured, and StG6PDH4 was down-regulated in injured potato tubers. 【Conclusion】A total of four potato StG6PDHs gene family members are identified, unevenly distributed on four chromosomes, of which one is cytoplasmic and three are plastidic. The promoter region of StG6PDHs includes light, hormone, and stress response elements. The expression of StG6PDHs in wounded tubers is differential, and the members synergistically regulate the responses of potato tubers to damage stress.

Promotion of StHY5 in the Synthesis of SGAs during Tuber Turning-green of Potato

WANG Chao, BAI Ru-qian, GUAN Jun-mei, LUO Ji-lin, HE Xue-jiao, CHI Shao-yi, MA Ling

2024, 40(9): 113-122. doi:10.13560/j.cnki.biotech.bull.1985.2024-0514

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【Objective】Post-harvest storage, transportation, shelving, and other factors can lead to the “turning green” of potato, resulting in a significant accumulation of SGAs in tuber. HY5 plays a crucial role as a key mediator of light signals in plants. Investigating its involvement in the turning-green of tuber can establish a molecular foundation for understanding SGAs accumulation induced by light in post-harvest potato. 【Method】Through comprehensive gene expression analysis, transcriptome profiling, targeted metabolite analysis, subcellular localization studies, yeast single hybridization and double luciferase experiments, the involvement of StHY5 in tuber turning green following light exposure was preliminarily elucidated.【Result】The StHY5 gene was expressed in both the peel and flesh of potato, but with different expression patterns. During the treatment of potato greening, the expression of the StHY5 gene in potato meat correlated with changes in SGAs content, while the expression of SGAs synthesizing genes StSGT1/GAME1 and StGAME4 was significantly up-regulated. Yeast single hybridization and double luciferase experiments demonstrated that StHY5 directly up-regulated the expression of StSGT1/GAME1 and StGAME4.【Conclusion】During the process of potato greening, the StHY5 plays a role in promoting the accumulation of SGAs by directly up-regulating the expression of StSGT1/GAME1 and StGAME4. This finding provides a solid foundation for further investigation into the function of StHY5 in potato greening.

Quick Detection of Potato Pollen Viability Based on Deep Learning

XIA Shi-xuan, GENG Ze-dong, ZHU Guang-tao, ZHANG Chun-zhi, LI Da-wei

2024, 40(9): 123-130. doi:10.13560/j.cnki.biotech.bull.1985.2024-0511

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【Objective】Traditional methods for detecting potato pollen viability rely on visual counting, which can be inefficient and inaccurate. In this study, a method for quickly detecting pollen viability was proposed based on PaddlePaddle deep learning framework by comparing different models.【Method】First, the pollens were stained with 2,3,5-triphenyltetrazolium chloride（TTC）and imaged using a microscope. The images were annotated by Photoshop（PS）. Viable and total pollens were labeled respectively, then the label images were converted into single-channel images. Three models, SegFormer, U-Net and DeepLabV3, were used for training to distinguish viable pollens and total pollens. Finally, a Python OpenCV program was used to count the pollen number and calculate pollen viability. 【Result】Compared with other models, SegFormer demonstrated the best performance in various evaluation indexes of the two datasets. Compared with manual recognition, the OpenCV program enabled fast and batch counting with less error.【Conclusion】Potato pollen viability can be detected quickly and accurately by image processing technology. This method was used to quickly identify the pollen viability of 200 F₂ individuals, providing a soild foundation for the collection of pollen viability in potato.

Genetic Diversity Analysis of Potato Varieties Based on SSR Fluorescent Marker Capillary Electrophoresis and Construction of Molecular Identity Card

MAO Xiang-hong, LU Yao, FAN Xiang-bin, DU Pei-bing, BAI Xiao-dong

2024, 40(9): 131-140. doi:10.13560/j.cnki.biotech.bull.1985.2024-0113

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【Objective】 Genetic diversity analysis and evaluation of introduced potato varieties is a necessary method for screening good parental materials. 【Method】SSR fluorescent marker capillary electrophoresis was used to analyze the genetic diversity of 51 introduced potato varieties and construct molecular ID cards. 【Result】The 41 pairs of primers amplified a total of 327 alleles, and 311 alleles were differentiated at the same position, and the percentage of polymorphism was 95.11%. The primer polymorphism information index（PIC）ranged from 0 to 0.89, with an average of 0.71. Shannon's（I）ranged from 0 to 0.54, with an average of 0.32. The genetic similarity coefficient（GS）of the introduced potato varieties was 0.32. In the genetic similarity coefficient GS of 0.76, the test materials were categorized into six major groups: Group I having 14 varieties, group II having 30 varieties, group III having three varieties, group V having two varieties, and group IV and VI having one variety each. Cluster analysis revealed that four varieties, Vilas, Dingshu 4, Kexin 33 and Dongnong 312 could be used as potato breeding materials. 【Conclusion】Three pairs of core primers, STM0037, SSR0387 and SSR01054, were selected to construct fingerprints and molecular IDs of 51 potato varieties.

