农业生物技术学报

Research and Prospects for Genetically Engineered Agricultural Microorganisms

Huang Dafang

2003, 11(2): 111-114 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Since 1996, a remarkable progress in the area of genetically engineered agricultural microorganisms has been made in China. The recombinant phytase, diazotrophic Alcaligenes faecalis and modified Bacillus thuringiensis respectively used for feed additive, biofertilizer and biopesticide were approved to commercial production. For the development in next 5~10 years, enhancing basic research, especially on microbial resources and functional genomics would be extremely important. And the relevant strategy and measures were also suggested.

Construction and Characteristics of Poly-3-hydroxybutyrate Depolymerase Encoding Gene (phbD ) Mutant in Sinorhizobium meliloti

Dai Meixue 1 Wu Bo 2 Bai Xueliang 2 Zhang Chenggang 1 Ma Qingsheng 2

2003, 11(2): 115-120 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Rhizobia, as so many other bacteria, can synthesize the carbon storage compound, polyhydroxyalkanoates (PHAs), when non-carbon nutrient , such as N, P or O2, is limited to grow and excessive carbon is available. When subsequent conditions make carbon limit, the internal PHA stores may serve as sources of carbon and energy. Poly-3-hydroxybutyrate (PHB) is a common member of PHAs family.The ability to degrade PHAs depends on the production PHA depolymerases, which release dimers or monomers from the PHA polymer. Bacterial PHA depolymerases are classified as intracellular or extracellular and widely distributed among microorganisms. So far, intracellular PHA or PHB depolymerase mutants have only been described in two strains of bacteria: Pseudomonas oleovorans and Ralstonia eutropha. All Sinorhizobium meliloti PHB pathway mutants other than phbD mutant have been characterized. Creation of S. meliloti phbD mutant would be useful to study the role of phbD gene in the PHB pathway and to understand the PHB cycle in more detail. The S. meliloti phbD gene sequence was obtained from the S.meliloti 1021 genome sequencing project. A homologue of the Ralstonia eutropha phaZ gene, encoding intracellular PHB depolymerase, was identified and designated to be phbD. Using a pair of primers designed by the sequence of S. meliloti Rm1021 genome which is homologous to phaZ gene of R. eutropha , an 835 bp fragment of phbD gene was amplified by PCR from S. meliloti genome and cloned onto vector pGEM?襆-T Easy and generated the recombinant plasmid pDC45. The insert was sequenced to ensure that the cloned fragment does contain the desired sequence. A unique Kpn21 site was identified to be located within the coding region at 299 bp from the translation start codon. Interposon ΩSmSp was inserted into phbD gene at the Kpn21 site and generated the plasmid pDC48. phbD :: ΩSmSp fragment was excised from pDC48 as an Eco RⅠfragment and ligated with Eco RⅠdigested plasmid pK19mobsacB to get pDC50. Plasmid pDC50 was introduced into Rm5000 by conjugation and Rf RSmRSpR transconjugants selection. Putative double crossover homogenotes were selected on TY Sm Sp containing 5% sucrose and subsequently screened for the loss of vector-encoded NmR. A RfRSmRSpRNmS mutant was thus isolated and designated to be Rm11417. The insertion of Ω into phbD of Rm11417 was transduced into Rm1021 to generate mutant Rm11430.The putative phbD mutant Rm11430 was confirmed by Southern blotting analysis using theDIG-labelled phbD PCR product as probe. The probe hybridized to an about 1.3 kb Eco RⅠfragment of genomic DNA from the wild-type strain, while with genomic DNA from the putative mutant Rm11430, the hybribizing fragment was about 3.3 kb, consistent with the presence of a 2 kb interposon insertion within the about 1.3 kb Eco RⅠfragment. The S. meliloti phbD mutant accumulated 1~2.6 times PHB more than that of wild-type strain and showed non-mucoid colony on YMA and TY plates but mucoid colony on M9 minimum media with acetoacetate (AA) or 3-hydroxybutyrate (HB) as sole carbon source. The alkaline phosphatase in phbD mutant assay results indicated that exoF-phoA fusion introduced low activity expression in YMB but high activities in M9-AA and M9-HB.

