Table of Content

26 June 2023, Volume 39 Issue 6

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Research Progress in Uniconazole Alleviating Plant Drought Damage

DING Kai-xin, WANG Li-chun, TIAN Guo-kui, WANG Hai-yan, LI Feng-yun, PAN Yang, PANG Ze, SHAN Ying

2023, 39(6):  1-11.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1352

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The abnormal global climate change has caused dramatic variation in the water cycle worldwide, and extreme weather and drought and flood disasters have occurred frequently. Drought has become one of the most common non-stress biological stresses in agricultural production. Drought stress can directly or indirectly affect the photosynthesis, osmotic regulation and endogenous hormone levels of plants, thereby reducing crop yield and quality and seriously restricting agricultural production. Uniconazole has the characteristics of high efficiency, broad spectrum and rapidity. It has the functions of dwarfing plants, preventing lodging and increasing the content of chlorophyll. It also plays an important role in plant tolerance and resistance to stress. Exogenous uniconazole alleviates the damage of drought stress on plant physical and chemical processes. In this review we systematically summarized the effects of drought stress on the physical and chemical processes of plants, and clarified the stress response of plants to drought stress from the aspects of photosynthesis, carbon metabolism, stress physiology, endogenous hormone levels and stress resistance gene expression. Further we analyzed the positive effects of exogenous uniconazole on regulating reactive oxygen species metabolism and antioxidant defense system, increasing osmotic adjustment substance content, regulating endogenous hormone levels and inducing gene expression under drought stress. Finally, we pointed out the research status and development trend of exogenous uniconazole alleviating drought stress, which provide direction and basis for future research on drought resistance of crop production.

Applications and Perspectives of Radiation Mutagenesis in Woody Plant Breeding

LI Yu-ling, MAO Xin, ZHANG Yuan-shuai, DONG Yuan-fu, LIU Cui-lan, DUAN Chun-hua, MAO Xiu-hong

2023, 39(6):  12-30.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1008

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Radiation mutagenesis plays an important role in the selection and genetic improvement of new plant varieties. In order to provide breeders with a rapid and accurate picture of the research and application of radiation mutagenesis on woody plants, based on the CNKI database and the official website of the International Atomic Energy Agency, a statistical analysis of the published literature related to radiation mutagenesis in woody plants after 2000 and the released radiation mutant species were carried out. A comprehensive review was sorted out from the aspects of radiation mutagenesis mechanism, factors affecting radiation efficiency, mutant identification and isolation, radiation breeding achievements in this field. The result shows that the mechanism of radiation mutagenesis is still unclear, the key factor affecting radiation efficiency is the appropriate irradiation dose, followed by attention to the sensitive variability of the irradiated material, and mutant identification methods are phenotypic, physiological-biochemical, cytological and molecular markers, which are used in combination for more accurate results. Overall, the number of radiation-breeding woody plant varieties is small and tends to decline gradually. The progress of radiation breeding research in woody plants is reviewed, and solutions to the problems of radiation mutagenesis, such as low mutagenesis efficiency, poor control of mutation direction and imperfect mutant identification methods, are given, and the future directions of radiation breeding research are also prospected, aiming to provide reference and new ideas for plant radiation breeding research and application.

Research Progress of Important Traits Genes in Cassava

XIAO Liang, WU Zheng-dan, LU Liu-ying, SHI Ping-li, SHANG Xiao-hong, CAO Sheng, ZENG Wen-dan, YAN Hua-bing

2023, 39(6):  31-48.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1274

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Cassava（Manihot esculenta Crantz）is an important food crop, cash crop and energy crop in global tropical regions, but the biology research and breeding progress have lagged behind major food crops. Molecular breeding is the important driving force for cassava genetic improvement. Discovery the genes related to important traits is the foundation and premise for the transformation from traditional breeding to molecular breeding. In this paper, systematically, we recapitulate recent progress on the genes related to the traits, such as plant morphology, yield, tuber quality, stress resistance, in cassava, also functional characterization of some genes. We further point out that both of constructing self-crossing population and multiple-omics data integrated are important ways to discovery the key genes in cassava in the future. This paper aims to provide reference for promoting the application of results of functional genome studies in the construction of cassava breeding technology system, and provide theoretical guidance for cassava genetic improvement.

Research Progress in Bioremediation of Cr（VI）

XV Ru-yue, WANG Zi-xiao, SHEN Lu, WU Rong-rong, YAO Fang-ting, TAN Zhong-yuan, LIU Heng-wei, ZHANG Wen-chao

2023, 39(6):  49-60.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1331

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Chromium is a major inorganic pollutant released into soil and water bodies through natural processes and anthropogenic activities, such as rock weathering, volcanic eruptions and the metallurgy, chemical and leather industries. Chromium mainly exists in two different stable oxidation states: Cr（III）and Cr（VI）. Cr（VI）is a widely recognized class I carcinogen due to its high toxicity, strong fluidity and high ecological risk. Therefore, the research on the treatment of Cr（VI）pollution has received extensive attention. Traditional physical and chemical remediation technologies have some disadvantages, such as high execution cost, low efficiency, toxic by-products and inability to be implemented on a large scale. Bioremediation technology improves the limitations of traditional remediation technologies and is a more economical, more environmentally friendly and sustainable green remediation technology. In this paper, the pollution sources and toxicity characteristics of chromium are introduced. The process and mechanism of microbial remediation and phytoremediation are described in detail. Several suggestions on bioremediation of chromium contamination are summarized, aiming to provide theoretical guidance and scientific basis for the application of bioremediation of Cr（VI）contamination.