High-throughput Sample Preparation Method for the Identification of Potato Ploidy Using Flow Cytometry

YUAN Lan, HUANG Ya-nan, ZHANG Bei-ni, XIONG Yu-meng, WANG Hong-yang

2024, 40(9): 141-147. doi:10.13560/j.cnki.biotech.bull.1985.2024-0611

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【Objective】Chromosome ploidy identification is an important part of the evaluation of potato germplasm resources. The establishment of a high-throughput sample preparation method for the identification of potato chromosome ploidy based on flow cytometry would lay a foundation for the subsequent large-scale identification of potato ploidy.【Method】The 30 parthenogenetic offspring of potato were used to compare the effects of low-temperature steel-bullet beating, liquid nitrogen grinding and blade shredding to prepare nuclear suspensions. The ploidy of diploid potato IVP101 and tetraploid potato HTJ349-3 was identified.【Result】The suspension of cell nuclei prepared by the low-temperature steel-bullet beating method had obvious fluorescence signal, sufficient cell lysis and few impurities. Due to the simple and easy operation of the low-temperature steel-bullet beating method, the sample preparation time was greatly shortened, and the identification efficiency was 45-60 times higher than that of the liquid nitrogen grinding method, and 105-180 times higher than that of the blade shredding method. The chromosome ploidy of potato IVP101 and HTJ349-3 was accurately detected by this low-temperature steel-bullet beating method. 【Conclusion】The results by the low-temperature steel-bullet beating method are as accurate as that by the liquid nitrogen grinding method and the blade cutting method, which can achieve high-throughput identification of potato chromosome ploidy.

Roles of bZIP Transcription Factor in the Response to Stresses, and Growth and Development in Plants

MA Bo-tao, WU Guo-qiang, WEI Ming

2024, 40(9): 148-160. doi:10.13560/j.cnki.biotech.bull.1985.2023-1176

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bZIP，one of the largest transcription factor families in plants, plays an important role in the response to stress and growth and development of plants. It has a conserved domain consisting of about 60-80 amino acids, including a highly conserved alkaline region and a relatively variable leucine zipper region. bZIP regulates the expressions of target genes by specifically binding to DNA sequences containing ACGT cores in target gene promoters in homologous or heterodimeric forms. When plants are stimulated by stress signals, bZIP is phosphorylated by the upstream signal-responsive kinases, and its stability is enhanced through phosphorylation. Under stresses（such as drought, salt, temperature, light, heavy metal, and plant disease）conditions, bZIP binds to the promoter regions of stress-related genes and interacts with other proteins to promote or inhibit the expressions of these genes, thus positively or negatively regulating the responses to abiotic stress and biotic stress. In addition, bZIP is involved in the synthesis and metabolism of many substances（such as anthocyanins, terpenes, flavonoids, and alkaloids）during plant growth and development, and also mediates the regulation of hormones（such as abscisic acid, salicylic acid, and jasmonic acid）signaling pathways. In this paper, the discovery, structure, classification, regulation of the bZIP transcription factors and their roles in plant stress response and growth and development are reviewed, and the future research directions are also prospected. This paper may provide theoretical basis and technical support for genetic improvement of crops stress resistance.

Research Progress in Microbial Herbicides

LIU Lu, ZHU Zhe-yuan, LI Ying-xi, WANG Jie, PENG Di

2024, 40(9): 161-171. doi:10.13560/j.cnki.biotech.bull.1985.2024-0063

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Weeds are one of the most serious pests to agricultural production, and their control mainly depends on chemical herbicides. Long-term and large-scale use of chemical herbicides will lead to the increase of weed resistance, environmental security and food security. Microbial herbicides are biological herbicides developed by using microorganisms themselves or their metabolites, which are characterized by abundant resources, environmental friendliness, not easy to have resistant weeds, which are the development direction of green weed biocontrol in the world. In this paper, we summarized the types of microbial herbicides and listed the biocontrol microorganisms and toxins with herbicidal activity. Then, we reviewed the mechanism of microbial weed control. Microorganisms invaded the weed host through infection, causing the tube closure of the weed, and finally resulting in plant wilting and dying. In otherwise, they affected the cell structure integrity, biofilm function, lipid stability, energy transfer, photosynthetic pigment synthesis, lipid synthesis and amino acid synthesis of the weed host through effective active substances in metabolites, to achieve the purpose of killing weeds. In addition, we summarized the application status of microbial herbicides, including commercialization, constraints in application and improvement strategies of control effect; as well as the application of omics technology in the field of microbial herbicides. Finally, we prospected the development direction and application prospect of microbial herbicides，aiming to provide reference for the research and development of new herbicides.

