农业生物技术学报

Mechanism of Fungal Pathogenesis in Insect and
Strain Improvement by Gene Engineering

Pei Yan Fang Weiguo Zhang Yongjun

2003, 11(3): 221-226 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: With the increasing of widely polluted environmental concerns and health risks associated with the use of synthetic chemical insecticides, research and development of mycoinsecticide have been paid much attention to. However, there are some barriers to exploit entomopathogenic fungi further because of their poor performance in field. In order to widen the acceptance of mycoinsecticide products in market, the molecular biology basis of fungal pathogenesis in insect should be elucidated to identify important virulent genes, which could be then used to improve the strain performance by genetic engineering. In this paper, molecular basis of appresorium formation, cuticle degrading and the role of toxins in fungal pathogenesis in insect were introduced. Meanwhile, the gene transferring methods and its usage in strain improvement of entomopathogenic fungi were also summarized.

Cloning of germin Gene and Its Expression in Tobacco

Wang Bingshan1 Dou Daolong2 Zhang Meng3 Wang Zhixing1 Jia Shirong1

2003, 11(3): 233-235 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Oxalic acid was produced by several plant pathogenic fungi and thought to ploy a primary role in the pathogenicity of fungal species, including the wide host-range pathogen Sclerotinia sclerotiorum. In the current work, a germin gene from wheat (Triticum aestivum )was cloned. A plant expression vector containing the germin gene was constructed by CaMV 35S promoter, which was used for Agrobacterium tumefaciens-mediated transformation of tobacco(Nicotiana tabacum ). Southern blotting assay indicated that the target gene was integrated into tobacco genome. Enzymatic activity assay of oxalate oxidase in transgenic tobacco showed that the germin gene was expressed, which resulted in the formation of homohexamer with enzymatic activity correctly, and the transgenic plants were tolerant of the exogenously supplied oxalic acid.

To Transfer Anti-herbicide Gene into Upland Rice (Oryza sativa)Cultivars

La Honggui1 Wang Huaqi1** Huang Danian2 Hua Zhihua2 Yan Meixian2 Gao Zhenyu2 Feng Xiujing1

2003, 11(3): 227-232 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The anti-herbicide gene bar was transferred into immature embryos calli from elite cultivars of upland rice（Oryza sativa L. ）297, 10 and so on with help of particle bombardment. Some regeneration plantlets were acquired after two selections on media containing herbicide Basta and differentiation. Further molecular test by both PCR amplification and Southern blotting and experiments in fields demonstrated that extrogenous gene bar was integrated into T0 genomes and inherited to T1. The tests of induction and differentiation media indicated that MB and MS were better for induction of calli and the modified media of RMB2 and RMS2 promoted differentiation frequency of calli remarkably. The obtained transgenic plants and established genetic transformation system of the experiment were available for further anti-herbicide molecular breeding and other genetic transformation.

Expression of a Novel Potato Patatin ClassⅠ cDNA in Transgenic Tobacco and Its Lipid Acylhydrolase Activity