Research Progress in the Microalgal Growth and Accumulation of Target Products Regulated by Exogenous Phytohormone

LI Yuan-hong, GUO Yu-hao, CAO Yan, ZHU Zhen-zhou, WANG Fei-fei

2023, 39(6):  61-72.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1166

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Microalgae are widely used in bioenergy, functional food, medicine and health care owing to their unique growth advantages and richness in oils, proteins, carotenoids, unsaturated fatty acids and other substances. The use of abiotic stresses（nitrogen deficiency, high light intensity, high temperature, high salinity, heavy metals, etc.）is an effective and traditional means of inducing rapid enrichment of lipids and other metabolites in algal cells; however, it is usually at the expense of the growth of microalgae, which limits the efficient accumulation of target products at the expense of growth. Phytohormones are small molecule signaling substances that regulate algal cell growth and metabolism, including promoting microalgal cell proliferation，enhancing stress tolerance，elevating photosynthetic activity and promoting the accumulation of important secondary metabolites. Therefore, the combination of phytohormones and abiotic stresses can further promote the synthesis of target products and improve the tolerance of microalgae to abiotic stress conditions. Based on this, this paper summarized the types, biosynthetic pathways and physiological functions of phytohormones that have been applied to microalgae culture systems in recent years, analyzed their roles in the responses of microalgale to abiotic stresses and their effects on cell growth and target product synthesis, and discussed the internal mechanism of microalgae resistances to different abiotic stresses under the regulation of phytohormones and the possible mechanism of phytohormone-mediated stress tolerance and lipid accumulation in microalgae. In addition, the opportunities and challenges of exogenous phytohormones are also prospected in the development of microalgae industry, aiming to provide theoretical basis and technical support for the efficient cultivation of microalgae and the accumulation of high value-added products.

Research Progress in Thermophilic Microorganisms for Cellulose Degradation

ZHANG Jing, ZHANG Hao-rui, CAO Yun, HUANG Hong-ying, QU Ping, ZHANG Zhi-ping

2023, 39(6):  73-87.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1240

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Cellulosic biomass is a geographically abundant renewable resource, showing enormous potential to contribute to alleviate the escalating energy and environmental problems. Cellulase is the key to bioconversion of cellulose, and its catalytic effect determines the commercial potential of using cellulose for bioenergy production. In view of the fact that it is difficult for conventional mesophilic enzymes are to adapt to harsh conditions of industrial processing, cellulase from thermophilic microorganisms has attracted the attention of researchers. In this paper, recent progress in thermophilic cellulolytic microorganisms, cellulase types and cellulose degradation strategies was reviewed. Further, the role and application status of thermophilic cellulase in industrial production were discussed. Finally, the technical bottleneck and research emphases of large-scale application of thermophilic cellulose were presented, and the commercial prospect of thermophilic cellulose was prospected.

Research Progress in the Structure of Tailed Bacteriophage and Its Receptors

LI Tuo, LI Long-ping, QU Lei

2023, 39(6):  88-101.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1329

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With the emergence and rapid spread of “super drug-resistant” bacteria, phages have become a research hot spot for antibiotic alternatives and a new way to solve the problem of antibiotic resistance and promote the healthy development of the farming industry. The key to the therapeutic role of phages is their ability of specifically lysing their host bacteria, while the specificity of phage lysis of bacteria depends on the recognition and adsorption of phage receptor-binding proteins to the receptor. Tailed bacteriophages use a broad range of receptor-binding proteins, such as tail fiber, tail spikes and the central tail spike, to target their cognate bacterial cell surface receptors lipopolysaccharide, outer membrane protein, capsule, flagella and pili, etc.，and finally the bacteria are lysed. In the present review, we systematically summarized the research advances in the types and structures of tailed bacteriophage and its receptors. We also discussed the selection strategies of phage therapeutic agents based on the research foundation of phage-host interaction mechanism. It is aimed to provide a solid theoretical foundation for the further study of the mechanism of interaction between phages and their host bacteria, modification of phages and creation of phage biocidal agents.

Research Progress in the Molecular Mechanism of MCR-1 Mediated Polymyxin Resistance

CHEN Yong, LI Ya-xin, WANG Ya-xuan, LIANG Lu-jie, FENG Si-yuan, Tian Guo-bao

2023, 39(6):  102-108.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1058

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The emergence of the polymyxin resistance gene mcr-1 has brought new challenges to the treatment of clinical infections. mcr-1 has been reported from 61 different countries or territories on 6 continents after its discovery. In order to curb the spreading of mcr-1, Chinese Ministry of Agriculture and Rural Affairs proposed a compulsory official banning of polymyxin as a feed additives. Although previous studies have shown that the withdrawal of polymyxin from an animal feed additive may effectively reduce the prevalence of mcr-1 positive bacteria in animal, environmental and human samples, the mcr-1 prevalence fluctuated at a low level. Up to now, 34 mcr-1 variants and 9 different MCR family proteins have been found. It remains to be seen whether they will evolve into a more prevalent MCR subtypes. In addition, recent studies have explored the molecular mechanism of mcr-1-mediated polymyxin resistance and its effect on the bacterial cell wall. This article briefly reviews the latest progress of mcr-1 on epidemic, drug resistance mechanism, and mechanism affecting bacterial fitness cost, aiming to provide a reference for curbing the spread of the polymyxin resistance gene mcr-1.

Research Advances in the Treatment of Inflammation Bowel Disease Using Escherichia coli Nissle 1917

CHEN Cai-ping, REN Hao, LONG Teng-fei, HE Bing, LU Zhao-xiang, SUN Jian

2023, 39(6):  109-118.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1027

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Inflammatory bowel disease（IBD）is a general term of a group of specific intestinal diseases, mainly manifested as chronic and recurrent intestinal inflammation. The burden of IBD has been a global health concern since its incidence and prevalence are elevating rapidly, which has seriously affected people's life and health quality. Over the past decades, tremendous studies underlined the association between incidence of IBD with imbalance of intestinal flora. Therefore, the treatment strategy based on probiotics has become the focus, among of them Escherichia coli Nissle 1917（EcN）has received accumulative attentions. In the current work, we focused on the application and mechanisms of EcN in the treatment of IBD, systematically reviewed the probiotic properties of EcN and its application for treating IBD through regulating epithelial integrity, immune-modulation, mucus protection and gut microbiota homeostasis. Furthermore, the future perspectives on formulating novel therapies based on engineered EcN were discussed, highlighting the applicable and feasible strategies of EcN in IBD curing and prevention. Besides, the prospects for further research in this field were given, providing ideas and references for further in-depth research on the EcN treatment of IBD.

