Superintended by: Ministry of Agriculture and Rural Affairs of the People’s Republic of China
Sponsored by: Agricultural Information Institute of CAAS
Editor in Chief: XIE Qi
Monthly, Started in 1985
ISSN 1002-5464
CN 11-2396/Q
Biotechnology Bulletin has been indexed by the following databases: Chemical Abstracts (CA),JST SinoMed, Chinese Science Citation Database(CSCD), Chinese Science and Technology Paper Citation Database(CSTPCD), CNKI.
26 January 2024, Volume 40 Issue 1
Research Progress in the Compartmentalization of Plant Specialized Metabolism
GUAN Zhi-jing, SUN Chao
2024, 40(1):  1-11.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0510
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Plant specialized metabolism(PSM)can be compartmentalized at multiple levels,which plays important roles in plant growth and development,as well as responses to environmental stress. Here we reviewed the recent advance in the compartmentalization of PSM at the molecular,subcellular,cellular and organ levels. Due to the compartmentation of PSM,the intermediates of biosynthesis pathway have to shuttle between different compartments. Therefore,the transporters are an important part of the compartmentalized biosynthesis system of specialized metabolites. The studies of the compartmentalization of PSM and its related transporters not only expand our knowledge about PSM,but also provide new targets and research strategies for the synthetic biology of natural products.

Research Progress in the Composition Analysis and Ecological Function of Plant Root Exudates Under Salt Stress
WANG Yu-qing, MA Zi-qi, HOU Jia-xin, ZONG Yu-qi, HAO Han-rui, LIU Guo-yuan, WEI Hui, LIAN Bo-lin, CHEN Yan-hong, ZHANG Jian
2024, 40(1):  12-23.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0516
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Salt stress as one of the common abiotic stresses can affect plant growth and development. Root exudates are considered as an important medium for “information” exchange between plants and rhizosphere environment. When plants sufferred in salt stress, the composition and content of root exudates would change to affect the growth and development of plants. This article summarizes the components, detection methods, mechanism of action and changing trends. Root exudates mainly contained amino acids, sugars, organic acids and phenolic acids, etc. The high performance liquid chromatography(HPLC), gas chromatography-mass spectrometry(GC-MS), liquid chromatography-mass spectrometry(LC-MS)and nuclear magnetic resonance(NMR)are widely used to identify and detect compositions and contents of root exudates. Combined with previous studies, the changes of composition and content of amino acids, sugars, organic acids, etc. under salt stress were summarized. From the aspect of physical and chemical environment and rhizosphere microorganisms, the putative roles of root exudates under salt stress were also summarized, including: 1)acting as signal molecules in plant suffering to salt tolerance; 2)maintaining the homeostasis of root cells; and 3)affecting rhizosphere environment. The change mechanism of amino acids and sugars in salt tolerance of plants were enumerated. Moreover, we pointed out that, the identification of root exudates function and their mechanisms of action on salt stress need further being studied. This review aims to analyze the ecological effects of plant root exudates under salt stress, and provides a reference for further exploring the mechanism of plant response to salt stress in the future.

Advances in Genetic and Molecular Mechanisms of Pre-harvest Sprouting in Rice
LIN Xin-yan, ZHANG Chuan-zhong, DAI Bing, WANG Xin-heng, LIU Jian-feng, WEN Li, XU Xing-jian, FANG Jun
2024, 40(1):  24-31.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0786
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Pre-harvest sprouting refers to the phenomenon that rice tends to induce seed germination on the spike if it encounters continuous cloudy and rainy weather or high-temperature and humid environmental conditions before harvest. Pre-harvest sprouting leads to decreased seed vigor and quality of rice, resulting in huge losses to rice production. In addition to expanding the screening of pre-harvest sprouting mutants, excavating and cloning some new genes controlling pre-harvest sprouting and analyzing their spike germination regulatory mechanisms are important tasks for breeding rice for pre-harvest sprouting resistance. During the process of pre-harvest sprouting, rice amylase activity is enhanced, soluble sugar content is elevated, and the content and balance of phytohormones ABA and GA in rice seeds are the key factors determining pre-harvest sprouting. Some key genes such as OsVP1 control rice seed dormancy through the ABA signaling pathway, while GA regulates seed germination through the activation of GA-associated transcription factors. The article reviews the four areas, i.e., internal physiological factors and environmental conditions of rice pre-harvest sprouting, genetic mechanisms of rice pre-harvest sprouting, molecular mechanisms of rice pre-harvest sprouting, and trait improvement of rice pre-harvest sprouting, and thus aiming to provide theoretical references for the selection of varieties that are resistant to pre-harvest sprouting in rice.

Research Progresses and Application in the Growth-promoting Effect of Symbiotic and Epiphytic Bacteria on Green Tide-causing Ulva prolifera
BI Fang-ling, ZHAO Shuang, LI Bin, LI Ai-qin, ZHANG Jian-heng, HE Pei-min
2024, 40(1):  32-44.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0652
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The migration and large-scale outbreak of green tides are not only related to the biological characteristics such as the strong stress resistance of algae and the environmental conditions of the sea area, but also inseparable from the participation of algal microorganisms. The microbial community plays an important role in algal growth and extinction. Under different environmental conditions, the community structure of symbiotic and epiphytic microorganisms of Ulva prolifera are diverse. The promotion to algae growth is mainly reflected in the processes of algae morphogenesis, growth, nutrient absorption and photosynthesis. During the occurrence of algal blooms, U. prolifera could help the algae to efficiently absorb nutrients, promote algae proliferation, and thus increase the formation of green tides. Based on previous researching results, we summarized the current research status of symbiotic and epiphytic bacteria of U. prolifera, the dominant species of green tide, described the establishment process of the separation technology of symbiotic and epiphytic bacteria of U. prolifera, and pointed out the importance of molecular biology techniques such as high-throughput sequencing in the analysis of microbial community structure and diversity, analyzed the influence of these bacteria on the physiological characteristics of the growth, reproduction, morphogenesis of U. prolifera and the outbreak of the green tides, and explored the utilization value of symbiotic and epiphytic bacteria in future production and life, aiming to express new ideas for predicting and preventing and controlling the outbreak of green tides and for the algal utilization as a resource.