Si Huaijun1 Liu Jun2 Xie Conghua1**

2003, 11(3): 236-240 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Patatin is the trivial name for a family of glycoproteins that accounts for up to 40% of the soluble protein in potato (Solanum tuberosum L.) tubers. Unlike most other storage proteins, patatin has a lipid acylhydrolase (LAH) activity. It is proposed that patatin may be involved in particular biological processes, especially tuber formation. Despite a detailed molecular and biochemical analysis of patatin, its physiological role and function, especially in potato tuber formation and regulation have not been elucidated. In order to approach this question, we have isolated a classⅠpatatin cDNA (GenBank accession No. AF498099) from a cDNA library constructed from wild diploid potato species Solanum chaconse under tuber inducing conditions. We constructed the expression vector pBSSP by fusing the patatin class I cDNA with CaMV 35S promoter and introduced it into tobacco without patatin gene to prove its expression and LAH activity.
The expression vector pBSSP was introduced into Agrobacterium tumefaciens strain LBA4404 by electroporation and then transferred into tobacco (Nicotiana tabacum ) via Agrobacterium tumefaciens system. Leaves from 4-week-old plant grown in vitro of tobacco line T1 were used for transformation. Transformants were selected on MS medium containing 2.25 mg/L BA, 0.3 mg/L NAA, 100 mg/L kanamycin and 400 mg/L carbenicillin. When green shoots reached 1~1.5 cm, transferred them to the selective rooting MS medium supplemented with 50 mg/L kanamycin and 200 mg/L carbenicillin. A total of 45 kanamycin resistance plants were obtained. In order to prove patatin class Ⅰ cDNA was integrated into genome of tobacco, DNA was isolated from putative transformed and control tobacco plants. PCR analysis using patatin class Ⅰ cDNA specific primers was performed on 45 putatively transformed plants. There were 40 transformed plants showed a 500 bp amplification product which was missing in non-transformed control plants. The PCR amplification results were conformed by PCR-Southern blot analysis. The PCR products were electrophoresed on 1% agarose gel and transferred to positively charged nylon membranes (Boehringer Mannheim, Germany). Labeling, hybridization and detection were carried out using a DIG High Prime DNA Labeling and Detection Starter KitⅠ(Roche, Germany) following the manufacturer’s instructions. The results showed that the PCR product was really patatin cDNA fragment and patatin cDNA has been integrated intogenome of tobacco. Expression of the class I patatin cDNA in the transformed tobacco plants was also verified by Northern hybridization analysis using DIG RNA Labeling Kit (Roche, Germany). The results showed the predicted transcript of class I patatin cDNA in the transformed plants while no transcript in the control ones. This indicated that the class I patatin cDNA was well transcribed in the transgenic tobacco plants.The total soluble protein content of transformed plant leaves was measured by Bradford method. The highest protein content of transformed plant increased up to 6.4 mg/g FW that was 64.1% higher than that of the control plant. Lipid acyl hydrolase (LAH) analysis demonstrated that LAH activity was increased obviously in leaves of the transformed tobacco plants. The average LAH activity of 6 different transformed plants was increased by 67.7% compared to the control plant.The present experiment proved that this new classⅠpatatin cDNA expresses accurately and has normal function with LAH activity, and provides a foundation for a further study on physiological role and function of patatin to look into its relationship with tuber formation and regulation.

Genetic Analysis and Mapping of Blast-resistance Genes in japonica Rice
Yunyin from Yunnan Province

Zhang Jianfu 1 Wang Guoying 1** Xie Hua'an2 Ling zhongzhuan3

2003, 11(3): 241-244 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: A new blast-resistant, japonica rice, Yunyin, was identified with 8 races of Pyricularia grisea (Cooke) Sacc). The results showed that Yunyin was resistant to 8 blast races. Yunyin was crossed with a blast-susceptible rice variety Lijiangxintuanheigu. Both parents and their progeny (F1, F2 and F3) were inoculated with 8 races of Magnaporthe grisea by injection in the field. The results showed that the blast-resistance to the 8 races in Yunyin was controlled by single gene. A blast-resistant locus to Sichuan-43 was mapped in chromosome 11 by SSR marker. The linkage value was 3.8 cM with RM202.