Related Inflammatory Diseases Caused by Mitochondrial Dysfunction and Targeted Therapy to Them

MA Xue-hu, MA Li-hua, GOU Yan, MA Yan-fen

2023, 39(6):  119-125.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1146

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As an essential organelle in eukaryotic cells, mitochondria play a central role in energy metabolism, maintenance of redox balance and regulation of apoptosis. Mitochondrial dysfunction, whether caused by mitochondrial damage, mitochondrial DNA gene mutation or defective mitochondrial electron transport chain, oxidative stress, or abnormal genes and inflammatory signal transduction, will cause certain damages to tissues or organs or lead to inflammation, such as kidney, liver, breast and other organs or tissues. This paper summarized the functions of important components in mitochondria, including mitochondrial DNA, cytochrome c and cardiolipin, and the inflammatory diseases caused by mitochondrial dysfunction, as well as the treatment of mitochondrial dysfunction, aiming to provide more technical support for mitochondrial dysfunction, inflammatory diseases and targeted therapy.

Progress in Selection and Application of Antibacterial Aptamers

LI Dian-dian, SU Yuan, LI Jie, XU Wen-tao, ZHU Long-jiao

2023, 39(6):  126-132.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0860

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One of the major challenges in modern medicine and molecular biotechnology is to explore new therapeutic options for bacterial infections. The increasing rates of multidrug-resistant bacteria, overlaid with the efficacy, development process, and cost of antimicrobial agents, has made the treatments of bacterial infections even more challenging. In response to this situation, the most promising solution is to find alternative sources of antibacterial drugs, and antibacterial aptamers obtained through SELEX screening are promising alternatives. Antibacterial aptamers can reduce the pathogenic abilities of bacteria by interfering with the biochemical processes of bacteria, controlling the formations of bacterial membrane and blocking the infections of toxins. Currently, aptamer-based antibacterial strategies mainly include single aptamer antibacteria, aptamer composite nanomaterials, aptamer composite antibiotics, etc. In this paper, the screening strategies of aptamers for common pathogenic cells or their related components are summarized, and the adaptation and modification of aptamers are discussed from the aspects of antimicrobial mechanism and its application in antimicrobial therapy. The development prospect of antimicrobial strategies based on antibacterial aptamers in the treatment of infection is prospected, aiming to provide new ideas for the diagnosis and treatment of infectious diseases caused by pathogenic bacteria.

High-throughput Specific Detection Methods for Transgenic Maize Based on the KASP Platform

ZHU Shao-xi, JIN Zhao-yang, GE Jian-rong, WANG Rui, WANG Feng-ge, LU Yun-cai

2023, 39(6):  133-140.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1191

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In order to promote the industrialization of transgenic maize and accelerate the transfer of excellent trait transformants and backbone inbred lines, high-throughput foreground selection methods for backcross population need to be developed urgently. In this study, the transgenic maize DBN9936 was used as the material, according to its exogenous insert fragment and its flanking sequence, 6 pairs of specific primers were designed using the primer5 software, combined with the internal standard gene zSSIIb primer for evaluation. After obtaining the optimal primer combination, further the specificity verification, detection limit testing and multi-sample verification were carried out. The results showed that the optimal primer combination was DBN9936-LB1*zSSIIb-k1; the results of KASP genotyping of 10 DBN9936 BC₁ seeds were completely consistent with those of agarose gel electrophoresis; the results of 10 transformants DBN9858, DBN9501, C0010.1.3, C0010.3.1, C0010.3.7, C0030.2.5, BT11, GA21, MIR162 and 2A-7 were negative on the dual platform. The detection limit of transgenic maize DBN9936 was stable at 10%; and the KASP genotyping results of 48 different DBN9936 hybrids were positive. In summary, this method can be used for the foreground selection in the process of backcrossing of transgenic maize DBN9936 with high sample throughput, which provides a technical reference for target gene detection of other crops in transgenic breeding.

SNP Markers Development and Genetic Relationship Analysis of Dendrobium Germplasms Using SLAF-seq Technology

CUI Xue-qiang, HUANG Chang-yan, DENG Jie-ling, LI Xian-min, LI Xiu-ling, ZHANG Zi-bin

2023, 39(6):  141-148.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1386

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The genetic relationship of the collected Dendrobium germplasm resources was analyzed by SNP marker technology to provide theoretical basis for the selection of new varieties breeding parents. Total 60 Dendrobium germplasm resources were used for SNP marker development and genetic relationship analysis using specific-locus amplified fragment sequencing technology（SLAF-seq）. The sequencing data of each sample were statistically analyzed, and 157.34 Mb Clean Reads data were obtained. The Reads data of each sample ranged from 576 195 to 5 359 710. The average sequencing quality value（Q30）and GC content of samples was 93.85% and 40.16%. Through sequencing data analysis, a total of 1 337 217 SLAF tags and 1 049 638 polymorphic SLAF tags were obtained. The average sequencing depth of the tags was 9.63×. A total of 11 248 186 population SNP markers were developed. The number of SNP markers in each sample ranged from 694 015 to 6 367 379. The integrity ratio was 2.71% to 24.89%, and the hetloci ratio was 1.13% to 5.74%. The population SNPs were filtered, and a total of 31 499 highly consistent and effective SNP markers were obtained. The phylogenetic tree was constructed by using the SNP markers obtained. The 60 Dendrobium germplasm resources were divided into 3 subgroups. These three subgroups contained germplasm resources: Q1（3）, Q2（21）, and Q3（36）. The results of germplasm clustering were basically consistent with the morphological classification. Using SLAF-seq technology can efficiently and accurately develop SNP markers suitable for the genetic analysis of Dendrobium. The SNP markers developed may provide molecular basis for Dendrobium breeding, genetic map construction, variety identification and association analysis of agronomic traits.