Research Progress in Three Major Mycotoxins and Their Toxin-degrading Enzymes
XIANG Xia, ZHU En-heng, HAN Nan-yu
2024, 40(1):  45-56.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0596
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Feed during production and storage is often contaminated with mycotoxins, mainly including aflatoxin, zearalenone, vomitoxin, fumonisin B1, ochratoxin A and T-2 toxins. Mycotoxins cause serious physical injury or even death to livestock and poultry, and the coexistence of mycotoxins will lead to greater economic losses. The degradation of mycotoxins mainly includes chemical degradation, physical degradation and biological enzymatic hydrolysis. Biological enzymatic hydrolysisis more environmentally friendly and efficient than the other two methods, thus it has attracted much attention. In this paper, we analyzed the harmful mechanisms, degradation pathways and related mycotoxin-degrading enzymes of aflatoxin, zearalenone and vomitoxin with strong toxicity and wide pollution in detail. We revealed the interaction between toxin small molecules and mycotoxin-degrading enzymes in degradation reactions by molecular docking and other means, and screened the key amino acids in the degradation process. Although enzymatic hydrolysis possesses advantages in removing mycotoxins, its current application is still limited due to high cost and other reasons, and further research and development are urgently needed. Therefore, optimizing the process and conditions of enzymatic hydrolysis to achieve efficient and cost-effective removal of mycotoxins will be the focus of future research. This study provides an important reference to guide the design and optimization of mycotoxin-degrading enzymes.

Research Progress in the Regulation of Chlorogenic Acid Biosynthesis and Its Application
CHEN Zhi-min, LI Cui, WEI Ji-tian, LI Xin-ran, LIU Yi, GUO Qiang
2024, 40(1):  57-71.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0728
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Chlorogenic acid(CGA)is an important phenolic acid secondary metabolite widely found in plant kingdom. CGA plays an important role in plant growth and development, resistance to biotic and abiotic stresses. In addition, it has a multiple biological activities and pharmacological functions, and plays an important role in application potential in anti-inflammatory, antibacterial and hypoglycemic. However, the content of CGA is usually very low in plants, which seriously restricts its development and utilization values. Therefore, effectively improving CGA content in plants is particularly crucial. Recently, many researchers have made important advances on improving the CGA content in plants by genetic engineering, stress and hormone treatment. On this basis, researchers have also initiated new explorations on the biosynthesis of CGA and its molecular mechanism. This would provide new ideas for improving the CGA content in plants. In view of this, this paper reviews the research progresses on the structure and function, biosynthesis and regulation of CGA, and systematically analyzes the effects of key rate-limiting enzymes, such as PAL, C4H and 4CL, is involved in the synthesis of CGA. Moreover, we elucidate the role of transcription factors MYB, WRKY and bHLH in regulating the biosynthesis of CGA. Meanwhile, we comprehensively summarize the effects of exogenous factors, e.g., biotic/abiotic stresses, plant hormones, light quality and photoperiod on the CGA content and biosynthesis in plants,and introduce the mechanism of CGA in improving animal and human health. Finally, we explore and prospect the unsolved problems and future research directions in the CGA. It is aimed to offer some valuable references for the rational development and utilization of CGA and the improvement of crop resistance.

Research Progress in the Production of Polyunsaturated Fatty Acids by Yarrowia lipolytica Cell Factories
HE Si-cheng, ZHANG Zi-yuan, HAN Yu-qing, MIAO Lin, ZHANG Cui-ying, YU Ai-qun
2024, 40(1):  72-85.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0657
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Polyunsaturated fatty acids(PUFA)stand as linear olefinic fatty acids that assume a pivotal role as structural constituents within biofilms in living organisms. They wield the power to govern glucose, lipid, and hormone metabolism, yielding an array of physiological functions. Their capabilities encompass fostering developmental progress, bolstering immunity, preventing disease, and the essential maintenance of overall bodily well-being. Consequently, PUFA hold significant intrinsic worth and expansive developmental potential across diverse fields such as functional food, medicine, and animal feed. In contrast to traditional marine biological extraction techniques, microbial synthesis approaches offer distinct advantages, encompassing shortened production cycles, streamlined procedures, and environmental compatibility. In recent years, the utilization of microbial cell factories for generating PUFA and other microbial oils has emerged as a focal point within both scientific and industrial communities. A standout player in this arena is Yarrowia lipolytica, an unconventional oleaginous yeast that stands out due to its inherent high-yield lipid and fatty acid production capacity. As substantiated by the publication of Y. lipolytica's complete genome sequence and the rapid advancements in gene expression vectors, genetic transformation methods, synthetic biology components, and gene editing technologies, the domain of metabolic engineering focused on Y. lipolytica as a chassis cell for microbial production is undergoing gradual expansion. This article commences by meticulously reviewing the origins and intrinsic pathways that underpin the natural synthesis of PUFA. It subsequently encapsulates the contemporary landscape of research, pinpointing metabolic engineering strategies applied to reconfigure Y. lipolytica into a PUFA producer. It further presents an in-depth examination of the central hurdles associated with the deployment of the engineered Y. lipolytica strains for PUFA production. Culminating with foresight, the article sketches out the overarching developmental trends in harnessing Y. lipolytica cell factories for large-scale industrial PUFA production, aiming to provide theoretical support and ideas for establishing the microbial cell factories in efficiently synthesizing PUFA.

Research Progress in the Production of Ergothioneine by Biosynthesis
LI Liang, XU Shan-shan, JIANG Yan-jun
2024, 40(1):  86-99.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0663
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Ergothioneine(ERG), a rare natural sulfur-containing histidine derivative, has been proved to have strong antioxidant property and many biological functions. Therefore, ERG has been received much more attention from researchers and product developers. Currently, ERG has been widely used in food, cosmetics and medical industries. Research shows that ERG only can be synthesized by a few bacteria and fungi. Plants, animals and humans cannot synthesize ERG directly, but it could be obtained by other sources. ERG can be obtained by bioextraction, chemical synthesis, and biosynthesis. However, because of the low yield and poor production efficiency of the traditional methods(bioextraction and chemical synthesis), the large-scale production and application of ERG is limited. Therefore, there is an urgent need for an efficient, economical, safe and reliable ERG synthesis method to meet market needs. With the rapid development of synthetic biology, the use of genetic engineering, protein engineering and metabolic engineering to improve the ability of ERG biosynthesis has gradually become an increasingly favored method. This paper will elaborate the biological characteristics and functions, briefly introduce the biosynthetic pathways of ERG and application prospects of ERG in food, cosmetics and medical industries, compare the main production methods of ERG, and summarize and sort the research progress of adopting various engineering strategies to produce ERG by biosynthesis in recent years, and propose several engineering strategies on how to use genetic engineering, protein engineering and metabolic engineering to increase the yield of ERG, which aims to provide theoretical reference and research ideas for biosynthesis high-yield of ERG.