Cloning of a New Sperm Membrane Protein sp18 Gene and Its Expression in E. coli

Bo Jialin1 Gou Kemian1,2 An Xiaorong1,2 Li Ruiguo1 Hou Jian1,2 Chen Yongfu1,2**

2003, 11(3): 268-272 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: cDNA encoding for a sperm membrane antigen, designated sperm membrane protein sp18, was cloned and sequenced from murine testis λgt11-cDNA expression library using sp18 mAb. Computr-generated translation analysis of 1 468 bp cDNA yielded an open reading frame(ORF) of 429 bp which deduced 142 amino acids with the first ATG(Met) start codon at nucleotide 694 bp, the stop codon TAA at nucleotide 1 122 bp and a small polyadenylation tail at nucleotide from 1 245 to 1 250 bp. The translated protein, which consists of 142 amino acids, has a calculated molecular mass of 15.7 kD, 8 potential N-linked glycosylation sites and 12 O-linked glycosylation sites. The hydropathy plot generated from deduced amino acids sequence indicated it to be a sperm span-membrane peptide. Comparing the nucleotides of sp18 cDNA with other nucleotides deposited in GenBank , the 10 to 651 bp of sp18 cDNA and 532 to 1 173 bp of human Hr44, the 10 to 816 bp and 864 to 1 444 bp of sp18 cDNA and 1 408 to 2 208 bp and 2 250 to 2 309 bp of bovine Hr44 had 98% similarity respectively. This indicated that sp18 cDNA was a new gene(GenBank accession number:AF129872) specifically localized in reproductive system.The sp18 cDNA was subcloned into pET-30a(+) vector and expressed in BL-21(DE3) of E.coli with molecular mass of 16.5 kD. Western blot indicated that sp18 mAb could recognize sp18 protein and protein possesse immunogenicity. All above laid a foundation of further investigating the function of sp18 gene.

Preparation and Regenaration of Drechslera monoceras Protoplast

Duan Guifang Huang Shiwen** Yan Qiusheng Yu Liuqing

2003, 11(3): 245-248 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Factors, which affect the protoplast formation of barnyardgrass (Echinochloa spp.) pathogen Drechslera monoceras, such as mycelium age, lytic enzyme concentration, osmotic pressure of stabilizer, pH value of buffer solution, digesting temperature, period and so on, were studied. Maximum yield (1×106/mL) of protoplast could be obtained with 42 h old mycelia by using 2% lywallzyme in combination with 2% cellulase which containing 0.7 mol/L KCl as osmotic stabilizer and digesting for 4 h at 30 ℃ in pH 4.4~5.8 condition. The regeneration rate of the protoplast cultured on the solid media containing osmotic stabilizer was 0.15%.

Impact of TL-DNA on Content of Ginsenosides in Ginseng Hairy Roots and Cloning and Sequence Analysis of rolC Gene

Zhao Shoujing1 Li Changyu2 Qian Yanchun1 Shi Jing1 Liu Qingchang3

2003, 11(3): 249-252 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Ginseng （Panax ginseng C.A.Mey）hairy roots were induced from the root explants of ginseng by the co-culture of Agrobacterium rhizogenes and ginseng roots with the method of direct inoculation. The content of ginsenosides in ginseng hairy roots after 4 weeks culture basically reached the level of that in 3-year-old cultivated ginseng roots, and the content of single ginsenoside-Rb1 had an evident increase. The result indicated that TL-DNA had a function of influencing the biosynthesis of ginsenosides. Based on the analytical results of RiA4TL-DNA sequence, rolC gene affecting the synthesis of ginsenosides was amplified and cloned from the ginseng hairy roots by PCR method. Comparison with previously published nucleotide sequence showed that the homology was 99.9%.

Influences of Nitrogen Ratio and Carbon Sources on Adventitious Shoots Regeneration of Malus zumi Leaves In Vitro

Li Bao Han Zhenhai**

2003, 11(3): 253-258 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstracts: Factors that affect regeneration of adventitious shoots of Malus zumi Mats leaves in vitro were examined. The results showed that the changes of NAA concentrations were more responsive than that of BA; different ratios of NO3-:NH4+ in MS medium had different results, 2∶1 was the best, followed by 3∶1, 4∶1 and 1∶1; Fructose improved the regeneration of adventitious shoots significantly among the four tested carbon sources which were fructose, sucrose, glucose and sorbitol; And the optimimum medium was consisted of MS salt and vitamins and supplemented with BA (2.5 mg/L), NAA (0.25 mg/L), fructose (30 g/L), agar (8 g/L), and the pH was 5.8. The action mechanism of carbon sources was discussed in the paper.