GC-MS Analysis and Antioxidant Activities of Liposoluble Components from Three Sanghuangporus Fruit Bodies

LUO Yang-lan, CAO Nai-xin, YANG Yu-mei, HUANG Li-ling, YAN Yong, HUANG Shi-Lv

2023, 39(6):  149-157.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1275

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This work aims to study components and antioxidant activities of liposoluble components from different Sanghuangporus sp. fruiting bodies. Soxhlet extraction method was applied to extract the liposoluble components from different Sanghuangporus sp. fruiting bodies and their chemical compositions were identified by GC-MS. Its reducing ability was determined, and the antioxidant activity was evaluated by the reducing capacity, 1,1-diphenyl 1-diphenyl-2-picrylhydrazyl（DPPH）free radical scavenging ability and hydroxyl free radical scavenging ability. The GC-MS results suggested that there were 176 identified liposoluble components in total. Liposoluble components of JM1, JM2 and JM3 were 68, 75, 71 compounds, respectively. After classification and analysis, the compounds mainly contained alkane, ester, aromatic group and other substances. There are 8 common components, and their relative contents account for 12.88%, 9.85% and 9.54% of JM1, JM2 and JM3 extracts, respectively, which were 2,4-dimethylheptane, ethylbenzene, 1,4-xylene, styrene, 6-dodecanone, methyl hexadecanoate, methyl tetracosanoate and tetratetracontane. JM1 liposoluble components had the best antioxidant activities. The liposoluble components of three Sanghuangporus sp. fruiting bodies contained a variety of active substances and had various biological activities, which can provide a reference for the further study of their pharmacological efficacy and resource development.

Development of Single Cell Transcriptome Sequencing Technology and Its Application in Caenorhabditis elegans

ZHAO Jin-ling, AN Lei, REN Xiao-liang

2023, 39(6):  158-170.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1190

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Single cell transcriptome sequencing technology enables people to get rid of the interference of cell heterogeneity，thus realizing the exploration of gene expression and transcriptional regulation mechanism at single cell level，as well as the recognition of different cell subpopulations and special cell types，which is of great significance to the research in the field of phylogeny. The traditional model organism Caenorhabditis elegans has become an important model for single-cell transcriptomic research in recent years because of its fixed number of somatic cells and clear trajectory of cell differentiation. This paper summarizes the development of single-cell transcriptome sequencing technology and its application in the research of cell lineage analysis，single-cell trajectory inference and neuronal cell Atlas of C. elegans，and further prospects the future research direction.

Construction and Functional Study of RagA Transgenic Drosophila

MENG Guo-qiang, GUAN Jian-wen, NIU Chun-mei, ZHOU Ying, SHEN Su-lin, WEI You-heng

2023, 39(6):  171-180.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1341

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Rag GTPase is GTP-binding proteins belonging to a member of the Ras family and localizes on lysosomes. Drosophila RagA protein, a homologue of mammalian RagA/RagB protein, forms a complex with RagC to regulate TORC1 activity. Here we found that knockdown of RagA caused Drosophila development to stagnate at the pupal stage. To explore the function of RagA on Drosophila development, we constructed the overexpressing vector pUASp-RagA-wt with RagA RNA interfering target sites changed. On this basis, the GTP-binding RagA-overexpressed plasmid pUASp-RagA-Q61L and the GDP-binding RagA-overexpressed plasmid pUASp-RagA-T16N were constructed by point mutation. The pUASp-RagA-wt, pUASp-RagA-Q61L and pUASp-RagA-T16N plasmids were screened by microinject and screentransgene lines. The effects of different forms of RagA on the TORC1 activity were evaluated by the method of somatic cell cloning, RagA regulated the TORC1 activity, and it was in the activated state after binding with GTP, and inactivated after binding with GDP. To determine the effects of RagA activity on the Drosophila development under GTP/GDP state, the UASp transgene lines were crossed with Tub-Gal4/TM6 and the eclosion rate of offspring was counted. The results showed that Drosophila with RagA knockdown failed to develop into adults due to obstacles in the pupal stage. The overexpression of wild-type RagA completely avoided the death of Drosophila caused by RagA knockdown, confirming that depletion of RagA was the cause of developmental defects in RagA RNAi. The overexpression of GTP-bound RagA-Q61L partially reduced the lethal effect of Drosophila with RagA knockdown, while the overexpression of GDP-bound RagA-T16N could not decrease the lethal effect of Drosophila with RagA knockdown at all. Our work suggests that RagA plays an important role in the growth and development, and the binding of RagA and GTP/GDP needs to be in a dynamic equilibrium. Binding of RagA only to GTP or GDP would make TORC1 activity too high or too low, thus affecting fruit fly growth and development.

Construction and Identification of Huh7 Hepatoma Cell Line with ACE2 Gene Knockout Based on CRISPR/Cas9 Technology

ZHANG Zu-lin, LIU Fang-fang, ZHOU Qing-niao, ZHAO Rui-qiang, HE Shu-jia, LIN Wen-zhen

2023, 39(6):  181-188.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1251

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The experiment is aimed to establish ACE2（angiotensin converting enzyme 2）-knock out Huh7 liver cancer cells using CRISPR-Cas9, which could be used as cell model to study the role of ACE2 in HCC（hepatocellular carcinoma）. Firstly, the ACE2 domain was identified, and two sgRNAs targeting ACE2 exons were designed using an online website that destroyed all the domains. Secondly, the recombinant vector was constructed and transfected into HCC cell Huh7, and the monoclonal cell line was screened by puromycin. Finally, the knockout effect was identified by immunoblotting. The domain identification results showed that there were Zn-binding sites and activation sites at amino acids 340-520. According to the sgRNA target design principle, two pairs of sgRNA were designed for the ninth and tenth exons of ACE2 by fragment knockout method, and the recombinant plasmid PX459-ACE2-sgRNA was successfully constructed. The monoclonal cell line was successfully screened by puromycin, sequencing confirmed the occurrence of fragment knockout, and ACE2 protein was not expressed in the ACE2 knockout cell line. CRISPR-Cas9 technology was used to successfully construct ACE2 knockout Huh7 cell line, which lays a foundation for future research on the mechanism of ACE2 in HCC.

