Biotechnology Bulletin

Identification of Drought Tolerance of Sweet Sorghum at Booting Stage

YUAN Chuang, XU Xing, TANG San-yuan, MAO Gui-lian, ZHU Lin

2019, 35(12): 1-9. doi:10.13560/j.cnki.biotech.bull.1985.2019-0391

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The objective of this work is to select drought-tolerant sweet sorghum varieties at booting stage and their identification indexes. Total 28 sweet sorghum varieties were selected and 15 indexes such as plant height（PH）,stem diameter（SD）,leaf area（LA）,plant water content（WPC）,relative chlorophyll content（SPAD）,cell membrane permeability（CMP）,and photosynthetic rate（Pn）at booting stage were determined under normal irrigation（T1）and drought stress（T2）. Based on the drought tolerance coefficient of each index,the drought resistances of different sweet sorghum varieties at booting stage were comprehensively evaluated and classified by means of membership function,principal component analysis,and cluster analysis. Drought stress caused significant impact on all indexes,and the strong correlation analysis among 15 indexes existed. These 15 indexes were transformed into 6 new independent comprehensive indexes by principal component analysis,which represented the most of the original drought resistance information of all indexes. The D value of sweet sorghum of different varieties was calculated by membership function,and then 28 sweet sorghum varieties were divided into 4 categories by clustering analysis：category I consisting of 9 highly drought-resistant varieties,category II consisting of 10 drought-resistant varieties,category III consisting of 8 moderately drought-resistant varieties,and category IV consisting of one sensitive variety. Drought stress had an effect on all indexes of sweet sorghum at booting stage. Nine drought-resistant varieties were screened as following：F6036,F6080,F6099,F6043,F6149,F6059,F6096,F6271,and F6172.

Expression of FPPS Recombinant Protein from Pogostemon cablin and Screening of the Interaction Proteins

ZHONG Li-ting, CHEN Xiu-zhen, TANG Yun, LI Jun-ren, WANG Xiao-bing, LIU Yan-ting, ZHOU Xuan-xuan, ZHAN Ruo-ting, CHEN Li-kai

2019, 35(12): 10-15. doi:10.13560/j.cnki.biotech.bull.1985.2019-0475

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The purpose of this study is to express the recombinant protein of farnesyl pyrophosphatase synthase（PatFPPS）of Pogostemon cablin in prokaryotic cells and to screen its interaction proteins. The PatFPPS CDS was amplified and ligated into the pGEX-6P-1 vector by PCR. The plasmid confirmed by sequencing was obtained and transformed into BL21（DE3）expression strain,which was then induced by IPTG and the GST-tagged PatFPPS fusion protein was acquired. With GST Pull-Down technique,GST-tagged PatFPPS fusion protein was incubated with total proteins from the P. cablin leaves in vitro. The solution containing the protein complex was eluted and then identified by SDS-PAGE and LC-MS/MS. The results showed that the prokaryotic expression system of PatFPPS was successfully established and the purified recombinant protein was obtained,and some candidate interaction proteins were screened. In conclusion,soluble PatFPPS protein can be obtained with optimized prokaryotic expression system,and candidate proteins interacted with PatFPPS are identified.

Study on Antioxidant and Antitumor Activity of Essential Oil from Flowers of Syringa oblata

HU Jian-ran, LI Ping, TIE Jun, JIN Shan

2019, 35(12): 16-23. doi:10.13560/j.cnki.biotech.bull.1985.2019-0144

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Essential oil in the flowers of Syringa oblata at the bud stage was extracted by hydro distillation,and 3 different antioxidant tests were employed to evaluate the antioxidant activities of the essential oil. The scavenging effects of the essential oil at different concentrations on DPPH radical and hydroxyl free radicals were measured,and the total antioxidant capacity（T-AOC）of the essential oil was also measured. The results indicated that the essential oil had a favorable scavenging effect on DPPH free radical and hydroxyl radical in a dose-dependent manner. The T-AOC of the essential oil was 19.86±1.74 U/mg,higher than that of 3,5-Di-tert-butyl-4-hydroxytoluene（0.38±0.08 U/mg）. Gastric cancer lines MGC80-3 and HGC-27 were used to access the inhibitory effect of the essential oil by MTT（3-（4,5-Dimethyl-2-thiazolyl）-2,5-diphenyl-2H- tetrazolium bromide）method. The apoptotic index of the essential oil was detected by TUNEL assay. The results of MTT assay indicated that the essential oil significantly inhibited MGC80-3 and HGC-27 in a dose-dependent manner,and the IC₅₀ values,after 48 h exposure,were 0.282 mg/mL and 0.263 mg/mL respectively. TUNEL assay indicated the essential oil induced apoptosis also in a dose-dependent manner,and HGC-27 cells showed more sensitive than MGC80-3 cells. Therefore,essential oil in the flowers of S. oblata shows remarkably high antioxidative and antitumor activities in vitro,and demonstrated potential value of medical application.