Construction of a New Plant Transformation Vector pBG1112 with a Chitinase Gene SchiA from Serratia marcescens and Its Genetic Transformation in Rice

He Yingchun1 Li Xiaoxiang2 Gao Bida1

2003, 11(2): 121-126 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: A 2.8 kb CaMV 35S promoter/Schi A coding region/Nos terminator fusion gene was inserted into the polycloning site of a 11.8 kb binary vector pCAMBIA1301 to produce a new 14.6 kb plasmid pBG1112, which was used in rice (Oryza sativa L.) transformation by floral organ-mediated method. T3 rice plants were screened by hygromycin solution and the positive plants were examined by PCR for presence of transgene and by RT-PCR for matured mRNA. Bioassay for some of the T3 transgenic rice plants which displayed both hygromycin-resistance and RT-PCR positive showed the increased resistance to rice sheath blight (Rhizoctonia solani ) and rice blast（Pyricularia oryzae）. The sequencing result showed that the nucleotide acid sequences of RT-PCR products were the same as that of transgene analyzed by BLAST software. Chitinase activity of T4 transgenic rice was higher than that of non-transgenic rice,which showed that the transferred exogenous chitinase gene could be expressed normally.

Cloning of Ethylene Receptor Genes LeETR1 and LeETR4 and
Their Expression in Tomato Fruit

Wei Shaochong Zhu Benzhong Luo Yunbo** Yu Bianyun

2003, 11(2): 127-130 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract：To explore the relationship between ethylene and ethylene receptor genes in different developmental stages of wild type tomato（Lycopersicon esculentum Mill cv. Lichun） fruits, as well as in antisense ACS transgenic tomato fruits, LeETR1 and LeETR4 fragments were amplified by reverse -transcription - polymerase -chain -reaction (RT-PCR)from tomato fruit in the experiment. Northern blot analysis indicated that the expression of two ethylene receptor genes in wild type tomato fruit did not differ significantly during fruit ripening, whereas LeETR4 showed lower expression in outer pericarp compared with radial pericarp and columella at the same stage. The level of either LeETR1 mRNA or LeETR4 mRNA in antisense ACS transgenic tomato fruit was significantly lower than that in wild-type fruit. Exogenously applied ethylene could induce the expression of both LeETR1 and LeETR4. The results demonstrated that the expression of LeETR1 and LeETR4 genes could be regulated by ethylene.

Microsatellite Analysis of Landrace Rice Core Collection in Yunnan, China

Zhang Hongliang1 Li Zichao1** Liao Dengqun1 Liu Xia1,3 Zeng Yawen2
Shen Shiquan2 Mu Ping1 Yang Zhongyi2 Wang Xiangkun1

2003, 11(2): 131-139 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The distribution of genetic diversity between Oryza sativa L. ssp. indica and O. sativa L. ssp. japonica covered different ecological zones in Yunnan was Studied, and the specific markers of indica /japonica subspecies, paddy/upland rice and different ecological zones were screened, using 36 microsatellite primers and 113 accessions in Yunnan landrace rice core collection. The results showed that the genetic diversity of japonica was higher than that of indica , and the ecological zone with the highest and smallest genetic diversity lay in Southeast and Northeast of Yunnan respectively. This distribution was consistent with the results of the studies on whole Yunnan rice resources and core collection at morphological and isozyme levels. In addition, the results showed that, among 416 markers, there were 6indica /japonica-specific markers, 15 specific markers in paddy/upland and 3 specific markers in different ecological zones. So the conclusion was primarily that the landrace rice core collection in Yunnan genetically represented the whole landrace rice resources in Yunnan, the center of genetic diversity at DNA level lay in Southeast of Yunnan, and the DNA differentiation between indica and japonica was small. And microsatellite marker was a useful tool to study the genetic diversity, classification and ecotype of germplasm resources and their core collection.