SiYABBYs Involved in Rhamnoside Biosynthesis During the Flower Development of Setaria italica, Based on Metabolomics

HAN Hua-rui, YANG Yu-lu, MEN Yi-han, HAN Shang-ling, HAN Yuan-huai, HUO Yi-qiong, HOU Si-yu

2023, 39(6):  189-198.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1463

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Flower organ is the premise and basis of reproductive development, and also plays a decisive role at the final stage of spikelet formation. YABBY transcription factors play an important role in the development of flower organs. In order to clarify the function of YABBY gene family and improve the yield of foxtail millet (Setaria italica), we studied the sequence characteristics and expression patterns of YABBY gene family members in the foxtail millet. We analyzed genome-wide identification of YABBY gene family in the foxtail millet, the physical and chemical properties, and tissue expression patterns of SiYABBYs gene. Also, we aligned YABBY sequences of the foxtail millet, Arabidopsis and rice（Oryza sativa）for functional prediction. The results showed that 8 YABBY family members in the foxtail millet, encoding 150-303 amino acids. And we found these SiYABBYs genes were specifically expressed in panicles at heading and pollination stages, which may be involved in the development of vegetative tissues and the formation of flower organs in the foxtail millet. The expressions of SiYABBYs were correlated with the metabolome data at heading and pollination stages of the foxtail millet. The results showed that SiYABBY1/SiYABBY5/SiYABBY7/SiYABBY8 were closely related to rhamnoside metabolic pathway. Also, the results indicated that rhamnoside accumulation in the panicles during the pollination might be related to the fertility and seed setting rate of the foxtail millet.

Physiological Characteristics and Transcriptome Analysis of Sorghum bicolor × S. Sudanense Seedlings Under Salt-alkali Stress

KONG De-zhen, DUAN Zhen-yu, WANG Gang, ZHANG Xin, XI Lin-qiao

2023, 39(6):  199-207.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0758

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Sorghum bicolor × S. sudanense has the characteristics of drought resistance and salt-alkali tolerance, and has gradually become an important feed crop in animal husbandry. It is of great significance to clarify the molecular regulation mechanism of salt-tolerant and alkali-tolerant grass for molecular-assisted breeding. In this paper, the seeds of S. bicolor × S. sudanense were treated with different concentration of NaCl and Na₂CO₃ stress, and the germination rates of the seeds were analyzed under different concentrations. The seedlings were treated with 200 mmol/L NaCl and Na₂CO₃ stress. The physiological indexes of soluble sugar, proline（PRO）, catalase（CAT）, peroxidase（POD）and total superoxide dismutase（T-SOD）were determined and transcriptome expression was analyzed in different time periods. The results showed that the germination rate and root length of S. bicolor × S. sudanense under the same concentration of neutral salt stress were higher than those under alkaline salt stress. With the time prolonging, POD and CAT showed a gradually decreasing trend under neutral salt stress, but gradually increasing trend under alkaline salt stress. PRO, soluble sugar and T-SOD increased gradually under neutral salt stress, but decreased gradually under alkaline salt stress. Transcriptome analysis showed that 241, 293 and 149 DEGs were identified after 6, 12 and 24 h of neutral salt stress, and 664, 641 and 728 DEGs were identified under alkaline salt stress. GO and KEGG cluster analysis revealed that DEGs involved in oxidative reductase synthesis, osmotic stress, cell membrane composition, cell oxidation and detoxification played a key role in the responses to salt-alkali stress the grass in the seedling. DEG mainly focused on hormone signal transduction, photosynthetic metabolism, redox, glucose metabolism, nucleic acid repair, phenylpropane biosynthesis and other processes related to abiotic stress or stress. Under saline-alkali stress, the grass seedlings responded to environmental stimuli through hormone signal transduction and oxidoreductase detoxification. Glucose metabolism and reductase synthesis and transport play an important role in salt-tolerant varieties.

Cloning and Expression of AP2/ERF Transcription Factor Gene ShERF3 in Sugarcane and Subcellular Localization of Its Encoded Protein

ZHAO Xue-ting, GAO Li-yan, WANG Jun-gang, SHEN Qing-qing, ZHANG Shu-zhen, LI Fu-sheng

2023, 39(6):  208-216.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1400

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AP2/ERF transcription factors play an important role on regulating plant growth and development and involving in biotic and abiotic stresses resistances in plants. Cloning and gene function analysis of ShERF3 may provide gene resource for sugarcane resistance genetic improvement. The ShERF3 gene was cloned from‘ROC22’sugarcane plants based on the transcriptome data. Then the structure, subcellular location and gene expression pattern of ShERF3 were analyzed with real time PCR, bioinformation and rice protoplast subcellular location methods. The results showed that 1 142 bp cDNA sequence of ShERF3 was obtained. It contained a 1 053 bp integral opening reading frame encoding 350 amino acids. The predicted encoding protein of ShERF3 containing a conserved AP2 domain belonged to the ERF subgroup of AP2/ERF family. The homology analysis indicated that ShERF3 was highly homologous with ERF3 from Sorghum bicolor and Panicum virgatum and ERF118 from Panicum hallii and Setaria italica. It predicted that ShERF3 is a unstable hydrophobic protein. The subcellular location analysis showed that ShERF3 located in the nulear of rice protoplast cells. ShERF3 mainly expressed in sugarcane mature internodes and relatively lower expressed in the leaves and roots. In addition, the expression of ShERF3 was firstly down-regulated and then up-regulated under PEG treatment. And the expression of ShERF3 decreased with the extension of stress time under NaCl treatment. These results indicates that ShERF3 actively responds to draught and salt stresses. It may play a key role on regulating sugarcane stem development, draught and salt stresses responses.

Identification and Analysis of NAC Transcription Factor Family Genes in Helianthus tuberosus L.

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

2023, 39(6):  217-232.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1111

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The NAC transcription factors closely related to drought stress responses of Jerusalem artichoke（JA）（Helianthus tuberosus L.）leaves were screened by transcriptome method, which may lay a theoretical foundation for the cloning and functional verification of JA NAC gene in the future. In this study, the drought-resistant Qingyu No. 2 JA was used as experimental material. The leaves of JA were collected after treatment for 0, 18, 24 and 36 h, respectively, and the differentially expressed NAC transcription factors were screened and analyzed by transcriptomic sequencing. Referenced as RNA-Sequencing data, 70 NAC genes were identified in JA leaves with sequence length between 408-1 869 bp. The lengths of the detected motif ranged from 21 to 50 bases. Phylogenetic tree analysis showed that most HtNACs genes were clustered with other NAC genes. It was found that HtNAC90-3 was clustered with AtNAC72, HtNAC67-3 was clustered with AtNAC055, HtNAC2-1 was clustered with AtNAC019, HtNAC36 was clustered with SINAC6 through the clustering of NAC genes in other crops related to reported drought resistance. HtNAC83-1 was clustered with AtNAC96. Tissue-specific expression analysis showed relatively high HtNAC expression in seeds and petals. Drought-induced expression analysis showed that 38 and 11 HtNAC genes revealed the increasing expression trend after drought induction in JA leaves and roots, respectively. In this study, it was found that the expressions of HtNACs in seeds and petals was high, especially in petals. Subsequently, HtNACs of JA can be cloned from petals of JA and related functions can be verified.

