Table of Content

26 February 2023, Volume 39 Issue 2

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Research Progress in Signals and Molecular Mechanisms of Ovule Primordia Initiation

YU Shi-xia, JIANG Yu-tong, LIN Wen-hui

2023, 39(2):  1-9.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0247

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The seed is the reproductive organ for angiosperm. Seed production is crucial for plant reproductive development and crop yield. Ovule is the precursor of the seed. Ovule primordia initiation is process of seed organ formation, also the first step of plant forming seed. Different species have different patterns of placenta formation and ovule initiation. The regulatory mechanism of ovule initiation has been mainly studied in Arabidopsis. Arabidopsis is a multi-ovule ovaries plant, i.e., there is multiple seeds in a fruit, and ovule primordia initiation causes great impact on the single-fruit seed yield and seed yield. In Arabidopsis, ovule primordia are generated from placentae developed from the carpel margin meristem（CMM）. Transcription factors, regulators, and plant hormones are involved in regulating ovule initiation and ovule number by affecting placenta development. Currently, the rules of asynchronous ovule initiation have been identified, and the researcher also revealed that the dynamic of polar auxin transport（PAT）and auxin response determine the multiple rounds of ovule primordia initiation. In this review, we firstly introduce the process of CMM and placenta formation in different plants and their regulators, then summarize the current research progress on ovule initiation, including hormones signal network affecting ovule initiation and asynchronous ovule initiation. Finally, we raise the un-solved questions in ovule initiation, future research work, as well as application prospects in agricultural productions.

Research Progress and Prospects of Biological Studies on the Medicinal Plant Scutellaria baicalensis

ZHENG Min-min, LIU Jie, ZHAO Qing

2023, 39(2):  10-23.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0728

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Scutellaria baicalensis Georgi is a perennial herb of Scutellaria L in the Labiaceae, and has been used as medicine for a long history in China. Many studies have shown that flavones are the main bioactive compounds of S. baicalensis, especially baicalein, wogonin and its glycosides in the root, which have anti-tumor, anti-virus, anti-inflammation, anti-oxidation, liver protection and neuroprotection activities. This paper summarized the chemical components, pharmacological mechanisms of active substances, biotechnology, omics, metabolic biology and synthetic biology. Also the paper discussed the potential values, significances and existing issues of S. baicalensis and other plants in Scutellaria genus, aiming to provide reference for the development and utilization of other traditional medicinal plants.

Genetic Mechanisms of Plant-microbiome Interaction by Genome-wide Association Analysis Study

LI Kai-hang, WANG Hao-chen, CHENG Ke-xin, YANG Yan, JIN Yi, HE Xiao-qing

2023, 39(2):  24-34.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0557

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All the microorganisms that inhabit the plant host are called the plant microbiome. With the development of high-throughput sequencing, the plant microbiome as a complex ecosystem has been widely concerned. Despite increasing recognition of the structure and function of plant microbiome the interaction mechanism of plants and microbiomes remains largely elusive. As an effective method, the recent application of genome-wide association analysis study（GWAS）is instrumental in unravelling this issue.This review focuses on the latest research progresses by the following two aspects: the effect of plant on microbiome, and the genetic mechanism of plant-microbiome interaction. The selection of phenotypic data in plant-microbiome association analysis is elaborated, and genetic mechanisms by different hosts are summarized, aiming to elucidate the host regulation of the microbiome and enhance the understanding of plant-microbiome interactions.

Advances of Trichoderma in Controlling Root Knot Nematodes and Cyst Nematodes

LUO Ning, JIAO Yang, MAO Zhen-chuan, LI Hui-xia, XIE Bing-yan

2023, 39(2):  35-50.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0618

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Root-knot nematode（Meloidogyne spp.）and cyst nematode（Heterodera spp.）are two kinds of plant-parasitic nematodes that are the most agriculturally damaging group of plant-parasitic nematodes worldwide. They infect roots the of the host plant, obtaining nutrition through giant cells or syncytia, affecting plant growth and development, and causing severe economic losses to crops. Trichoderma spp. is an important biocontrol resource in agricultural production. With the improvement of environmental protection awareness, Trichoderma spp. has recently attracted more and more attention as a biocontrol resource of plant-parasitic nematodes. Here we discussed the biocontrol mechanism, action mode, influencing factors, existing problems, and production application of Trichoderma against root-knot nematode and cyst nematode. Finally, we analyzed the issues of Trichoderma in biological control and provided insight into their application.

Research Progress in the Approaches to in vivo RNA Secondary Structure Profiling in Plants

ZHOU Xi-wen, CHENG Ke, ZHU Hong-liang

2023, 39(2):  51-62.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0518

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RNAs fold into complex secondary and tertiary structures via hydrogen bond pairing to enable their diverse functions in cells after dynamic changes according to environment. RNA structure is considered to be related to the regulation of gene expression and ligand sensing in plants, and is also considered to be an important factor in post-transcriptional regulation. Structured RNA can also affect the dimensional structure of RNA binding proteins and their functions. Thus, the analysis of RNA structure is particularly critical in the study of RNA function. At present, various detection methods for RNA secondary structure（RSS）has been developed from X-ray to enzyme digestion and chemical probe labeling, which can be combined with high-throughput sequencing to construct models. In addition, machine learning and other methods also provide new ideas for the detection of RNA structure. In this review, we summarize the factors affecting the RNA structure in vivo and recent reported methods for probing RNA structure in vivo, discuss the significances of developing novel prediction methods for RSS under the limitations of current methods as well as the issues, and prospect the future development trend of computer prediction methods and experimental methods, aiming to provide a methodological reference for subsequent research on RNA structure.