Establishment of the Method for Identifying the
Extracellular Fatty Acid Binding Protein Genotypes with Allele-specific-PCR

Zhao Chunjiang Li Ning* Deng Xuemei

2003, 11(3): 273-275 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract： The genotypes of chick (Gallus gallus ) extracellular fatty acid binding protein (EX-FABP) are closely related to chick abdominal fat percentage. In the study, the method for identifying the EX-FABP genotypes with allele-specific PCR (AS-PCR) technique was established and the strategy of identifying single nucleotide polymorphisms with AS-PCR was discussed. A typical AS-PCR system consists of two allele-specific primers and ane common primer. PCR products can be resulted when a sample's DNA is amplified by the common primer and allele-specific primer which matches with the sequence of sample's DNA. There are not PCR products if the allele-specific primer does not match the sequence of the samples' DNA. According to whether there is PCR products or not, the genotypes of samples can be identified. For establishing an AS-PCR, it is very important to design proper number and kinds of mismatched bases at the 3′ end of the allele-specific primers where the mutation occurs and different genotypes are generated. When one mismatched base in the primers can not lead to different PCR results which can identify genotypes, the second or even the third mismatched bases should be added to the allele-specific primers. The different kinds of mismatched bases have different effect on the results of AS-PCR. If the mismatched bases are A-C or T-G, they have little effect on the amplifying efficiency of AS-PCR. But when the mismatched bases are A-G or C-C or C-G, they have dramatically negative effect on it. A modified AS-PCR, allele-specific and length-deferent PCR (ASLD-PCR) were established and introduced. In an ASLD-PCR system, two pairs of primer sites, each of them consists of an allele specific primer and a common primer,were used to amplify and identify genotypes. The PCR products of the two pairs of primers were different in the length and the different length of PCR products standed for different kinds of genotypes. So ASLD-PCR was more convenient to identify genotypes than AS-PCR.

Analysis of the Secreted Materials from Epithelial Cells Cultured on Mantle of Pearl Oyster（Pinctada martensii）

Wang Aimin1 Yan Bing2 Su Qiong2 Ye Li2 Chen Wenguang2

2003, 11(3): 285-290 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Using the tissue culture technique, the mantle of the pearl oyster (Pinctada martensii ) in vitro was cultured.The epithelial cells migrated out from mantle kept activity and function of the secretion. The secreted materials (SMs) were studied by polarizing microscope and scanning electron microscope. After 4-day culturing， round epithelial cells changed in shape, and began to secrete fine granules. During culture, the epithelial cells secreted increasingly，and the SMs became large granules. The birefringence were show under the polarizing microscope. After 34-day culture, the SMs accumulated into a mass of granules, appearing strong birefringences, and some moving birefringences. The SMs progressively increased into laminary layers after 43-days culture. The SMs, secreted by the epithelial cells in vitro, were very similar with the nacrum of the pearls and pearl shells. The contents of SMs during culture were detected by X-ray microanalyser. The SMs contained 59.43% Ca, 33.03% S and 1.6% P on the 10th day culture. Ca element in the SMs increased significantly on the 20th and 34th day culture, 71.86% and 92.03% respectively. At 43th day culture, containing 44.25% Ca, 4.98% S and 14.61% P were examined in the SMs. Other elelments, such as Si, K and Cl were also detected in the SMs，but containing little. The SMs were formed laminary layers made by CaCO3 crystals under the scanning electron microscope, and sticked by organic membrane. Because of the primary nacrum with large amounts of sulfur, it was related to nucleation and growth of CaCO3 crystals. This researching could provide help in culturing pearls in vitro or tube-pearls.

