Investigate the capability of spermatogonial stem cells(SSCs) differentiating into adipocytes under different experimental conditions in vitro. In present study, SSCs were isolated from chicken embryo testis on 16th hatching days, then cultured and subcultured in vitro. SSCs were induced into adipocytes in medium containing desamethason, insulin and IBMX. The differentiated cells were then identified by oil red-O staining. Results showed that SSCs were induced into adipocytes during 7～21d after cultured in induction medium. The programmer ⑥ showed the best result with 85% oil red-O staining positive SSCs.The induced adipocytes highly expressed PPARγ. The cooperative inductive effect was higher significantly than alone inductive effect of desamethason, insulin and IBMX（p<0.01）. Therefore, we concluded that Chicken embryonic SSCs have the capability of multipotential differentiation into adipocytes in vitro.

Experiments were conducted to determine the effect of lamb age, transport stimulation and repeated hormone superovulation on the number of collected oocytes, and to study the effect of the culture medium containing ethylene-diamine-tetra-acetic acid (EDTA) on the development of in vitro produced embryos from lamb oocytes. The results indicate that the mean number of collected and available oocytes from per 6- to 8- week lamb were 60.8±13.9 and 58.2±12.3 respectively, which were more than those (27.3±5.1 and 26.0±4.9 ) from per 12- to 14- week lamb(P<0.05); the transport stimulation didn’t decrease the number of collected oocytes from the superovulation lambs(P>0.05), however, the number of collected oocytes in the repeated superovulation group was reduced significantly compared with the control group(P<0.05); the embryonic culture medium containing 10 μmol EDTA could improved the development capability of in vitro produced embryo from lamb oocytes significantly(P<0.05), and the healthy lambs were born by the embryo transfer.

Abstract: Hepatitis E is an enterically transmitted zoonosis caused by hepatitis E virus that infectious to many animals includes swine. To understand the current status of HEV infection in swine liver, 581 swine liver samples collected from Heilongjiang, Jilin, Liaoning, Hebei, Beijing and Henan province was examined by Immunohistochemistry stain、 H.E and RT-PCR. The result showed that HEV Immunohistochemistry stain positive rate was beyond 90%, except Henan(33.3%), the highest positive rate was 100%. With a pair of primer design from HEV ORF2 gene, Nucleic Acid of HEV can be detected in liver cells with PCR. It suggests that hepatitis E was widely prevalent in Heilongjiang, Jilin, Liaoning, Hebei, Beijing and Henan province .

In the present study, based on the gene sequences encoding mature bovine antimicrobial peptides bac7、bac5 and - defense（LAP） as registered in Genbank, the fragment of the fusion gene Bac7-Bac5-βdefense was synthesized and cloned into the pFASTHTb vector of BAC-TO-BACTM recombinant baculovirus expression system to construct a recombinant expression vector, The plasmid pFASTBac-B7-B5- was then transformed into DH10BACTM competent cell. High molecular weight DNA, the recombinant bacmid, was prepared from the overnight cultures of selected E. coli DH10BACTM colonies. By transfecting Sf9 cells with the recombinant bacmid the recombinant baculovirus from the superenatant was obtained, which could further infect Sf9 cells obtained and express the target protein. The expression of rB7-B5- was confirmed by 12% SDS-PAGE and accounted for 10.6％ of total bacterial protein,the molecular weight of rB7-B5- was 20kD, a figure consistent with the predicted value. rB7-B5- showed antimicrobial activity in E. coli. The present study provides basis for the research and development of novel antibacterial preparations.

Abstract: With two melon hybrid F1s ‘Dongfangmi1’, ‘Dongfangmi2’ and their parental lines and some hybrids (having the same female and different male parents compared with the two hybrid F1s) as materials, the method of melon hybrids purity test quickly by SSR (Simple Sequence Repeat) markers was studied. Results showed that there was a high polymorphism between two melon hybrids and their parents by using SSR markers. Special SSR primers were selected that could identify female parent inbred lines from true hybrids quickly and correctly. The purity of 3 groups of melon hybrid seeds tested by SSR was identical to that tested by field. However, as to the two melon hybrid F1s and some of their uterine hybrids, they couldn’t be distinguished completely by single special SSR primer. Most of them could be distinguished by assemble SSR primers except the hybrids which had similar genetic background with the two hybrid F1s.