Cloning and Expression Analysis of Tobacco Peroxidase Gene NtPOD2

WANG Jing, LIU Lun, YANG Yong, HU Sheng, LI Li-qin

2019, 35(12): 24-30. doi:10.13560/j.cnki.biotech.bull.1985.2019-0395

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Peroxidase（POD）is widely present in various organs and different developmental stages of plants,and plays an important role in plant growth,development and coping with stress. In order to explore the function of POD gene family in tobacco,A peroxidase gene was cloned from the common tobacco K326 by gene cloning. The gene was 99% homologous of Nicotiana tomentosiformis POD2,thus it was named NtPOD2 gene. Bioinformatics analysis was used to predict the structure of the NtPOD2 gene,and qRT-PCR technique to determine the expression level of NtPOD2 in different tissues and abiotic stress. Results showed that the sequence contained an open reading frame（ORF）of 897 bp and encoded 298 amino acid residues,and NtPOD2 protein had a type Ⅲ peroxidase typical conserved domain. NtPOD2 gene was expressed in root,stem,leave and flower,but the highest expression in root,and rapidly responded to abiotic stress induction. This indicates that NtPOD2 may be involved in plant growth,development and abiotic stress.

Cloning and Expression Analysis of Peroxidase Gene（ScAPX1）from Sugarcane

ZHANG Bao-qing, SHAO Min, HUANG Yu-xin, HUANG Xing, SONG Xiu-peng, CHEN Hu, WANG Sheng, TAN Qin-liang, YANG Li-tao, LI Yang-rui

2019, 35(12): 31-37. doi:10.13560/j.cnki.biotech.bull.1985.2019-0396

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Here cloning the ScAPX1 of sugarcane and analyzing its expression under low temperature stress is to provide a basis for further studying the function of APX1 gene in sugarcane under low temperature stress and investigating the molecular mechanism of breeding cold stress-resistant sugarcane. Having total RNA of sugarcane leaf as template,RT-PCR technique was used to clone the complete ORF sequence of ScAPX1 from sugarcane leaf,bioinformatics software was to analyze the characteristics of the encoding protein,and quantitative real-time PCR（qRT-PCR）method was applied to study the expressions of ScAPX1 gene under low temperature stress in two sugarcane varieties GT28 and YL6 with widely different cold resistance. The results showed that the ScAPX1 gene（GenBank accession number：KC794939）in sugarcane was cloned,which contained a complete open reading frame of 759 bp and encoded 252 amino acids. The protein encoded by this gene contained no signal peptide and no transmembrane structure,was a soluble protein and located in cytoplasm,and its homology with sorghum amino acid was 98%. It was inferred that ScAPX1 gene was a cytoplasmic ascorbic acid peroxidase gene（ScAPX）. qRT-PCR analysis showed that with the extension of low temperature（0-4^oC）,the expression levels of ScAPX1 gene in two sugarcane varieties increased first and then decreased,but differed. In the whole process of cold stress,the relative expression level in GT28,a cold resistant variety,was always higher than that in YL6,a cold sensitive variety. This result suggests that the ScAPX1 gene of sugarcane is active in response to low temperature stress,and the induced expression of this gene is closely related to the cold resistance of sugarcane varieties.

Functional Analysis of Arginine N-methyltransferase Gene MoHMT1 in Magnaporthe oryzae

ZHANG Wen-ze, ZHANG Yan-Li, MEN Yan-ming, ZHANG Yu-jiao, SUN Zhi-xin, LI Wen-hui, LU Guo-dong, QI Yao-yao

2019, 35(12): 38-44. doi:10.13560/j.cnki.biotech.bull.1985.2018-0313

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Arginine methylation regulated by protein arginine methyltransferases（PRMTs）is a common post-translational modification in eukaryotic organisms. In order to analyze the functions of gene MoHMT1（hnRNP arginine N-methyltransferase,PRMT1 homologous）,Mohmt1-knockout mutants were obtained based on the principle of homologous recombination,and preliminarily phenotypic characterizations were carried out. The results showed that the deletion of Mohmt1 resulted in the smaller colony,thinner aerial hyphae,and significantly slower growth of Magnaporthe oryzae,compared to the wild type. The conidia amount produced by Mohmt1-deleted mutants demonstrated obviously reduction with only 20% of the wild type;however,the conidia produced by the mutants normally germinated to form appressorium and successfully penetrated the onion epidermal cells. Furthermore,using the conidia suspension to inoculate the tested rice Leaves,it was found that the number of Lesions produced by the MoHmt1-deleted mutants significantly decreased related to the wild type and deletion of MoHmt1 attenuated pathogenicity on susceptible rice. These results indicate that MoHMT1 protein may play important regulation role in the hyphal development and pathogenic process of M. oryzae.