Research Progress in Molecular Mechanism of Interaction Between Rice and Meloidogyne graminicola

ZHU Shi-fei, LIU Jing, ZHANG Jia-qian, HUANG Wen-kun, PENG De-liang, KONG Ling-an, PENG Huan

2024, 40(9): 172-180. doi:10.13560/j.cnki.biotech.bull.1985.2024-0207

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Meloidogyne graminicola is a significant pathogen of rice（Oryza sativa L）. The serious epidemic of M. graminicola is a significant global concern, as it significantly impacts the safe production of rice. Understanding the pathogenesis of M. graminicola and the resistance mechanisms of rice to it has been a key research focus in recent years, as it provides the foundation for developing new strategies for the prevention and control of rice root-knot nematodes. In recent years, with the rapid development of molecular biology and genomics sequencing technologies, the genome of M. graminicola has been successfully sequenced, and the multiple effectors have been explored. Additionally, the resistance of rice root knot nematodes was analyzed, and multiple quantitative trait loci（QTLs）were identified within its chromosomes, and the first rice gene, MG1, resistant to root knot nematodes has been successfully cloned. Furthermore, rice possesses the capability to modulate hormonal signaling cascades, including those involving jasmonic acid and ethylene. This activation initiates a regulatory cascade, enabling the rice plant to effectively counter the nematode's pathogenicity and parasitism. These significant research findings provide scientific evidence, deepening our understanding of M. graminicola's pathogenic mechanism and rice's resistance mechanism against root-knot nematodes. This paper presents a review of recent research progress on the pathogenic mechanisms of M. graminicola and the response mechanisms of rice and highlights potential future directions in the interaction between M. graminicola and its host.

Screening and Identification of a Bacillus licheniformis Strain with High Electro-transfection Efficiency and Elevated Biomass

DU Wei, LI Zhi-min, XING Yan-ming, LIU Pu-lin, MIAO Li-hong

2024, 40(9): 181-189. doi:10.13560/j.cnki.biotech.bull.1985.2024-0080

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【Objective】Bacillus licheniformis is an important chassis cell for expressing heterologous proteins in the fermentation industry. Screening B. licheniformis strains with easy electro-transfection and high biomass provides strain resources for effectively improving the host modification efficiency and protein expression level. 【Method】Screening of strains with high homology to B. licheniformis from soil was carried out through enrichment, gyrB amplification, and 16S rDNA sequence analysis. The electro-transfection efficiency and biomass of the screened strains were compared and analyzed with the commonly used chassis cell B. licheniformis 2709 in the industry. The screened strains with easy electro-transfection and high biomass were identified morphologically and physiologically and biochemically as well as were subjected to antibiotic susceptibility assay and growth in high temperature and alkaline environment. 【Result】Ten strains with high homology to B. licheniformis were screened. Among them, the strain 1-33 presented the highest electro-transfection efficiency with a maximum value of 6 700 transformants per μg DNA, which was 13.7 times higher than that of B. licheniformis 2709. Cultured in semi-solid soymeal-corn flour medium and SR liquid medium, the maximum biomass of strain 1-33 was found to be 5.2×10¹⁰ CFU/mL and 6.8 g/L, respectively, surpassing B. licheniformis 2709 by 1.71 and 1.3-fold. Further morphological and physiological experiments identified strain 1-33 as Bacillus licheniformis, which had sensitivity to various commonly used antibiotics and good alkaline and heat resistance. 【Conclusion】A B. licheniformis strain 1-33 with high electro-transfection efficiency and elevated biomass is screened, thereby foundation for its further development as an efficient host for exogenous protein expression in industrial process is established.