山羊痘病毒;P32基因;重组PCR;表达

Objective To explore the correlation between the polymorphism of Single nucleotide polymorphisms(SNPs) of Resistin and the reguration of fat diposition. Methods Screening formutations in the entire coding region for the Resisin gene were Performed by the polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) in Lantang pig、Luchuan pig、Lingao pig、Landrace、Large white and duroc pigs. Results Detected results of genotype demonstrate that no polymorphism in exon-1 and exon-3,two genotypes in exon-2(CC、GG),three genotypes in promoter region(CC、TT、CT),six genotypes in intron-1(EE、FF、GG、EF、EG、FG) and intron-2(LL、MM、NN、LM、LN、MN). Variance analysis revealed that Resistin gene had a high linkage with their growth and fatness traits.Conclusion There are SNPs in resistin gene in pigs ,and have association of these SNPs with fat diposition and obesity in pigs.

This article determine the LXRα mRNA level of forced feed and normal group in the liver, subcutaneous fat, abdominal adipose and muscle of landres geese and analysis the relativity between the transcription level and alt et al serum parameters. The result show that compared to control, forced feed group had elevated mRNA level of LXRα in the liver and abdominal adipose（P<0.05）. In the subcutaneous fat, the expression of LXRα is mostly significantly higher in higher L/W than that of control （P<0.01）and lower L/W（P<0.05）. At the same time, the expression of LXRα in the liver, subcutaneous fat had a positive relation with L/W respectively. the correlation coefficient between TG, Che and the expression of LXRα in liver was 0.66, 0.70 (P<0.05).

Epidermal Growth Factor Receptor is becoming a perfect target to kill carcinoma cells specially because of its overexpression on the surface of carcinoma cells. Cationic antimicrobial peptides (CAP) have its own special membrane-lytic cytotoxicity mechanism. In this study, the membrane-lytic immunotoxin (IT), named as chimeric protein MEGFMEL, which composed of mouse epidermal growth factor (MEGF) as targeting part and melittin (MEL) as cytotoxic part, was constructed to kill carcinoma cells with epidermal growth factor receptor (EGFR) overexpression. Using E.coli BL21 as expression strain and pET30a as expression vector, through low-temperature inducing expression and thawing & freezing purification without cytolysis procedure, the MEGFMEL obtained was of 63.45μg/mL concentration and 68% purity. In vitro activity measurement showed the interested MEGFMEL had significant killing effect to A431 cell of squamous epithelium carcinoma which overexpresses EGFR on its surface, with LD50 value 52.6μg/mL.

In order to evaluate the genotype of CAST gene and its relationship with the muscle histology and other postmortem traits in Jinhua×Pietrain F2 pig family. 158 Jinpi F2 pigs were electrically stunned and exsanguinated. The blood and muscle samples were collected, and both postmortem and meat traits were recorded. PCR-RFLP, PAS and myosin heavy chain immunohistochemistry were employed to explore the relationship between the genotypes and the muscle histology. Myosin heavy chain immunohistochemistry could differentiated the fibers into slow fibers and fast fibers, the ratio of slow fibers is about 7.62:92.38%. The amplification products of the region between 6th and 7th exon of CAST gene were digested by MspI and discriminated 2 polymorphic sites(371bp and 628bp) in endonuclease map. Only 3 genotypes (AA, AB and BB) was distinguished out. The frequency of AA,AB and BB is 0.1795, 0.5897 and 0.2308 respectively. The frequency of A and B is 0.4743 and 0.5256 respectively. The PCR products of the MyoG gene were digested with MspI and 2 polymorphic sites(290bp and 462bp) were distinguished. The allele C was defined as 408+290+180bp, the allele D was defined as 462+408+180bp and 3 genotypes (CC, CD and DD) were discriminated. The frequency of CC,CD and DD is 0.3671,0.5696 and 0.06330 respectively. The frequency of C and D is 0.6519 and 0.3481 respectively. Different genotypes of these tow gene have no statistical significant effect on area, water holding capacity, pH45m value, conductivity of loin muscle. The results revealed that the MspI polymorphism of CAST gene had a significant effect on the aspect ratio of muscle and show a negative correlation with the percentage of intramuscular connective tissue. The MspI polymorphism of MyoG gene had a significant effect on the fiber size and fiber density. The individual with DD genotype had more big-sized fibers and the individuals with CC genotype had high fiber density.