Effect of Bacillus amyloliquefaciensYM6 on Growth Promotion of Maize Under Salt Stress

WANG Hua-xiao, LIU Huan, YANG Guo-ping, ZHANG Xiu, LI Zhuang, ZHANG Yan

2019, 35(12): 45-49. doi:10.13560/j.cnki.biotech.bull.1985.2019-0263

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Bacillus amyloliquefaciens strain YM6 isolated from soil is able to mitigate the salt stress on maize. More detailed effects of stress relieving by this strain need to be elucidated. Provide new bacterials for the production of non-halophyte such as Maize and the improvement of saline soils in related areas. Maize seedlings were growing in hydroponic jars containing plant nutrients solution amended different concentrations of NaCl. YM6 was inoculated to these salt stressed seedlings,and plant growth was measured and analyzed. Results showed under 75 mmol/L NaCl stress,compared to the un-inoculated control,plant height increased by 15.2% when YM6 was inoculated to roots of maize seedlings. Root length and plant dry weight of inoculated maize increased by 28.8% and 27.0% respectively as compared to un-inoculated control. In the bonsai experiments with saline-alkali soil,plant height and dry weight of maize treated with YM6 increased by 37.8% and 43.9% respectively as compared to the un-inoculated control. Strain YM6 was also found to colonize maize root.Our preliminary results indicated that YM6 is able to alleviate the adverse effects of salt stress on maize.

Expression Analysis of Sl-miR482 in Tomato Fruit and the Construction of STTM Silencing Vector

MO Xian-lan, SHI Lie-qin, LU Qiu-li, WANG Xiao-min, REN Zhen-xin

2019, 35(12): 50-56. doi:10.13560/j.cnki.biotech.bull.1985.2019-0566

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The aim of this study is to investigate the specific expression pattern of tomato gene Sl-miR482 in fruit ripening stage,and to conduct bioinformatics analysis of its function and to construct STTM silencing vector. Quantitative PCR was used for gene expression detection,promoter analysis software and target gene prediction website was to perform bioinformatics analysis,PCR amplification,restriction enzyme digestion and ligation reaction were applied to construct STTM vector,and Agrobacterium was transformed. The expression level of Sl-miR482 increased gradually during flowering,and reached the peak in the immature green stage,and then gradually decreased. Further bioinformatics analysis showed that ethylene response elements were present in the Sl-miR482 promoter. The results of target gene prediction website showed that tomato pectin lyase Sl-PL13 was the target gene of Sl-miR482. Gene expression analysis showed that the expression levels of Sl-PL13 and Sl-miR482 demonstrated a trade-off relationship at different stages of fruit ripening. In sum,the STTM482 silencing vector is successfully constructed and transformed into Agrobacterium by PCR amplification and restriction enzyme ligation. The results of this study indicate that Sl-miR482 plays an important role in fruit ripening and softening.

Effects of Different Slope Positions on Soil Moisture and Physiological Indicators of Artemisia ordosica Root Zone in the Mu Us Sandy Land

MENG Wen-ting, WANG Tian-tian, ZHAO Xue-lin, ZHU Lin

2019, 35(12): 57-63. doi:10.13560/j.cnki.biotech.bull.1985.2019-0462

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This work aims to explore the variations in physiological indexes and soil moisture of Artemisia ordosica shrubs on different slopes for providing a theoretical basis and references in revealing the physiological and biochemical mechanisms of drought resistance of sandy plants,in the protection and restoration of sandy plants and in the response of desert plant communities under future environmental changes. Soil water content（SWC_0-300）,cell membrane permeability,malondialdehyde（MDA）and antioxidant enzymes of Artemisia ordosica on different slopes（bottom,middle and top of slopes）of total 0-300 cm soil layer in the Mu Us Sandy Land in Yanchi,Ningxia in 2018 were measured. The results showed that soil water content decreased with the increase of slope position and soil moisture content in 0-300 cm soil layer presented as middle slope > bottom slope > upper slope. With the increase of slope position,the cell membrane permeability of A. ordosica first decreased and then increased,and the superoxide dismutase（SOD）first increased and then decreased. MDA first increased and then decreased in July and August,but decreased with the increase of slope position in September and October. Peroxidase（POD）decreased with the increase of slope position in July and August,but first decreased and then increased in September and October. It is concluded that the correlation between cell membrane permeability and drought resistance of slope is the closest. A. ordosica has the greatest moisture content in the slope soil and thus strong drought resistance.