Structure and Biosynthesis of Surfactin as well as Its Role in Biological Control
WANG Jun-fang, HUANG Qiu-bin, ZHANG Piao-dan, ZHANG Peng-pai
2024, 40(1):  100-112.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0509
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As a lipopeptide produced by various strains of Bacillus spp., surfactin is composed of a cyclo-heptapeptide head group connected to β-hydroxy fatty acids with a chain length of 12 to 17 carbon atoms by endosomal bonds. It is a very powerful biosurfactant and has significant biological functions. In this paper, we reviewed the structure, biosynthesis model and the role of surfactin lipopeptides in biological control of plant diseases. Surfactin is synthesized by non-ribosomal peptide synthetases, which endows the structural diversity of surfactin family members. Previous studies found that the action mechanism of surfactin lipopeptides in biological control of plant disease mainly are in four ways. 1)It caused damage to the cell membrane of pathogen, resulting in its cell membrane rupture or osmotic pressure imbalance. 2)It inhibited the reproduction of pathogen. 3)It induced systemic resistance(ISR)in plants. 4)It promoted the colonization of biocontrol strains or the formation of their biofilms. The current information about surfactin using genetic engineering techniques is also highlighted to guide the biosynthesis of surfactin and to develop new derivatives. Surfactin is rich in physical and chemical properties due to its high structural diversity, which can be associated with a variety of biological activities and may have a wider range of applications in different fields. With the increasingly in-depth research on the biosynthesis of surfactin, more new lipopeptides with high performance and wide application will be developed, which will provide the basis for further research and biocontrol application of surfactin.

Effects of Intestinal Microbiota on Host Adaptive Immunity
LIU Yan, SUN Jing, GE Liang-peng, MA Ji-deng, ZHANG Jin-wei
2024, 40(1):  113-126.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0497
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The intestinal microbiota coevolves with the host, forming an inseparable host-microbe symbiosis. These symbiotic microbes play an important role in the host immune system by participating in the development and maintenance of adaptive immunity. The adaptive immune system maintains the body's homeostasis through B cell-mediated humoral immunity and T cell-mediated cellular immunity. Intestinal microbiota can directly regulate the differentiation and activation of B and T cells, thus protecting the host from pathogen infections. This article reviews the regulatory effects of intestinal microorganisms on host early immune system development, cellular immunity and humoral immunity, aiming to provide a theoretical reference for studying the regulatory effect of “microbe-host interaction” on host adaptive immunity.

Optimization of DNA Base Editing Technology and Its Application in Pig Genetic Modification
YANG Shuai-peng, QU Zi-xiao, ZHU Xiang-xing, TANG Dong-sheng
2024, 40(1):  127-144.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0445
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Base editing technology originated from CRISPR/Cas system is the latest site-specific gene modification technology at present. According to the function characteristics of the base editor, the base editors can be divided into cytosine base editor(CBE), adenine base editor(ABE), glycosylase base editor(GBE), adenine translation base editor(AYBE), dual base editor(DBE)and prime editor(PE). Since the emerging of base editing technology, it has been widely used in the research of animals and plants. And it has been proven to have great application value in animals and plants for genetic improvement or disease treatment. As an important agricultural economic animal and excellent disease model, genetic improvement to pig has become very important. Base editing technology has been rapidly applied in genetic improvement of animals and plants due to its convenient operation, high efficiency, fewer by-products, and high cost-effectiveness, and even it have a high prospect of providing important support for human gene therapy. In this paper, we focus on the development, optimization, application characteristics, existing issues and future prospects of base editing technology, and briefly summarize its application in pigs. It is expected to provide reference for relevant scientific workers to understand the base editing technology.

Application Progress of Spatially Resolved Metabolomics in Disease Diagnosis Research
HE Shi-yu, ZENG Zhong-da, LI Bo-yan
2024, 40(1):  145-159.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0351
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Mass spectrometry imaging(MSI)based on spatially resolved metabolomics(SRM)has been recently developed to be a powerful technology. SRM can not only directly identify and quantify the metabolites in animal and plant tissues or cells in situ, but also use overall or local features of the image to determine the structure, content, spatiotemporal dynamic change, and spatial distribution of metabolites. It also may identify potential metabolic biomarkers. This technology has the advantages of high sensitivity, high resolution, judicious visualization and label-free, and therefore has been more and more widely applied in clinical disease diagnosis and research. This review summarizes some of the technological advances of SRM in recent years, and its emerging application in the fields of tumor, neuropsychiatric disorder, diabetes mellitus, among others, in an attempt to guide future application of SRM in disease diagnosis.

A Novel SNP Marker for the Identification of Persimmon(Diospyros kaki)Cultivars
LIANG Jin-jun, ZHU Su-yuan, ZHANG Yu-qin, ZHANG Peng-fei, WEN Peng-fei, YANG Yun-liang
2024, 40(1):  160-167.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0363
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【Objective】In order to identify the genetic differences among persimmon(Diospyros kaki)cultivars, a simple and efficient SNP molecular marker based on nrDNA internally transcribed spacer sequence was developed, which may provide reference for the collection, utilization and promotion of germplasm. 【Method】The leaves of 18 hexaploid cultivated persimmon cultivars were used as experimental materials, and the ITS regions of 18 cultivars were amplified and sequenced, and sequence differences were analyzed. Finally, Sau96 I restriction endonuclides were used to verify 151 heterozygous sites. 【Result】Sequencing results showed that ITS length of 18 persimmon varieties was 730 bp. Six heterozygous loci were found in the sequencing peak map, which were 151, 168, 205, 278, 279 and 622 bp. The proportion of base peak area of hexaploid persimmon heterozygous sites had special pattern, that is, C:T= 2:1, C:T= 1:1, C:T= 1:2 and A:G= 1:1. The 18 cultivated persimmon varieties could be divided into 11 categories according to the differences of heterozygous sites and the proportion of peak area. 【Conclusion】The results of enzyme digestion at 151 sites verified that the concentration of enzyme digestion products was consistent with the proportion of its peak map area. The results showed that the SNP molecular labeling method based on nrDNA ITS sequence could divide 18 cultivated persimmon varieties into 11 groups.