Melanocortin- 4 receptor (MC4R) is a member of the superfamily of G protein coupled receptors (GPCRs), which affects body weight, energy homeostasis and food intake in human and mouse. In this study, the Asp298Asn polymorphism of MC4R gene in Laiwu, Yorkshire×Laiwu and commercial crossbred pig populations was investigated using PCR-RFLP method, and, the relationship of this mutation with backfat thickness was analyzed. The results indicated that only genotype 11 exist in 33 individuals of Laiwu pigs, whereas in Yorkshire×Laiwu and commercial crossbred populations, three genotypes of 11, 12 and 22 were detected. The distributions of allele and genotype frequencies in Yorkshire×Laiwu and commercial crossbred populations were similar with the frequency of allele 1 being higher than that of allele 2. All of the three populations were at Hardy-Weinberg equilibrium with respect to this mutation site（P>0.05）. In commercial crossbred pigs, individuals with genotype 22 had significant higher backfat thickness than the ones with genotypes 12 and 11 (P < 0.05). This study provide evidence that the Asp298Asn polymorphism of MC4R is associated with backfat thickness in commercial crossbred pigs with western pigs as parental lines, and therefore, can be used as a DNA marker for pig breeding in such kind of populations.

Abstract:Osteopontin gene was regard as a candidate gene for reproductive traits of pig in recent years, in this study, genetic polymorphism of OPN gene and correlation with reproductive traits was studied in Landrace pig, Large White pig and Beijing Black Pig. The polymorphism was found by the PCR, which was induced by a deletion mutation of 305bp in the sixth intron of OPN gene after sequencing. AA, AB and BB genotypes were found in Landrace pig and Large White pig populations, which showed a moderate polymorphism. The statistical results of χ2-test indicated that this locus fitted Hardy-Weinberg equilibrium. While polymorphism of this locus was not found in Beijing Black pig, and the BB genotype was only present. Meanwhile, the effect of polymorphism for OPN gene on total number born (TNB), number born alive (NBA) and born weight (WB) were analyzed, the results indicated that the individuals of the first parity Landrace pig with AA genotype showed more TNB and NBA significantly than that with AB and BB genotype (P<0.05), the individual of multiparous sows with AB genotype showed more TNB and NBA significantly than that with AA and BB genotype (P<0.05), and A allele might have the positive effective on TNB, NBA and WB. While in Large White pig, the TNB and NBA of sow with BB and AB genotype were more than those with genotype AA, but the results showed no significant difference (P>0.05), and B allele might have the positive effective on TNB and NBA.

BADH gene was tested in 26 species of Compositae and 2 chrysanthemum cultivars by PCR-Southern. 18 species and 2 cultivars showed clear positive signal. The PCR products of 7 species and 1 cultivar were sequenced（GenBank accession No. EF683587-683595）. 2 homologous fragments of BADH gene , which were very different in length, were obtained from Inula japonica. Sequence analysis showed that the cloned exon regions were identical to each other in length, the homology among them was above 83% , and the homology among the deduced amino acid sequence was above 90%. The second site changed from threonine to alanine in the conserved decapeptide in the longer fragment from Innula japonica. The length of intron region was very different among the cloned sequences. There was not apparent homogology among the intron regions from Eupatorium lindleyanum, Stemmacantha uniflora and the intron region of the longer sequence from Inula japonica. And there was not apparent homology between the 3 intron regions and any other intron regions. However, there was apparent homology among the other intron regions. And the dendrogram of gene relationships from the cloned intron sequences was almost identical to that from exon sequences.