Chloroplast Genomic Characterization and Phylogenetic Analysis of Colocasia esculenta L. Schoot var. cormosus cv. ‘Hongyayu’ from Jiangxi Yanshan

YIN Ming-hua, YU Huan-yuan, XIAO Xin-yi, WANG Yu-ting

2023, 39(6):  233-247.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1369

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In order to analyze the structure and composition of the chloroplast genome of Colocasia esculenta L. Schoot var. cormosus cv. ‘Hongyayu’ from Jiangxi Yanshan, its evolutionary position in Colocasia genus and its difference from the chloroplast genome of the same genus were determined, which may provide relevant basis for species identification, genetic diversity analysis and resource protection of Colocasia genus. The Illumina NovaSeq 6000 sequencing platform was used to sequence the genome of the chloroplast of C. esculenta L. Schoot var. cormosus cv. ‘Hongyayu’ from Jiangxi Yanshan. Bioinformatics analysis methods were to conduct sequence assembly, annotation and feature analysis, and bioinformatics software such as Geneious, MISA, MAFF, FASTREE, CUSP, Chips and Codon W were used to analyze the genome structure and number, codon preference, sequence duplication, SSR sites and phylogeny. The results showed that the chloroplast genome size of C. esculenta L. Schoot var. cormosus cv. ‘Hongyayu’ from Jiangxi Yanshan was 162 544 bp, showing a tetrad structure. It contained 130 genes, including 84 protein coding genes, 37 tRNA genes, 8 ribosomal rRNA genes and 1 pseudogene. By codon preference analysis, the average number of effective codons was 45.60, and 39 genes with ENC value < 45, indicating strong codon preference. Through SSR analysis, 101 SSR sites were detected, of which single nucleotide repeats were the most（about 83.17%）, and single nucleotide was mainly A/T type. Compared with related species, the chloroplast genome sequence was highly conserved, especially the protein coding sequence was highly similar. In addition, the phylogenetic analysis showed that C. esculenta L. Schoot var. cormosus cv. ‘Hongyayu’ from Jiangxi Yanshan was clustered into one branch with Colocasia genus and Remusatia genus. This study obtained the basic information of chloroplast genome and phylogenetic location of C. esculenta L. Schoot var. cormosus cv. ‘Hongyayu’ from Jiangxi Yanshan, and provided a preliminary study for its species identification, genetic diversity of natural population and functional genomics.

Identification and Expression Analysis of the Tobacco TCP Gene Family

ZHANG Lu-yang, HAN Wen-long, XU Xiao-wen, YAO Jian, LI Fang-fang, TIAN Xiao-yuan, ZHANG Zhi-qiang

2023, 39(6):  248-258.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1141

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TCP gene is a plant-specific transcription factor, and plays an important role in plant growth and development. The identification of tobacco TCP gene family may provide theoretical basis for the study of tobacco TCP gene function and genetic improvement. Based on the whole genome data of tobacco（Nicotiana tabacum L.）, the tobacco TCP gene family was identified by BLAST, and the physicochemical properties, gene structure, protein domain, chromosome distribution, phylogeny evolution and promoter analysis of the family members were analyzed by bioinformatics. The expressions of TCP gene family in various tissues of different flue-cured tobacco varieties were verified via RT-qPCR. A total of 20 TCP genes were identified in tobacco, and they were divided into two categories, Class I and Class II. The known TCP genes were located on different chromosomes and were unevenly distributed, and all TCP genes had conserved domains. The cis-elements of growth, development and hormone response were significantly enriched in the promoter regions of the TCP gene family, and some TCP genes also had low-temperature stress elements; 20 NtTCP genes were expressed to different degrees in different tissues and leaves of K326 and Ti706, and NtTCP genes were expressed in tobacco K326 and Ti706, and the expressions were tissue-specific. The expressions of subfamily Class I genes were higher in 6 tissues and leaves at seedling stage, while the expressions of subfamily Class II genes were higher in seedling leaves, upper leaves and middle leaves. The results revealed that the members of tobacco TCP gene family play an important role in the growth and development of tobacco, which provides a basis for exploring the biological functions of tobacco TCP gene family.

Transcriptome Analysis of Interaction Between Gossypium barbadense and Fusarium oxysporum f. sp. vasinfectum

YANG Yang, ZHU Jin-cheng, LOU Hui, HAN Ze-gang, ZHANG Wei

2023, 39(6):  259-273.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1316

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Cotton Fusarium wilt is a common disease in cotton production, especially very seriously in island cotton（Gossypium barbadense）, affecting its yield and quality, which pose a great threat to the development of island cotton industry. To understand the molecular mechanism of the interaction between cotton and Fusarium oxysporum, RNA-seq sequencing technology was applied to analyze the gene expression characteristics of the interaction between Fusarium oxysporum and cotton using the resistant and susceptible cotton root tissues and pathogenic bodies infected by Fusarium oxysporum for 48 h as materials. The results showed that 15 218 and 9 358 differentially expressed genes were detected in resistant and susceptible cotton varieties respectively, 3 708 and 3 656 differentially expressed genes were identified in Fusarium oxysporum after infecting the resistant and susceptible cotton varieties. Through GO enrichment analysis, we found that the main processes in cotton after interaction were oxidative stress, auxin-activated signaling pathway, response to stimulus, response to injury and transcription factor activity. KEGG pathways were significantly enriched in endocytosis, plant hormone signal transduction, amino acid biosynthesis, carbon metabolism, plant-pathogen interactions, phenylpropane biosynthesis and other metabolic pathways. Disease-resistant cultivars were significantly up-regulated in responses to stimulation and injury. In the Fusarium oxysporum after interaction, GO enrichment analysis found that differentially expressed genes were mostly involved in membrane components, catalytic activity regulation, ATP binding etc. KEGG was significantly enriched in peroxisome, mitogen-activated protein kinase signaling pathways, valine, leucine and L-Isoleucine degradation, glycine, serine and threonine metabolism, carbon metabolism, amino acid biosynthesis, starch and sucrose metabolism and other metabolic pathways. This study provids an abundant gene resources for studying the responses of cotton to Fusarium wilt and the pathogenicity of F. oxysporum, and laid a foundation for an in-depth analysis of the mechanism of interaction between Fusarium oxysporum and cotton.