Creation of Glyphosate-tolerant Rice by Cytosine Base Editing

LU Zhen-wan, LI Xue-qi, HUANG Jin-guang, ZHOU Huan-bin

2023, 39(2):  63-69.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0848

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Glyphosate is the most widely used broad-spectrum herbicide. Breeding glyphosate-resistant rice(Oryza sativa L.)will help to improve the chemical control effect of weeds in rice fields, reduce the dosage of herbicide and simplify the control measures. In this study, cytosine base editing technology was used to conduct base directional substitution on the proline codon at position 177 of rice endogenous glyphosate target protein gene OsEPSPS. OsEPSPS（P177L）with base C replaced by T was obtained by Agrobacterium-mediated genetic transformation as editing material. A homozygous edited rice material OsEPSPS（P177L）was isolated and identified from its self-crossing progenies. The results showed that OsEPSPS（P177L）presented significant resistance to glyphosate and tolerated four times of the recommended concentration of glyphosate in the field. Further investigation discovered that P177L mutation did not interfere with plant height, germination rate and other economic traits. The new glyphosate-resistant rice base editing germplasm OsEPSPS（P177L）obtained in this study provides a new approach for improving glyphosate-resistant rice and weed control in rice fields.

Construction of Soybean Genetic Map Based on SLAF Markers and QTL Mapping Analysis of Salt Tolerance at Seedling Stage

CHEN Yi-bo, YANG Wan-ming, YUE Ai-qin, WANG Li-xiang, DU Wei-jun, WANG Min

2023, 39(2):  70-79.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0579

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QTL mapping of salt tolerance traits in soybean seedling stage was carried out for two consecutive years to provide new gene loci for salt tolerance traits in soybean seedling stage, and to provide basis for in-depth understanding of genetic effects controlling salt tolerance and its location on chromosomes. The recombinant inbred line population（RIL）derived from the crossing of cultivated soybean variety Jinda 53 as female parent and wild soybean variety Pingnan as male parent was used as materials. At the seedling stage of soybean, the plant death concentration（PDC）was used as salt tolerance index after salt treatment, combined with the soybean genetic map constructed based on SLAF markers, the QTL loci of salt tolerance traits in soybean seedlings were detected by composite interval mapping method. The results showed that a total of 12 QTL loci were detected in different environments in two years, which were distributed on chromosome 2, 3, 5, 7, 9, 11, 13, 15, 18 and 20. The interpretation rate of genetic variation was 23.24%-38.28%. Among them, 2 QTL loci were distributed on chromosome 3, 2 QTL loci were distributed on chromosome 7, and the other 8 QTL loci were distributed in different chromosome segments. Among the 12 QTLs, 5 were consistent with the results of previous studies, and the remaining 7 QTL loci were new findings of salt tolerance in soybean seedlings.

Targeted Editing of BoZDS in Broccoli by CRISPR/Cas9 Technology

HUANG Wen-li, LI Xiang-xiang, ZHOU Wen-ting, LUO Sha, YAO Wei-jia, MA Jie, ZHANG Fen, SHEN Yu-sen, GU Hong-hui, WANG Jian-sheng, SUN Bo

2023, 39(2):  80-87.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0823

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ζ-Carotene desaturase（ZDS）is one of the key enzymes regulating carotenoids biosynthesis. Here having the high-generation inbred broccoli（Brassica oleracea var. italica）line ‘ZN09’ as testing materials and BoZDS as target gene, then two target sites on the first exon of BoZDS were selected to construct CRISPR/Cas9 vector for stable genetic transformation. For the target site 1, the transformation efficiency and the mutation rate were 0.80% and 15.79%, respectively, and three heterozygous mutants were generated. For the target site 2, the transformation efficiency was 0.84%, and the mutation rate was 36.84%, and seven mutants were generated, including two homozygous, two heterozygous, and three chimeric mutants. All of the mutant plants showed albino or mottled phenotype. The L* and a* values of mutant plants were significantly higher than those of wild-type plants, while the b* value decreased. To sum up, a stable genetic system for CRISPR/Cas9-mediated gene editing in broccoli was established, and BoZDS was effectively edited. Our results provide the technical support for gene function study and germplasm innovation of broccoli by using gene editing technology.

OsDIS1 Negatively Regulates Rice Drought Tolerance Through Antioxidant Pathways

YANG Mao, LIN Yu-feng, DAI Yang-shuo, PAN Su-jun, PENG Wei-ye, YAN Ming-xiong, LI Wei, WANG Bing, DAI Liang-ying

2023, 39(2):  88-95.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0538

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Drought, as an abiotic stress factor, seriously affects the yield and quality of rice. Exploring drought-related functional genes will help to breed new drought-resistance varieties. In this study, the Nipponbare OsDIS1-RNAi transgenic rice was constructed by Agrobacterium-mediated transformation method, and a series of physiological and biochemical experiments and agronomic traits analysis were conducted on it and wild type. The results showed that the activities of antioxidant enzymes after drought treatment significantly enhanced, the content of malondialdehyde significantly reduced and the expression of reactive oxygen species clearance gene significantly increased in OsDIS1-RNAi transgenic rice compared with the wild type. After ABA treatment, OsDIS1-RNAi transgenic plants did not show significantly morphological changes; these results indicated that OsDIS1-mediated drought tolerance may be independent of the ABA pathway. Meanwhile, agronomic traits such as plant height, spike length and yield of OsDIS1-RNAi rice plants did not change. The above results indicate that OsDIS1 negatively regulates drought tolerance in rice by activating the antioxidant pathways to increase the intracellular scavenging capacity of reactive oxygen species, and it may have potential applications for drought tolerance breeding in rice.

