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    26 January 2023, Volume 39 Issue 1
    Commercialization Strategy of Transgenic Soybean in China
    YU Hui-lin, WU Kong-ming
    2023, 39(1):  1-15.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0667
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    Soybean is one of the most important agricultural products that are closely related to people's life and economic and social development. At present, the improvement of soybean production level and increasing soybean self-sufficiency rate are major problems that must be solved in China's agricultural development. Due to the shortage of cultivated land resources in China, scientific and technological innovation is the only way to improve soybean production capacity. Transgenic breeding is a key technology to promote the development of soybean production, and it has played an important role in the development of soybean industry in the world's major producers such as the United States, Brazil and Argentina. In last 20 years, the breeding techniques of herbicide-tolerant and insect-resistant soybean have obtained a great progress and some products can be used for commercialization in China. And the commercial planting of transgenic soybeans can significantly reduce productive cost and raise soybean yield. Based on the developmental progress of transgenic soybean techniques and the characteristics of soybean production in China, the following strategy is suggested to boost the commercialization scientifically and orderly. Firstly, in terms of product application, based on the single trait product for glyphosate tolerance, and the products with multiple genes for two or more herbicide tolerant traits such as glyphosate and glufosinate tolerance, as well as for stacking of herbicide tolerance and insect resistance traits, the commercialization of the above seeds should be conducted in order. Secondly, in terms of product regional layout, based on occurrence and regional distribution characteristics of target weeds and pests, top-level design of growing zones to herbicide-tolerant and insect-resistant soybean products should be performed. Thirdly, in terms of biosafety management, population monitoring and management techniques for resistant weeds and pests should be developed to extend service period of genetically modified products. In the meantime, the conservation of wild soybean germplasm resources should be strengthened to reduce the negative impact on biodiversity of wild soybean caused by gene flow from transgenic soybean in natural eco-system.

    Commercialization of Biological Breeding in China: Opportunities and Policy Issues
    XIE Wei, LIU Chun-ming
    2023, 39(1):  16-20.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0808
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    Along with the pilot projects on cultivating genetically modified(GM)maize and soybean in China, the large-scale commercialization of GM crops has reached a critical moment in the country. To orderly promote the commercialization of GM crops in China, this study first reviews the global and China's history of the commercialization of GM crops. Second, it analyzes two major opportunities of commercializing GM crops in China: 1)The increasing import and indirect consumption of GM crops in China have improved the acceptance of downstream processing industries and consumers to GM crops; 2)China's R&D investment and technical reserves of GM technology over the last decades have made excellent foundation for the country's commercialization of GM crops. Finally, this study proposes improving consumers' awareness of GM crops, making the best use of the technical reserves of GM technology, and strengthening the supervision to the whole process of commercializing GM crops to extend the cultivating area of GM soybean and maize in China.

    Identification of Target Traits and Genetic Stability of Transgenic Maize 2HVB5
    LI Sheng-yan, LI Xiang-yin, LI Peng-cheng, ZHANG Ming-jun, ZHANG Jie, LANG Zhi-hong
    2023, 39(1):  21-30.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0536
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    In order to confirm the target traits and genetic stability of transgenic maize 2HVB5,Southern blot, ELISA, bioassay in laboratory and field, target herbicide-tolerance analysis and agronomic traits investigation were carried out on BC5S1 and BC5S2 transgenic maize 2HVB5 plants which had been backcrossed to Zheng 58. The results showed that the target genes cry2Ah-vp and bar were integrated into maize genome in the form of single copy and steady inheritance in 2HVB5 plants. Cry2Ah-VP and PAT proteins were expressed in different stages and different tissues in 2HVB5 plants, and the expressions in the leaves were relatively high, reaching 2-3.5 μg/g fresh weight(FW)and 8-17 μg/g FW respectively. The bioassay results in laboratory showed that 2HVB5 plants had high resistance to armyworm(Mythimna separate)and cotton bollworm(Helicoverpa armigera), the corrected mortality rates of armyworm and cotton bollworm larvae reached 100% on 4-5 d, and the 2HVB5 plants presented a significant weight inhibiton to fall armyworm(Spodoptera frugiperda). The bioassay results in field also showed that 2HVB5 plants had high resistances to armyworm and cotton bollworm, the average resistance indexes were 1.19-1.29 and 0.60-0.73, respectively. The transgenic maize 2HVB5 tolerated 4 times the recommended medium dose for glufosinate application in field, and there were no significant differences in agronomic traits compared with the control Zheng 58. These indicate that transgenic maize 2HVB5 is of genetic stability and high insect resistance and herbicide resistance. It can be used for the control of corn pests, especially Noctuidae pests, and has a prospect of commercial application.

    Spatio-temporal Expression of Cry1Ab/Cry2Aj Insecticidal Protein in Genetically Modified Maize Ruifeng 125 with Stacked Insect and Herbicide Resistance Traits
    LI Dong-yang, XIAO Bing, WANG Chen-yao, YANG Xian-ming, LIANG Jin-gang, WU Kong-ming
    2023, 39(1):  31-39.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0339
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    Maize is one of the most important food crops in China, but pests reduce the quality and yield of maize production. Genetically modified(GM)insect resistant maize expressing Bacillus thuringiensisBt)toxins are new tools for integrated pest management. The insecticidal effect of Bt-transgenic maize is closely related to the expression of exogenous insecticidal protein. Evaluating the expression of Bt insecticidal protein in different growth stages and tissues is of significance to the integrated pest management and biosafety management of agricultural GM organisms. In this paper, the expression of Cry1Ab/Cry2Aj insecticidal protein in different tissues of Ruifeng 125 planted in 9 places in two years was determined by ELISA. The Bt insecticidal protein expressions in the leaves at jointing stage(V6-V8), tassels at tasseling stage(VT), leaves and filaments at silking stage(R1), leaves and grains at maturity stage(R4)were comparatively analyzed. It was found that the expressions of Bt insecticidal protein in different tissues of maize varied largely. The results showed that the expression of Cry1Ab/Cry2Aj insecticidal protein in the leaves was higher than that in the kernels. The expressions of Cry1Ab/Cry2Aj insecticidal protein of Ruifeng 125 from 9 different places were different, but the overall difference was slight.