Expression of annfaf Gene Expressed Specifically in Strawberry
Fruit in E.coli and Preparation of Its Antiserum

Wang Guanlin1 Xia Ran1 Fang Hong-Jun1 Yang Huaiyi2 Na Jie1

2003, 11(2): 140-143 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The fusion expression vector and the non-fusion expression vector were constructed by the open reading frame (ORF) of annfaf expressed specifically in strawberry (Fragaria ananassa Duch.)fruit subcloning to plasmid pET-30a. The recombinant vectors were transformed and expressed high-effectually in E.coli BL21(DE3), and the target protein was detected by SDS-PAGE with a molecular weight of 35 kD. The SDS-PAGE also indicated that expression product was inclusion body. The study of effective expression showed that the optimum culture conditions of E.coli were 37 ℃ for 4 h, and the target protein content of the total expression proteins was increased clearly after adding rifamycin in the medium. The target protein recovered from SDS-PAGE was used to immunize rabbit, and antiserum with high titer was obtained. The ELISA and Western bloting analysis indicated that the reaction between antiserum and antigen was highly specific and effectual. The experement results wear a foundation of the study on function and location of annafaf protein in the cells of strawberry fruit.

Transformation and Functional Analysis of Antisense Ethylene
Receptor LeETR2 Gene in Tomato

Zhang Mingfang1 Xiang Qingning1 Ying Tiejin2 ** Yang Huqing2 Zheng Tiesong 3 Du Rongmao2

2003, 11(2): 144-147 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract： An antisense ethylene receptor gene LeETR2 was introduced into tomato (Lycopersicon esculentum ) through Agrobacberium tumefaciens -mediated method. Plantlets were regenerated in vitro by resistance selection on MS medium containing various concentrations of kanamycin. Approximately 73.5% of calli and 38.5% of leaves from plantlets exhibited intense blue color reaction by histochemical GUS assays. By PCR-aided assays, transformed plants displayed 362 bp (antisense LeEtr2 gene)and 500 bp(NPTⅡ) amplification products, however, they were not detected in non-transformed controls. Furthermore, Southern hybridization of DNA from PCR-positive transformants with the NPTⅡ probe indicated that the inserted gene was single copied in genome, by which it was further confirmed that the antisense LeETR2 gene was transferred into tomato. The transformants in tested lines showed delayed abscission, less elongated internodes(6±1.5) cm in length for transgenic plants and (8±1.58) cm for control, and out-growed axillary buds. The endogenous ethylene levels in fruits at day 3 after breaker in transgenic lines 1~5 were 155.58%, 166.96%, 251.76%, 441.07% and 100.89% of those in the controls, respectively. It is still ambiguous that how ethylene interacts with other hormones, such as auxin, abscisic acid and cytokinin in regulation of plant development through signal pathways. Nevertheless, the ethylene receptor LeETR2 might play a negative regulatory role in the signal transduction, and possibly involve other hormones in the control of plant development. It is essential to make clear how the antisense LeETR2 gene acts at transcriptional or translational level in the regulation of ethylene receptor LeETR2 gene, and whether it has regulatory side-effect on ethylene receptor LeETR1 gene due to their high sequence homologies.

Cloning and Expression of Self-incompatibility Related S-RNase Gene in Apricot

Qi Jie1,2 Gu Manru1 Shu Huairui1

2003, 11(2): 148-153 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract:Three cDNAs named PA1, PA2 and PA3 were isolated from self-incompatibility cultivars of Badanshui, Hongyu and self-compatibility Katy apricot(Prunus aremeniaca ). They encode 576 , 601 and 591 bp，respectively.There was 80.6% similarity among their amino acid sequences. The comparison of amino acid sequences of Rosaceae S-RNase showed highly divergent, and intra-subfamilial similaritiy was higher than that of inter-subfamilial one. Fourteen sites were perfectly conserved among encoded 187 amino acid residues , and in hyperaviable region(RHV) two amino acid residues (H, M) in PA1 and three amino acid residues(N, S, A) in PA3 were different from other S-RNase, which indicated the different amino acid residues might play a role of S-allele gene special recognition. From the phylogentic tree about Rosaceae, Solanaceous and some S-like RNase, apple and pear belong to Maloideae, but cherry, almond and apricot belong to Amygdaloideae, and three sequences were identified to be homology to S-RNase not to S-like RNase from other families. Northern blotting analyses indicated that PA3 was expressed predominatly in pistils of Kate apricot, which molecular weight was 0.9 kb, but not expressed in root, stem and leaf of Katy apricot. According to the sequence analyses and blast in GenBank, the result showed the obtained three genes were expression genes of S-RNase.