Tissue Culture and Component Analysis of Pinellia ternata

Guo Yulong Jia Yongfang Yang Xingyong* Li Mingyang Pei Yan

2003, 11(3): 259-262 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Pinellia ternata Breit, a Chinese traditional herb, is facing problems of resource declining and requirement increasing. The segments of leaves and petioles from P. ternata were used as explants to induce small tuber. The highest inducing frequency reached 100% on MS medium supplemented with BA 0.5~2.0 mg/L and NAA 0.2 mg/L. One tuber propagated to 16.9 tubers evenly within 3 weeks on MS medium with BA 2.0 mg/L and NAA 0.2 mg/L.The small tubers could be easily induced to plantlets and transplanted to field. The experiment showed that it was practicable to produce large numbers of high quality plantlets by tissue culture. HPLC was employed to analyze the extracts of 80% methanol, 0.2 mol/L H2SO4 and distilled water from wild and tissue culture tubers. The results showed that the total content of the components tested in the tissue culture tubers were higher than that in the wild, and the main chemical composition were similarity between them.

Culture , Isolation and Passage of Porcine Embryonic Stem Cells

Dong Xiao1,3 Feng Shutang1** Wang Zhanhe1,2 Wang Duanyun1 Zheng Xing3

2003, 11(3): 263-267 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abctract: Embryonic stem (ES) cells are pluripotential cells isolated from culture of preimplantation embryos in vitro. Experiments were undertaken to identify preimplantation embryonic stages and culture conditions under which pluripotential porcine embryo-derived cell lines could be isolated. Porcine embryos from D5,6,7,9,10 after the first day of estrus and insemination (day 0) were recovered by a surgical uterine flush technique with phosphate buffered saline (PBS) supplemented with 1% fetal bovine serum (FBS). A total of 124 porcine embryos were collected. Embryos were cultured at 38 ℃ on the feeder layers prepared from mitotically inactivated permanent mouse embryonic fibroblasts (STO) in the same medium[DMEM(Dulbecco's modified eagle medium) + 10 % NBS (newborn bovine serum)+ 10 %FBS+1 000 IU/mL LIF (leukemia inhibitory factor)+ 30 ng/mL SCF（stem cell growth factor）], the ICM(inner cell masses) growing rate of the embryos collected on day 5, 6, 7, 9, 10 was 0, 12.5%, 59.2%, 100%, 100% respectively; When embryos were cultured in two different mediums [Medium 1:DMEM + 10 % NBS + 10 % FBS + 1 000 IU/mL LIF + 30 ng/mL SCF; Medium 2: DMEM + 10 % NBS + 10 % FBS + 1 000 IU/mL LIF + 30 ng/mL SCF+ 20 ng/mL bFGF (basic fibroblast growth factor)], the result showed that bFGF was not essential in the culture and passage of ES cells; When the diameter of Day10 embryos was considered, we got that the larger embryos were better in ES cell culture, and the highest ES cells passages rate was gotten when the embryos have a diameter over 100 μm. In this experiment, we got ES-like colonies passed 4 times thatexpressed alkaline phosphatase activity.

Expression of Mutant Tumor Suppressor Gene p53 in Egg-type Chicken
Infected with Avian Leukosis Viruse Subgroup J

Xu Binrui1 Dong Weixing2 He Zhaoqing1 Yu Chunming1 Li Ning1
Feng Xiaoyu 1 L.F. Lee3 Maoxiang Li3

2003, 11(3): 276-279 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract：Avian leukosis in egg-type chickens is infectious neoplastic disease caused by avian leukosis viruse subgroup J( ALV-J). Expression of mutant tumor suppressor gene p53 in egg-type chicken with avian leukosis subgroup J was detected to study cancericidally molecular mechanism of ALV-J. Using immunohistochemistry method of strep avidin-biotin complex (SABC), many organs of illed egg-type chickens, involved liver, kidney, ventriculus glandularis, tumor, heart, pancreas, bone marrow, spleen, oviduct, lung, duodenum, thymus and bursa of fabricius were studied. And large numbers of mutant p53 protein were expressed in liver, kidney, ventriculus glandularis, tumor, oviduct. Moderate expression were detected in heart, pancreas, bone marrow, spleen, lung, duodenum, thymus and bursa of fabricius. The result suggested that expression of tumor suppressor gene p53 was concerned with occurrence and development of avian leukosis subgroup J.