    Insect Resistance Identification and Agronomy Traits Analysis of Transgenic Maize HGK60 with Different Genetic Backgrounds
    LI Peng-cheng, ZHANG Ming-jun, WANG Yin-xiao, LI Xiang-yin, LI Sheng-yan, LANG Zhi-hong
    2023, 39(1):  40-47.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1227
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    In order to study the genetic stability and insect resistance of transgenic maize HGK60 under different genetic backgrounds, HGK60 transgenic maize harboring Bt cry1Ah gene was used as donor, and the cry1Ah gene was introduced into inbred line Zheng 58, Chang 7-2, lx05-4 and lx03-2 by successive backcrossing respectively, and transgenic maize inbred line HGK60-Zheng 58, HGK60-Chang 7-2, HGK60-lx03-2, and HGK60-lx05-4 were acquired. The hybrid line HGK60-zhengdan 958(HGK60-Zheng 58 with HGK60-Chang 7-2)and HGK60-Ludan 9066(HGK60-lx03-2 with HGK60-lx05-4)were obtained by crossing. The foreign cry1Ah gene was confirmed to be transferred into the maize with different genetic background by event-special PCR detection. The expressions of Cry1Ah proteins in the transgenic maize leaves of the inbred and hybrid lines under different genetic backgrounds were insignificantly different by ELISA detection. Bioassay in field and in laboratory showed that the transgenic maize with different genetic backgrounds had same high resistance to Asia corn borer, and the mortality rate reached 100% at 4 d after indoor inoculation. The agronomic traits of the transgenic maize HGK60 with different genetic backgrounds demonstrated that there was no significant difference in agronomic traits between the transgenic maize and the non-transgenic control maize, and transgenic maize HGK60 may be used for the breeding of insect-resistant maize varieties.

    Research Progress in the Regulation of Development and Stress Response by Long Non-coding RNAs in Plants
    LI Jian-jian, HE Chen-jing, HUANG Xiao-ping, XIANG Tai-he
    2023, 39(1):  48-58.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0053
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    Long non-coding RNAs(lncRNAs)are a class of endogenous RNAs that are longer than 200 nucleotides, and lack apparent protein-coding capacity without open reading frame. Due to the low expression level and weak conservation, lncRNAs are considered to be transcription by-product, which play no biological functions in vivo. With the further research on non-coding RNAs, they are confirmed to be an important component regulating other type RNAs, which are involved in development and stress response biological processes. In this review, we summarize the generation and classification, mechanism, the biological functions of lncRNAs in development and stress response, aiming to provide reference for the application of lncRNAs in crop production and breeding.

    Research Progress in Na+ Antiport and Physiological Growth Mechanisms of Differernt Halophytes Adapted to Salt Stress
    LIU Jia-xin, ZHANG Hui-long, ZOU Rong-song, YANG Xiu-yan, ZHU Jian-feng, ZHANG Hua-xin
    2023, 39(1):  59-72.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0342
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    Halophytes refer to plants that can grow and complete their life cycle in habitats with an ion concentration of at least 200 mmol/L. Halophytes can be classified into three types:euhalophytes, recretohalophytes and pseudohalophytes. This review summarizes the different strategies and research progress of the three types of halophytes in response to salt stress from the three aspects of growth morphology, physiology and molecule. It is found that halophytes mainly use Na+ transporters genes and energy-providing genes to respond to excessive Na+ at the molecular level, which may be an important factor that causes halophytes to be different from non-halophytes in physiology and growth morphology. Euhalophytes respond to salt stress mainly through vacuolar ion compartmentalization and have succulent growth morphology. Recretohalophytes respond to salt through by expelling salt from their bodies and evolve unique physiological structures-salt glands or salt vesicles. Pseudohalophytes reduce the upward transport of Na+ by accumulating salt ions in the vacuoles of cortical cells and parenchyma cells of root xylem; meanwhile, root is having suberization to reduce the absorption of Na+. The purpose of the review is to provide relevant basis for the study of halophytes and their salt tolerance mechanism and lay a foundation for plant salt tolerance molecular breeding in the future.

    Research Progress in the Effects of Ubiquitin-proteasome System on Plant Agronomic Traits
    WANG Cui-cui, FU Da-qi
    2023, 39(1):  72-83.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0160
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    At present, problems such as population explosion, environmental pollution and ecological destruction emerge one after another. Improving agronomic traits, such as increasing yield and enhancing stress tolerance, is the most important goal of crop genetic improvement and the basis of promoting high-quality agricultural development. The ubiquitin-proteasome system(UPS)is a rapid and selective system for the hydrolysis of redundant and damaged proteins produced by plant. Now, studies have found that the ubiquitin-proteasome system affects plant development, reproduction, and important agronomic traits, such as pathogen responses, induction of flowering, seed size, and so on. This review focuses on recent studies illustrating the important functions of the UPS components and subunits of the proteasome and describes how the UPS affect plant agronomic traits. Finally, the review discusses the future research hotspots and the potential for crop improvement with UPS.

    Research Progress in Gibberellin Regulation on Maize Seed Vigor
    JIN Yun-qian, WANG Bin, GUO Shu-lei, ZHAO Lin-xi, HAN Zan-ping
    2023, 39(1):  84-94.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0473
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    Maize is the main crop with the highest total yield and average yield per unit area in China, which plays an important role in guaranteeing national food security. Seed vigor is a key index to measure seed quality and application value. High vigor seed is the basis to ensure high and stable yield of crops. Gibberellin is an important plant growth regulator, which can relieve seed dormancy and promote germination. The application of exogenous gibberellin has been widely used in agricultural production to improve crop yield. At present, researches about the effects of GA on the maize seed vigor are mainly focused on the physiological indicators by applying exogenous GA. However, the molecular mechanism of maize seed vigor regulated by GA needs to be further studied. This review is mainly about the research progress of GA biosynthesis, signal transduction, mechanism of GA and its effect on the seed vigor of maize and other crops, aiming to provide reference for further exploring the regulation mechanism of GA on maize seed vigor and the creation of new maize germplasm with high vigor in maize breeding practice.