Generation of Virus-free TRAC-knocked-in T Cells Using Cas9TX

CUI Hai-yang, TAN Miao, QUAN Zhuang, CHEN Hong-li, DONG Yan-min, TANG Li-chun

2024, 40(9): 190-197. doi:10.13560/j.cnki.biotech.bull.1985.2024-0321

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【Objective】Cas9TX, a Cas9 exo-endonuclease, can significantly reduce the chromosomal translocation and greatly improve the safety of gene editing. Thus this work aims to study the feasibility of Cas9TX replacing Cas9 for gene loci knockin.【Method】 Firstly, His-tagged Cas9TX protein was prepared and purified. Exo-endonuclease function of Cas9TX was verified using the curves of EC50 and kinetics of nuclease cleavage. Secondly, in vitro activated human T cells were edited via electroporating with Cas9TX RNP and dsDNA. The knockin efficiencies were measured by FACS. Finally, the feasibility of stability-modifications to donor templates for enhancing site-directed knock-in efficiency was explored. 【Result】 The knock-out efficiencies of the prepared Cas9TX at three TRAC-targeted site A, R and S of the T cells were 71.8%, 81.0% and 79.9%, respectively, which were equivalent to that by Cas9. However, the efficiencies of targeted integration of 2A-GFP（dsDNA donor templates designed）or +GTC bp（ssDNA donor templates）into the first exon of TRAC by Cas9TX were much lower than that by Cas9. DNA modifications that inhibit TREX2 exonuclease digestion could not improve the target knock-in efficiency of Cas9TX RNP. 【Conclusion】 Here we uncovered that Cas9TX RNP may be used for replacement of Cas9 RNP in the gene knock-out in three targets of TRAC，while the targeted integration efficiency using Cas9TX RNP is about half of using Cas9 RNP. This work serves as a valuable reference for using Cas9TX in targeted knock-in strategies.

Identification and Expression Analysis of UGT Gene Family in Pepper

WU Hui-qin, WANG Yan-hong, LIU Han, SI Zheng, LIU Xue-qing, WANG Jing, YANG Yi, CHENG Yan

2024, 40(9): 198-211. doi:10.13560/j.cnki.biotech.bull.1985.2024-0269

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【Objective】The structure and function of UGT gene family members in pepper were identified and analyzed, laying the foundation for subsequent research.【Method】BioEdit, BLASTP and Pfam were used to align and search for UGT members in pepper. CDD and HMMER databases were used to verify conserved domains. ExPASy, Cell PLOC, MAGA X, MG2C, GSDS, and STRING were used to analyze and predict protein physicochemical properties, phylogeny, chromosome localization, gene structure and protein interaction. Transcriptome data and real-time PCR were used to analyze the expression pattern of UGT gene in various organs during the development of various organs.【Result】The prediction results showed that the 140 CaUGTs family members identified from the pepper gene family encoded amino acids in the range of 253-534 and the isoelectric point in the range of 4.7-8.45, most of the CaUGTs were located outside the cell, and a few were located in the plasma membrane and chloroplasts, and were distributed in 17 groups and mapped on 13 chromosomes, of which 25 members were located in chrom12, and all members contained the conserved domains motif 1 and motif 3. They responded to the action elements related to plant growth and development, stress. Real-time quantitative PCR analysis of the expression patterns of UGT gene in pepper under different tissues, stress and hormone response showed that UGT gene was of tissue-specific expression differences, and the expressionof UGT gene significantly increased or decreased under the conditions of ABA, GA3, high and low temperature stress, and CaUGTs members might play different roles in specific stages of development, and may be involved in regulating the defense response under stress in pepper. 【Conclusion】The distribution and structure of the 140 pepper UGT members are diverse, while the members within the group are highly similar, indicating that the CaUGTs gene may respond to abiotic stress during the growth and development of pepper plants.

Screening Genes Related to Male Sterile in Welsh Onion（Allium fistulosum L.） Based on Transcriptomic Profiling and WGCNA