Study on Expressing A Segment of Infectious Bursal
Deasease Virus to Milk in Transgenic Mice

Lin Aixing Zhou Yun Hu Hongyu Hou Jian An Xiaorong Chen Yongfu**

2003, 11(3): 280-284 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract The A segment of infectious bursal desease virus (IBDV) was ligated to the mammary gland expression vector p7GMB53. This transgenic construct was linearized to perform transgenic experiments, total 879 fertilized eggs were microinjected and transferred into 26 recipient mice, 39 mice were born with 4.4%(39/879) embryo survival rate. Results of PCR showed that 4 of 39 were positive. Southern hybridization showed 2 of 4 were positive, in which the copy numbers of IBDV integrated were 3(No.8) and 9(No.3) respectively. The female mouse No.3 was mated with nontransgenic male mouse and transgenic male mouse No.8. The ratio of positive mice was 52% (10/19) and 100%(5/5) respectively and the result indicated that the No.3 was a germ pure line. The No.8 was mated with nontransgenic female mouse and the ratio of positive mice was 11%(2/18), indicated the No.8 was a germ chimera. The milks of No.3 (female), No.6(the female offspring of No.3 and a nontransgenic mouse) and No.10 (the female offspring of the No.3 and No.8) were performed by Western blot and ELISA. Both of the mice expressed the foreign gene in mammary glands. We concluded preliminarily that this vector, containing matrix attachment region seguence(MARs), could drive foreign gene to express to milk independent effects of position in transgenic animals.

Effects of L-NAME and L-Arg on Meiotic Maturation of Mouse Oocyte in Vivo

Bu Shumin1,2 Xia Guoliang1** Wang Songbo1 Wang Jianhong1,3

2003, 11(3): 291-294 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The effect of nitric oxide (NO)on meiotic maturation of mouse oocyte in vivo was investigated by injecting L-NAME alone or co-injecting L-NAME and L-Arg intraperitoneally. The results showed that: (1) 5, 10 and 20 mg/kg bw (body weight) of L-NAME inhibited significantly the extrusion of first polar body of mouse oocyte(P <0.01), especially 10 mg/kg bw. But they all had little impact on germinal vesicle breakdown (P >0.05). (2) The inhibitory effect of L-NAME (10 mg/kg bw) on the extrusion of first polar body of mouse oocyte could be reversed by 100 mg/kg bw or 200 mg/kg bw L-Arg.(3) The abnormality caused by L-NAME was related to the culture time, but L-NAME had no significant effect on viability of mouse oocytes. It could be concluded that NO was involved in regulation of mouse oocyte meiotic maturation in vivo.

Detection of a New Virulence Associated Gene in Streptococcus suis
Type 2 Jiangsu Isolate

Li Ganwu Yao Huochun Lu Chengping**

2003, 11(3): 295-298 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: Streptococcus suis type 2 is an important pathogen for pig and human. It has different virulence factors including muramidase-released protein(MRP), extracellular factor(EF), suilysin and capsular polysaccharide. Smith H E et al(2001) had successfully detected a new virulence associated sequence orf2 by using the method in vivo complementation. It was interesting to investigate the new orf2 in swine streptococcal strains isolated in China. The results showed that the new orf2 was detected from genomic DNA of Streptococcus suis type 2 Jiangsu isolate HA9801 by polymerase chain reaction(PCR) technique and the PCR product from isolate HA9801 was choned into plasmid vector pET-32a(+) via restriction endonuclease Bam HⅠand Hind Ⅲ. The recombinant fragment was verified by restriction endonuclease analysis and sequenced. The nucleotide sequence was 100% homological to that of the new hypothetical virulence assolated sequence orf2 reported by Smith H E. By the same method, this orf2 was also detected from genomic DNA of Streptococcus suis type 2 strains and other streptococcal isolates from swine in China. 7/8 of Streptococcus suis type 2 isolates and 9/23 of other streptococcal isolates from pigs were positive. The positive ratio of the orf2 in all isolates including Lancefield group A, B, C, D, R and other serotypes of Streptococcus was 51.6%(16/31). It suggested that the new orf2 distributed widely among streptococcal strains of pigs from different areas in China. The pathogenicity of the orf2 would be further studied