    Research Status and Prospect of Hybrid Wheat Seed Production
    KONG De-zhen, NIE Ying-bin, CUI Feng-juan, SANG Wei, XU Hong-jun, TIAN Xiao-ming
    2023, 39(1):  95-103.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0340
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    The utilization of heterosis plays an important role in improving wheat yield. Hybrid breeding in wheat is an important development direction for seed production. At present, great progress has been achieved in the strong heterosis combination of hybrid wheat. There are issues such as lack of excellent parents for hybrid combination and hybrid seed production system for high yield and high efficiency wheat, which leads to low seed production efficiency in large area. Therefore, to explore the development of hybrid seed production technology system, germplasm improvement of parental resources and seed production process standards will be conducive to summarizing the overall trend of the development bottleneck and future development direction of hybrid seed production. In this paper, the influencing factors of hybrid breeding in wheat are reviewed. Through the comparison between commercial hybrid crops and superior seed from novel hybrid wheat, some effective suggestions for rapid commercialization of hybrid wheat are put forward, and its future development trend is forecasted.

    Molecular Evolutionary Relationship and Protein Structure of Glycoside Hydrolases from GH79 Family
    CHEN Quan-bing, CAO Wei-jie, LI Chun, LV Bo
    2023, 39(1):  104-114.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0417
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    The glycoside hydrolases of the GH79 family have extensive physiological activities and important application prospects in carbohydrate modification, cellular immune recognition and signal transduction. However, the diverse catalysis mechanism of GH79 family remains unclear, and the structural basis and molecular mechanism of substrate recognition are still unclear. This review summarizes the research progress of the GH79 family, systematically analyzes the origin and distribution of GH79 family enzymes, and deeply expounds their sequence features, molecular evolutionary relationships, and the crystal structures analysis, aiming to lay a foundation for protein engineering and functional metabolic mechanism of GH79 family.

    Construction of Multi-gene Interference System for Plant and Analysis of Its Application Efficiency
    ZHOU Jia-yan, ZOU Jian, CHEN Wei-ying, WU Yi-chao, CHEN Xi-tong, WANG Qian, ZENG Wen-jing, HU Nan, YANG Jun
    2023, 39(1):  115-126.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0378
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    Most important functional genes belong to polygenic families, and abundant functional redundancy is enriched among those family members. An efficient multi-gene interference system is of great significance for investigating the biological function and molecular regulation mechanism of polygenic family members. The pCAMBIA1301 vector was modified to construct multi-gene interference systems pCAMBIA1301m and pCAMBIA1301s for plants. The four genes interference vector, pCAMBIA1301m:35S∷SlPP2C1-2-3-4,containing PP2C1PP2C2PP2C3 and PP2C4 from tomato PP2C family A group, was constructed using this system and then introduced into tomato by genetic transformation. The positive plants were detected by GUS staining and PCR, the interference efficiency in T1 and T2 transgenic plants was tested by RT-qPCR, and the sensitivity of T2 transgenic seeds to ABA was analyzed. The results showed that the transgenic plant 35S∷SlPP2C1-2-3-4 with four interfered genes was successfully obtained by using this multi-gene interference system. In the transgenic plants, the expressions of the four target genes were significantly lower than that in wild plants, and the interference efficiency was above 70%. The achieved transgenic seeds demonstrated strong insensitivity to exogenous ABA. The multi-gene interference system may effectively and synchronously silence multiple target genes.

    Establishment of Droplet Digital PCR Assay for Quantitative Detection of Pseudomonas cocovenenans in Foods
    LI Hui-jie, DONG Lian-hua, CHEN Gui-fang, LIU Si-yuan, YANG Jia-yi, YANG Jing-ya
    2023, 39(1):  127-136.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0254
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    Pseudomonas cocovenenans contaminates a variety of foods including fermented rice and noodles. It produces two toxins that cause severe food poisoning, which threatens human health. Based on digital PCR technology, the quantitative method targeting to P. cocovenenans was established. It demonstrated good specificity and repeatability. The detection limit in polluted glutinous rice soup was 361 CFU/mL. Compared with plate counting method, the detection time was shortened obviously via the digital PCR quantitative method, which was conducive to the rapid and accurate measurement of P. cocovenenans. Thus, it may be applied in the fields of food safety monitoring and poisoning analysis in the future.

    Application of Synthetic Biology Based Whole-cell Biosensor Technology in the Rapid Detection of Food Safety
    CHEN Xiao-lin, LIU Yang-er, XU Wen-tao, GUO Ming-zhang, LIU Hui-lin
    2023, 39(1):  137-149.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0389
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    Synthetic biology-based whole-cell sensing technology provides an alternative approach for rapid and on-site detection of heavy metals. Because the intracellular environment is relatively stable, whole-cell biosensor presents strong anti-interference ability; the biosensor cells can proliferate with cell self-replication, so the whole-cell biosensor has the characteristics of simple, cheap and fast in production. Therefore, whole-cell biosensor demonstrates a promising application prospect in the rapid detection of food pollutants. This article, the composition, construction methods, and types of core elements for whole-cell biosensor are summarized. Then synthetic biology-based gene circuits for multi-function detection technologies are introduced; further the commercialized applications of whole-cell biosensor in rapid food safety testing are enumerated; and finally the challenges and development prospects of cell biosensors in the rapid detection of food safety are illustrated.