YUE Li-xin, WANG Qing-hua, LIU Ze-zhou, KONG Su-ping, GAO Li-min

2024, 40(9): 212-224. doi:10.13560/j.cnki.biotech.bull.1985.2024-0276

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【Objective】The aim of this study is to identify the co-expression modules of male sterile Welsh onion（Allium fistulosum L.）and to screen key hub genes, so as to provide deeper insights into the molecular mechanisms of male sterile in the Welsh onion. 【Method】Twenty-four cDNA libraries were constructed and sequenced for different development stages of flower buds from the male-sterile（MS）plants and male-fertile（MF）plants. Then weighted gene co-expression network analysis（WGCNA）was performed to reveal the key modules and hub genes. 【Result】A total of eighteen co-expression modules were identified based on 8,320 genes, among which three modules（blue, midnightblue, and black）were significantly correlated with pollen abortion of the MS plant. Functional enrichment analysis suggested that several pathways associated with the “phenylpropanoid biosynthesis” “cutin, suberine, and wax biosynthesis” “starch and sucrose metabolism” “alpha-linolenic acid metabolism” and “fatty acid elongation” were found to be involved in pollen abortion of the MS plant. By considering gene significance and intramodular connectivity, several hub genes including atpB, TPS9, TPD1, and BHLH35, were identified, which might play key roles in pollen development of the Welsh onion. Then hub genes related to pollen development response were verified for quantitative real-time PCR（RT-qPCR）analysis, and the expression patterns of RNA-Seq and RT-qPCR were very consistent. 【Conclusion】Three key modules of male sterile Welsh Onion are obtained, and hub genes like atpB, TPS9, and TPD1 are identified. It is found that the pollen abortion of the MS plant mainly is involved the processes of phenylpropanoid biosynthesis, cutin, suberine, and wax biosynthesis, starch and sucrose metabolism, alpha-Linolenic acid metabolism, and fatty acid elongation.

Effects of Phosphate-solubilizing Bacteria on the Rhizosphere Soil Properties and Microbial Community Structure of Maize in Lead-contaminated Soil

WEN Shao-fu, JIANG Run-hai, ZHU Cheng-qiang, ZHANG Mei, YU Xiao-qin, YANG Jie-hui, YANG Xiao-rong, HOU Xiu-li

2024, 40(9): 225-237. doi:10.13560/j.cnki.biotech.bull.1985.2024-0313

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【Objective】To explore the effects of phosphate-solubilizing bacteria（PSB）and their fermentation products on the properties of maize（Zea mays L.）rhizosphere soil and the composition and diversity of microbial community in lead-contaminated soil. 【Method】Based on the screening of Klebsiella pasteurii with lead-resistant and phosphate-solubilizing function, corn was cultivated in lead-contaminated soil by pot experiment. LB medium, supernatant（bacterial secretion）, bacterial solution（only bacterial cells）and fermentation broth（supernatant + bacterial cells）were applied to the rhizosphere, and sterile water control was set to explore the effects of phosphate-solubilizing bacteria on the physical and chemical properties of rhizosphere soil and microbial community structure.【Result】The supernatant, bacterial liquid and fermentation liquid of K.baumannii had no significant effect on the diversity of bacterial community in maize rhizosphere, while the bacterial liquid significantly increased the Shannon index and Chao index of soil fungal community. The supernatant, bacterial liquid and fermentation liquid of the strain increased the relative abundances of heavy metal-resistant microbial groups such as Bacteroidetes and Actinobacteria, while the supernatant and fermentation liquid increased the abundances of Proteobacteria and Mortierellomycota. The supernatant increased the relative abundances of Sphingomonas, Blastococcus, Bradyrhizobium and Archaeorhizomyces. In addition, Pearson correlation analysis of differential genera in the maize rhizosphere soil showed that there were positive correlations among 7 groups of differential genera, which revealed that different microbial genera tended to form mutually beneficial symbiotic relationships. The supernatant, bacterial liquid and fermentation liquid of the strain significantly increased the activities of soil acid phosphatase（Acp）. Among them, the Acp activity of maize rhizosphere soil in the fermentation liquid treatment group was the highest（574.44 mg/g, 24^-1）. The application of supernatant and bacterial liquid significantly increased the content of alkali-hydrolyzed nitrogen（AN）in the rhizosphere soil, which was 47.4% and 39.5% higher than that of the control, respectively. The three treatment groups significantly reduced the soil pH value. Through redundancy analysis（RDA）, it was found that soil AN, Acp, pH value and available phosphorus（AP）were the main factors affecting the microbial community structure.【Conclusion】This study reveales that exogenous application of PSB and their fermentation products was beneficial to improving the fertility of lead-contaminated soil, and affectes the composition and structure of soil microbial community, which provides a theoretical basis for inoculating PSB and improving soil nutrients and soil microbial community structure in lead-contaminated farmland.