Scaling up Production of pDNA Plasmids in Disposable Bioreactors
YANG Hong-yan, HAN Xiao, YANG Jian-jun
2024, 40(1):  168-175.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0691
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【Objective】The objective is to explore the feasibility of using WAVE bioreactor for Escherichia coli strain amplification after the strain recovered without using shake flask and expanding the seed cells into a disposable XDR bioreactor for plasmid production, as well as to establish a process for GMP grade plasmids production on a fully disposable upstream platform. 【Method】Optimizing the carbon to nitrogen ratio for E.coli growth, basic culture medium and supplementary culture media that do not contain any animal derived components were screened.Directly inoculating E.coli strain to shake flasks and WAVE bioreactor after strain thawing, the feasibility of low-density innoculation was investigated, and the differences of E.coli strain amplification in shake flask and in WAVE bioreactor was compared. The expansion process of E.coli strains in WAVE bioreactor was established, and then the seed cells were amplified into a 50L XDR bioreactor for plasmid production. 【Result】Compared with traditional LB medium, the optimized basic medium without any animal origin ingredients increased the maximum OD600 and plasmid yield by 43% and 77% respectively. After E.coli strain thawing, WAVE25 bioreactor was directly inoculated with a low density of 1:1 000-1:8 000. The specific growth rate of the E.coli strain in WAVE25 bioreactor reached (0.65±0.065)/h. WAVE bioreactor showed better capability for controlling process parameters, and the seed cells from one WAVE bioreactor directly inoculated 50-200 L production fermentor for plasmid production. The maximum OD600 and plasmid titer reached 53 OD and 340 mg/L respectively, the specific plasmid yield reached 6.42 mg/L/OD600, which was more than 2 times higher than that of conventional plasmid process. The proportion of supercoiled plasmids reached 90% at upstream harvest, which made it possible for downstream two-step chromatography purification of plasmids. 【Conclusion】In conclusion, a full disposable production platform has been established to directly amplify E. coli strain through WAVE bioreactor, and the seed cells from one WAVE bioreactor can be used to inoculate 50-200 L production fermentor for GMP grade plasmid production.

Advances in the Biological Modification of Galactose Oxidase
XIA Guang-li, CAO Na, SUN Hui-hui, ZHAO Ling, CAO Rong
2024, 40(1):  176-185.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0699
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As an important biological catalyst, galactose oxidase has the characteristics of high catalytic efficiency and environmental protection. It has been widely used in biosensors, food, medical treatment, chemical industry and other fields. However, natural galactose oxidase has some inherent defects such as poor stability, low catalytic efficiency, and high recovery cost, which greatly limit its application in food safety, biomedicine, and other fields. With the development of genetic engineering and protein engineering, galactose oxidase can be purposefully modified to improve the performance of the enzyme or to obtain recombinant enzymes with new functions. Therefore, the pursuit of galactose oxidase with important catalytic capacity and powerful functions has become a research hotspot in this field. In this paper, the molecular structure and catalytic mechanism of galactose oxidase are reviewed, and the relevant cases of biological modification of galactose oxidase are analyzed and summarized, aiming to provide new ideas and methods for galactose oxidase related researchers.

Aptamer of Pseudomonas aeruginosa: Acquiring and Application
LIU Xing-yu, LI Jie, ZHU Long-jiao, LI Xiang-yang, XU Wen-tao
2024, 40(1):  186-193.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0336
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Pseudomonas aeruginosa is an obligate aerobic Gram-negative bacterium, which is the most important microorganism causing opportunistic infections in humans and poses a variety of health threats to humans. Thus, rapid, sensitive and specific detection is crucial for the prevention and treatment of P. aeruginosa infection. At present, there are problems for conventional detection methods,such as time-consuming, high cost and difficult operation, thus more efficient and economical detection methods are needed. Aptamers are oligonucleotides screened from random libraries that can form a variety of complex secondary structures. In recent years, aptamers have attracted widespread attention due to their ease of synthesis and modification, high affinity and strong specificity, and numerous studies of detection, the prevention and treatment of P. aeruginosa based on aptamers have emerged. In this review, the current progresses in screening and tailoring of P. aeruginosa aptamers were summarized, and the application of its aptamers were introduced from two aspects of detection and control of P. aeruginosa, aiming to provide a reference for the development and application of P. aeruginosa aptamer.

Functional Analysis of TaMYB1 Gene in Wheat Under Cadmium Stress
ZHANG Yi, ZHANG Xin-ru, ZHANG Jin-ke, HU Li-zong, SHANGGUAN Xin-xin, ZHENG Xiao-hong, HU Juan-juan, ZHANG Cong-cong, MU Gui-qing, LI Cheng-wei
2024, 40(1):  194-206.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0672
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【Objective】Members of the MYB family play key roles in diverse biological processes including regulation of plant growth and development, biotic and abiotic stress. However, there were few researches specifically focusing on the function of MYB transcription factors in the response to cadmium(Cd)stress in Triticum aestivum. Therefore, identifying Cd-responsive genes and illustrating their biological function may lay a foundation for wheat Cd-resistant or low-Cd breeding. 【Method】Firstly, the yeast cDNA library of wheat roots under Cd stress was constructed, and then the Cd-resistant gene was screened. Then, the expression patterns of Cd-resistant gene in different tissues under Cd stress were examined by quantitative real-time PCR(RT-qPCR). The function of the gene was verified by virus-induced gene silencing, and physiological and biochemical indicators such as the Cd content, chlorophyll content, malondialdehyde(MDA)content, superoxide dismutase(SOD)and peroxidase(POD)activities of the control and gene-silenced plants were detected. Finally, the candidate Cd-resistant gene and its homologous genes in other plants were analyzed by evolutionary relationship, gene structure and expression profile in detail. 【Result】The results indicated that root development of wheat was mainly inhibited under Cd stress. Total 18 positive transformants were screened by cDNA library on a tolerance of 0.002 mol/L Cd, and five transformants encoded TaMYB1 protein, which grew well while the control yeast strains restrained in high Cd medium. Results of RT-qPCR indicated that TaMYB1 responded to Cd stress in the wheat. Compared to the control, TaMYB1 expression in TaMYB1-silencing wheat significantly decreased and the concentration of Cd in the roots and leaves as well MDA content were remarkably lower than that in the control plants, while the contents of chlorophyll and the activities of SOD and POD increased apparently in TaMYB1-silencing plants. Meanwhile, bioinformatics analysis found that TaMYB1 belonged to the 1R-MYB family with highly conserved MYB and CC domains, containing seven exons and six introns. TaMYB1 expressed the highest in the inflorescences of two nodes or internodes visible stage and maximum stem length reached stage, while the lowest in in pre-filling seeds and mid- and late-filling seeds. 【Conclusion】TaMYB1 plays a important role in wheat response to Cd stress.

