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Chinese Science Bulletin 1991年第11期36卷 936-941页

作者：谢明唐吴伯骥 laboratory of plant cell non-carrier genetic manipulation Chengdu Institute of Biology Academia Sinica Chengdu 610015 PRC

Ribulose-1, 5-bisphosphate carboxylase/oxygenase (EC 4, 1, 1, 39, abbreviation: RuBPcase)is a bifunction enzyme. Because of its properties in genetic function and structure, RuBPcase has been intensively studied. In the absence of certain effective genetic markers for identifying hybrids, the analysis of the isoelectrofocusing pattern of RuBPcase has become a standard procedure to verify the nature of the hybrid plants.

关键词： RuBPcase isoelectrofocusing RuBPcase immunochemistry spinach+ tobacco hybrid.

Expressing activity of promoter elements of large intergenic region from cotton leaf curl virus in host plant

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Science China(Life Sciences) 2001年第1期44卷 8-17页

作者：谢迎秋刘玉乐朱祯 1. laboratory of plant genetic manipulation Institute of Genetics Chinese Academy of Sciences 100101 Beijing China 2. Institute of Microbiology Chinese Academy of Sciences 100080 Beijing China

Cotton leaf curl virus (CLCuV) is a type of single-stranded DMA virus, belonging to geminivirus of subgroup Ⅲ. In order to determine the function of CLCuV large intergenic region (LIR), total DNA of CLCuV-infected cotton leaves was used as template, and fragment of LIR was obtained by PCR and inserted into clone vector. The fragment of LIR was fused with gus reporter gene and nos terminator in the orientation of transcription of virion sense and complementary sense respectively, and the plant expression vectors were constructed. GUS activity of Agrobacte-rium-mediated transgenic tobacco was measured. The result indicated that LIR showed strong promoter activity in complementary sense gene orientation. Average GUS activity of the complementary sense promoter was 5-6 times that of CaMV 35S promoter, and the highest GUS activity of individual plant was ten times of that of CaMV 35S promoter. Histochemical localization confirmed its activity in both mesophyll and vascular tissues. Activity of virion sense of LIR was rather low. Thus LIR isolated from CLCuV could be used as a novel strong promoter in plant genetic manipulation.

关键词： geminivirus large intergenic region promoter tobacco.

Heterologous expression of Lolium perenne antifreeze protein confers chilling tolerance in tomato

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Journal of Integrative Agriculture 2018年第5期17卷 1128-1136页

作者： Srinivasan Balamurugan Jayan Susan Ann Inchakalody P Varghese Shanmugaraj Bala Murugan Mani ChANDra Harish Sarma Rajeev Kumar Ramalingam Sathishkumar plant genetic Engineering laboratory Department of BiotechnologyBharathiar University

Antifreeze proteins（AFP） are produced by certain plants, animals, fungi and bacteria that enable them to survive upon extremely low temperature. Perennial rye grass, Lolium perenne, was reported to possess AFP which protects them from cold environments. In the present investigation, we isolated AFP gene from L. perenne and expressed it in tomato plants to elucidate its role upon chilling stress. The T1 transgenic tomato lines were selected and subjected to molecular, biochemical and physiological analyses. Stable integration and transcription of Lp AFP in transgenic tomato plants was confirmed by Southern blot hybridization and RT-PCR, respectively. Physiological analyses under chilling conditions showed that the chilling stress induced physiological damage in wild type（WT） plants, while the transgenic plants remained healthy. Total sugar content increased gradually in both WT and transgenic plants throughout the chilling treatment. Interestingly, transgenic plants exhibited remarkable alterations in terms of relative water content（RWC） and electrolyte leakage index（ELI） than those of WT. RWC increased significantly by 3-fold and the electrolyte leakage was reduced by 2.6-fold in transgenic plants comparing with WT. Overall, this report proved that Lp AFP gene confers chilling tolerance in transgenic tomato plants and it could be a potential candidate to extrapolate the chilling tolerance on other chilling-sensitive food crops.

关键词： Lolium perenne antifreeze protein chilling tolerance genetic transformation transgenic tomato

Protection of Mice with a Divalent Tuberculosis DNA Vaccine Encoding Antigens Ag85B and MPT64

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Acta Biochimica et Biophysica Sinica 2004年第4期36卷 269-276页

作者： Xia TIAN, Hong CAI, and Yu-Xian ZHU* The National laboratory of Protein Engineering and plant genetic Engineering, Peking University, Beijing 100871, China The National laboratory of Protein Engineering and plant genetic Engineering Peking UniversityBeijing 100871 China