    Role of the AtERF49 in the Responses to Salt-alkali Stress in Arabidopsis
    RUAN Hang, DUO Hao-yuan, FAN Wen-yan, LV Qing-han, JIANG Shu-jun, ZHU Sheng-wei
    2023, 39(1):  150-156.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0479
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    Saline-alkali stress is one of the main environmental constraints that cause crop failures. AP2/ERF(APELATA2/ethylene response factors)transcription factors play important roles in plant growth, development and response to abiotic stress. Investigating the responses of AtERF49 to Saline-alkali stress would lay a foundation for explaining the molecular mechanism of plant AtERF49 involving in Saline-alkali stress. Arabidopsis wild-type, namely, Col-0, AtERF49-overexpressed lines of Arabidopsis and CRISPR/Cas9 mutant erf49 of Arabidopsis were treated with 150 mmol/L Saline-alkali solution(NaHCO3 and Na2CO3 molar ratio 9∶1 mixed). The RT-qPCR assays were used to analyze the basic characteristics and expression pattern of this gene under saline-alkali stress and photosynthetic response. Results showed that the leaves of erf49 mutant were wilt and bleach under Saline-alkali stress, and the leaves of AtERF49-overexpressed lines turned to be yellow slightly. Moreover, the overexpression of AtERF49 up-regulated the expression of Saline-alkali stress inducible genes(RD29A and RAB18)and photosynthetic gene rbcL under Saline-alkali stress. Chlorophyll fluorescence parameters assay of Arabidopsis leaves revealed that actual photochemical efficiency(Y(II))and non-photochemical quenching coefficient(qP)in AtERF49-overexpressed plants were significantly higher than that on Col-0, and the photodamage(NO)and photochemical quenching coefficient(qN)were significantly lower compared to Col-0. However, the opposite trend was apparent in erf49 mutant. These results suggested that AtERF49 might participate in response to Saline-alkali stress through regulating its downstream Saline-alkali stress-responsive genes or affecting photosynthesis efficiency.

    Functional Analysis of ACOL8 Gene in the Ethylene Synthesis and Response in Arabidopsis thaliana
    LIN Rong, ZHENG Yue-ping, XU Xue-zhen, LI Dan-dan, ZHENG Zhi-fu
    2023, 39(1):  157-165.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0560
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    The plant hormone ethylene plays pivotal roles in various physiological and biochemical processes. However, the mechanisms controlling its synthesis in certain tissues and organs are not completely understood. There are 12 ACC oxidase homologs(ACO-like homolog, ACOL)of unknown functions in Arabidopsis thaliana. The loss-of-function mutants of ACOL8 gene were constructed using site-directed gene editing technology. It was found that the mutations of this gene attenuated the classical ethylene triple response. Compared with wild type, the mutants showed significant increases in the length of hypocotyls and primary roots of etiolated seedlings, which was consistent with the observation that the mutants had decreased sensitivity to exogenous ACC. Additionally, it was evident that the expression of this gene was subjected to positive feedback regulation of ethylene signaling, as exemplified by the finding that overexpression of EIN3 increased the expression of ACOL8 gene, whereas the etr1-3 muation exerted the opposite effect. Furthermore, the mutation of ACOL8 gene did not appear to affect the growth of A. thaliana under normal conditions, whereas the root-shoot ratio of the mutants decreased significantly under salt stress conditions, suggesting its involvement in plant salt stress response. Collectively, these results indicate that ACOL8 may function as an ACC oxidase to medaite ethylene synthesis and response.

    Expression and Functional Analysis of OsPT1 Gene in Rice Under Cadmium Stress
    JIANG Nan, SHI Yang, ZHAO Zhi-hui, LI Bin, ZHAO Yi-hui, YANG Jun-biao, YAN Jia-ming, JIN Yu-fan, CHEN Ji, HUANG Jin
    2023, 39(1):  166-174.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0489
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    OsPT1-encoded rice phosphate transporter protein plays important regulatory roles in growth and abiotic stress response in rice. Previous data showed that the expression of OsPT1 gene was induced by cadmium(Cd)stress, however, the function and mechanism of OsPT1 under Cd stress remains unclear. Therefore, the aim of this study is to clarify the function of OsPT1 under Cd stress and lay a foundation for the breeding of low-Cd rice cultivars. By using bioinformatics methods, the sequence feature, structure and function of OsPT1 gene were analyzed or predicted. The expression profiles of OsPT1 in different tissues and at different time points under Cd stress were examined by quantitative real-time PCR(RT-qPCR)as well. Simultaneously, PCR was used to clone the encoding sequence of OsPT1, and the recombinant plasmid pGADT7-OsPT1 was constructed and transferred into the the Cd-sensitive yeast strain ∆ycf BY4741 for determining the effect of OsPT1 on the tolerance of yeast to Cd. The results showed that OsPT1 is a protein of 527 amino acids with the molecular weight of 57.46 kD, which was encoded by a CDS of 1 584 bp. In rice genome, the cis-acting elements such as environmental factors like light and anaerobic, as well as hormones including methyl jasmonate were found in the promoter region of this gene. The results of phylogenetic analysis showed that OsPT1 was most closely related with sorghum SbPT1. The results of Cd-responsive gene expression analysis showed that the expression levels of OsPT1 in rice shoots increased to 1.31, 1.34 and 2.46 folds comparing to the untreated control under 100 μmol/L Cd treatment for 1, 6 and 12 h, respectively. The expressions in the roots increased to 1.28 and 1.14 folds comparing the untreated control under 100 μmol/L Cd treatment for 1 or 6 h, Cd treatment, however, it was down-regulated to 0.62 folds after 12 h Cd treatment. The result of Cd tolerance examination showed that the yeast transfered with OsPT1 demonstrated the decreased tolerance to Cd when treated with 25 μmol/L Cd when compared with the control(0 Cd). All together, OsPT1 may play a certain role in the respondses of rice to Cd stress.