Safety Evaluation of Staphylococcus aureus Bacteriophage Based on Transcriptome

GENG Hui-jun, ZOU Wei, CUI Hui-jing, LI Xiao-yu, WANG Li-li, XU Yong-ping

2019, 35(12): 64-75. doi:10.13560/j.cnki.biotech.bull.1985.2019-0696

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The emergence of multidrug-resistant bacteria has forced us to consider bacteriophage therapy as one of the possible alternative therapies. The aim of this study is to investigate the safety and long-term use condition of Staphylococcus aureus bacteriophage in mammary perfusion therapy in mice by transcriptional sequencing. Twenty-four Kunming female mice lactating for 10 d were randomly divided into three groups,including healthy control group（T01）,bacteriophage VBSM-A1 perfusion group（T02）and bacteriophage cocktail perfusion group（T03）. S. aureus phage suspension of different components was injected into the fourth pair of mammary glands（abdomen）of mice by non-invasive method,and then the mice and the changes of histopathology and TNF-α in the breast tissue were observed. Meanwhile,transcriptome sequencing was used to analyze the mammary gland of mice. The results showed that phage infusion caused slight inflammation in the mammary glands of mice,but did not cause physiological state changes of the mice. At the same time,the number of phages decreased significantly（started：5×10⁷ PFU/gland;24 h latter：10^3.6 and 10^4.4 PFU/gland respectively）during 24 h,suggesting that the way and dosage of phage administration needed further exploration. The prospect of combined application of phages and antibiotics to reduce the phage immune response and antibiotic resistance is proposed,which provides a theoretical and experimental basis for the use of subsequent phages at the transcriptome level.

RNA-seq Analysis of Drug-resistant Genes Associated with baeSR and acrB Double Gene Deletion Strains of Salmonella typhimurium

XU Jun, WANG Rui, LI Rui, GAO Hai-xia, ZHANG Rui-liang, WANG Wen-jing, ZHAO Xia, LI Lin

2019, 35(12): 76-84. doi:10.13560/j.cnki.biotech.bull.1985.2019-0464

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The transcriptome sequencing technology（RNA-seq）was used to screen the target genes related to baeSR and acrB of Salmonella typhimurium,which laid a foundation for further study on the drug resistance mechanism of S. typhimurium. RNA-Seq was applied to screen the related drug-resistant genes with varied expressions after the deletion of baeSR and acrB genes,GO enrichment and KEGG pathways to explore their functions,and qRT-PCR to verify some differential genes. Under the condition of |log₂（Fold Change）| > 1 and q value < 0.005,a total 1 320 differentially expressed genes were screened by CR△acrB/CR as comparison group,of which 894 were down-regulated and 426 were up-regulated. A total 1 377 differentially expressed genes were screened by CR△baeSR△acrB/CR,of which 972 genes were down-regulated and 405 genes were up-regulated. GO enrichment classification results showed that the differential genes were mainly concentrated in transmembrane transport,cell adhesion,cilia or flagellum movement and other functions. KEGG database showed that the differential genes were mainly concentrated in metabolic pathways,ABC transport system,two-component signal transduction system,flagellum assembly,beta-lactam resistance and so on. Among them,ompR,csgD,fliA and fliG regulated their resistances to various antibiotics by regulating the ability of bacterial biofilm formation. emrA,mdtC and yejE belonged to efflux pump-related genes,which mediated the resistance of bacteria to colistin and neomycin. STM3031 endowed Salmonella with ceftriaxone resistance,and pmrF regulated bacterial resistance to polymyxins. In conclusion,the deletion of acrB and baeSR genes in S. typhimurium may affect the expressions of related resistance genes.

Bioinformatics Analysis of the Extracellular Region of NA Protein in Novel H7N9 Avian Influenza Virus and Preparation of Polyclonal Antibodies

QIU Shu-xing, YIN Xing, SU Shu-juan, YIN Jun-lei, ZHANG Jia-you, LIU Xue-he, JIA Kun-yi, YANG Xiao-ming