Functional Analysis of Soybean GmPDAT1 Genes in the Oil Biosynthesis and Response to Abiotic Stresses

MIAO Shu-nan, GAO Yu, LI Xin-ru, CAI Gui-ping, ZHANG Fei, XUE Jin-ai, JI Chun-li, LI Run-zhi

2023, 39(2):  96-106.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1171

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The present study was conducted to investigate functions of GmPDAT1（phospholipid: diacylglycerol acyltransferase）genes in soybean oil biosynthesis and abiotic stress response, providing a new scientific reference for soybean oil improvement and stress resistance molecular breeding. The omics tools were employed to GmPDAT1 genes in soybean. Quantitative real-time RT-PCR was used to examine expression patterns of GmPDAT1 genes in various tissues and under three abiotic stresses. Functional complementarity assay was performed with TAG-deficient yeast（Saccharomyces cerevisiae）mutant H1246. Six soybean GmPDAT1 gene family members（GmPDAT1-A-GmPDAT1-F）were identified. GmPDAT1-F had eight exons while the remaining five GmPDAT1 genes contained six exons. Multiple stress-responsive cis- elements were detected in GmPDAT1 promoter regions. The sequence length of GmPDAT1-encoded proteins was between 582-668 aa and their isoelectric point（pI）proteins ranged from 5.91 to 8.59. All GmPDAT1 proteins had PLN02517 superfamily protein domains, classifying as the membrane-binding proteins. The secondary structure of GmPDAT1 proteins mainly consisted of α-helix and ranclom coiled coil. The six GmPDAT1 proteins were phylogenetically clustered into three subgroups, which were closely related to Arachis hypogaea AhPDAT1, Jatropha curcas JcPDAT1 and Ricinus communis RcPDAT1-2, respectively. The members of GmPDAT1 gene family had tissue-specific expression patterns. Of them, GmPDAT1-B was expressed in various tissues with the highest expression in developmental seeds, suggesting that GmPDAT1-B may function crucially for TAG biosynthesis in soybean seeds. Functional complementarity assay using TAG-deficient yeast（Saccharomyces cerevisiae）mutant H1246 evidenced that GmPDAT1-B had the high enzyme activity to catalyze TAG synthesis. Under the treatment of low temperature, drought and salt stress, GmPDAT1 gene members demonstrated different expression patterns, indicating that they differentially participated in different stress responses in soybean. In particular, GmPDAT1-B possibly mediates soybean responses to the three different stresses. In conclusion, GmPDAT1-B may have dual functions in promoting soybean oil synthesis and stress resistance.

Cloning of BrHsfA3 in Chinese Flowering Cabbage and Its Responses to Heat Stress

PANG Qiang-qiang, SUN Xiao-dong, ZHOU Man, CAI Xing-lai, ZHANG Wen, WANG Ya-qiang

2023, 39(2):  107-115.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0568

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In order to study the response mechanism to heat stress and explore related genes of Chinese flowering cabbage（Brassica campestris L. ssp. chinensis var. utilis Tsen et Lee）, a differentially expressed gene screened from the transcriptome database under heat stress in the early stage was cloned by PCR-based accurate synthesis（PAS）. Then bioinformatics analysis was performed, and the expression profiles of the gene under heat stress were analyzed by RT-qPCR. The results showed that the gene with an ORF of 906 bp was obtained and named as BrHsfA3, encoding 301 amino acid residues, the relative molecular weight was 34.12 kD with a theoretical isoelectric point being 5.00. BrHsfA3 protein was a hydrophilic protein with three phosphorylation sites of serine, threonine and tyrosine. Sequence analysis indicated that BrHsfA3 possessed a Hsf conserved domain, a DNA-binding domain（DBD）, a oligomerization domain（OD）, a nuclear localization signal（NLS）, and an activator domain（AHA）, the secondary structure were mainly alpha helix and random coil. Evolutionary analysis revealed that BrHsfA3 gene had the closest genetic relationship with Brassica napus. The RT-qPCR analysis results showed that the expression of BrHsfA3 was highly induced in the leaves and flowers of heat-resistant self-inbred line CX1-7 by heat stress and significantly higher than in heat-sensitive self-inbred line CX7-3, but the expression in the roots of CX7-3 was high under the same heat treatment, and which was significantly up-regulated after heat stress. These results provide a foundation for the high temperature response mechanism of BrHsfA3 in Chinese flowering cabbage.

Identification and Analysis of WRKY Transcription Factor Family Genes in Helianthus tuberosus

ZHAO Meng-liang, GUO Yi-ting, REN Yan-jing

2023, 39(2):  116-125.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0551

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Jerusalem artichoke（JA）（Helianthus tuberosus L.）is a drought-resistant species. Through analyzing the transcriptomic data of JA, WRKY transcription factor genes closely related to drought stress response of JA were screened and analyzed, laying a theoretical foundation for clarifying the function of WRKY family genes of JA. Bioinformatics methods were applied to analyze the physicochemical properties, conserved domains, tertiary protein structures of WRKY gene family members in JA and their phylogenetic relationships with other species. RT-qPCR was used to detect the relative expression levels of WRKY genes in the seeds, roots, stems, leaves, petals and tubers under drought stress, and the transcription levels of WRKY gene family members in JA. A total of 74 WRKY genes were identified in JA leaves, with the sequence length ranging from 510 to 2 040 bp. The detected motif lengths ranged from 25 to 50 bases. Phylogenetic tree analysis showed that the most of the HtWRKY genes clustered with other WRKY genes. The results of tissue-specific expression analysis showed that the relative expressions of HtWRKY genes were higher in petals and tubers, and the drought-induced expression analysis showed that 20 and 19 HtWRKY genes were up-regulated in the leaves and roots of JA, respectively. HtWRKY is involved in the process against drought stress.