Sex identification was essential for the management, human-assisted breeding, prevention and cure of genetic disease and evolutionary studies of birds, especially for endangered birds. Red-Crowned Crane is a first class protected animal in China and a kind of monomorphic bird, so it’s difficult to identify the sex with morphological method, this situation challenged the couple and human-assisted breeding of Red-Crowned Crane. Genome DNA was extracted from feathers by using nondestructive sampling method and three combinations of primers were selected to amplify the EE0.6 sequence of Red-Crowned Crane specifically. The results showed that male birds displayed only one distinct amplification band, whereas two bands were amplified for female ones. With this molecular method in the article, the sex of Red-Crowned Crane could be identified successfully and accurately and then solved the key problem during the human-assisted breeding of Red-Crowned Crane. This method also gave a guide and reference for the accurate sex identification of other endangered birds.

To investigate mechanism of fluoride-resistance to highly resistant silkworm, using fluorescent different display technique, a differentially expressed cDNA amplicon A14-6 was identified and denoted Bmbcl-2(AB008449), which encodes antiapoptosis protein. Bioinformatics analyses indicates that Bmbcl-2 encodes 965 amino acid with relative molecular mass being 108.800kD，and that isoelectric point was 6.370. The protein has no hydrophobic transmembrane anchor region at the C-terminus and has 19 exons and 18 introns, and splice sites conform to GT/AG rule. Protein analyses indicated that BCL-2 family proteins contain four evolutionarily conserved regions known as BH1、 BH2、 BH3 and BH4, but low homologues between BmBCL-2 and other BCL-2 family proteins by using ClustalX1.83 and MEGA3.1 software. RT-PCR results showed that the Bmbcl-2 expressed in five tissues of the 48h of 5th instar silkworm，but its expression was higher in midgut and fat body in 441DZ. Highly fluoride-resistant strain 441 and highly susceptible strain 440 were used. After treated with water and 200mg•L-1, the transcription level of Bmbcl-2 was higher in 441F than in 440F, while there was no significant change in water treatment. The results indicated that Bmbcl-2 may be related with fluoride-resistance.

The results of bioinformatics analysis showed that the transmembrane segments, signal peptide, protein secondary structure, the amounts of phosphorylation sites and Palmitoylation sites were different in the four caragana intermedia fad2 proteins. All of these were very important to study the functions and regulation of these genes further.

The production of recombinant human pharmaceuticals in the milk of transgenic farm animals has many advantages when compared with microbial bioreactors or animal cell bioreactors. At the same time, somatic cell nuclear transfer (SCNT) has provided an alternative avenue to the production of transgenic animals. The purpose of this study was to establish human lactoferrin cDNA transgenic donor cell lines and to prepare competent donor cells for the production of transgenic animals by SCNT. Human lactoferrin cDNA was obtained by RT-PCR while 6.5 kb caprine β-casein regulatory elements were amplified by Long and Acute PCR. Subsequently the two fragments were inserted into the corresponding site of the plasmid pEGFP-C1, resulting in the mammary specific expression vector p6.5hLF-EGFP. Then the caprine fetal fibroblast cells were transfected with p6.5hLF-EGFP by LipofectamineTM-2000 and selected by G418 for 3 to 4 weeks. The G418 resistant transfectants were identified by PCR and EGFP detection. The results indicated that the transgene was stably integrated into the open region of the chromatin of G418 resistant fibroblast cells. This study may provide competent transgenic donor cells for the production of transgenic animals by SCNT and improve the efficiency of transgenic cloning.