    Genome-wide Identification and Expression Analysis of the RNAi-related Gene Families in Setaria viridis
    LUO Hao-tian, WANG Long, WANG Yu-qian, WANG Yue, LI Jia-zhen, YANG Meng-ke, ZHANG Jie, DENG Xin, WANG Hong-yan
    2023, 39(1):  175-186.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0478
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    RNAi pathway is a derived pathway of RdDM pathway, proteins AGOs, DCLs and RDRs of which play important roles in plant growth and development and in responses to abiotic/biotic stresses. In order to study the sequence and structural characteristics of these three main proteins of the RNAi pathway in Setaria viridis, we identified 13 AGO genes, seven DCL genes and four RDR genes in S. viridis by comparative genomics. Further we predicted the protein subcellular localization, phylogenetic relationship and conserved domain of these proteins. Meanwhile, the transcriptome data were used to analyze the expression patterns of three types of genes families in different organs in 16 different growth stages and different growth conditions of S. viridis. Protein conserved domain analysis revealed that SvDCL3b and SvRDR3 were lack of important domains. Transcriptome analysis showed that SvAGO1b, SvDCL1a and SvRDR1 were highly expressed in each family, which might play a major role in the RNAi pathway, and the expression patterns of homologous genes of S. viridis and S. italica were basically the same. In conclusion, this study provides a preliminary theoretical basis for understanding the functions and roles of the three main genes of the RNAi pathway in regulating the epigenetic modification of S. viridis, and provides a reference for the molecular mechanism of domestication between S. viridis and S. italica.

    Identification of Zinc Finger Protein DnaJ-Like Gene Family in Capsicum annuum and Its Expression Analysis Responses to High Temperature Stress
    DUAN Min-jie, LI Yi-fei, YANG Xiao-miao, WANG Chun-ping, HUANG Qi-zhong, HUANG Ren-zhong, ZHANG Shi-cai
    2023, 39(1):  187-198.  doi:10.13560/j.cnki.biotech.bull.1985.2022-1033
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    Plant DanJ-Like zine finger protein(DNAJE)is one of the important protein found in recent years, which plays an important role in photosynthesis, development and motility of plastids and the responses of plants to stresses. Here, the DNAJE whole gene family(CaDNAJE)was identified by the whole genome data of pepper(Capsicum annuum, and the characteristic of gene and protein, comparative evolution, collinearity and transcriptional expression under high temperature stress were analyzed. Totally, 28 members of CaDNAJE genes were identified, and they were distributed unevenly on 10 chromosomes and 3 Scaffolds. The amino acid length, molecular mass and isoelectric point were between 99-470 aa, 10.05-52.26 kD and 4.75-9.64, respectively. Moreover, these encoded proteins were mainly located in the chloroplast and nucleus. The comparative evolution of pepper, tomato(Solanum lycopersicum)and Arabidopsis analysis can be divided into 2 subgroups: GroupⅠ and GroupⅡ, and the CaDNAJE protein in the same subgroup had similar conserved motifs and gene structures. Cis-acting element analysis in the promoter region showed that CaDNAJEs contained a large numbers of functional elements related to light response, hormone response and stress. There were 10 pairs of collinearity genes between pepper and Arabidopsis, but only one pair within pepper species. The transcriptome analysis of different pepper tissues indicated that most members of GroupⅠ were highly expressed in all tissues, while most members of GroupⅡ had low or no expression except for CaDNAJE2 and CaDNAJE9. The content of hydrogen peroxide in pepper leaves increased rapidly and then decreased. The content of malondialdehyde kept increasing and peroxidase activity also had been improved to different extent. High temperature stress induced the high expression of the heat shock factor/heat shock protein(Hsf/HSP)and CaDNAJE genes, which played a regulation role in different stress time. It is suggested that CaDNAJE family genes are involved in response to high temperature stress and then increased tolerance to reduce cell damage.

    Metabolomics Analysis of Germinating Peanut Seed Under Salt Stress
    XU Yang, DING Hong, ZHANG Guan-chu, GUO Qing, ZHANG Zhi-meng, DAI Liang-xiang
    2023, 39(1):  199-213.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0438
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    Salt stress causes seed vigor reduction and seed germination inhibition, which limits peanut production in Saline-alkali land. The metabolic process of seed germination plays an important role in seed germination and plant morphogenesis, which has gradually become an important indicator to evaluate seed vigor and quality. Having the peanut seed in different germination as study object, changes of basic nutrients and the differential metabolites along peanut seed germination under salt stress were analyzed by physiological indexes and liquid chromatography tandem-mass spectrometry(LC-MS/MS). Seed germination after absorbing water prompted the catabolism of fats, proteins and soluble sugars. The levels of fats and soluble sugars decreased gradually with the germination time prolonging, while those of soluble protein first decreased and then increased. Principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA)showed that the metabolic profiles of those groups were different, indicating that salt stress largely impacted peanut seed metabolism. VIP analysis and KEGG pathway prediction analysis showed that there was little differentiated metabolites and none was enriched in any KEGG pathway during water absorption period, but they were enriched in 12 KEGG pathways during radicle elongation period under normal condition, indicating that the metabolism was more vigorous in the late germination period than in the early germination period. Salt stress dramatically increased levels of different metabolites, among which levels of osmotic protectors betaine and proline were significantly induced. In addition, differentiated metabolites significantly increased during water absorption period and radicle elongation period under salt stress, and they were enriched 26 and 31 KEGG pathways, respectively. Salt stress significantly promoted the related pathways such as energy metabolism, glycerophospholipid metabolism, glutathione metabolism, and glucosinolate biosynthesis, suggesting that may be associated with stress tolerance. Betaine and proline are likely two key metabolites, and glycerophospholipid metabolism, glutathione metabolism and glucosinolates biosynthesis may be important metabolic regulatory pathways for the adaptation to salt stress during germination. This study provides a theoretical basis and reference value for exploring new methods to promote peanut seed germination and emergence under salt stress and improving peanut germination rate in Saline-alkali land.