Growth Promoting of Pinus massoniana Seedlings Regulated by Rhizosphere Phosphate-solubilizing Paraburkholderia spp.

XU Hong-Yun, LV Jun, YU Cun

2023, 39(6):  274-285.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1226

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It is aimed to screen phosphate-solubilizing rhizobacteria（PSB）and to clarify its growth-promoting effect on Pinus massoniana seedlings and the mechanism. The soil-dilution plate method was used to isolate and purify the PSB strains. The PSB strains were identified by morphology and 16s rDNA sequencing methods. Finally, the masson pine seedlings were inoculated with PSB strains for 60 d, then the seedling growth and physiological indexes, soil physical and chemical properties, and rhizosphere bacterial community were determined. The results showed that three PSB strains（WJ10, WJ25 and WJ41）with strong phosphate-solubilizing abilities were isolated from the P. massoniana rhizosphere soil and belonged to Paraburkholderia spp. The three PSB strains had the strongest phosphate-solubilizing abilities to aluminum phosphate, followed by tricalcium phosphate, calcium monophosphate and ferric phosphate. Based on the pot experiment, the three PSB strains effectively promoted seedlings growth, with the best effects on the seedling height and root length promotion for WJ25, followed by WJ41 and WJ10. The main mechanisms of seedling promotion of the three PSB strains include: 1）The PSB strains increased the root activity, chlorophyll b, soluble protein, nitrogen, phosphorus and potassium contents in masson pine seedlings; 2）meanwhile, PSBs enhanced the contents of available phosphorus, available potassium, active nitrogen, soil urease and phosphatase in rhizosphere soil; 3）in addition, the addition of three PSB strains also affected the composition and diversity of the rhizosphere bacterial community, and promoted the significant enrichment of beneficial bacteria such as Bacillus, Nitrosospira, Gemmata and Cytophaga in the rhizosphere soil. In conclusion, the three Paraburkholderia spp. strains could promote masson pine seedlings growth by regulating plant physiology and changing rhizosphere micro-environment. This study provides a theoretical basis for the development and application of rhizosphere PSB fertilizer of P. massoniana.

Functional Analysis of the Type III Secreted Effector Gene aop2 in Acidovorax citrulli

CHEN Bao-qiang, LI Ying-ying, MA Bo-ya, ROUZHAGULI Malike, YOULITUZI Naibi, SONG Jin-di, LIU Jun, WANG Xi-dong

2023, 39(6):  286-297.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1303

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Type III secreted effectors are the key pathogenic factors secreted by Acidovorax citrulli, the pathogen of bacterial fruit blotch（BFB）. To identify the T3SE gene aop2 with GNAT（Gcn5-related N-acetyltransferase）superfamily domain, which is specific for Acidovorax citrulli, and to analyze the way that its encoded protein affects plant immunity, will lay a foundation for further understanding the role of this gene in pathogen pathogenesis. Bioinformatics was applied to analyze the sequence characteristics. Fluorescence quantitative PCR was used to analyze the expression regulation of aop2 and its relationship with the expression of disease-resistant genes. Gene mutation and gene function complementation was to analyze the gene function, including pathogenicity and host reactive oxygen species accumulation. Transient expression technique was used to investigate the ability of Aop2 inhibitory elicitor to induce necrosis and its subcellular localization. There was a core gene binding site of Type III secretion system（T3SS）in the promoter region of aop2 gene, and there was no signal peptide or transmembrane helical region in the encoded protein, and there was a GNAT family acetyltransferase domain but no homologous protein. The expression of aop2 gene in hrpG/hrpX mutant of T3SS significantly decreased. The mutant with aop2 gene deletion decreased the pathogenicity of host cucumber, but increased the accumulation of reactive oxygen species in cucumber cotyledon. Aop2 can be localized to whole tobacco cells and inhibited programmed cell death（PCD）induced by NIP. The expression of Aop2 enhanced the expression of genes involved in SA and JA signaling pathways triggered by pathogen-related molecular patterns in tobacco leaves. The results showed that Aop2 was a specific T3SE containing GNAT domain in A. citrulli. Aop2 played the function of virulence factor in the interaction with cucumber and was involved in the regulation of plant cell death and PTI and phytohormone-related disease resistance in the interaction with tobacco.

Screening Identification and Degradation Characteristics of Three Iprodione-degrading Strains

PAN Hu, ZHOU Zi-qiong, TIAN Yun

2023, 39(6):  298-307.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1372

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To isolate the iprodione-degrading microbial resources suitable for Qinghai-Tibet Plateau, three efficient iprodione-degrading strain Y-20, Y-29 and Y-32 were isolated from the greenhouse soil in Tibet by enrichment culture method. These strains were preliminarily identified based on the morphological characteristics, physiological and biochemical characteristics, and 16S rRNA gene sequence analysis. The effects of NaCl concentration（m/V）, pH value, temperature, inoculation amount（V/V）and initial iprodione concentration on the iprodione-degradation rates of these strains were also investigated. The metabolic pathways of iprodione and ipaH gene in these strains were analyzed using gas chromatography method and polymerase chain reaction technique, respectively. The results showed that strain Y-20, Y-29 and Y-32 were identified as three species of Microbacterium genus. Under the optimal iprodione-degrading condition of 1.0% NaCl（m/V）, pH 7.0, 25-30℃, 5% inoculation amount（V/V）and 100 mg/L initial iprodione concentration, 100 mg/L iprodione was completely degraded by these strains within 8-12 h. These strains decomposed iprodione into N-（3,5-dichlorophenyl）-2,4-dioximidazolidine and isopropyl carbamate, and had a highly similar ipaH gene（99.14%-99.69%）. This study provides the strain resources and theoretical basis for the bioremediation of iprodione-contaminated environment at high altitude area.