DNA vaccine may be a promising tool for controlling tuberculosis development. However, vaccines encoding single antigens of mycobacterium did not produce protective effect as BCG did. In the present study, we evaluated the immunogenicity and protective efficacy of a divalent DNA vaccine encoding two immunodominant antigens Ag85B and MPT64 of Mycobacterium tuberculosis. We found that both humoral and Th1-type (high IFN-γ, low IL-4) cellular responses obtained from the divalent DNA vaccine group were significantly higher than that conferred by BCG. RT-PCR results showed that antigens were expressed differentially in various organs in divalent DNA vaccine group. The survival rate for mice treated with the divalent DNA vaccine after challenging with high doses of virulent M. tuberculosis H37Rv was significantly higher than that of the BCG group or any of the single DNA vaccine group. Significant differ- ences were also found between the single and divalent DNA vaccinated mice in terms of body, spleen and lung weight. Bacterial loading decreased about 2000-fold in lungs and about 100-fold in spleens of divalent DNA vaccinated mice when compared with that of the control group. We conclude that our divalent DNA vaccine may be a better choice for controlling tuberculosis disease in animals.

关键词： tuberculosis divalent DNA vaccine immunogenicity protection

Effect of cDNA fragments in different length derived from Potato Virus Y coat protein gene on the induction of RNA-mediated virus resistance

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Science China(Life Sciences) 2004年第4期47卷 382-388页

作者： ZHU Junhua ZHU Xiaoping WEN Fujiang BAI Qingrong ZHU Changxiang SONG Yunzhi laboratory of plant genetic Engineering College of Plant ProtectionShandong Agricultural UniversityTaian 271018China

We have reported that cDNA derived from entire coat protein (CP) gene of potato virus Y (PVY) could induce resistance to PVY infection in transgenic tobacco plants, and the resistance was further demonstrated to be RNA-mediated rather than coat protein-mediated. In this study, we cloned cDNA fragments of 202 bp, 417 bp, and 603 bp in length derived from the 3′ end of the PVY CP gene, and the cDNA fragments were introduced into tobacco (var. NC89) plants via Agrobacterium-mediated transformation system. The results of resistance assay showed that the CP cDNA fragments of 417 bp and 603 bp could confer resistance of the trans-genic plants to PVY infection, but the fragment of 202 bp in length could not. Molecular analysis revealed that the resistance was RNA-mediated, which is believed to be a result of post-transcriptional gene silencing. The results indicate that the length of cDNA fragments needed for resistance induction was located somewhere between 202 bp and 417 bp from the 3′ end of PVY CP gene.

关键词： PVY partial length of CP gene fragments transgenic tobacco plants RNA-mediated resistance post-transcrip-tional gene silencing.

Chikungunya infection:A potential re-emerging global threat

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Asian Pacific Journal of Tropical Medicine 2016年第10期9卷 910-915页

作者： Shanmugaraj Bala Murugan Ramalingam Sathishkumar plant genetic Engineering laboratory Department of BiotechnologyBharathiar University

Infectious diseases are indeed a lifelong threat to everyone irrespective of age, sex, lifestyle and socio-economic status. The infectious diseases have persisted among the prominent causes of death globally. Recently, re-emergence of Chikungunya viral infection harmed many in Asian and African countries. Chikungunya was considered as a major threat in developing and underdeveloped countries; the recent epidemiological outbreak of Chikungunya in La Reunion urges the global researchers to develop effective vaccine against this viral disease. In this review, Chikungunya, pathogenesis and epidemiology were briefly described.

关键词： Aedes mosquitoes Alphavirus Arthropod-borne disease Chikungunya non-Structural Proteins Structural Proteins

ROOT PROTOPLAST ISOLATION AND CULTURE IN HIGHER plantS

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Science China Chemistry 1985年第4期28卷 386-393页

作者：许智宏,M. R. DAVEY,E. C. COCKING Shanghai Institute of plant Physioloyy Academia Sinica plant genetic manipulation Group Department of Botany University of Nottingham Nottingham NG7 2RD U.K. plant genetic manipulation Group Department of Botany University of Nottingham Nottingham NG7 2RD U.K.

A simple enzymatic method has been developed for the isolation of protoplasts in workable quantities from radicles of 1—3 days germinating seeds of twelve plant species, including eight legumes (Cajanus cajan, Glycine max, Medicago sativa, Phaseolus aureus, P. mungo, Pisum sativum, Trigonella foenum-graecum, and Vicia fabs) and four members of the Cruciferae (Brassica alba (Sinapsis alba), B. campestris, B. napus and B. oleracea). Root protoplasts showed high division potential, with callus formation in most species tested except C. cajan and V. faba, where only a few cell divisions were observed. plantlets were regenerated from root protop]ast-derived tissues via shoot formation in B. napus and B. oleracea, and somatic embryogenesis in M. sativa, demonstrating the totipotency of root protoplasts. Root protoplasts provide a useful experimental system for cultural studies and genetic manipulations, especially for those species which have previously been recalcitrant in culture. The advantages of root pr