Differential Display and Sequence Analysis of Senescence-associated
Genes in Cut Roses

Bai Shangyi1 Liu Qinglin1* Ouyang Qing2 Cai Wenqi2

2003, 11(2): 154-157 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Using DDRT-PCR, twenty-two cDNA specific fragments were amplified from petal total RNA of Rosa cv. Pavarotti with longer vase life. Nine of those cDNA specific fragments were cloned, which were b21 , b32（b33）, b43 , b51（b52 , b814）, b618（b101 , b108 , b813）, b624 , b131 , b134 and b186 respectively. Sequences blasted in GenBank showed that b618 and b624 derived from recombination plasmid b6 were two different sequences. b618 , which had 439 bp and 137 deduced amino acid, was 71%~79% homologous to that of corresponding encoding regions of chalcone isomerase(CHI), such as Arabisopsis thaliana, Citrus sinensis, Raphanus sativus, Malus sp., Vitis vinifera and Elaeagnus umbellata . While b624 , which had 440 bp and 134 deduced amino acid,was 86%~90% identical to the cholinephosphate cytidylyltransferase (CPCT) with Arabidopsis thaliana, Pisum sativum and Brassica napus. RT-PCR showed b618 and b624 were positive results. CHI was an enzyme involved in flavonoid biosynthesis pathway, while CPCT is responsible to CDP-choline pathway, both were related to the senescence process.

Genetic Diversity Analyses of Three Wild Populations of Pinctada martensii
( Dunker.) Using RAPD Technique

Wang Aimin1,2 Yan Bing1 Ye Li1 Deng Fengjiao3 Zhang Xiyuan3 Wu Bo4

2003, 11(2): 163-168 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract : In order to understand genetic diversity of pearl oyster Pinctada martensii （Dunker.）, Random amplified polymorphic DNA (RAPD) analysis was applied in investigating three wild populations，SW population from Sanya,Hainan, DW population from Daya Bay ,Guangdong, and BW population from Beihai,Guangxi. 18 random primers, each containing 10 nucleotide were used. Six of the primers could carry on RAPD amplification，producing the amplified bands of DNA fragments with 0.2 kb to 3.0 kb. All the six primers produced polymorphic products among individuals of both the different populations and the same populations. The genetic similarity index of three wild populations were 0.641 (SW) ,0.672(DW), and 0.688 (BW) respectively, and the Shannon's index of phenotypic diversity(H) in three populations were 0.266 (SW),0.211 (DW) and 0.174(BW) respectively.The genetic similarity of sequence was SW< DW < BW , and the genetic diversity was SW > DW >BW . The relative genetic distances were 0.104 between DW and SW, 0.094 between DW and BW, and 0.212 between SW and BW. The difference of genetic distances in these three wild populations were relative not only to their geographical distances，but also to their different habitants. According to genetic distances between populations, it was predicted that the heterosis of progenies from SW crossing BW, and SW crossing DW were higher than that from BW crossing DW. The result which genetic diversity in BW was significantly lower than that in SW and DW was from the artificial breeding and large scale farming in BW habitant. The reproduction of culturing pearl oysters from the artificial breeding disturbed the diversity of BW population. It will be paid attention to the influence of aquaculture on the genetic structure of wild populations .

Studies on Improving T-A Cloning Efficiency of the PCR Products

Li Ruiguo An Xiaorong Gou Kemian Bo Jialin Hou Jian Chen Yongfu**

2003, 11(2): 158-162 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract:Three kinds of T-A cloning methods were tested to compare the efficiency of four different length PCR products. The results indicated that high cloning efficiency was obtained using common T-A cloning method for short PCR product(Length is about 500 bp), the cloning efficiency was improved using temperature cycle T-A cloning method for moderate PCR product(Length is about 3 000 bp) and the cloning efficiency was increased using two-step recombinant T-A cloning method for long PCR product(Length is about 6 000 bp).