    Transformation and Functional Identification of the Key Genes Associated with Steviol Glycosides Biosynthesis in Stevia rebaudiana
    ZHANG Jun, ZHANG Hong, ZHANG Rui, LU Guo-dong, YONG Jing-jiao, LANG Si-rui, CHEN Ren
    2023, 39(1):  214-223.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0388
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    Steviol glycosides produced by Stevia rebaudiana Bertoni are regarded as the most promising substitute for sucrose with a wide range of potential applications and high economic value due to their high sweetness, low caloric energy, non-participation in human metabolism and health care functions. Three key genes from Stevia leaves, SrUGT85C2, SrUGT91D2m and SrUGT76G1 in the biosynthetic pathway of steviol glycosides, were cloned, and overexpression vector of plant genes were constructed. In individual or in combined way, these genes were transferred into the S. rebaudiana, and the transgenic plants were obtained. Compared with the control of wild-type plant, the content of steviolmonoside increased in the transgenic plant transferred SrUGT85C2 alone, despite little changes in the total content of steviol glycosides, rebaudioside A, and the content ratio of rebaudioside A to the total steviol glycosides. In the transgenic plant transferred SrUGT91D2m alone, the content of steviolmonoside decreased, while the content of steviolbioside increased significantly. In the other hand, in the transgenic plant transferred SrUGT76G1 alone, the total amount of steviol glycosides significantly increased, especially the content of rebaudioside A reached to 10%, which was nearly two times than that in the control, whereas the content of stevioside reduced by half. The transgenic plant transferred the three genes in combination was similar to the transgenic plant transferred SrUGT76G1 alone, and the total content of steviol glycosides, rebaudioside A, and the content ratio of rebaudioside A to the total steviol glycosides increased significantly. These results provides the theoretical basis and technical methods for regulating the expressions of key genes in the biosynthesis of steviol glycosides and cultivating new high-quality S. rebaudiana strains with high content of rebaudioside A through molecular biology techniques.

    Cold Resistance Function Analysis of Cassava MeMYC2.2
    YU Xiao-ling, LI Wen-bin, LI Zhi-bo, RUAN Meng-bin
    2023, 39(1):  224-231.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0461
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    Jasmonic acid(JA)signal transduction is a key pathway in plant response to stresses, in which MYC2(MYeloCytomatosis)plays an important role. This study aims to investigate the function of MeMYC2.2 from cassava(Manihot esculenta Crantz)under cold stress. Bioinformatics was used to identify the structures and physicochemical properties of two MYC2 gene, namely MeMYC2.1 and MeMYC2.2 from cassava. Quantitative PCR was to analyze the responses of them in the leaves of tissue-cultured cassava seedling to cold stresses. Furthermore, the function of MeMYC2.2 for cold resistance was verified in transgenic Arabidopsis. As result, the expressions of two MeMYC2 genes were dramatically induced under early cold stress in the leaves of tissue-cultured cassava seedling, and the differential expression of MeMYC2.2 was more significant compared to MeMYC2.1. The MeMYC2.2 protein was predominantly localized in the nucleus and presented of transcriptional activation ability in yeast, suggesting this protein probably was characterized of a transcription factor. Over-expression of MeMYC2.2 resulted in significant improvement of freezing tolerance in transgenic Arabidopsis compared to wild one. Furthermore, the expression of CBF3 was significantly up-regulated by MeMYC2.2 transgenic in Arabidopsis under cold conditions. However, expressions of other three CBF genes were down-regulated by MeMYC2.2 transgenic Arabidopsis under cold conditions. The expression of MeMYC2.2 was regulated by cold and jasmonic acid, which may increase its cold tolerance and affect the responses of CBF genes to cold stress. This study lays a theoretical foundation in using MeMYC2.2 to improve cold resistance in cassava cultivars.

    Comparative and Phylogenetic Analyses of Complete Chloroplast Genomes in Ardisia crenata
    LIU Xiong-wei, LIU Chang, ZENG Xian-fa, YANG Xiao-ying, FENG Ting-ting, ZHAO Jie-hong, ZHOU Ying
    2023, 39(1):  232-242.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0471
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    Illumina sequencing platform was used to obtain the whole chloroplast genome sequences of Ardisia crenata. Compared with the structural characteristics and variation degree of chloroplast genomes based on bioinformatics methods, this study is aimed to clarify the characteristics and differences of the chloroplast genomes of A. crenata and Ardisia crenata var. bicolor, to compare and analyze the chloroplast genomes, and to provide reference for the phylogenetic study of Myrsinaceae. As results, both of them were composed of one large single copy region(LSC), one small single copy region(SSC)and a pair of reverse repeat regions(IRa/IRb). Total 132 genes were annotated, and their repeat sequence types and distribution patterns were similar, but the number was different. The results of comparative genomics showed that the coding regions of psbA, matK, rpoC2, ropB, ndhK, accD, ndhF, ndhD, ndhH and ycf1 genes were different, and they provided new loci for molecular identification of Ardisia. The chloroplast genome of A. crenata and A. crenata var. bicolor was highly conserved, and there was no rearrangement or inversion between chloroplast genomes. The variation of IR region sequence was the lowest, and the variation degree of SSC region was the highest. In the phylogenetic tree, the A. crenata and A. crenata var. bicolor were sister groups, which clearly reflected the genetic relationship, and provided a scientific explanation for the variety of A. crenata from molecular level. The Myrsinaceae and Primulaceae can be divided into two branches according based on the support rate. In this study, the chloroplast genome structure of A. crenata was analyzed, and the phylogenetic relationship among the genera of Myrsinaceae was discussed, which lays a foundation for the study of taxonomic identification, phylogenetic evolution and resource exploitation and utilization of medicinal plants of Myrsinaceae.