Mechanism of Exogenous Calcium Alleviating Salt Stress Toxicity in Wheat Seedlings
JIAO Jin-lan, WANG Wen-wen, JIE Xin-rui, WANG Hua-zhong, YUE Jie-yu
2024, 40(1):  207-221.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0583
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【Objective】This paper aims to explore the mechanism that exogenous calcium maintains the homeostasis of wheat seedlings under salt stress when autophagy participates in regulating. These results will provide molecular evidence for revealing the response mechanism of plants to salt stress. 【Method】The wheat variety ‘Henong 6425' was used as experimental material. The wheat seedlings were cultured hydroponically. And then some physiological, biochemical, and molecular biology methods were adopted to carry out the research, including chlorophyll fluorescence parameter analysis, in-situ staining of tissues, quantitative real-time PCR(RT-qPCR), and confocal laser scanning microscope technique. 【Result】The results indicated that 150 mmol/L NaCl stress significantly inhibited the growth of wheat seedlings, led to damage to photosystem II(PSII), weakened antioxidant capacity, decreased cell viability, and increased autophagy activity. With the extension of NaCl treatment time, the number of autophagosomes in the roots and leaves of wheat seedlings significantly increased, and the level of programmed cell death(PCD)in the roots and leaves also increased. Exogenous application of Ca2+ alleviated the damage of NaCl stress to wheat seedlings and regulated the autophagy level in the wheat roots and leaves. Under normal growth conditions, exogenous application of Ca2+ treatment had no significant effect on the number of autophagosomes in the roots and leaves of wheat seedlings. Under NaCl stress, exogenous Ca2+ treatment maintained autophagy in the roots and leaves of wheat seedlings at a pro-survival level induced by NaCl stress, and reduced the PCD level in the roots and leaves of wheat seedlings, and slowed down the toxic effect of NaCl stress on wheat seedlings. 【Conclusion】These results indicate that Ca2+ controls the scale of PCD by regulating autophagy levels in the roots and leaves of wheat seedlings, alleviating the damage of NaCl stress to wheat seedlings.

Genome-wide Association Study of Seed Glucosinolate Content in Brassica napus
ZHOU Hui-wen, WU Lan-hua, HAN De-peng, ZHENG Wei, YU Pao-lan, WU Yang, XIAO Xiao-jun
2024, 40(1):  222-230.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0611
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【Objective】To explore SNPs and candidate genes significantly associated with seed glucosinolate content in rapeseed is helpful to improving rapeseed quality and cultivating high quality rapeseed varieties. 【Method】Total 300 oilseed rapeseed inbred lines grew in the experimental field of Jiangxi Agricultural University(JXAU)and Jiangxi Institute of Red Soil and Germplasm Resources(JXIRS), and the seed glucosinolate content(SGC)was analyzed after harvested. A total of 201 817 SNPs developed by SLAF-seq in all populations were used for genome-wide association with study(GWAS)of SGC. The 100 kb flanking regions on either side of significantly associated SNPs were used to identify candidate genes, and to perform functional annotations of these genes. 【Result】The SGC of these populations showed phenotypic variations under two environments. Based on the general linear model(GLM)the mixed linear model(MLM), 209 SNPs were detected to have significant association with SGC, and 41 SNPs on A05(1), A09(36)and C09(4)were detected by GLM and MLM under JXAU and JXIRS. The functional annotation of candidate genes showed that eight candidate genes participated in the glucosinolate biosynthetic process(GO:0019761), including regulating the glucosinolate synthesis related genes MYB28BnaC09g05290D and BnaC09g05300D), MYB34BnaA09g05480D), and the glucosinolate transporter 2 related genes(BnaA09g06180D and BnaA09g06190D). 【Conclusion】The SNPs significantly associated with SGC were detected under two environments by GLM and MLM, and the candidate genes were mined at the flanking of SNPs, which is conducive to resolving the genetic variation of SGC and providing a basis for the genetic improvement of new rapeseed varieties with low SGC.

Screening Multi-functional Rhizobacteria from Maize Rhizosphere and Their Ehancing Effects on Winter Wheat-Summer Maize Rotation System
CHANG Lu-yin, WANG Zhong-hua, LI Feng-min, GAO Zi-yuan, ZHANG Hui-hong, WANG Yi, LI Fang, HAN Yan-lai, JIANG Ying
2024, 40(1):  231-242.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0690
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【Objective】In China's wheat-maize production region, the Huang-Huai-Hai Plain primarily employs a winter wheat-summer maize rotation cropping system. Sandy loam soil, widely distributed in the Huang-Huai-Hai region, demonstrates various properties and associated challenges, including poor structural integrity, and weak water retention and nutrient-holding capabilities. To improve fertilizer utilization, enhance soil fertility, optimize crop yields, and improve quality, we identified a multi-functional plant growth-promoting rhizobacteria(PGPR)and validated its broad-spectrum growth-promoting effects within this rotation system. 【Method】From the sandy loam soil within the maize rhizosphere, we isolated a multi-functional PGPR bacterium and evaluated its ability of producing indole-3-acetic acid(IAA), organic phosphorus solubilization, and potassium release ability. We identified the bacterium at the species level using morphological, physiological, biochemical, and 16S rDNA sequence analysis. Under shaken culture conditions, we determined the optimal conditions for IAA production by PGPR YM3. Subsequently, we assessed its growth-promoting ability through maize pot experiments. We verified its broad-spectrum yield-increasing effects in a field trial within the winter wheat-summer maize rotation system. 【Result】1)a multi-functional root-associated growth-promoting bacterium named YM3 was isolated, identified as Bacillus subtilis, and found to produce 59.21 mg/L IAA, solubilizing 0.72 mg/L organic phosphoruing, and releasing 18.56 mg/L potassium. The optimal conditions for IAA production by YM3 were achieved when using maltose and peptone as carbon and nitrogen sources, respectively, within a pH range of 6-8 and a liquid volume of 25 mL/250 mL. 2)Maize pot experiments revealed that soil IAA, available phosphorus, and available potassium levels significantly increased by 75.00%, 48.66%, and 20.00%, respectively, compared to soils treated with heat-inactivated YM3 bacterial suspension. Maize seedlings exhibited substantial improvements in root length, root surface area, root volume, root tip number, and root branch number, by 67.95%, 59.21%, 51.13%, 71.34%, and 92.06%, respectively. Additionally, maize plants had significant increases in fresh weight, plant height, relative chlorophyll content, total nitrogen, total phosphorus, and total potassium, with enhancements of 39.86%, 23.51%, 18.27%, 17.68%, 52.26%, and 36.53%, respectively. 3)Field trials within the winter wheat-summer maize rotation system demonstrated that soil available nitrogen, available phosphorus, and available potassium levels significantly increased by 9.08%, 13.78%, and 16.66%, respectively, with a yield increase rate of 42.18% upon YM3 bacterium agent application. Maize field soil showed notable increments of 19.18% and 15.95% in available phosphorus and available potassium, respectively, with a yield increase rate of 13.22%. 【Conclusion】The isolated B. subtilis strain YM3, possessing the ability to produce IAA, solubilize organic phosphorus, and release potassium, demonstrates strong adaptability and broad-spectrum growth-promoting effects in sandy loam soil of the Huang-Huai-Hai Plain. Moreover, it enhances soil fertility and elevates yields within the winter wheat-summer maize rotation system.