Cloning of Bombyx mori Cytoplasimic Actin Gene Promoter and Construction of piggyBac Transposon Expression Vector

Li Wei1 Wang Yu1 Zhang Shiying1 Zhu Lingqiao1 Huang Min1 Liu Huifen2

2003, 11(2): 173-178 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: In order to construct silkworm (Bombyx mori ) transposon expression vector as a tool in research on the stable transgenesis of B. mori , the cytoplasmic actin A3 gene promoter ( named A3 ) from silkworm genome DNA was amplified by PCR and sequenced, the results showed that two typical constructive expression regulated elements: SRE (serum response element) and ActE1(active element 1) were included. Its sequence similarity compared with cytoplasmic actin A3 gene from European B. mori strain homology is 94%. Promoter A3 was fused with recombined transposase gene. And a nonautonomous helper plasmid was obtained. Promoter A3 fragment contained signal sequence and partial coding region was fused with green fluorescent protein (gfp ) gene, The fused fragment with A3 promoter and gfp was then inserted into a cloning site between two synthetic transposon piggyBac terminal inverted repeats in a plasmid pXLBac. A silkworm transposon piggyBac vector was constructed successfully. The transposon expression vector consists of the piggyBac inverted terminal repeats, between which the recombined gfp gene derived by A3 promoter was flanked. Both transposon vector and helper plasmid were co-injected into the embryos of Bombyx mori , the constructive expression transposase in the helper palsmid might function and make the DNA fragment between the piggyBac inverted terminal repeats of transposon vector inserted into the host genome chromosome. This bipartite vector-helper system had advantages as followed: the transposon vector was only 6 400 bp and might facilitate further DNA manipulation; between the terminal inverted repeats, the transposon vector was designed with a multiply clone site, which was more convenient to be inserted exogenous gene of interest and construct expression plasmid. Nowadays, an interesting gene was inserted into the transposon vector and was tested for gene transfer vector function as a part of bipartite vector-helper system in Bombyx mori.

AFLP Markers for Pig Genomic DNA Fingerprinting

Ren Jun1 Huang Lusheng1** Gary Evens2 Gao Jun1 Ai Huashui1
Chen Kefei1 Ding Nengshui1 Deng Suhua1

2003, 11(2): 179-182 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The optimization of AFLP technique for porcine（Sus scrofa）genomic DNA fingerprinting was studied, including enzyme digestion, adapter ligation, preamplification, selective amplification, denatured polyacrylamide gel electrophoresis, silver staining and multicolor fluorescent detection. The Eco RⅠ+AAC/TaqⅠ+AAC selective primer combinations labeled by IRD 800 and IRD 700 fluorescent dyes were used. Gel running was performed in both Licor 4200 DNA sequencer and 4.5% PAGE(2600 V, 45 W, 50　℃), and the electrophoresis images were analysed by the AFLP QuantarTM Pro software of Keygene(the Netherland). The result showed that the template DNA concentrations did not affect the AFLP quality, templates with 250 ng, 500 ng and 1 000 ng DNA had the same AFLP fingerprinting. And 28 polymorphic markers were detected in the same pooled genomic DNA of 45 pig breeds (populations) by E32/T32 primer combinations.