    Regulation of Fusarium wilt Resistance in Cotton by Exogenous Melatonin
    ZHU Jin-cheng, YANG Yang, LOU Hui, ZHANG Wei
    2023, 39(1):  243-252.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0419
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    To investigate the effect and mechanism of exogenous melatonin on cotton resistance against Fusarium wilt, Gossypium barbadense Xinhai 14 treated with foliar application of various concentrations of melatonin(0, 10, 25, 50, 75 and 100 μmol/L)was used to identify disease resistance, to identify the activity of disease resistance related enzymes, and to analyze gene expression in cotton seedlings after inoculation with Fusarium oxysporum f.sp. vasinfectum(Fov). The results showed that exogenous melatonin did not inhibit the growth of cotton blight, and they 10-100 μmol/L all increased the cotton resistance to Fov to some extent, the best with 50 μmol/L melatonin. Compared with control, 50 μmol/L melatonin pretreatment reduced the hydrogen peroxide(H2O2)content and superoxide anion(O2·-)production rate in the cotton leaves after inoculation, and enhanced the expressions of peroxidase(POD), catalase(CAT), superoxide dismutase(SOD), ascorbate peroxidase(APX), chitinase(CHT), β-1,3 glucanase(GLU), polyphenol oxidase(PPO), phenylalanine ammonia lyase(PAL)activities, and significantly increased the expressions of key genes of the lignin synthesis pathway(GbPAL, Gb4CL and GbCAD)and key genes of the flavonoid synthesis pathway(GbCHI, GbDFR and GbTT7). This showed that 50 μmol/L melatonin treatment may improve the antioxidant capacity of cotton after inoculation, enhance the activities of defense enzymes, regulate the expressions of genes related to the lignin and flavonoid synthesis pathways, and thus improve the resistance of cotton to Fusarium wilt.

    Screening of Antagonistic Bacteria for Bacterial Fruit Blotch of Cucurbits and Its Antibacterial Effects
    ZHANG Ling, ZHANG Rong-yi, LIU Sheng-ke, TAN Zhi-qiong
    2023, 39(1):  253-263.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0449
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    The antagonistic strains against bacterial fruit blotch(BFB)of cantaloupe were screened from soil, their taxonomic status was clarified, and their growth conditions and disease prevention effects were determined. Separation and screening were carried out by plate dilution method and confrontation method, and the species was identified by comprehensive morphological, physiological, biochemical and molecular identification. After determining the species of biocontrol bacteria, the optimal growth conditions were determined, and the bacteriostatic effect was determined by the plate confrontation method. The leaves and potted plants of biocontrol bacteria were measured by acupuncture inoculation method and foliar spraying method respectively. In this study, 196 strains of bacteria were isolated from soil samples, 20 strains of them were found to have bacteriostatic effect via confrontation method. Among them, two strains numbered 131 and 791 had better plate antagonism effect on the pathogen of BFB, and the diameters of the inhibition zones were(19.03±0.13)mm and(17.55±0.29)mm, respectively. The strain 131 was identified as Bacillus safensis and strain 791 as B. velezensis. The optimal growth condition of strain 131 was NB medium, cultured for 18 h, temperature was 37℃, pH 7 and the inoculum was 2%. The optimal growth condition of strain 791 was KB medium, cultured for 18 h, temperature was 37℃, pH 6 and the inoculation amount was 1%. Strain 131 and 791 were 83.33% and 87.53% in vitro, 69.86% and 77.99% in indoor pots, respectively; meanwhile they had inhibitory effects on various pathogens. In summary, strain 131 and 791 are Bacillus with good control effect on BFB, which provides theoretical supplements for the prevention and control of BFB and lays a solid foundation for BFB.

    Isolation, Identification and Biocontrol Potential of Rhizospheric Fungus of Saposhnikovia divaricata
    SUN Zhuo, WANG Yan, HAN Zhong-ming, WANG Yun-he, ZHAO Shu-jie, YANG Li-min
    2023, 39(1):  264-273.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0501
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    Fusarium oxysporum and F. equiseti worldwide are the common fungal pathogens that cause a lot of damages on the growths of economic crops or medicinal plants. One ideal strategy for controlling the fungal diseases from F. oxysporum and F. equiseti is to utilize rhizospheric microorganisms. In order to obtain the biocontrol microorganisms against F. oxysporum and F. equiseti, 104 fungi from rhizospheric soil of Saposhnikovia divaricata plants were screened and isolated. Subsequently, strain MR-43 was selected as a potential stock for biocontrol due to the significantly antibiotic activity against F. oxysporum and F. equiseti. Based on the analysis of morphological traits and rDNA internal transcribed spacers(ITS), strain MR-43 was identified as Sirastachys castanedae(GenBank No. OK287148.1), belonging to a species in section Sirastachys of Stachybotryaceae, as a new record of S. castanedae from rhizospheric soil of S. divaricata. Based on the pot experiment under natural outdoor conditions, colonization rule in the soil of S. divaricate growing was studied, the antifungal efficacy of MR-43 against Fusarium wilt caused by F. oxysporum and root rot disease caused by F. equiseti of S. divaricata were assessed, and its growth promotion abilities of S. divaricata was also determined. The results indicated that MR-43 showed the strongest inhibition rate against F. oxysporum and F. equiseti, which showed up to 57% inhibition. The multiple test confirmed that the antifungal effect was relatively stable. The S. castanedae MR-43 successfully colonized and formed a stable population in soil where S. divaricata was growing. Inoculation of the MR-43 spore suspension delayed the occurrence of S. divaricata Fusarium wilt and root rot disease, suppressed disease lesions with a strong control efficacy of 68.52%, the control effect was not significantly different compared with fungicide treatment of 70% Mancozeb WP and 50% Carbendazim WP. Moreover, MR-43 presented a consistently positive effect on the growth promoting of S. divaricata plants. In nutshell, S. castanedae MR-43 could be considered for the developing potential biocontrol agent for the management of S. divaricata Fusarium wilt and root rot disease.