House-keeping Genes Screening and Expression Patterns Analysis of Genes Involved in Alkaloid Biosynthesis in Uncaria rhynchophylla

MU De-tian, WAN Ling-yun, ZHANG Yao, WEI Shu-gen, LU Ying, FU Jin-e, TIAN Yi, PAN Li-mei, TANG Qi

2023, 39(2):  126-138.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0567

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This work aims to study the expression pattern of the most suitable house-keeping genes in different parts of Uncaria rhynchophylla and the key enzymes genes in the upstream synthesis pathway of U. rhynchophylla indole alkaloids. The root, stem hook, leaf and capulse from U. rhynchophylla were used as experimental materials. The expression stabilities of 12 candidate house-keeping genes（18S, SAM, TUA, TUB, EF-1β, EF-1α, RNA L13, GAPDH, Actin6, PAL, CYP, and cdc73）were evaluated by RT-qPCR, GeNorm, NormFinder, Bestkeeper, △Ct, and RefFinder. The results showed that SAM can be used as the most suitable house-keeping genes for gene expression analysis in U. rhynchophylla. Having SAM as house-keeping gene, 15 key enzyme genes involved in alkaloid biosynthesis upstream pathway, i.e., G8H, 8-HGO, IS, CYP76A26, 7-DLGT, 7-DLH, LAMT, SLS, AS, AnPRT, IGPS, TSA, TSB, TDC, and STR were screened based on the co-expression analysis of “genome + transcriptome + metabolome”, and their expression levels were analyzed. The results showed that the expressions of these genes were consistent with the content trend, indicating that the genes are likely involved in the synthesis of terpenoid indole alkaloids.

Biological Function of Gene MiISU1 for Fe-S Cluster Assembly in Mangifera indica

CUI Ji-jie, CAI Wen-bo, ZHUANG Qing-hui, GAO Ai-ping, HUANG Jian-feng, CHEN Ya-hui, SONG Zhi-zhong

2023, 39(2):  139-146.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0616

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Iron-sulfur（Fe-S）cluster assembly genes play important roles in plant growth and development. We focused on the cloning and functional determination of MiISU1 involved in mitochondrial Fe-S cluster assembly in mango（Mangifera indica）and analyzed its biological functions, which may provide gene resources and theoretical foundation for the molecular mechanisms of Fe-S cluster assembly in tropical crops. The MiISU1 was isolated by homology cloning technology from mango. The tissue-specific expression characteristics and their responses to different Fe supplies were detected by quantitative real-time PCR. Functional verification of MiISU1 was carried out via creating over-expressed transgenic Arabidopsis seedlings. Results showed that MiISU1 was isolated from diploid mango ‘Guire 82’, whose expression was the highest in young fruits, followed by mature fruits, full-bloom flowers and new leaves, but was low in new roots and new phloem. In addition, Fe depletion significantly reduced the expression of MiISU1 in the roots of 1-year-old grafted seedlings, while high Fe toxicity significantly enhanced the expression of MiISU1 in the whole tested seedlings. Moreover, over-expression of MiISU1 in Arabidopsis significantly enhanced plant growth, accompanied with enhanced Fe accumulation, leaf total chlorophyll, nitrite reductase（NIR）, aconitase（ACO）and succinate dehydrogenase（SDH）activities in transgenic lines, and improved plant resistance to Fe depletion via enhancing the root growth and development. MiISU1 plays a key role in Fe nutrition and metabolism of mango, which is regulated by external Fe status.

Genome-wide Identification of XTH Gene Family and Their Interacting miRNAs and Possible Roles in Low Temperature Adaptation in Jatropha curcas L.

LV Yu-jing, WU Dan-dan, KONG Chun-yan, YANG Yu, GONG Ming

2023, 39(2):  147-160.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0746

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Jatropha curcas L. has been widely considered as a promising and multi-purpose energy plant, also a kind of thermophilic chilling-sensitive plants, and low temperature severely affects their growth, development and utilization. It is known that xyloglucan endotransglucosylase/hydrolases（XTH）act as cell wall modifying enzymes and are involved in response and adaptation of plants to stresses. The previous work showed that chill-hardening at 12℃ significantly enhanced chilling resistance of J. curcas plants, and XTH genes demonstrated high-response to the chill-hardening. In the present study, genome-wide identification, bioinformatics analysis, expression profile analysis of XTH gene family, and identification and analysis of their interacting miRNAs were performed based on the obtained data of transcriptome, miRNAome and degradome of J. curcas during the chill-hardening. The results showed that 29 members of the XTH gene family were identified in J. curcas, encoding 269 to 341 amino acids, their proteins were mainly located to cell walls, and contained Glyco_hyho_16 and XET_C domains and the conserved site ExDxE. The 29 members of JcXTH family could be divided into three groups, located on nine chromosomes, and there were 8 pairs of gene duplication events. Expression profiling and RT-qPCR analysis demonstrated there were significantly differential expression patterns in different organs of J. curcas plants and under chilling-hardening, drought and salt stresses, implying that there were differential roles in various members of the XTH gene family. Analysis of sequencing data of miRNAome and degradome revealed that 68 miRNAs were found to target 26 members of JcXTH family, and analysis of expression profile and their correlation of these miRNAs and the targeting JcXTH genes highly responsive to the chill-hardening demonstrated a totally negatively regulated relationship, indicating these miRNAs were involved in a negative regulation of JcXTH gene expression. These results will be conducive to elucidating the functions of JcXTH gene family and how the interaction between the miRNAs and the JcXTH genes targeted to was involved in adaptation of J. curcas to low temperature stress, which will provide useful reference for further molecular breeding to improve chilling resistance in J. curcas.