Studies on Tobacco Disease Resistances Mediated by Elicitor
Gene of Cryptogein fromPhytophthora cryptogea

Jiang Donghua1,2 Guo Zejian1 Chen Xujun1 Chen Zhiqiang1 Zheng Zhong1

2003, 11(3): 299-304 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract: The cryptogein (Crypt ) gene was obtained by PCR amplification of genomic DNA of Phytophthora cryptogea and verified by DNA sequencing. A promoter of a rice (Oryza sativa ) phenylalanine ammonia-lyase (PAL ) gene, which was low-level constitutive induced by pathogen, and specially expressed in epidermal tissues, was selected to control the expression of Crypt gene. This promoter may exhibit a potential to inhibit the infection of pathogen from epidermal tissues. Meanwhile, for functional interaction of cryptogein protein with its outside membrane binding sites, the Crypt gene was fused to signal sequence of the extracellular pathogenesis-related PR1b protein of tobacco(Nicotiana tabacum ), The final construction was subcloned into a binary vector and transformed into tobacco by useing Agrobacterium-mediated transformation method. Twenty-two kanamycin-resistant tobacco plants were obtained by screening in selective medium with 100 mg/L kanamycin. PCR amplification and Southern blot analysis demonstrated that Crypt gene was integrated into tobacco genome. The transgenic plants were challenged separately, with black shank fungi (Phytophthora parasitica var. nicotianae ) , brown spot fungi (Alternaria alternata ), and wild fire bacterium (Pseudomonas syringae pv. tabaci ). More than half of the plants（68.2%）showed that the resistance was strongly enhanced to these pathogens. Results suggested that a low-level constitutive expression of Crypt gene in tobacco should have a potential use to generate a broad-spectrum disease resistant plants.

Progress of the Research on Chicken Growth Axis-related Genes

Nie Qinghua Zhang Xiquan Yang Guanfu

2003, 11(3): 305-312 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract：Neurocrine axis or hypothalamus-pituitary growth axis plays a major role of regulation in the progress of animal growth and development. It consists of somatostatin(SS) and growth hormone releasing hormone(GHRH) secreted by hypothalamus, growth hormone secreted by pituitary, chicken growth hormone receptor and insulin-like growth factor-Ⅰsecreted by target organs(such as liver), as well as the path of hypothalamus-pituitary-target organs. Analysis of related genes in chicken growth axis and their interaction mechanism were reviewed in order to understand more about the regulation mechanism of poultry growth.

Advances of the Research on Isolation and Culture for the Domestic Preantral Follicles In Vitro

Liu Ruihua1 Li Ming1,2 Jiao Lihong1 Zhen Xing2 Wang Hong1**

2003, 11(3): 313-317 | doi: | Full text (HTML) (1 KB) | PDF

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Abstract

Abstract：The exact mechanisms regulating in vivo folliculogenesis in mammalians have only been partly unravelled. Some processes, such as the initiation of growth of primordial follicles are still poorly understood. This increases the difficulty to culture follicles in vitro. There are important species differences in regulation and timing of maturation, which makes it difficult to transpose techniques. Over recent decades, different animal follicle culture systems have been developed, and a better insight has been gained in the process of folliculogenesis, but the yield of good quality mature oocytes has been very limited. Until now, only within the mouse model, the birth of live young have been reported. With the exception of a unique experiment, where the starting tissue was composed of primordial follicles, all other culture systems could only sustain oocyte growth and development, when follicles were retrieved from the preantral follicle. In the domestic animal models (bovine, pig), the success of folliculogenesis in vitro has been very limited. At most the growth in vitro of preantral follicles could be achieved in the bovine and pig. The purpose of this review is to define the characteristics of preantral follicles growth and develop the isolation methods and culture system in vitro in domestic animals