    Whole-Genome Sequencing Identification of Phosphate-solubilizing Bacteria PSB-R and Analysis of Its Phosphate-solubilizing Properties
    WANG Shuai, LV Hong-rui, ZHANG Hao, WU Zhan-wen, XIAO Cui-hong, SUN Dong-mei
    2023, 39(1):  274-283.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0448
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    The phosphate-solubilizing bacterium PSB-R was identified as Serratia marcescens by physiological and biochemical indicators combined with molecular biology technology. Second-generation sequencing platform Illumina NovaSeq PE150 was used to have the whole genome of PSB-R sequenced, and to analyze and predict the composition of genes related to phosphate-solubilizing ability and other plant growth-promoting genes. The response surface optimization test showed that the maximum phosphorus dissolving capacity of PSB-R was 805.199 mg/L. After 10 generations of continuous culture, the phosphorus dissolving capacity was stable and had the ability to dissolve a variety of insoluble phosphates. This study provides a genomic data basis for the further study of the mechanism of phosphate solubilization by phosphate solubilizing bacteria, and confirms that there is a potential for PSB-R to be applied to bacterial fertilizer, which provides a research basis for the subsequent development of phosphate-solubilizing bacterial fertilizer.

    Screening, Identification, and Optimization of Culture Conditions of a Melanin High-yielding Strain of Hypoxylon sp.
    HUANG Hai-chen, LI Xiao-min, XUE Fan-zheng, WU Xiao-ping, ZHANG Jun-li, FU Jun-sheng
    2023, 39(1):  284-294.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0119
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    In order to screen a strain with strong melanin-producing ability and optimize its culture conditions, 11 tested strains were identified by ITS sequencing, and a ash-producing fungus with strong black-producing ability was screened based on mycelial growth rate, plate L value and other indicators, and the carbon source, nitrogen source, pH and other culture conditions required for its growth were optimized. The study showed that all 11 test strains were Hypoxylon sp. Among them, Hp.sp0006 had faster mycelial growth, larger and more uniform balls, the lowest L value, and the highest melanin content in the fermentation broth. The optimal culture conditions for this strain were glucose as the carbon source, beef extract as the nitrogen source, a 20∶1 carbon to nitrogen ratio and the addition of 10 mg vitamin B1, with a high yield of melanin up to(1.21±0.17)g/L melanin. Hp.sp0006 is a strain producing higher melanin. The optimized medium is more conducive to the synthesis of melanin, which lays the foundation for the development and utilization of melanin in Hypoxylon sp.

    Comparative Analysis of Rumen and Fecal Microbial Characteristics Associated with Residual Feed Intake in Beef Cattle
    ZHANG Yan-feng, DING Yan-ling, MA Ying, ZHOU Xiao-nan, YANG Chao-yun, SHI Yuan-gang, KANG Xiao-long
    2023, 39(1):  295-304.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0752
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    The aim of this experiment is to investigate the composition and relative abundance of microbial species in the gastrointestinal tract of beef cattle associated with different residual feed intake(RFI), and the functional annotation and enrichment characteristics of microbial genes. Thirty cattle were selected for the 81-day feeding experiment, and at the end of the experiment, five individuals each with extreme RFI were selected for slaughter, and rumen fluid and intestinal end fecal samples were collected for macrogenome sequencing. The results showed that a total of 259 045.47 Mb valid data were obtained, 4 318 393 Scaftigs were assembled, and 7 008 053 open reading frames(ORFs)were obtained from gene prediction. Species annotation revealed no difference in the relative abundance of Top 10 species in the fecal samples between High Residual Feed Intake(HRFI)and Low Residual Feed Intake(LRFI)groups(P>0.05), and the relative abundance of Top 10 microorganisms in the rumen fluid in the LRFI group was lower than in the HRFI group; the dominant phylum in both feces and rumen fluid was the phylum Bacillus and the thick-walled phylum; the dominant genus in the feces was Bacteroides and the dominant genus in the rumen fluid was Prevotella. LefSe analysis showed a significant enrichment of Erysipelotrichia in the feces in the LRFI group(P<0.05), the most significant difference in rumen fluid was in the Methanobacterium class, and the relative abundance of this organism in the HRFI group was significantly higher than in the LRFI group(P<0.05). Functional annotation analysis using the KEGG, eggNOG and CAZy databases showed that the abundances of some functional genes of microorganisms in the gastrointestinal tract was associated with the grouping of RFI. In conclusion, the microbial structures in the rumen fluid and feces of beef cattle with different RFI varied significantly, and the Erysipelotrichia and Methanobacterium phyla may be among the potential biomarkers for differentiating feed efficiency in beef cattle.

    Novel Coronavirus Subunit Vaccine and Screening of Its Efficient Immune Enhancer
    WANG Xiang-kun, SONG Xue-hong, LIU Jin-long, GUO Pei-hong, ZHUANG Xiao-feng, WEI Liang-meng, ZHOU Fan, ZHANG Shu-yu, GAO Pan-pan, WEI Kai
    2023, 39(1):  305-314.  doi:10.13560/j.cnki.biotech.bull.1985.2022-0408
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    This work aims to promote the development of novel coronavirus(2019-nCoV)subunit vaccine and explore the screening of high-efficiency immune enhancers suitable for the vaccine. We induced the expressions of 2019-nCoV-S and 2019-nCoV-N proteins respectively, determined the contents of the target protein after purification, different concentrations of pine pollen polysaccharide(PPPS)(200, 400, 800 mg/mL)were added as adjuvants to prepare genetic engineering subunit vaccine, and astragalus polysaccharide adjuvant was used as control. One hundred SPF BALB/c mice were randomly divided into ten groups and each subunit vaccine was used in five groups. After immunization, the immune indexes of mice in each group were detected to explore the immune enhancement effect of PPPS on 2019-nCoV-S and 2019-nCoV-N subunit vaccines. The results showed that the recombinant expression and purification of the target protein showed a single band at 55.68 and 45.64 kD, respectively, and the expression was correct. The protein concentration was 1.12 and 0.66 mg/mL, respectively. After immunizing mice with the adjuvant subunit vaccine, it was found that 400 mg/mL PPPS significantly improve the immune effect of the two vaccines, and the effect was better than that of astragalus polysaccharide adjuvant. In summary, the two recombinant proteins can induce higher antibody levels, and PPPS can be used as a candidate adjuvant for 2019-nCoV subunit vaccine.