Study on the Expression Pattern of Genes in Lignin Biosynthesis Pathway of Cassava Resisting to Tetranychus urticae

YAO Xiao-wen, LIANG Xiao, CHEN Qing, WU Chun-ling, LIU Ying, LIU Xiao-qiang, SHUI Jun, QIAO Yang, MAO Yi-ming, CHEN Yin-hua, ZHANG Yin-dong

2023, 39(2):  161-171.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0500

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Lignin is an important physicochemical barrier for crops to defend against pests, however, its mechanism of cassava resistance to insect remains unclear. In order to explore the role of lignin biosynthesis pathway genes in cassava resisting to Tetranychus urticae（two-spotted spider mite, TSSM）, the mite-resistant cassava cultivar C1115, Myanmar, SC9 and mite-susceptible cassava cultivar SC205, Bread and BRA900 were applied during mite-cassava interaction. Furthermore, real-time quantitative PCR was used to analyze the expression pattern of lignin biosynthesis pathway genes（PAL, 4CL, C4H, HCT, CSE, COMT, CCoAOMT, F5H, CCR and CAD）among different cultivars infested by TSSM at different times of 0, 1, and 4 d. The results showed that the expressions of C4H, HCT, CSE, F5H and CAD in TSSM-infested mite-resistant and mite-susceptible cassava cultivars generally increased compared with those before infestation, but they did not always show significant differences, in addition, there was no significant difference in the expressions of these five genes among the mite-resistant and mite-susceptible cassava cultivars at the identical TSSM infested time. On the contrary, compared with those before infestation, the expressions of PAL, 4CL, CCoAOMT and CCR significantly increased in the TSSM infested mite-resistant cassava cultivars. However, the expressions of those four genes in mite-susceptible cultivars were either unchanged or significantly decreased, moreover, during the same mite-infested time, the expressions of these four genes in mite-resistant cassava cultivars were generally higher than those in mite-infested cassava cultivars, especially when the cassava plants were infested by TSSM at 4 d. Further correlation analysis found that the increase in the expressions of these four genes was significantly and positively correlated with the resistance of cassava to mite. To sum up, the above results speculated that the expressions of lignin biosynthesis pathway genes PAL, 4CL, CCoAOMT and CCR may be probably related to the resistance level of cassava cultivars.

Overexpression of CaCP1 Enhances Salt Stress Sensibility in Transgenic Tobacco

DU Qing-jie, ZHOU Lu-yao, YANG Si-zhen, ZHANG Jia-xin, CHEN Chun-lin, LI Juan-qi, LI Meng, ZHAO Shi-wen, XIAO Huai-juan, WANG Ji-qing

2023, 39(2):  172-182.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0945

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Cysteine proteinases（CPs）play significant roles in salt stress responses of plants, thus the function of CaCP1 in salt resistance was studied by cloning CaCP1, which may provide reference for the breeding of pepper salt resistant varieties. First, the CaCP1 promoter of pepper（Capsicum annuum L.）was cloned, then recombinant vector with different length promoter fragments was constructed based on cis-regulatory elements associated with salt stress, and transient transformation of tobacco was performed. Salt treatment was carried out on T₃ generation tobacco transgenic lines overexpressing CaCP1, phenotype symptoms were observed, physiological indexes were measured, and the expressions of stress-related genes were analyzed. The results revealed that in response to salt treatment, the GUS expression in -410 to -1 bp region was the strongest. The leaves of CaCP1 transgenic plants wilted and yellowed earlier. The chlorophyll and relative water content were significantly lower than those of wild type, while MDA and electrolyte leakage rates were significantly higher than those of wild-type. ROS scavenger enzymes CAT and SOD activities decreased, while POD activity increased. Compared to wild-type, the expressions of ROS-scavenging genes（NtSOD, NtPOD, NtCAT, and NtAPX）and salt stress-related genes（NtLEA5, NtNHX1, NtP5CS1, and NtSOS1）significantly decreased. CaCP1 acts as a negative regulator to mediate the plant defense response to salt stress.

Molecular Cytology at Meiosis in Allotriploid Nicotiana tabacum（SST）

WANG Ge-ge, QIU Shi-rui, ZHANG Lin-han, YANG Guo-wei, XU Xiao-yun, WANG Ai-ling, ZENG Shu-hua, LIU Ya-jie

2023, 39(2):  183-192.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0668

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The genus Nicotiana is an important germplasm source for cultivated tobacco varieties improvement, the creation of tobacco interspecific hybrids and their molecular cytological analysis during the meiotic phase will provide the basis for the subsequent efficient transfer and utilization of excellent wild germplasm. Allotriploid interspecific hybrids were created from the parents of Nicotiana sylvestris Q67 and common Nicotiana tabacum L. cv. K326. Their chromosome composition, chromosomal behavior during the meiosis and expression of related genes were investigated by routine cytological observation, genomic in situ hybridization and real-time quantitative PCR. In this study, 68 allotriploid hybrids（SST）with 36 chromosomes were identified by phenotypic and cytological analysis from crossing Nicotiana sylvestris with Nicotiana tabacum. The results of general cytology and genomic in situ hybridization showed that 29.26% of the pollen mother cells of interspecific hybrids were abnormal in meiosis. The main abnormal behavior was the backward chromosomes from the T chromosome group in metaphase I and II, with 1-3 lagging chromosomes. The cells with the most lagging types in metaphase I and II were the cells with 1 and 2 lagging T chromosomes respectively. They accounted for 29.09% and 30.88% of the total number of cells carrying backward chromosomes, respectively. The abnormal types of anaphase I, anaphase II and telophase II were mainly unequal segregation, backward chromosomes and micronucleus. In anaphase I, a variety of abnormal segregation ratios were counted, but the chromosome composition after segregation was still close to 2∶1（S∶T）. The results of RT-qPCR showed that in prophase I, except for the significant up-regulation of CDKA:5319 and CYCA:6720 from S chromosome group, the expressions of other genes were significantly down regulated. From the metaphase I to the telophase II, compared with the control, except that MIS12 and CDKA:0239 from T chromosome group had no significant changes, other genes（CENH3, CDKA:5319, CYCA:0111 and CYCA:6720）were significantly or extremely significantly up-regulated, indicating that the disorder expression of meiosis related genes may be causing the abnormality of meiosis.