关键词： root

Purification and Immunity Analysis of Recombinant 6His-HPT Protein Expressed in ***

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Biomedical and Environmental Sciences 2003年第2期16卷 149-156页

作者： LI-CHEN YANG ZHEN ZHU XIAO-GUANG YANG plant genetic manipulation laboratory Institute ofGeneticsChinese Academy of SciencesBeijing 100101China Key laboratory of Trace Element Nutrition ofthe Ministry of Health ChinaInstitute of Nutrition and Food SafetyChinese Centerfor Diseases Control and PreventionBeijing 100050Chin

To obtain HPT protein (Hygromycin B Phosphotransferase), a kind of plant selective maker gene product expressed from *** and to prepare the polyclonal antibody (pAbs) against it. Methods HPT cDNA fragment was obtained by PCR and was inserted into the prokaryotic expressing vector pBV222. Then the constructed recombinant plasmid pBV222-HPT was transfered into *** DH5?for HPT expression. The recombinant expressing system was confirmed by restriction endonuclease digestion, DNA sequencing and protein expression. *** cells were lysed by sonication and detergent dissolution. After cell membrane was extracted, the inclusion bodies were denatured by 8 mol/L Urea and purified with metal chelate affinity chromatography on Ni-NTA agarose under denaturing condition. The purified 6His-HPT was characterized by SDS-PAGE, and used to immunize rabbit. The titer and specificity of antisera were detected by ELISA and Western blot respecitively. Results Analysis of DNA sequence and restricted enzymes showed that the sequence of PBV222-HPT plasmid was correct. The amount of recombinant HPT expressed in *** accounted for 30% of total cellular proteins. From 1 liter of fermentative bacteria about 22 milligrams of pure recombinant HPT was isolated with purity above 95%. The recombinant HPT protein could produce high titer antiserum in rabbits and show good immunity activity. Western blot showed specific binding reaction between the antiserum to the purified 6His-HPT protein and their expressed products (plants protein and bacterial protein). Conclusion HPT protein can be expressed and purified from *** by a relatively simple method, which has high immunity activity.

关键词： Organisms genetically Modified DNA Bacterial Electrophoresis Polyacrylamide Gel Escherichia coli genetic Engineering genetic Markers Phosphotransferases (Alcohol Group Acceptor) Plasmids Polymerase Chain Reaction Recombinant Proteins Research Support non-U.S. Gov't

Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

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Science China(Life Sciences) 2003年第4期46卷 389-398页

作者：徐云剑顾雪松李珺李晴 PeterJ.Davies 朱玉贤 The National laboratory of Protein Engineering and plant genetic Engineering Peking University Department of plant Biology Cornell University Ithaca

The AAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficient E. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with the AAIR expression vector. Further study revealed that overexpression of the pea AAIR cDNA in acetohydroxy acid isomeroreductase deficient E. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of the AAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulate AAIR gene expression.

关键词： AAIR G2 pea A/T rich region enzymatic activity senescence. D

Molecular Biological and Biochemical Studies Reveal New Pathways Important for Cotton Fiber Development

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Journal of Integrative plant Biology 2007年第1期49卷 69-74页

作者： Yu Xu Hong-Bin Li Yu-Xian Zhu National laboratory of Protein Engineering and plant genetic Engineering Peking University Beijing 100871 China

As one of the longest single-celled seed trichomes, fibers provide an excellent model for studying fundamental biological processes such as cell differentiation, cell expansion, and cell wall biosynthesis. In this review, we summarize recent progress in cotton functional genomic studies that characterize the dynamic changes in the transcriptomes of fiber cells. Extensive expression profilings of cotton fiber transcriptomes have provided comprehensive information, as quite a number of transcription factors and enzyme-coding genes have been shown to express preferentially during the fiber elongation period. Biosynthesis of the plant hormone ethylene is found significantly upregulated during the fiber growth period as revealed by both microarray analysis and by biochemical and physiological studies. It is suggested that genetic engineering of the ethylene pathway may improve the quality and the productivity of cotton lint. Many metabolic pathways, such as biosynthesis of cellulose and matrix polysaccharides are preferentially expressed in actively growing fiber cells. Five gene families, including proline-rich proteins （PRP）, arabinogalactan proteins （AGP）, expansins, tubulins and lipid transfer proteins （LTP） are activated during early fiber development, indicating that they may also be needed for cell elongation. In conclusion, we identify a few areas of future research for cotton functional genomic studies.

关键词： cotton fiber functional genomics plant hormone transcriptome transcription factors