Cloning and Expression Analysis of ClPP2C3 in Citrullus lanatus
ZHU Yi, LIU Tang-jing, GONG Guo-yi, ZHANG Jie, WANG Jin-fang, ZHANG Hai-ying
2024, 40(1):  243-249.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0709
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【Objective】Protein phosphatase (PP2C) is a type of protein phosphatase that exists in both animals and plants, In order to explore its important role in the ripening process of watermelon fruits.【Method】In this study, a PP2C gene ClPP2C3 was cloned in watermelon (Citrullus lanatus) by reverse transcription- polymerase chain reaction (RT-PCR), and its bioinformatics, expression pattern and subcellular localization analysis were conducted. 【Result】The cDNA sequence length of ClPP2C3 in watermelon was 1 317 bp and it encoded 438 amino acids. The molecular weight of ClPP2C3 protein was 47.81 kD and its isoelectric point was 5.12. The ClPP2C3 protein contained one PP2C conserved domain and it had high homology with tomato SlPP2C3 in tomato and FaABI1 in strawberry that negatively regulated fruit ripening. ClPP2C3 in the watermelon was located in the nucleus. The gene quantitative expression analysis showed that the expression of ClPP2C3 in high-sugar-containing watermelons was significantly higher than that in low-sugar-containing watermelons. The 2 kb promoter (up 2 kb to ATG) activity of cultivated watermelon was significantly higher than that in ancestral watermelons, while there was no significant difference in the 1 kb promoter (up 1 kb to ATG) activity detected between these two species. 【Conclusion】The SNPs in 1-2 kb promoter region might result in the difference of promoter activity in different varieties.

Identification and Analysis of WRKY Gene Family in Rheum palmatum L.
WU Zhen, ZHANG Ming-Ying, YAN Feng, LI Yi-min, GAO Jing, YAN Yong-Gang, ZHANG Gang
2024, 40(1):  250-261.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0326
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【Objective】This study provides a basis for the functional verification of WRKY genes, and then provide a scientific basis for the transcriptional regulation mechanism research into rhubarb quality formation. 【Method】In this study, we identified WRKY gene family members in R. palmatum L. based on full-length transcriptome data, and performed a series of bioinformatics analysis, including physicochemical properties, protein domains and phylogenetic tree. RNA-seq was used for gene tissue expression analysis, along with expression profile in response to MeJA treatment. 【Result】53 gene members in RpWRKYs family with complete ORFs, and the numbers of amino acids encoding the protein ranged from 192 to 748 bp, all were hydrophilic proteins, and the predicted localization was all in the nucleus. RpWRKYs combined with A. thaliana WRKYs were clustered into three groups, i.e., subgroup I, II and III, and group II with the highest number of WRKYs, accounting for 58.49%. The RNA-seq analysis revealed that RpWRKYs were differentially expressed in the roots, rhizomes and leaves of R. palmatum L., seven of them genes were highly expressed in the roots and rhizomes. Twelve RpWRKY genes showed differential expressions in the response to MeJA treatment, ten of them were significantly induced by MeJA. The expression profile of RpWRKY5, RpWRKY6, RpWRKY9 and RpWRKY33 verified by qPCR was almost consistent with the transcriptome results. RpWRKY2, RpWRKY16, RpWRKY20, RpWRKY22 and RpWRKY23 interacted with chalcone synthase (CHS) gene. 【Conclusion】Expression profiles of 53 R. palmatum L. WRKY genes, bioinformatics, tissues and response to MeJA were obtained, five of which may be related to the synthesis of rhubarb anthraquinones.

Structural and Functional Analysis of MtCIM Gene in Medicago truncatula
XIE Hong, ZHOU Li-ying, LI Shu-wen, WANG Meng-di, AI Ye, CHAO Yue-hui
2024, 40(1):  262-269.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0484
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【Objective】CIM(cytokinin induce messager)belongs to the Expansin protein family, which plays a critical role in plant growth and development. To analyze the structure, expression, subcellular localization, and function of MtCIM during Medicago truncatula growth and development. 【Method】Bioinformatics tools were employed to conduct a comprehensive study of molecular weight, theoretical isoelectric point, instability index, and protein structure prediction and analysis. Meanwhile, RT-qPCR method was used to measure the gene expressions under different hormone treatments(IAA, ABA and GA3). Fusion protein transient expression and yeast expression methods were used to identify its subcellular localization and transcriptional self-activation, respectively. Analysis of MtCIM-transgenic plants was performed in terms of their adaptation to a low phosphorus environment. 【Result】Bioinformatics analysis showed that the CIM protein had a relative molecular weight of 29.563 kD, theoretical isoelectric point of 5.54, and instability index of 41.62, indicating its protein property were unstable. The secondary and tertiary structure predictions showed that the protein was mainly composed of irregular curls. Exogenous IAA significantly increased the expression of MtCIM, while the expression of MtCIM showed a trend of rising and then decreasing after exogenous ABA and GA3. The subcellular localization results showed that it was located in the cytoplasm. Upon analysis of transcriptional self-activation, it was found that there existed no self-activation activity in MtCIM. Analysis of the 20 obtained transgenic plants revealed that the cell walls of the leaves from these transgenic lines were thicker. Moreover, the transgenic plants demonstrated superior adaptability to low-phosphorus environments. 【Conclusion】A comprehensive analysis of the MtCIM gene in M. truncatula revealed its key role as a member of the Expansin protein family in plant growth and development. The phenotype of the transgenic plants demonstrated that MtCIM enhanced the adaptability of plants in low-phosphorus environments. These findings provide new insights for further understanding the role of the Expansin protein family in plant growth and development.

Cloning and Expression Pattern Analysis of Jasmonic Acid Receptor Gene McCOI1a in Mentha canadensis L.
TANG Wei-lin, KANG Qin, WANG Xia, SHEN Ming-yang, SUN Xin-jiang, WANG Ke, HOU Kai, WU Wei, XU Dong-bei
2024, 40(1):  270-280.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0789
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【Objective】The jasmonic acid(JA)receptor COI1 plays a vital role in regulating plant growth and development, and stress response in plants. Cloning and analysis of the protein characterization, and analyzing expressions of McCOI1a from Mentha canadensis L. may provide the gene resources for its molecular breeding. 【Method】The McCOI1a gene was cloned from the leaves of M. canadensis based on transcriptome data, and then the characteristics, subcellular localization, and expression profiles of McCOI1a were analyzed through bioinformatics analysis, transient expression in the leaves of tobacco, and real-time quantitative PCR. 【Result】The full-length of McCOI1a gene was 1 842 bp and it encoded 613 amino acids. McCOI1a protein possessed the conserved F-box and LRR domains and that was highly homologous with Salvia miltiorrhiza SmCOI1. Subcellular localization results showed that McCOI1a protein was located in the nucleus. The qRT-PCR results showed that McCOI1a was broadly expressed in different tissues, with the highest expression in the root; McCOI1a expression gradually increased during leaf development. In addition, the expressions of McCOI1a in the leaves were up-regulated to varying degrees under MeJA, drought, NaCl, AlCl3, CdCl2, and CuCl2 treatments, and the up-regulation was the most obvious under AlCl3 treatment, with the highest expression reaching 1 206-fold. And McCOI1a expression in the root was firstly down-regulated and then up-regulated under CuCl2 treatment, while the expression of McCOI1a showed varying degrees of down-regulation under other treatments. 【Conclusion】McCOI1a responds to MeJA and abiotic stress, and may play an important role in regulating the growth and development, and stress response in M. canadensis.