ABSTRACT Aim The population doubling number(70～80 times) of human fetal bone marrow mesenchymal stem cells(BMMSCs) is about two times more than that (30～40 times) of the adult BMMSCs, and their differentiation capacity is superior to that of their adult counterparts. Especially, human BMMSCs from first-trimester fetus are closer to embryonic stem cells, show greater telomerase activity, more plasticity and faster propagation than the mid- and late-trimester fetal BMMSCs, thus being the ideal seed cells for human tissue engineering and regeneration medicine. The purposes of this study are to establish a method for isolating human BMMSCs from 2- to 3-month-old abortuses, select culture system for the cells, identify their biological properties and biosafety and provide seed cells for their tissue engineering and induced differentiation in vitro. Methods This experiment was conducted in Shaanxi Branch of National Stem Cell Engineering and Technology Centre of China from April to December 2005. BMMSCs were isolated from 2- to 3-month-old human abortuses by scissoring their long bones lengthwise, followed by rinsing and culturing whole marrow cells. Basic medium and serum concentration for BMMSCs culture were optimized and growth curves made, both with MTT reduction assay. Isolated cells were identified with flow-cytometry and immunocytochemistry for their antigen markers. The biosafety of isolated cells was evaluated by karyotype analysis and tumor forming experiment. Results Lengthwise scissoring of fetal long bones and rinsing their marrow cells was practical and useful to recover the BMMSCs from human abortuses at the age of 2-3 months. In the present experiment, α-MEM+20%FBS was the best system for culturing such BMMSCs in vitro. The third passage BMMSCs expressed Oct4, SSEA3 and SSEA4 beside the surface markers of their adult counterparts. The population doubling time of the BMMSCs of passage 6, 12 and 24 were 34 h、36 h and 40h respectively. The cells in all the passages showed diploid karyotype and formed no tumor in the nude mice. Conclusion The BMMSCs of human abortuses at the age of 2-3 months could be isolated and cultured in vitro and expressed some of the antigen markers of embryonic stem cells. Compared with their adult counterparts, fetal BMMSCs grew faster and their population doubling level was higher. They were proved to be biologically safe and ideal seed cells for researches on human tissue engineering and regeneration medicine.

Using an introgression lines population of Chinese common wild rice（O. rufipogon Griff.）from Yuanjiang, Yunnan Province, with the background of Teqing (O. sativa ssp. indica), Four quantitative trait loci (QTLs) for heat tolerance at the flowering stage were identified by the method of single-point regression analysis. Two QTLs (qHT1and qHT3) mapped on chromosome 1 and 3, explained 12% and 6% of the phenotypic variance, respectively, and the O.rufipogon-derived alleles contributed desired effects on Teqing background, which could increase 9.13% and 6.71% of percent seed set. The other two QTLs （qHT8 and qHT10）detected on chromosome 8 and 10, explained 6% and 6% of the phenotypic variance, respectively, and the O. rufipogon-derived alleles can decrease percent seed set on Teqing background. The results obtained in this study might be helpful for improvement of heat tolerance by marker-assisted selection in rice. It was also suggested that there was enormous potential for improving the tolerance to abiotic stress of cultivated rice utilizing the favorable genes of wild species in rice breeding program.

For development of the Ee chromosome specific molecular markers for diploid species of L.elongatum, amplified fragment length polymorphism (AFLP) analysis was performed on common wheat Chinese Spring (CS), CS—L. elongatum disomic substitution and additional lines using 5 primer combinations, and 28 AFLP markers, which were specific for 1Ee to 7Ee chromosome of the diploid species of L.elongatum, respectively, were obtained. After cloning and sequencing of these AFLP fragments, four AFLP markers of E-ATC/M-CGTA341bp, E-ATT/M-CTAG265bp, E-AAT/M-CCAG139bp and E-ATT/M-CTAG205bp were successfully converted into reliable sequence tagged markers (STS). These four markers were further subject to amplification other five accessions of the L.elongatum and other eight common wheats without L.elongatum sources for validating their specificity and polymorphism. The results showed that these markers could amplification the same size of the fragment from L.elongatum accessions but couldn’t amplification it from other eight common wheat, suggesting that these markers could be used for identify and tracing the Ee chromosomes in wheat- L.elongatum substitution and additional lines.