2019, 35(12): 85-93. doi:10.13560/j.cnki.biotech.bull.1985.2019-0762

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This work aims to express and purify the extracellular region of NA（neuraminidase）protein in novel H7N9 avian influenza virus（Anhui Strain）in prokaryotic expression system and to prepare its polyclonal antibodies. Firstly,bioinformatics analysis of the extracellular region of NA protein in novel H7N9 avian influenza virus was conducted. The codon optimization was performed according to the codon preferences in the Escherichia coli expression system,and the gene encoding this protein was synthetized. Subsequently the recombinant plasmid pET28b-tN9（truncated N9）carrying chemically synthesized extracellular region of NA gene was transformed into E. coli BL21（DE3）,E. coli Rosetta and E. coli Arctic Express（DE3）and their expressions were induced with IPTG,and the SDS-PAGE identification was conducted. The recombinant E. coli BL21（DE3）was cultured for more mass,induced by IPTG,the induced products were purified by Ni column,analyzed using SDS-PAGE,and identified by mass spectrometry. The purified protein was used to immunize rabbit to prepare polyclonal antibodies. Western blotting and an indirect ELISA assay were performed to determine specificity and titer of the polyclonal antibody. As results,the recombinant protein was successfully expressed and purified. Western blotting analysis showed that the prepared polyclonal antibody specifically recognized the recombinant protein and H7N9 avian influenza virus（Shanghai Strain）. The titer of the antibody was about 1：256000 detected by indirect ELISA. In conclusion,the specific polyclonal antibodies with high titer were successfully prepared by immunizing the rabbit using the purified tN9 protein in in prokaryotic expression system,laying a foundation for further studying the structure and function of the NA protein,pathogenesis and building rapid detection methods of H7N9 avian influenza virus.

Eukaryotic Expression of BMPR1B in Sheep and Identification of Its Interaction Proteins

JIA Jian-lei, CHEN Qian, JIN Ji-peng, YUAN Zan, ZHANG Li-ping

2019, 35(12): 94-104. doi:10.13560/j.cnki.biotech.bull.1985.2019-0447

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This work aims to study the roles and pathways of BMPR1B protein and its interactive protein in the development and ovulation of sheep ovarian oocyte. By constructing the eukaryotic expression system of BMPR1B gene and characterizing BMPR1B protein,CoIP-MS technology was used to identify the specifically interactive proteins with BMPR1B in ewe ovarian extract,and the target protein interaction network was established. Then we used bioinformatics to analyze and predict the interaction pathways of BMPR1B protein and its interaction proteins in the development and ovulation of sheep ovarian oocyte. There were 23 proteins from ewes ovary extract that were correlated with BMPR1B,while 6 target proteins（BMP2,BMP4,GDF5,GDF9,RhoD and HSP10）specifically interacted with BMPR1B. Bioinformatics analysis showed that the target protein constituted complex and close interactions with TGF-β signal transduction pathway,ovarian steroid generation pathway and MAPK signal transduction pathway,and the metabolic pathways of BMPR1B protein regulating the development and ovulation of sheep oocytes was designed based on the results of their interactions. BMPR1B protein interacts with Smads family proteins in lambing trait pathway and plays an important role in the development and ovulation of sheep oocyte.

Deletion of the Marker Gene in Transgenic Goat Mammary Epithelial Cells by Cre/Loxp

SONG Shao-zheng, YU Kang-ying, LU Rui, ZHANG Ting, CHEN Chao-jun, PAN Sheng-qiang, CHENG Yong, ZHOU Ming-ming

2019, 35(12): 105-111. doi:10.13560/j.cnki.biotech.bull.1985.2019-0544

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In order to eliminate the potential biosafety risk of marker genes in transgenic animals by nuclear transfer,the Cre/Loxp system was used to delete the marker genes of the transgenic goat mammary epithelial cells（GMECs）with human lactoferrin（hLF）,so as to explore the effect of marker gene deletion on the mammary gland specific expression of functional genes. First,the transgenic hLF goat mammary epithelial cells were revived and purified. Then,the purified PBS185 plasmid was electrotransfected into mammary epithelial cells. After 10-14 d culture,the monoclonal cells were selected by self-made mouth-controlled transfer needle and trypsinase digested cells. Finally,whether or not the deletion of the marker gene was detected by PCR,prolactin nduced expression and the expression level of functional protein hLF was detected by ELISA and Western Blot. The results showed that a total of 65 monoclonal mammary epithelial cells were obtained after electrotransfection,and 18 of the cells in fine condition were detected by PCR. Among them,3 cell lines were deleted marker gene,and the deletion efficiency was 16.7%（3/18）. Moreover,the expression level of marker gene-deleted cell lines increased about 8 times （2.85 g·L^-1/0.35 g·L^-1）,the size of protein band was consistent with the target protein（80 kD）. The above results prove that the Cre/Loxp system can effectively delete the marker gene in GMECs genome and the expression level of cell lines significantly increase. Moreover,a system for the deletion of the marker gene in transgenic GMECs is successfully established,which also lays a foundation for the further expanding the production of transgenic goats without the marker gene.