Exploring on the Pathogenesis of Root Rot of Lycium barbarum cv. ‘Ningqi-5' Based on the Rhizoplane Fungal Community and Pathogens Identification

ZHANG Ya-ya, LI Pan-pan, GAO Hui-hui, JIA Chen-bo, XU Chun-yan

2024, 40(9): 238-248. doi:10.13560/j.cnki.biotech.bull.1985.2024-0187

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【Objective】 This paper is to compare the composition of rhizoplane fungal communities in susceptible strain ‘Ningqi-5’ between the healthy and diseased plants in the two planting areas（QXBZ and QTBZ）, as well as the isolated rot root fungi. It is also to clarify the pathogens causing root rot disease in ‘Ningqi-5’, and to explore the reasons for the root rot disease in ‘Ningqi-5’.【Method】High-throughput technology was applied to investigate the compositional characteristics of the rhizoplane fungal community. Tissue isolation method was to isolate the rot-root fungi the decayed roots of Lycium barbarum. They were taxonomically identified based on the morphological characteristics and the sequences of ITS and EF-1α gene fragments. The pathogenicity bythe rotten-root fungi was verified based on Koch's law.【Result】 The root rhizoplane fungal community composition differed significantly between the healthy and diseased plants of ‘Ningqi-5’ in both planting areas, showing the increase in relative abundance of Fusarium and decrease in relative abundance of Mortierella in the diseased plants, accompanying with the increase of unknown taxa like Fusicolla and Pseudogymnoascus. While the fungal community structure on the rhizoplane of the healthy plants in both planting areas tended to be consistent. The 36 strains isolated from the rotten rot of QXBZ were classified as Fusarium oxysporum, F. solani, Earliella scabrosa, Rhizoctonia solani, and Penicillium pimiteouiense. The 29 strains isolated from the rotten rot of QTBZ were classified as F. oxysporum, F. solani, E. scabrosa, Clonostachys rosea, P. pimiteouiense, F. andiyazi and Acremonium persicinum. Isolation frequency of F. solani was the highest in both planting areas, followed by F. oxysporum. Based on the artificial infection test, F. solani, F. oxysporum, R. solani, and F. andiyazi were identified as the pathogens of ‘Ningqi-5’ root rot disease, among which F. andiyazi was a newly discovered pathogen of L. barbarum root rot.【Conclusion】In this study, it is clarified that the occurrence of ‘Ningqi-5’ root rot disease is mainly closely related to the changes in the structure of the root rhizoplane fungal community and pathogens. F. solani, F. oxysporum, F. andiyazi, and R. Solani are the pathogens of ‘Ningqi-5’ root rot disease.

Paenibacillus polymyxa New Strain X-11 and Its Growth-promoting Effects on Tomato and Rice

LIU Wen-zhi, HE Dan, LI Peng, FU Ying-lin, ZHANG Yi-xin, WEN Hua-jie, YU Wen-qing

2024, 40(9): 249-259. doi:10.13560/j.cnki.biotech.bull.1985.2024-0253

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【Objective】A plant-growth-promoting rhizobacterium strain（named X-11）was screened from rhizosphere soil of wheat（Triticum aestivum L）. Which may provide a basis for the application of the strain in plant growth-promoting.【Method】Strain X-11 was identified by morphological characteristics, physiological and biochemical characteristics, 16S rDNA sequence and genome sequencing, and its effects on the growth and defense enzyme activities of tomato（Solanum lycopersicum）and rice（Oryza sativa）were measured. 【Result】The colonies of strain X-11 are round, swell, neat edge, translucent, and sticky. And the cells are rod-shaped, can form spores, gram negative reaction. And it has the functions of fixing nitrogen, reducing nitrate, producing indole and siderophore, degrading organic phosphorus. Strain X-11 was classified as Paenibacillus polymyxa group based on 16S rDNA sequence, while it was revealed to be a new strain with 55.7% genetic similarity to its closest relative strain through gene comparison in non-redundant database. Tomato（Solanum lycopersicum）seeds soaked with X-11 bacterial suspension increased germination rate and radicle length significantly. Moreover, peroxidase（POD）activity in the leaves significantly increased by X-11 bacterial suspension irrigating roots（R）and irrigating roots combined with spraying leaf surface（R+L）. Meanwhile, the R+L treatment increased superoxide dismutase（SOD）activity by 34.58%. X-11 bacterial suspension increased the seedling root length by 6.8% when applied at one-leaf phase of rice（Oryza sativa）. Additionally, the SOD activity of the seedlings increased by 90.4% and 51.8%, when the X-11 bacterial suspension was applied at vertical phase or one-leaf phase of rice. 【Conclusion】 Strain X-11 was identified as a new strain of P. polymyxa, which is the effective plant-growth promoting rhizobacterium with multiple functions and has potential for development and application in plant growth promotion.

Control Potential of Bacillus thuringiensis 4BM1 Strain to Sclerotiniose in Brassica campestris L.