OPW02-1756, OPO11-964, OPY13-661, OPB11-520, OPW05-766, OPV03-1365 and OPJ16-759, seven RAPD markers linked to the resistant powdery mildew genes in Chinese wild Vitis were reclaimed, cloned, sequenced and analyzed. The results showed that the real length of seven RAPD markers was 1756bp, 964bp, 661bp, 520bp, 766bp, 1365bp and 759bp respectively. Marker OPW02-1756 shows 94% to 98% identity with 3 EST sequences from European grapes. OPO11-964 shows 93 % identity with 1 EST sequence from Cabernet Sauvignon, shows 90% and 87% identity respectively with 2 EST sequences from green grape berries library, shows 83% identity with 1 EST sequence from multiple pathogen-infected sweet orange. OPO11-964 sequence has a biggest open reading frame of 444bp encoding 147 amino acids, has partial identity with 21 protein sequences of unknown functions from other lives. OPY13-661 sequence has identity with 16 EST sequences from European grapes, and shows 89% identity with 2 EST sequences from abiotic stressed leaves of Vitis vinifera var. Chardonnay. OPB11-520 shows 94% identity with 4 genomic sequences of unknown functions of Arabidopsis thaliana. OPW05-766 shows 50% identity with GAG-POL precursor of wine grape. OPV03-1365 shows 81% to 88% identity with 6 rice genomic sequences, 84% to 91% identity with 3 sequences from Arabidopsis thaliana. OPJ16-759 shows 83% to 100% identity with 14 EST sequences from abiotic stressed leaves of Vitis vinifera var. Chardonnay, 94% to 100% identity with 3 EST sequences from water stressed berry of Vitis vinifera var. Cabsau.

Northern corn leaf blight caused by Exserohilum turcicum was inhibited by spraying leaves with salicylic acid (SA) solution in greenhouse test. The tests indicated that: the lesion amount and lesion area on the leaf could be reduced in the maize seedlings (OH43, OH43Ht1, Huangzao4, Huangzao4Ht1) sprayed continuously for 6 days with 10mM SA, which had the effect of inhibition above56% generally. The study on physiological mechanism of resistance to Northern Corn Leaf Blight showed the infection speed of E. turcicum was slowed in the maize leaves pretreated by 10mM SA. Haustorium formation from papilla and the growth of haustoriun in the leaf cells were delayed compared with the water-control. The pretreatment of SA also induced and promoted cell death in the infected cell as well as the adjacent. The activities of phenylalanine amonnialyase (PAL), chitinase and β-1,3-glucanase of maize leaves were enhanced significantly with SA treatment or treatment of E. turcicum inoculation after SA pretreatment, the content of lignin and DAMBOA in the maize leaves were also increased. Increasing of H2O2 levels was also observed in the maize leaves with the treatment of E. turcicum inoculation after SA inducing.

Abstract: Using 16S rDNA-PCR-DGGE, electron microscopy and conventional plating method, an attempt had been made to explore the genetic diversity and phenotype polymorphism of the endophytic bacteria within A.microphylla. Based on 16S rDNA-PCR-DGGE profile it is suggested that there is a complex and divergent bacterial community with Bacillus cereus and its variants as dominant species within Azolla-cyanobacteria association, which was supported by the fact that the endobacterial cells exhibited distinct ultrastructural characteristics in vivo and the bacteria appeared various colonies with different size, shape and colors in vitro. The advantage and limitation of using DGGE in uncovering genetic diversity and the potential role of endophtic bacteria in phylogeny and synchronic evolution among the Azolla, cyanobacteria and bacteria were discussed.