Responses of Choloroplast Specific Protein Profile to Different Stomatal Densities in Nicotiana benthamiana

SA Shi-juan, WU Han-yu, WEN Yuan, CHEN Xue-na, ZHENG Rui, YAO Xin-ling

2023, 39(2):  193-202.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0506

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CO₂ enters chloroplast through stomata. How chloroplast is in response to stomatal density in molecular level is unknown yet. In this study, transgenic Nicotiana benthamiana lines, showing either significantly higher（HSD）or lower（LSD）leaf stomatal density, respectively, were generated through regulation of NtEPF 2（Nicotiana benthamiana EPIDERMAL PATTERNING FACTOR 2）expression in vivo. Transgenic lines were characterized with chlorophyll accumulation, photosynthetic parameters and leaf chloroplast differential expression proteins（DEPs）based on iTRAQ（isobaric tags for relative and absolute quantitation）. The response of chloroplast composition to stomatal density variation was expected to be revealed. The results showed that accumulation of DEPs, metabolizing PSI-PSII-LHCII complex formation and accelerating its electron transfer were up-regulated significantly, following down-regulation of DEPs for PSI photoprotection and light damage repair in HSD lines. Therefore, chlorophyll accumulation and net photosynthetic rate were 43% and 67% higher in HSD lines than control. On the other hand, attributing to strong photophosphorylation ATP biosynthesis at the early stage of leaf development, chlorophyll accumulation and photosynthetic parameter in LSD lines were significantly higher than in control. Chlorophyll accumulation went down along with growing leaf age. The result indicated that the increase in stomatal density accelerated PSI-PSII-LHCII complex formation and its electron transfer, and reduced photoprotection. Thus, chlorophyll accumulations in leaf at all ages were maintained at a higher level. Reversely, the decrease in stomatal density resulted in an upside-down mode for chlorophyll accumulations along with leaf ages owing to unbalance between ATP biosynthesis and carbon fixation. The result not only contributes to further understanding how stomata work, also broadens our view to rise carbon fixation through stomata regulation.

Isolation and Identification of Saline-alkali Tolerant Aspergillus terreus SYAT-1 and Its Activities Against Plant Pathogenic Fungi

WANG Feng-ting, WANG Yan, SUN Ying, CUI Wen-jing, QIAO Kai-bin, PAN Hong-yu, LIU Jin-liang

2023, 39(2):  203-210.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0582

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Microorganisms co-exist and evolve in saline environment for a long time and have unique adaptability and biological properties, which make them the first choice in the microbial improvement of saline land. A saline-alkali tolerant fungal strain SYAT-1 was isolated from a typical soda saline soil in Songyuan city, Jilin province. It was identified by morphological characteristics observation and ITS-rDNA sequence analysis. The growth characteristics at different pH, salt tolerance, antagonistic activity against phytopathogenic fungi and potential pathogenicity of the strain were investigated. The results showed that it was identified as Aspergillus terreus and grew under alkaline（pH=7.0-12.0）medium conditions, with an optimal pH range of 7.0-9.0. It grew well on both 0-17.5 mg/mL NaCl PDA medium and 0-4mg/mL NaHCO₃ PDA medium. Antagonistic phytopathogenic fungi assay results revealed that strain SYAT-1 presented some inhibitory effect on the test phytopathogenic fungi. Potential pathogenicity test results demonstrated that strain SYAT-1 had no potential pathogenicity to the test plants. In sum, A. terreus strain SYAT-1 belongs to a fungus with high salt tolerance characteristics and suppressing phytopathogenic fungi. This study provides new strain resources and theoretical support for the use of microorganisms to improve saline soils and biological control of plant diseases.

Screening, Identification and Growth-promoting Effect of Antagonistic Bacillus spp. Against Cucumber Fusarium solani

YANG Dong-ya, QI Rui-xue LI, Zhao-xuan , LIN Wei, MA Hui, ZHANG Xue-yan

2023, 39(2):  211-220.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0522

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Cucumber root rot caused by Fusarium solani seriously affects the sustainable development of cucumber industry. In order to obtain high-efficiency antagonistic Bacillus spp. against cucumber root rot and to clarify its growth-promoting effect, Bacillus strains were isolated from the rhizosphere soil of cucumber plants, and the strains with high antagonistics to F. solani were screened, and the morphological, physiological and biochemical, genetic characteristics and plant growth-promoting characteristics were evaluated. The results showed that strain XY-1, XY-13 and XY-53 demonstrated significant inhibitory effects on F. solani, with inhibitory rates of 65.90%, 66.13% and 60.83%, respectively. XY-1 and XY-13 were identified as B. amyloliquefaciens, and XY-53 as B. subtilis. All the three strains showed phosphate-solubilizing ability, nitrogen-fixing ability, and produced protease and ACC deaminase. In the seedlings growth promotion test, inoculation with strain XY-1, XY-13 and XY-53 effectively promoted the growth of cucumber seedlings, and the seedlings’ stem lengths increased by 37.17%, 27.14% and 34.94%, respectively compared with control, and the chlorophyll content increased by 31.37%, 40.48% and 43.43%, the fresh weight of above ground increased by 33.03%, 38.81% and 51.52%, and the dry weight of above ground increased by 28.66%, 29.03% and 46.27%, respectively. In pot experiment for control efficiency, the concentrations of 10⁸ CFU/mL showed the best control efficiency on the cucumber root rot after 10 d of inoculation with XY-1, XY-13 and XY-53 different concentrations bacteria suspensions, and it was 65.12%, 72.09% and 82.86%, respectively. Moreover, the strain XY-1, XY-13 and XY-53 promoted the increase of seedlings stem length and chlorophyll content under the infection of pathogenic fungi. In conclusion, XY-1, XY-13 and XY-53 can be used as potential biocontrol resources to control cucumber seedlings root rot caused by F. solani and to simultaneously promote the growth of seedlings for sustainable and efficient cucumber production.