Cloning, Sequence analysis and Prokaryotic Expression of
Human Pancreas Kallikrein cDNA

Shi Weiqing Zhang Lei Sun Huaichang**

2003, 11(2): 169-172 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: To get monoclonal antibodies for human kallikrein (KLK1), a pair of primers were synthesized according to the previously published sequence for human pancreas KLK1 mRNA and used to amplify the double-stranded cDNA from pooled human pancreas single-stranded cDNAs by PCR. Agarose gel electrophoresis of the PCR product showed a single band of 0.8 kb. After cloning into pGEM-T vector, 5 recombinant plasmids were sequenced, one of which was identical to the previously published human renal/pancreas/salivary KLK1 cDNAs or there was only 3 bp difference. The cDNA was excised from the pGEM-T vector by digestion with restriction endonucleases Pvu Ⅱ and Xho Ⅰ to remove its signal peptide sequence and subcloned downstream into the Sma Ⅰ/Xho Ⅰ site of prokaryotic expression vector pGEX-4T-3. The recombinant plasmid was transformed to BL21 E.coli cells for expression by IPTG induction. SDS-PAGE analysis of the transformant showed an expected size of protein band of 48 kD, which was mainly present in the insoluble fraction of the cell lysate. Western blotting of both cell lysate and Glutathione Sepharose 4B-purified expression product showed that the fusion protein was able to be recognized by a monoclonal antibody specific for glutathione-S-transferase. The antiserum, with an ELISA titer of 1∶1600, was obtained by immunizing mice with the SDS-PAGE purified fusion protein. These results demonstrated both cloned cDNA and its expression product were correct and could be used to prepare specific monoclonal antibodies for human KLK1.

A Secreted Expression Vector Construction of Pokeweed Antiviral Protein Gene and Its Expression in Pachia pastoris　

Chen Dinghu Wang Xifeng Li Li Zhou Guanghe**

2003, 11(2): 183-186 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract：The total RNA was isolated from pokeweed (Phytolacca americana ) leaves using the method of guanidine isothiocyante and used as template for amplification of the deleted mutant pokeweed antiviral protein (PAP ) gene by RT-PCR and then the amplified fragment was cloned into secreted expression pPIC9K vector to form pPIC9K-p, then the vector was transferred into Pachia pastoris GS115 strain. The specific expression protein was secreted into the medium after inducing with methanol and the protein amount reached about 50~60μg /mL measured by UV-absorbed methods in the supernatant of the medium via high density fermentation. SDS-PAGE results showed that there was one main protein band with molecular weight about 34 kD in the fermentation supernatant and it could specifically react with France PAP antiserum in Western blotting. Activity tests revealed that this protein could inhibit plant virus infection with high efficiency.

Cloning, Sequence Analysis and Prokaryotic Expression of the vRNA3
NS3 Gene in Rice Grassy Stunt Virus Shaxian Isolate

Lin Liming Wu Zujian Xie Lianhui** Lin Qiying

2003, 11(2): 187-191 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Rice grassy stunt virus (RGSV) is classified as a member of Tenuivirus . The filamentous particles of RGSV are ribonucleo-proteins (RNPs), which are composed of a single nucleocapsid (NC) protein and genomic ssRNA segment. RGSV caused rice yield to lose a lot in South and Southeast Asia during the 1970's. It also occurred in South of China, such as Fujian, Taiwan, Guangdong, Guangxi and Hainan Provinces. The whole sequence of RGSV genome was determined in 1998 by Toriyama et al, and the result revealed that RGSV has six genomic RNA segments, RNA segments 1, 2, 5 and 6 correspond to RNA 1-4 of other tenuiviruses. And all six RNA segments had an ambisense coding strategy, which is unique not only in genus Tenuivirus but also in plant viruses. The unique RNA segment 3 contains an ORF on each of vRNA and vcRNA. However, the functions of RNA3 are still unknown. The potential function of vRNA3 was studied by cloning, sequencing and expression of RGSV vRNA3 NS3 gene. Based on the known RNA sequence of RGSV-IR isolate, the cDNA of the vRNA3 NS3 gene was obtained by RT-PCR with genomic RNAs of RGSV-SX isolate as template. The cDNA was then cloned and sequenced. The results showed that NS3 gene was composed of 588 nt and the sequence identities were 99.1% and 96.2% at the nucleotide level, 98.4% and 96.4% at the amino acid level, comparing with those of RGSV- IR and SC published isolates. The 22.9 kD protein encoded by RGSV-SX vRNA3 showed 33.0% identity between 80 amino acids compared with the 21.6 kD protein encoded by vRNA5 of RGSV, and no other significant matches were found in GenBank. The similarity between the 22.9 kD protein and the 21.6 kD protein suggested that vRNA3 of RGSV might have sort of recombination with other RGSV RNA segments and/or unknown RNAs. Using recombinant plasmids containing vRNA3 NS3 gene and vector of pGEX-2T, we constructed prokaryotic expression plasmids pGTNS3 which produced 49.0 kD fusion protein of GST-NS3 in E. coli, prepared the antiserum against the fusion protein and used it for the detection of RGSV by Western blot. The encoded protein was detected only in infected rice (Oryza sativa ), nothing in purified virus and viruliferous planthopper vectors. These results would provide more information for the further study on the functions of the vRNA3 NS3 gene and its encoded protein.