Cloning of bHLH96 Gene and Its Roles in Regulating the Biosynthesis of Peppermint Terpenes
WANG Bin, YUAN Xiao, JIANG Yuan-yuan, WANG Yu-kun, XIAO Yan-hui, HE Jin-ming
2024, 40(1):  281-293.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0641
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【Objective】Peppermint essential oil is an important raw material in the cosmetics, pharmaceutical, and food industries. bHLH transcription factors(TFs)play a crucial role in regulating the biosynthesis of volatile secondary metabolites(VSMs)in plants. Exploring the role of bHLH TFs in the regulation of VSM biosynthesis in peppermint may provide important gene resources for improving peppermint germplasm through metabolic or genetic engineering, and expand our understanding of the pathways and regulatory mechanisms of plant VSM biosynthesis. 【Method】The expression pattern of bHLH96 in peppermint roots, stems, and leaves was analyzed using transcriptome sequencing and qRT-PCR techniques. The full-length of bHLH96 coding sequence(CDS)was cloned using the RT-PCR method. Subsequently, a plant expression vector, pBI121-bHLH96, was constructed using DNA recombination technology, and the overexpression of bHLH96 in the peppermint leaves was achieved through Agrobacterium-mediated transformation. The effects of bHLH96 overexpression on the content of terpenoid compounds and the expression of related genes involved in terpenoid biosynthesis in the peppermint leaves were examined. 【Result】The results showed that the full length of bHLH96 CDS was 861 bp encoding 286 amino acids. Peppermint bHLH96 protein localized at the nucleus, and the sequence similarity among plant bHLH96 proteins ranged from 45.45% to 73.68%. Peppermint bHLH96 had a close relationship with plant bHLH proteins from the Labiatae family such as Lavandula angustifolia LaMYC4 and Salvia miltiorrhiza SmbHLH94. Transient overexpression of the bHLH96 gene in peppermint leaves significantly influenced relative contents of 15 terpenoid compounds. bHLH96 overexpression significantly upregulated the expressions of 5 terpene synthesis-related genes, such as OC2, SM1, KS1, ND, and EAO1, while downregulated the expressions of NLS1, LS1 and iPR. 【Conclusion】Peppermint bHLH96 might regulate terpene synthesis by modulating the expressions of terpene synthesis-related genes and influencing the activities of terpene synthases.

Isolation and Identification of Bacteria in Forest Rhizosphere Soil and Their Biological Activity Screening
FENG Lu-yao, ZHAO Jiang-yuan, SHI Zhu-feng, MO Yan-fang, YANG Tong-yu, SHEN Yun-xin, HE Fei-fei, LI Ming-gang, YANG Pei-wen
2024, 40(1):  294-307.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0586
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【Objective】Functional strains with multiple biological activities were excavated from the forest rhizosphere soil of Wuliangshan National Nature Reserve, and their potential for development and application was explored. 【Method】The 25 rhizosphere soil samples from different regions and plants in Wuliangshan National Nature Reserve were collected. The active strains with functions such as phosphate dissolution, nitrogen fixation, zinc dissolution, and antibacterial activity were isolated and identified using selective culture medium. The biological activities of these strains, such as secreted siderophore, ACC deaminase, and indoleacetic acid, were tested while verifying their effectiveness in promoting tomato seed germination and plant growth. 【Result】70 phosphorus-dissolving bacteria, 27 nitrogen-fixing bacteria, 8 potassium-dissolving bacteria, and 51 antibacterial bacteria were isolated and identified. Among them, YIM B08401 and YIM B08402 were identified as Burkholderia alba and Pseudomonas qingdaonensis based on morphological, physiological, biochemical characteristics, and 16S rDNA sequencing. The two strains both had the activities of phosphate lysis, nitrogen fixation, zinc lysis and siderophore secretion. Their maximum soluble phosphorus content was(455.63±59.65)mg/L and(878.95±64.78)mg/L. The results of seed germination promotion tests of the two strains were similar, and the germination rates of the two strains were maintained at 82%-93% after applying the fermentation supernatant diluted 10 times, 102 times and 103 times, which was significantly higher than 56% and 49% in the control group. The length of the treatment group with the fermentation liquid of the strain significantly increased compared with the blank control group. Pot experiment showed that the above ground length, fresh weight, dry weight, stem diameter, root length, fresh weight and dry weight of the two strains in the treatment group with the most obvious growth promotion effect were significantly better than that in the control group. Compared with the control group, the above indexes of YIM B08401 significantly increased by 89%, 495%, 268%, 62%, 53%, 385% and 469%, and the above indexes of YIM B08402 significantly increased by 118%, 528% and 477%, 55%, 37%, 413% and 747%, respectively. In addition, strain YIM B08401 also had activities of antagonizing pathogenic bacteria and secreting ACC deaminase, and YIM B08402 also had the activity of secreting indoleacetic acid. 【Conclusion】The rhizosphere soil in the forest of Wuliang National Nature Reserves contained a variety of strains with biological activity. B. alba YIM B08401 and P. qingdaonensis YIM B08402 demonstrates the potential for the development and application of new microbial fertilizers.