Effects of Biochar on the Soil Properties and Fungal Community Structure under Continuous Cropping of Panax notoginseng

SUN Hai-hang, GUAN Hui-lin, WANG Xu, WANG Tong, LI Hong-lin, PENG Wen-jie, LIU Bo-zhen, FAN Fang-ling

2023, 39(2):  221-231.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0583

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Study the effects of biochar on the physical and chemical properties of Panax notoginseng rhizosphere soil and fungal community structure will be conducive to deeply understanding the soil condition of continuous cropping of P. notoginseng, and to providing scientific basis and theoretical guidance for soil improvement and ecological planting of P. notoginseng. In this study, tobacco stalk carbon TB, rice husk carbon RB were selected as experimental materials, and the treatment without biochar as control CK. A field experiment was conducted to study the effects of two kinds of biochar on the physical and chemical properties of biennial P. notoginseng rhizosphere soil and the structure of fungal community. Analysis was performed by high-throughput sequencing technology. The results showed that the application of biochar improved the survival rate of P. notoginseng, and the pH value, soil available phosphorus, microbial biomass carbon and available potassium promoted the survival rate of P. notoginseng, while ammonium nitrogen inhibited the survival rate of P. notoginseng. Soil pH value presented a positive contribution to acid phosphatase and urease activities, but had an inhibitory effect on invertase. Through the analysis of fungal community diversity, adding two biochar significantly improved the fungal community structure in P. notoginseng continuous cropping land, and improved the diversity and richness of soil fungal in the rhizosphere of P. notoginseng. There was obvious differentiation in the community structures under different treatments. After applying biochar, the relative abundance of Ascomycota significantly increased, and the relative abundance of Basidiomycota and Mortierellomycota significantly reduced. At the genus level, compared with the CK, the relative abundance of Fusarium, Aspergillus and Alternaria decreased significantly（P< 0.05）. The abundance of Botryotinia increased significantly（P< 0.01）. Combined with Pearson correlation analysis and RDA, pH value, nitrate nitrogen, available phosphorus and available potassium were the main environmental factors causing the community structure and diversity of P. notoginseng fungi. Biochar results in the survival rate increasing of P. notoginseng by increasing soil pH value, available phosphorus, microbial biomass carbon content and fungal community diversity and reducing the abundance of pathogenic bacteria.

Whole Genome Sequencing and Analysis of an Anti Gram-positive Bacterium Gordonia WA4-43

HE Meng-ying, LIU Wen-bin, LIN Zhen-ming, LI Er-tong, WANG Jie, JIN Xiao-bao

2023, 39(2):  232-242.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0612

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WA4-43 from the intestinal tract of cockroaches was the first Gordonia that was resistant to gram-positive bacteria. Whole-genome sequencing and comparative genome analysis of WA4-43 revealed the commonness and difference between WA4-43 and other Gordonia, providing a basis for subsequent research. Strain WA4-43 had inhibitory activity only against gram-positive bacteria. The strain WA4-43 was identified as G. terrae by morphological characteristics and molecular biological identification. Genome-wide bioinformatics analysis showed that the strain had a genome size of 5.44 Mb, GC content of 67.76%, and 4 963 protein coding genes, including 3 563, 4 178 and 1 870 genes annotated in GO, COG and KEGG databases, respectively. AntiSMASH predicted 13 secondary metabolite gene clusters in 8 types with an average homology of（22.67±24.72）%. PRISM 4 was used to predict compounds in gene clusters. Comparative genome analysis showed that the G. terrae gene cluster was conserved, but its homology was significantly lower than that of the known gene cluster, and it had the potential to synthesize new compounds.

Phenotypic Characterization of Blue Photoreceptor Plant Type Cryptochrome CRY Mutant in Chlamydomonas reinhardtii

LI Wang-ning, ZHANG Hao-jie, LI Ya-nan, LIANG Meng-jing, JI Chun-li, Zhang Chun-hui, LI Run-zhi, CUI Yu-lin, QIN Song, CUI Hong-li

2023, 39(2):  243-253.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0611

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The phenotypic differences of Chlamydomonas reinhardtii wild strain（CC5325）and CRY mutant strain（crcry）under normal CK（Control）and Blue BL（Blue light）cultures were investigated. The PCR verification showed that the crcry mutant was inserted with paromomycin resistance gene AphVIII expression box in the coding region. AphVIII resistance gene was inserted into crcry mutant and successfully expressed in plate and liquid culture system with paromomycin as screening condition. Phenotypic identification showed that there were no significant differences in growth, pigment, photosynthesis and lipid synthesis between wild strain CC5325 and mutant strain crcry under CK culture condition. However, under BL condition, the growth of mutant crcry was significantly inhibited, and the color of algae liquid turned yellow. Chlorophyll a, chlorophyll b and total pigment contents of unit cells decreased significantly, while total carotenoid contents increased significantly. The photosynthetic system was severely suppressed and the total lipid content significantly reduced. In conclusion, plant cry is involved in the blue light response of C. reinhardtii.

Role of agr System in the Antimicrobial Resistance and Biofilm Formation of Listeria monocytogenes

REN Si-yu, JIANG Cong-yi, YU Tao, KANG Rui, JIANG Xiao-bing

2023, 39(2):  254-262.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0421

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This work aims to investigate the role of agr system in antimicrobial resistance and biofilm formation of Listeria monocytogenes（Lm）. Gene deletion mutants of agr system were constructed and antimicrobial susceptibility of the mutants was determined. The microplate method was used to measure the biofilm biomass at different temperature, and the inverted microscope was applied to observe the biofilm structure. Plate migration method was to investigate the swimming and swarming motilities of strains. RT-qPCR was adapted to detect the relative expressions of motility-associated genes. Reduced antimicrobial resistances to cefotaxime, ciprofloxacin and benzalkonium chloride were observed in the agr deletion mutants. Compared with the wild type EGD-e, the mutant strains showed decreasing biofilm biomass at 37℃, 20℃ and 4℃; and increased swarming motility was observed in ∆agrC. Results from RT-qPCR showed that the transcription level of flaA in the agr deletion mutants decreased. agr system is involved in antimicrobial resistance and biofilm formation of L. monocytogenes.