WANG Mei-ling, GENG Li-li, FANG Yu, SHU Chang-long, ZHANG Jie

2024, 40(9): 260-269. doi:10.13560/j.cnki.biotech.bull.1985.2024-0279

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【Objective】In the previous study, Bacillus thuringiensis 4BM1 strains effectively suppressed Sclerotinia sclerotiorum growth by inducing systemic resistance in Brassica campestris. The induced systemic resistance mechanism and biocontrol potential were studied, which may provide strain resources for the biological control of sclerotiniose.【Method】Quantitative real-time PCR and transcriptome sequencing were used to analyze the transcription levels of defense-related genes in B. campestris leaves after roots were exposed to the Bt 4BM1 strain. The disease-resistance related secondary metabolites biosynthesis gene clusters were predicted by using the complete genome sequence of Bt 4BM1 strain and the antiSMASH 2.0 software. The 4BM1 strain was inoculated into the potted B. campestris seedlings by fermentation liquid irrigation method, and its colonization ability and growth promotion effect were analyzed. 【Result】4BM1 strain triggered hypersensitive response in B. campestris leaves. Genes involved in salicylic acid, jasmonic acid, ethylene signaling and brassinosteroid synthesis pathways of B. campestris leaves were upregulated after the roots were exposed to the Bt 4BM1 strain. The candidate gene clusters involved in exopolysaccharide and 13 other disease-resistance related secondary metabolites biosynthesis were predicted in 4BM1 strain chromosome. The 4BM1 strain colonized in the rhizosphere of B. campestris and promoted the growth of B. campestris. 【Conclusion】4BM1 strain can be used as a potential biological control resource to control sclerotiniose and promote the growth of B. campestris seedlings.

Metagenomic and Metatranscriptomic Analysis of Methanogenesis from Coal Degradation by Compounded Microflora

LIU Ding-rui, GUO Hong-guang, GONG Kai-yi

2024, 40(9): 270-281. doi:10.13560/j.cnki.biotech.bull.1985.2024-0050

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【Objective】Aromatic compound metabolism is considered a limiting factor for the generation of biogenic coalbed methane. To increase biomethane production from coal, the compounded microflora（CM）was obtained by compounding raw inoculum（RI）with ability of producing methane by degrading coal and phenanthrene-degrading functional microflora enriched from produced water. 【Method】A combined metagenome and metatranscriptome approach was used to analyze the differences in the community structure and metabolic pathways of CM and RI. 【Result】The methane yield of CM increased significantly by 114.55%. Compounding significantly increased the proportion of aromatics-degrading bacteria, such as Pseudomonas, accounting for 63.49%, and also increased the metabolic activity of the dominant bacteria as well as the synthesis and expression of the key enzymes in the metabolic pathway of aromatic compounds. Gene abundance of the aromatics-degrading pathway in CM was 1.65 times higher than that of RI, and the gene expression abundance was 6.34 times higher than that of RI（P<0.05）. The gene abundance and expression abundance of EC:1.13.11.2 were 2.24 and 62 times higher than those of RI, respectively. The increased expression abundance of these enzymes prompted the transformation of aromatic compounds to pyruvate. Compounding also enhanced the gene expression during the process of pyruvate metabolism to acetyl-CoA. The expression abundance of EC:1.2.4.1 reached 14.70-fold of the RI in CM. The gene expression abundance of the respective methanogenic pathways in CM was 2.66 to 7.10-fold of the RI. 【Conclusion】The compounding enriched the aromatics-degrading bacteria and significantly increased gene abundance, especially gene expression abundance, in the whole metabolic pathway of degrading aromatics to methane generation, resulting in the increased methane production.

Roles of RNA m¹A and m⁵C Methylation Modifications in the Fumonisin Biosynthesis of Fusarium verticillioides