Screening, Identification and Degradation Characteristics of Nicotine-degrading Bacteria in Lasioderma serricorne

WANG Yu, YIN Ming-shen, YIN Xiao-yan, XI Jia-qin, YANG Jian-wei, NIU Qiu-hong

2023, 39(6):  308-315.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1198

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The nicotine-degrading bacteria were screened and identified from Lasioderma serricorne, and their degradation characteristics were explored. The nicotine-degrading bacteria were primarily isolated and screened from L. serricorne by gradient dilution method, and re-screening was carried out using the four kinds of medium containing tobacco leaves. The strains were identified based on 16S rRNA gene sequencing combined with physiological and biochemical analysis. The whole genome sequence of the bacteria with degradation capabilities was analyzed to explore the degradation characteristics. Then, the potential degradation genes were explored through molecular docking and RT-qRCR. One bacterial strain J1 was isolated from L. serricorne, which significantly degraded nicotine. The strain J1 was identified as Mixta by 16S rRNA gene homology combined with physiological and biochemical characteristics. The results on the enzyme activities analysis showed that J1 did not produce β-glucosidase, fiber Enzymes, proteases, and amylases. The metabolism pathway and the key genes or enzymes involved in degrading nicotine in J1 were explored through functional annotation in the database. The nicotine dehydrogenase（NDH）possessed by J1 in the pyridine pathway was selected for molecular docking and functional verification. It was verified by RT-qPCR that the relative expression of ndh gene was up-regulated by 15.978 times using 1 g/L nicotine induction for 24 h compared with the control group. Additionally, the relative expression of ndh gene was up-regulated by 1.117 times when J1 was induced by 0.8% tobacco leaf extract for 24 h. The high-efficiency nicotine-degrading bacteria J1 was isolated and screened in L. serricorne. The degradation-related genes were verified. The results provided a new idea for the screening of nicotine-degrading bacteria and simultaneously enriched the nicotine-degrading microbial resources.

Increasing the Expression of FAD-dependent Glucose Dehydrogenase by Recombinant Pichia pastoris Using a Combined Strategy

DONG Cong, GAO Qing-hua, WANG Yue, LUO Tong-yang, WANG Qing-qing

2023, 39(6):  316-324.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1255

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In order to increase the expression of FAD-dependent glucose dehydrogenase（FAD-GDH）in Pichia pastoris, the recombinant strains of co-expressing different copy numbers of molecular chaperone genes were constructed to achieve high-efficiency expression through the G418 concentration gradient screening, and the shake flask fermentation conditions were optimized. One high-yielding recombinant strain was obtained by G418 concentration gradient screening. On this basis, the recombinant strains co-expressing 1-4 copies of three molecular chaperones, HAC1, Ero1 and PDI, were constructed respectively, and the results of RT-qPCR were in line with expectations. The enzyme activity at the test tube level showed that 3 copies of PDI, 3 copies of HAC1 and 2 copies of Ero1 increased by 83%, 77% and 51%, respectively, compared with 1 copy. The recombinant strain with 3 copies of PDI had the highest enzyme activity, which was 198% higher than the control. The optimal conditions of the recombinant strain were as follows: initial pH 7.0, liquid loading volume 50 mL, the methanol addition amount 1%, and induction temperature 28℃. The efficient expression of FAD-GDH in P. pastoris is achieved by screening of antibiotic concentration and increasing the gene copy number of molecular chaperones.

Effects of Knockout of G0S2 Gene in Ovarian Granulosa Cell Proliferation, Steroids Hormones and Related Gene Expression

MA Yu-jing, DUAN Chun-hui, HE Ming-yang, ZHANG Ying-jie, YANG Ruo-chen, WANG Yong, LIU Yue-qin

2023, 39(6):  325-334.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1398

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The objective of this research is to explore the effects of the G0S2 gene on the cell proliferation of ovarian granulosa cells, the secretion of estrogen（E₂）and progesterone（P₄）, as well as the expressions of steroid secretion-related genes and apoptosis genes. Fresh ovaries of small-tailed sheep were collected,and the follicular fluid gathered from small and medium-sized follicles by the suction method was centrifuged to separate the granulosa cells into experimental and control groups. The G0S2 gene in the experimental group was knocked out by CRISPR-Cas9 gene editing technology and the obtained recombinant expression vector PX458-sgRNA-G0S2 was transfected to the granulosa cells. The PX458 plasmid in the control group was transfected to the granulosa cells. The viability and apoptosis of the granulosa cells were detected, as well as the E₂ and P₄ concentrations and associated gene expression. The results showed that the relative abundance and the protein level of G0S2 mRNA in the experimental group were lower（P<0.01）than those in the control group, reduced by 66% and 70% respectively, proving that the G0S2 was successfully knocked out. The proliferation activity of the granulosa cells in the experimental group was higher（P<0.05）than the control group, and the apoptosis rate reduced（P<0.01）by 56%. Compared with the control group, the E₂ concentration was higher（P<0.05）and the P₄ concentration was lower（P<0.05）in the experimental group. The expressions of genes StAR, CYP11 and 3β-HSD in the experimental group was lower（P<0.01）than the control group, and the expressions of CYP19 was significantly higher（P<0.01）than the control group. Compared with the control group, the expression of Caspase3 and Bax in the experimental group of granulosa cells was significantly downregulated（P<0.01）, and the expressions of Bcl-2 was significantly upregulated（P<0.01）. Our results demonstrate that knockout of the G0S2 gene can promote the proliferation of ovarian granulosa cells, inhibit their apoptosis, and affect the secretion of E₂ and P₄ by downregulating the expression of genes StAR, CYP11, 3β-HSD and upregulating CYP19 in sheep.