Whole Mitochondrial Genome and Phylogeny Analysis of Anomala corpulenta

QU Chun-juan, ZHU Yue, JIANG Chen, QU Ming-jing, WANG Xiang-yu, LI Xiao

2023, 39(2):  263-273.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0689

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Anomala corpulenta is an important agricultural and forestry insect pest. In the present study, high-throughput sequencing of A. corpulenta mitochondrial genome was performed using the Illumina HiSeq X platform and the genome sequences were assembled and annotated. Then the structure features and base composition of A. corpulenta mitochondrial genome were analyzed. Based on the nucleotide sequences of 13 protein-coding genes（PCGs）, the maximum likelihood and Bayesian methods were used to reconstruct the phylogenetic relationship of Scarabaeidae. The results showed that the complete mitogenome of Anomala corpulenta was 16 673 bp in size, including 13 PCGs, 22 tRNA genes, 2 rRNA genes and an A+T region with the length of 1 074 bp. The gene arrangement is consistent with those of the known insect species of Rutelinae, and follows the ancestral pattern without gene rearrangement. Phylogenetic analysis showed that all species of Rutelinae belonged to the same branch, confirming Rutelinae to be a monophyly. Genus Anomala was more closely related to Mimela than to Popillia or Adoretus. This is the first report of a complete mitochondrial genome of genus Anomala, the largest genus of Rutelinae, and it will contribute to increasing our knowledge about the mitogenomics and the phylogeny of Rutelinae.

Effects of Escherichia coli Dairy Cow Mastitis on the Expressions of Milk-producing Trait Related Genes

LI Yan-xia, WANG Jin-peng, FENG Fen, BAO Bin-wu, DONG Yi-wen, WANG Xing-ping, LUORENG Zhuo-ma

2023, 39(2):  274-282.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0826

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This work aims to explore the reasons of milk yield and milk quality declining from dairy cows with Escherichia coli mastitis at the genetic level. This experiment established a cell inflammation model by lipopolysaccharide(LPS)-induced bovine mammary epithelial cells（bMECs）. The expressions of milk yield and milk protein-related genes（CSN1S1, CSN2 and LALBA）, milk fat regulation-related genes（FABP3 and LPL）and inflammation and milk yield-related gene MFGE8 were detected by real-time quantitative PCR（qPCR）. The results showed that the expressions of the above 6 genes were significantly down-regulated in the mammary tissues of dairy cows with E.coli mastitis（P < 0.01）compared with the control group（0 h）. In addition, the expressions of the six genes were also significantly down-regulated in LPS-induced bMECs at 3, 6, 12 and 24 h（P < 0.01）. This indicated that the expressions of the six genes were inhibited after E. coli mastitis or bMECs inflammation occurred, which explained the reasons for E.coli mastitis reducing milk yield, milk protein percentage and milk fat percentage.

Establishment and Application of Multigene Engineering Transformation Strategy for CHO Cells

CHENG Jing-wen, CAO Lei, ZHANG Yan-min, YE Qian, CHEN Min, TAN Wen-song, ZHAO Liang

2023, 39(2):  283-291.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0863

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In recent years, CHO（Chinese hamster ovary）cell engineering transformation mainly changes a single function of cells by knock-in or knock-out genes, but the knock-in and knock-out genes often cannot simultaneously play the corresponding functions in a single experimental operation, which limits the application of multigene synchronous transformation. In this study, Bcl-2, the anti-apoptotic gene in the apoptosis pathway, and FUT8, the key enzyme gene of fucose synthesis in the protein fucosylation pathway, were selected as the knock-in and knock-out genes. CRISPR/Cas9 technology was used to establish the site-specific synchronous knock-in and knock-out gene editing strategy. The monoclonal cell line obtained by using this strategy overexpressed Bcl-2 protein and lost FUT8 protease function. After subculture, it was found that the gene expressions of the cell lines obtained by the established site-specific synchronous knock-in and knock-out gene editing strategy were stable within 60 d. Compared with the original wild-type cells, the cell line demonstrated higher tolerance to serum deprivation and stronger resistance to death. This reveals that the gene editing strategy based on site-specific synchronous knock-in and knock-out is feasible and can be used to construct CHO engineering cell lines for recombinant protein production.

Development Status of Synthetic Biology in Globe and Its Enlightenment

WANG Xiao-mei, YANG Xiao-wei, LI Hui-shang, HE Wei, XIN Zhu-lin

2023, 39(2):  292-302.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0352

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Synthetic biology is a new interdisciplinary frontier science, demonstrating broad application prospects in the fields of life science, energy science and technology, medical health, material chemical industry and agricultural science and technology. The research on the development status of synthetic biology is of great significance for government departments, scientific and industrial communities to jointly promote its development. The current situation of synthetic biology policies, scientific research and industry in globe was systematically summarized. Combined with the development status and existing issues of synthetic biology in China, strategic suggestions for its development are put forward. The study showed followings. 1）Synthetic biology has entered a period of rapid development of global consensus, cooperation and competition. Many countries have assisted the scientific research and application innovation of synthetic biology, and produced many new technologies and applications with field characteristics through the top-down research and development system. 2）China is accelerating the improvement of the top-level design of synthetic biology, and a series of original discoveries and innovative achievements in the field has been achieved. However, there are also some shortcomings, such as lagging medium and long-term development planning, insufficient scientific research and innovation ability, the dislocation of application research and development subjects and the limitation of industrial application scenario expansion. 3）It is suggested to promote the development of synthetic biology in China from the aspects of strengthening macro policy guidance, building an efficient research system, cultivating high-quality industrial subjects and expanding the application scenarios of achievements.

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2023, 39(2):  303-303. 

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2023, 39(2):  304-304. 

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