HOU Zhi-han, HAO Nan, LI Jia-qi, ZHAO Bin, LIU Ying-chao

2024, 40(9): 282-290. doi:10.13560/j.cnki.biotech.bull.1985.2024-0213

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【Objective】Fusarium verticillioides is a serious plant pathogen fungus, which greatly reduces grain yield. It also produces class 2B carcinogen fumonisin, which threatens human and animal health. To explore the relationship between RNA methylation modification and fumonisin, the role of RNA methylated transferase in fumonisin synthesis was analyzed.【Method】 HPLC was applied to determine the fumonisin synthesis ability of F. verticillium strains from different regions. Six kinds of RNA methylation modification detection methods, including m⁶A, m^lA, m⁵C, Gm, m⁷G, and Um, were established by QuEChERS pretreatment combined with ultra-high performance liquid chromatograph-tandem mass spectrometry（UPLC-MS/MS）. Then the RNA methylation modifications of different toxin-producing fungi were detected, and the RNA methylation modification genes related to fumonisin synthesis were identified by bioinformatics and RT-qPCR.【Result】It was found that there were significant differences in fumonisin synthesis ability of F. verticillium strains in different regions, the detection method for RNA methylation modification was successfully constructed, the methylation of RNA m^lA and m⁵C was negatively correlated with fumonisin synthesis, and RT-qPCR showed that Fvalyref gene negatively regulated fumonisin biosynthesis.【Conclusion】The methylation modification of RNA m⁵C and its Reader gene Fvalyref negatively regulate fumonisin biosynthesis.

Cloning, Expression, Characterization and Application of the Pectin Esterase MtCE12-1 from Myceliophthora thermophila

ZHANG Man-yu, DONG Jia-cheng, GOU Fu-fan, GONG Chao-hui, LIU Qian, SUN Wen-liang, KONG zhen, HAO Jie, WANG Min, TIAN Chao-guang

2024, 40(9): 291-300. doi:10.13560/j.cnki.biotech.bull.1985.2024-0336

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【Objective】 To explore a novel pectin esterase enzyme, a homologous pectin esterase significantly up-regulated in tobacco biomass induction was highly expressed in Myceliophthora thermophila. The enzymatic properties of this pectin esterase were investigated, along with its role in assisting the degradation of tobacco biomass. 【Method】 RT-qPCR was used to analyze the expression of the pectin esterase gene Mtce12-1 in M. thermophila under the condition of tobacco biomass. Using a viral 2A peptide-mediated expression screening system, the pectin esterase gene Mtce12-1 was highly expressed in the M. thermophila wild-type strain ATCC 42462. The positive transformants overexpressing MtCE12-1-His-2A-GFP were subjected to recombinant enzyme production and protein purification, and the enzymatic properties of the pectin esterase MtCE12-1 were characterized. 【Result】 The transcription level of the pectin esterase gene Mtce12-1 was significantly up-regulated by about 109-110 folds under tobacco biomass conditions as compared to glucose condition. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis, copy number determination and Western blotting indicated that the recombinant proteins were successfully expressed and secreted in M. thermophila, with expressions reaching as the 464.08 U/mL. The MtCE12-1 presented the highest activity at 75℃ and pH 8.0. It had good enzymatic activity in the range of 50-80℃ and pH 7.0-9.0 and demonstrated excellent thermal stability. The addition of 100-300 µg of MtCE12-1 to the degradation system resulted in an increase in cellulose degradation efficiency in tobacco bar and tobacco stem by 18.5%-30.7% and 14.6%-30.5%, respectively. 【Conclusion】 The use of the 2A peptide-mediated expression system in M. thermophila facilitates efficient target protein expression and purification. The alkaline pectin esterase MtCE12-1 not only shows good temperature stability but also demonstrates exceptional effectiveness in tobacco biomass degradation, providing potential high-quality enzyme resources for tobacco industry applications.

Exploring the Protective Effect of Puerarin on Deoxynivalenol-induced C6 Cell Injury Based on WGCNA

ZHAO Hai-ping, LIU Lin, WANG Xin-lu, YUE Peng-fei, KONG Wei-jun, WANG Meng

2024, 40(9): 301-310. doi:10.13560/j.cnki.biotech.bull.1985.2024-0130

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【Objective】The experiment used deoxynivalenol（DON）to construct a C6 cell injury model and analyzed the protective mechanism of puerarin（PUE）in alleviating DON-induced C6 cell injury. 【Method】By using RNA sequencing（RNA-seq）and weighted gene co expression network analysis（WGCNA）, we aimed to identify the key modules and core target genes, which was most closely related to the alleviation of PUE on DON-induced C6 cell injury. Furthermore, we explored the molecular mechanism of PUE in alleviating DON- induced neurotoxicity. 【Result】The PUE significantly inhibited the decrease in the cell viability induced by DON, restored the morphology of damaged C6 cells, and effectively alleviated DON-induced cytotoxicity. The RNA seq and WGCNA results indicated that the Blue module was the most relevant key module to the alleviation of PUE on DON-induced C6 cell injury, and S100a11, Pdia3 and Hsp90b1 genes were selected as the key core genes. 【Conclusion】These results suggest that PUE may alleviate DON-induced C6 cell injury by targeting key core genes and regulating in endoplasmic reticulum signaling pathway.

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