Structural Characteristics of Sarcomyxa edulis Polysaccharide and Its Mechanism of Regulating Macrophage Immunomodulatory Activity
WANG Zi-xuan, SUI Yu, SHANG Xue-yu, MA Si-jia, WU Tian-xiang, LIU Yang, WANG Qi
2024, 40(1):  308-321.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0483
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【Objective】In order to clarify the structural characteristics of Sarcomyxa edulis polysaccharides and explore its mechanism of regulating macrophage immunomodulatory activity. 【Method】The crude polysaccharide of S. edulis was obtained by water extraction and ethanol precipitation, and the purified polysaccharide component SEP-0a was obtained by DEAE-52 ion exchange column chromatography and Sephacryl S-400 glucan gel column chromatography. The physicochemical properties of SEP-0a were investigated by high performance gel permeation chromatography evaporative light-scattering detector, ion exchange chromatography high performance liquid chromatography and Fourier transform infrared spectrometer. On this basis, the immunomodulatory effect of polysaccharide SEP-0a from S. edulis on macrophage cell RAW264.7 was further studied by CCK-8, ELISA, qPCR, Western Blot and other methods. 【Result】The S. edulis polysaccharide SEP-0a was composed of galactose, mannose, glucose and fucose with a relative molecular weight of 4.23×104 Da, and had α and β configurations. The results of in vitro immunocompetence showed that S. edulis polysaccharide SEP-0a significantly increased the proliferation and phagocytic ability of RAW264.7 cells, promoted the secretion of reactive oxygen species(ROS), nitric oxide(NO)and increased the expressions of tumor necrosis factor-α(TNF-α), interleukin(IL)-1β, and IL-6 in these cells. Western blot results showed that this polysaccharide significantly increased p65 protein phosphorylation expression in the nuclear factor-κB(NF-κB)signaling pathway. 【Conclusion】The polysaccharide SEP-0a of S. edulis may regulates the immune activity of macrophages by NF-κB signal pathway.

Site-directed Saturation Mutagenesis to Improve the Catalytic Performance of 11α-hydroxylase from Aspergillus ochraceus
SHI Jing-hui, CHEN Wen-hui, LU Kun, ZHENG Ting-ting, REN Zhi-yuan, BAO Guo-qing, WANG Min, LUO Jian-mei
2024, 40(1):  322-331.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0751
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【Objective】11α,17α-dihydroxyprogesterone is an important steroid hormone drug intermediate with high application value. The 11α hydroxylase CYP68J5 from Aspergillus ochraceus CICC 41473 is a key enzyme for the bioconversion from 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone, but its catalytic performance needs to be further improved. 【Methods】Based on the key amino acid sites D118, F216 and M488 identified in our previous works, the excellent mutants were screened by site-directed saturation mutagenesis and substrate transformation experiments. Molecular docking between enzymes and substrates was performed using AutoDock, and the intermolecular interaction forces and molecular dynamic simulations were studied by Discovery Studio and Gromacs, respectively. 【Results】Mutants D118V, F216W, M488L, and M488W possessed catalytic properties, among which, D118V showed the highest activity. At a substrate concentration of 0.5 g/L, the production intensity of D118V was 431.66 mg/(L·d), which was 2.12-fold higher than that of the wild type CYP68J5. This might be attributed to the fact that a new hydrophobic interaction was generated between the site and substrate when the aspartic acid at position 118 was changed to valine, consequently enhancing the hydrophobicity of the substrate-binding pocket of the enzyme. Molecular dynamics simulations showed more stable overall conformation of the mutant and tighter binding effect between enzyme and substrate. 【Conclusion】The excellent mutant D118V with significantly improved catalytic performance was obtained by site-directed saturation mutagenesis. The results provide an application case and theoretical guidance for the molecular modification of steroid 11α hydroxylase.

Effects of Commensal Microbiota on Intestinal Development, Metabolism, and Mitochondrial Function in Piglets
ZHANG Jin-wei, WU Yuan-xia, SUN Jing, LI Xiao-kai, LU Lu, LI Zhou-quan, GE Liang-peng
2024, 40(1):  332-343.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0492
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【Objective】Using pig as animal model to study the regulatory effects of commensal microbiota on host intestinal development and metabolism.【Method】Germ free (GF) piglets and specific pathogen free (SPF) piglets were cultivated through techniques such as aseptic cesarean section and aseptic feeding, and used to study the effects of commensal microbiota on intestinal morphology, metabolism, gene expression and mitochondrial function of piglets through morphological observation, liquid chromatography analysis, RNA seq, and other methods.【Result】Commensal microbiota affected the intestinal morphology and structure, short-chain fatty acid content, amino acid metabolism and mitochondrial content of intestinal cells. Commensal microbiota affected the overall gene expression of the liver, ileum and colon of piglets, and regulated the expression of genes related to biological processes such as mitochondrial oxidation and phosphorylation, amino acid metabolism and lipid metabolism, resulting in the changes in mitochondrial function, thus affected the absorption and metabolism of intestinal nutrients in piglets. 【Conclusion】Commensal microbiota affected the development and metabolism of piglet intestines by regulating the mitochondria of intestinal cells, which provided a theoretical foundation for the study on the regulation of "microbe-host interaction" in piglet’s intestinal health.

Effects of Bacillus pumilus on the Intestinal Inflammation and Barrier Function of Goat Kids
JIAO Shuai, FU Yu-ze, CUI Kai, ZHANG Ji-xian, WANG Jie, BI Yan-liang, DIAO Qi-yu, ZHANG Jian-xin, ZHANG Nai-feng
2024, 40(1):  344-352.  doi:10.13560/j.cnki.biotech.bull.1985.2023-0498
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【Objective】This experiment was conducted to investigate the effects of Bacillus pumilus on growth performance, diarrhea, serum indexes, colonic tissue morphology, inflammatory cytokines and mucosal protein contents of goat kids. 【Method】Thirty-six one-day-old male Saneng dairy goat kids with birth weight of about 3kg were randomly allocated to 4 groups with 9 replicates per group and 1 goat kid per replicate. The goat kids were fed milk replacer containing Bacillus pumilus solution 0(CON group), 1 mL(BP1 group), 5 mL(BP5 group)and 10 mL(BP10 group)every day, respectively. The viable count of B. pumilus was 1×108 CFU/mL. The experiment lasted for 14 d. 【Result】Feeding B. pumilus had no significant effect on the final weight, average daily gain, average daily feed intake and feed to gain ratio of goat kids(P > 0.05), but decreased the fecal score and diarrhea frequency(P < 0.05). The content of aspartate aminotransferase in the serum of BP1 group was lower than that of control group(P < 0.05), and the content of diamine oxidase was lower than that of control group and BP5 group(P < 0.05). Compared with control group, the content of D-lactic acid decreased in the other three groups(P < 0.05). The thickness of colonic muscle layer increased in BP1 group(P < 0.05). Compared with the control group, the contents of IL-1β, IL-6 and TNF-α in BP1 group, BP5 group and BP10 group decreased(P < 0.05). The contents of IL-10, TGF-β, IFN-γ, PPAR-γ, MUC2, Claudin-1, Claudin-4, Occludin and ZO-1 increased(P < 0.05), and neutrophil cell infiltration in colon tissue decreased. 【Conclusion】Feeding Bacillus pumilus to goat kids may reduce the frequency of diarrhea, relieve inflammatory reaction, and enhance mucosal barrier function of goat kids, but there is no significant effect on growth performance.