A high toxicity Bacillus thuringiensis (Bt) strain WZ-9 against larvae of Henosepilachna vigintioctomaculata was isolated from soil of Hebei province. Bipyramidal inclusions could be observed by scanning electron microscopy. SDS-PAGE analysis indicated that the strain produced a 130 kDa protein. Growth cycle of this strain was 24h, and pH of the culture varied with the strain growth. The bioassay results showed that the WZ-9 strain was highly toxic to the 2 instar larvae of H. vigintioctomaculata.. Correct mortality is 100% at 72h, and LC50 is 2.95×107cell/mL. Genotype identify showed this strain had cry7 gene. Sequence analysis showed that the encoding gene contained a ORF of 3414 bps and encoded 1138 amino acids residues. The deduced amino acid sequence is 99.65% identical to that of the reported Cry7Ab2 sequences. By Bt δ-endotoxin nomenclature committee this gene was designated as Cry7Ab3 with accession number BI 1015188 in GenBank database.

Bacillus cereus strain 905 isolated from wheat is an endophytic beneficial bacterium that can promote plant growth. In this study, the gene encoding CuZn-SOD in B. cereus 905 was cloned by using of PCR to analyze the toxicity of active oxygen species (AOS) to the bacteria during the colonization in plant. The sequence analysis shows that this gene contains 537 base pairs which encoding 179 amino acid residues. For studying the encoded peptide characteristics, the expression vector pET-sodC in Escherichia coli BL21(DE3) was constructed. Results showed the induced protein with IPTG was confirmed as a superoxide dismutase by dyed with NBT. Finally, the gene in B. cereus 905 encoding CuZn-SOD was identified on the basis of inhibited SOD activity by potassium cyanide (KCN) and hydrogen peroxide (H2O2).

In this experiment, we designed attB-flanked PCR primers and amplified coat protein gene (CP) and movement protein gene (CI) genes of Papaya ringspot virus with PCR, performed a BP recombination reaction with attB-PCR products and donor vector pDONRTM221 to generate an entry clone, then, processed an LR recombination reaction with the entry clone and destination vector pHellsgate12 of select to generate an expression clone system by screening of plate medium, and detected with PCR and enzyme digestion.

The coat protein (CP) gene of Rice stripe virus (RSV) and Rubisco SSU leader peptide gene of rice chloroplast were amplified by PCR。Depending on the expression vector pGEX-4T-1 and pET-29a, fusion gene PR-CP and PR-S-CP was constructed. The two fusion genes were confirmed correct by sequencing and analysis. The recombinant pGEX-PR-CP and pET-PR-S-CP were transformed into E. coli BL21 (DE3), and then induced to express by IPTG. The expression products of fusion gene were identified by SDS-PAGE electrophoresis and Western bolt. The result suggested that the fusion proteins were the same as we expected and could be specifically recognized by the antibody to RSV CP. This suggested that RSV CP gene in the fusion gene expressed correctly and maintained its antigen activity, so we could conclude that the expression products of fusion gene in the organism maybe could maintain its structure and function separately.

Yeast two-hybrid system is a method of researching interaction between protein and protein in vitro. This review summarizes the application of this technology to plant-pathogen recognition and signal transduction, including (1) The principle of yeast two-hybrid technology; (2) Special recognition between plant and pathogens; (3) Identification of elements in plant signal transduction pathway against pathogens.

To study cis-regulatory elements in AtEXPA1 (Arabidopsis thaliana expansinA1)promoter，a full length of 897 bp genomic fragment and a series of end deletions fragments, isolated from Arabidopsis genome by PCR, were fused to the beta-glucuronidase reporter gene (AtEXPA1::GUS) and transformed into Arabidopsis leaves by particle bombardement and tobacco protoplasts by PEG-mediated method , respectively. The results of histochemical GUS staining and fluorometric measurements showed that some enhancing transcription elements existed in -144bp to ATG. We treated protoplasts with external stimuli including light, cold, abscisic acid(ABA) and salinity. Quantity analysis of GUS activity suggested,(1)some regulatory elements response to light widely existed in AtEXPA1 promoter;(2)-897～-626bp contained negative elements response to cold;(3)-626～-444bp contained negative elements response to ABA;(4) -626～-282bp contained negative elements response to salinity.