Defense Response in Plants Induced by HarpinXoo，An Elicitor of Hypersensitive Response From Xanthomonas oryzae pv. oryzae

Wen Weigang Shao Min Chen Gongyou Wang Jinsheng **

2003, 11(2): 192-197 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Harpin proteins with the elicitor activity are encoded by particular genes in hrp gene clusters of Gram negtive plant pathogenic bacteria. As HarpinEa of Erwinia amylovora, HarpinXoo (15.6 kD) is encoded by gene hrfAXoo (420 bp) positioned in hrp cluster of Xanthomonas oryzae pv. oryzae and shows the ability for induction of hypersensitive reaction of plants. HarpinXoo could induce plant resistance against some diseases and the relevant mediated mechanisms by the expressed product of hrfAxoo. HarpinXoo was expressed in recombinant bacteria E.coli BLHR4/pHRF4(pET30a(+)::hrfAxoo) and the protein expression was at peak level after IPTG induction for 16 h. After applying HarpinXoo to tobacco (Nicotiana tabacum L), rape (Brassica campestris L.) and tomato (Lycopersicon esculentum Mill.), the resistance was induced with remarkable control effects to tobacco mosaic virus (TMV), sclerotinia stem blight of rape (Sclirotinia sclerotiorum (Lib.) de Bary) and tomato gray mold rot (Botrytis cinerea Pers.) respectively. Comparing with water treatment control, 66.5% for TMV control efficiency, 79.39% for contol of Sclerotinia sclerotiarum of rape and more than 75% for control of Botrytis fruit rot of tomato were achieved. Biochemical and molecular technologies were used to reveal resistance mechanisms in tobacco (Xanthi) as model plant. The activities of defense-related enzymes involving phenylalanine ammonia lyase (PAL), peroxidase (PO) and polyphenoloxidase (PPO) were determined in tobacco leaves treated with HarpinXoo and the result showed that their activites were 110.1%, 211.2% and 273.0% higher than that treated with water control respectively. Two pathogenesis-related genes PR-1b and PR-1a were expressed conspicuously at 24 h after induction with HarpinXoo and showed the highest level of expression at 36 h after treatments by RT-PCR methodology. These results indicated that HarpinXoo similar to HarpinEa could induce the resistance of plants by various signal transduction pathways.

RNA Silencing and Its Application In Gene Function Research

Peng Hao Lu Tiegang Jia Shirong Huang Dafang**

2003, 11(2): 198-206 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Existing in eukaryotes extensively, RNA silencing suppressed sequence-specific gene expression on post-transcriptional level. In recent years, fruitful results have been achieved in exploring the silencing mechanism and many related factors have been identified. RNA silencing was regarded as a defensive mechanism against destruction of important genes triggered by virus invasion and transposon moving in genomes. And RNA silencing became an important tool for functional genomics research.

Problems and Thoughts on Mammalian Somatic Cloning

Wang Hai Lian Zhengxing** Li Ning Wu Changxin

2003, 11(2): 207-211 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Mammalian somatic cloning got great progress in past few years, people have paid more attention to the researching and developing programs based on animal cloning platform. The article discussed some cloning problems about animals, which included synchronization of donor cells with recipient oocytes, abnormality and premature senescence of cloned animals, and disputed on somatic cell passages in vitro. Current progress of mammalian cloning was also introduced briefly. At last author's ideas on animal cloning were elucidated.