Expression of Recombinant Ovine FSH in the CHO-K1 Cell and Its Application

WANG Xing-long, LI Qian, MA Rui-xian, LI Sheng-jun, LI Xiang-rong, FENG Ruo-fei

2019, 35(12): 112-117. doi:10.13560/j.cnki.biotech.bull.1985.2019-0668

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This work aims to explore the expression of recombinant sheep FSH（follicle stimulating hormone）in CHO-K1 cells and its biological activity. The recombinant plasmid pcDNA3.1-FSHβα was constructed and electroporated into fully suspended CHO-K1 cells. The monoclonal cells with high expression of recombinant FSH protein were screened under G418 pressure,cultured in a bioreactor and concentrated by ultrafiltration. The concentrated recombinant sheep FSH was injected into mice,the ovarian mass of mice was weighed,and the contents of luteinizing hormone and progesterone in mouse serum were detected. The recombinant plasmid pcDNA3.1-FSHβα was successfully constructed and the secretory expression was successful in CHO-K1 cells,and the expression level was 297.97 ng/mL. The ovaries of mice injected with concentrated recombinant sheep FSH protein were significantly enlarged,and the contents of luteinizing hormone and progesterone were also significantly up-regulated. In sum,the recombinant sheep FSH protein is successfully expressed in CHO-K1 cells and presents certain biological activity.

Development Trend Analysis of Crop Genome Research Based on Bibliometrics

YUAN Xue, QIAN Wan-qiang, GUO Xi-chuan, LU Yao, YAN Yun

2019, 35(12): 118-128. doi:10.13560/j.cnki.biotech.bull.1985.2019-0228

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Genomics research is one of the fastest growing research areas in the world in recent years. The development of crop genomics research has played an important role in the effective use of modern molecular biology methods for genetic improvement of species. The sequencing of crop genomes of important crops such as rice,wheat,corn,soybean,rapeseed,cotton,vegetables,etc. has been conducted,on the basis of which the cloning and identification of genes controlling important agronomic traits is completed. The scientific literature objectively records the development overview of various disciplines or knowledge fields. The analysis of the discipline development trend based on bibliometrics may systematically summarize the current research status and hot issues of the field and evaluate the research strength and achievement output of the research institutions. This study comprehensively reveals the research and development status quo,important countries,important institutions and research topics of the crop genome in the past 10 years through the multi-dimensional and deep-level quantitative analysis of the funding of the Sino-US Natural Science Foundation projects and the SCI papers by the combination of quantitative analysis of literature and qualitative analysis by experts. The relevant research results may provide reference for future research and development layout and decision-making in related fields in China.

Research Progress on Family of Plant WRKY Transcription Factors

HUANG Xing, DING Feng, PENG Hong-xiang, PAN Jie-chun, HE Xin-hua, XU Jiong-zhi, LI Lin

2019, 35(12): 129-143. doi:10.13560/j.cnki.biotech.bull.1985.2019-0626

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WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form an integral part of signaling webs that modulates many plant processes. WKRY transcription factors have a variety of biological functions and play an important role in plant growth,development and senescence,abiotic and biotic stress and so on. At DNA level,WRKY transcription factors can bind to W-box TTGAC（C/T）in the promoter of its target genes and activate or inhibit the expression of downstream genes to regulate their response by self-regulation or cross-regulation. At protein level,WRKY transcription factors can regulate plant growth and development or various stress responses by interacting with a variety of proteins,including MAP kinases,histone deacetylase,resistant R proteins,and a variety of transcription factors. This paper reviews the research progress on the structure,biological function,regulatory mechanism and network of WRKY transcription factors,which will help us to understand their roles in plants more comprehensively.

The Role of NAC Transcription Factors in Plant Response to Abiotic Stress

ZHANG Dan, MA Yu-hua

2019, 35(12): 144-151. doi:10.13560/j.cnki.biotech.bull.1985.2019-0525

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Facing the deteriorating environment and the pressure of population growth,studying plant stress-resistance mechanisms and improving plant stress resistance are important for achieving sustainable development of agriculture. In recent years,the plant-specific transcription factors have been widely concerned for their important role in abiotic stress response. The NAC family is the largest one of plant-specific transcription factors and plays an important regulatory role in the growth and development of plants and in response to abiotic stresses. Here,the progress on the structural characteristics,mechanism and function of NAC transcription factors in abiotic stress are reviewed,aiming to provide a theoretical basis for the study of NAC-related genes and the cultivation of new stress-resistant plant varieties.

Research Progress in Fusion Modification of PAS

HAN Fei, JIANG Ming-feng, RAN Rong, WANG Gang

2019, 35(12): 152-158. doi:10.13560/j.cnki.biotech.bull.1985.2019-0519

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PAS protein modification technology refers to that functional polypeptides with polypeptide sequences composed of proline,alanine and serine by gene fusion are linked and fusion expression is conducted by gene engineering,thus polypeptide modification is achieved. At present,for small molecule polypeptide drugs,its smaller volume leads to a shorter half-life in plasma,and the clinical application of PEG to modify small molecule polypeptide drugs makes it have a larger hydrodynamic volume and thus its pharmacological activity is increased. However,with more and more PEG modified drugs entering the clinical application market in recent years,the safety drawbacks of PEG chemical polymers have been constantly uncovered. Therefore,it is necessary to seek more safe and effective methods besides PEG modification,and PAS has shown great advantages in small molecular polypeptide modification. In this paper,the design ideas and advantages of PAS protein modification technology,as well as the latest progress in application are described in detail.

Research Progress on Mesoporous Silica in Drug Delivery System and in vitro/in vivo

ZHANG Wen-jun, WU Meng-ting, LÜ Chun-yan, WANG Qing, CHEN Yong-lin

2019, 35(12): 159-168. doi:10.13560/j.cnki.biotech.bull.1985.2019-0481

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Mesoporous silica nanoparticles are widely concerned in biomedical field in recent years due to their specific characteristics,including large specific surface area,high porosity,adjustable mesoporous structure and easy surface modification. As a novel carrier,mesoporous silica can not only improve the solubility of poorly soluble drugs and effectively improve the bioavailability of drugs,but achieve the targeting of tumor cells by modifying or coating functional substances and control the adsorption and release of drug. Its stable framework structure can deliver and protect nucleic acid molecules for gene therapy purposes. However,in the application of mesoporous silica nanoparticles to the clinic,further work is needed to predict and assess potential toxicity and adverse reactions,to clarify their absorption,distribution,metabolism,excretion,biocompatibility and toxicity in vivo for ensuring their availability and safety in patients. This paper reviews the application of mesoporous silica nanoparticles in drug delivery system and gene therapy,mainly focuses on the research results on targeted and stimuli-responsive mesoporous silica,as well as deeply summarizes the pharmacokinetics,biocompatibility and toxicity of mesoporous silica,aiming at providing a reference for the application of mesoporous silica in biomedicine.

Application of Male Sterility Molecular Markers in Identification of Onion Haploid

YANG Yan-yan, HUO Yu-meng, WU Xiong, LIU Bing-jiang

2019, 35(12): 169-174. doi:10.13560/j.cnki.biotech.bull.1985.2019-0489

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In order to effectively identify and utilize onion haploid,the unfertilized flower buds of three onion hybrid cultivars were used as explants to induce regenerated plants through in vitro gynogenesis. Flow cytometry was used to identify ploidy of regenerated plants,and DNF-566,RNS-357 and AcSKP1 markers were used to detect homozygosity of the regenerated plants. The results showed that the genotypes of plants regenerated from ‘ATON’ and ‘Earth’ were homozygous at Ms locus. Of the sixteen regenerated plants of ‘TABAO’,thirteen were homozygous at Ms locus and three were heterozygous. These results suggest that homozygous plants are derived from megaspore mother cells,and heterozygous plants may be diploid from ovary wall or other somatic cells. Seven diploids were obtained,of which four were doubled-haploid. The doubled-haploid can be further used as parent material for hybrid breeding. The results further show that the molecular markers of Ms locus in onion can be used as an effective method to analyze the homology between haploid regenerated plants and their donor hybrids. Combining with flow cytometry,the double haploid could be identified quickly and accurately.

Multi-primer Multiplex PCR Detection Method for the Exogenous Multivalent Insect-resistant Bt Genes in Transgenic Rice

FANG Xin-yi, YANG Jing, WANG Jing-zhang, LI Yang-sheng

2019, 35(12): 175-183. doi:10.13560/j.cnki.biotech.bull.1985.2019-0383

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This work aims to explore a simple and effective method for detecting multivalent insect-resistant Bt genes in transgenic rice. Based on the insertion positions of 3 Bt genes（cry1Ab/Ac,cry2,and cry1C）from events（TT51-1,T2A-1,and T1C-19）,9 primers were designed and used to obtain 6 target bands in significant differences by multiplex primer PCR technique. Subsequently,the positive and negative bands representing the TT51-1 event were 937 bp and 718 bp,those representing the T2A-1 event were 600 bp and 434 bp,and those representing the T1C-19 event were 495 bp and 792 bp. The detection of homozygous,heterozygous and negative Bt transgenic rice plants was rapidly conducted by agarose gel electrophoresis. This technology overcame the technical difficulties such as mutual binding interference among the multiple primers in the PCR process and presented fine compatibility with different PCR reagents. It can cooperate with rapid DNA extraction of rice to rapidly analyze a large number of breeding offspring. In conclusion,the establishment of this method provides a simple and efficient gene detection technology for rice stem borer-resistant molecular breeding with multivalent Bt gene,which is beneficial to increase the detection efficiency of Btgenes and to accelerate the breeding process of rice stem borer-resistant transgenic rice.

Development of a Colloidal Gold Immunochromatographic Test Strip for the Detection of Potato Virus S

ZHANG Wei, LI Zhi-xin, FU Chun-jiang, LIU Wei-ping

2019, 35(12): 184-188. doi:10.13560/j.cnki.biotech.bull.1985.2019-0287

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This work aims to develop a colloidal gold immunochromatographic test strip for on-site rapid detection of potato（Solanum tuberosum L.）virus S（PVS）. Colloidal gold solution was prepared by trisodium citrate reduction method,its maximum absorption value was at the wavelength of 524 nm and the particle size was 20-30 nm. In the strip test,colloidal gold-labeled polyclonal sheep antibody against PVS was sprayed on the glass fiber as the detection antibody,and rabbit anti-goat IgG at the control line and anti-PVS at the test line on the nitrocellulose membrane of the test strip was served as the capture antibody. The positive result was easily judged by the presence of a red test line with naked eye within 2 min. The detection was still feasible when the samples was diluted by 10⁴ W/V,and test strip showed no cross effect when tested by other common 5 viruses（PVX,PVY,PLRV,PVA,and PVM）samples. The detection of potato leaves from field by the test strip was consistent with that by ELISA. Test strip is valid for 180 d while stored in dry sealed low-temperature 4℃. The test strip is convenient,simple and rapid in the field detection of PVS,no additional equipment is required,thus applicability and practicability are wide.

Overview on Efficient Methods for the Determination of Microalgae Lipid Content

LI Zhi, ZHOU Qiu-xiang, LI Qiu-ling, LIU Meng-ying, ZHOU Zhi-you, LI Han-guang

2019, 35(12): 189-195. doi:10.13560/j.cnki.biotech.bull.1985.2019-0364

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Due to the petroleum reserves diminishing and the concerns of global environmental protection rising,microalgae,as a sustainable and renewable energy source,has been attracted wide attention in recent years. The breeding of microalgae with high oil content is an important step in the development of microalgae bioenergy,and the efficient and rapid determination method of lipids is one of the key technologies in the breeding process. This paper reviews the basic principles and research status of several efficient,accurate and convenient determination methods of lipids,such as Nile red fluorescence spectrometry,BODIPY 505/515 fluorescence spectrometry,copper reagent method,Sudan black B staining method,Fourier transform infrared spectrometry,time-domain nuclear magnetic resonance method,and vis/near-infrared spectrometry. Furthermore,the advantages and methodological limitations of these methods are also compared,aiming at providing a grateful reference for determination of microalgae lipid and concurrently some scientific know-how for subsequent research on the production of microalgae oil.

Improve the Site-directed Mutagenesis Efficiency of Overlap Extension PCR by Outboard-primers

WANG Liu-yue, LI Hui-mei, MA Meng-qi, LIANG Ming-xing, HE Ru-yang, CHEN Hua-bo

2019, 35(12): 196-202. doi:10.13560/j.cnki.biotech.bull.1985.2019-0358

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Simplifying overlap extension PCR（OE-PCR）protocol in site-directed mutagenesis by parallel template is not valid. The low efficiency of restriction enzyme cleavage close to the end of DNA fragments is the key reason of restricting the site-directed mutagenesis by OE-PCR. The enzymatic digestion efficiency of DNA product is improved by Outboard-primers method,and as a result,the achievement ratio of site-directed mutagenesis increases. The matching loci in upstream and downstream primer were outward moved by 50-100 bp with a pair of outboard-primers;therefore,the enzymatic digestion sites at both ends of target gene were far from the ends of the second round PCR product. Thus,the subsequent enzyme digestion efficiency was improved and the reaction time was saved. Because the length of DNA was obviously shortened by enzymatic digestion at this time,the results of gel electrophoresis showed that the PCR product was fully digested in only 1 h. The number of clone after ligating the transformation of the products into receptive Escherichia coli also increased significantly,and the positive rate increased from 33%-70% to 100%. Comparing with traditional terminal primers,outboard-primers method not only saves time,but also remarkably increases the positive colony number and success rate in site-directed mutagenesis of overlap extension PCR.

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2019, 35(12): 203-203.

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