A Newcastle disease virus field strain PB9601 was isolated from a pigeon in Beijing which can form typical pathological changes through regression experiment after plaque-purification. NDV PB9601 was determined as a virulent strain with MDT of 50.5h , ICPI of 1.65, IVPI of 2.36 respectively. The F and HN gene were cloned and sequenced. The sequence analysis of F gene indicated that the amino acid sequence is 111 KRQKRF 117 in the F protein cleavage site in PB9601 strain which is identical to virulent strains of NDV. The homology analysis of F and HN gene sequences compared to reference isolates from GenBank indicated that: PB9601 belongs to genotype VI with YN-03 and TW-98；The F protein amino acid sequence has homologies of 88.6%,91.0%,90.6% with LaSota,V4,F48E9 and of 92.1%, 95.5%, 98.6% with isolates GX-05,YN-03,TW-98 in China and of93.3%、91.9%、94.6%、95.1%、93.6% with isolates Capital3-97、Tigre6-99、1166-00、IT-227-82、2736-00 abroad. When HN genes were compared, PB9601 demonstrated higher homologies of 91.9%, 93.3%, 94.6%, 95.1%, 93.6% with isolated strains Tigre6-99, Capital3-97, 1166-00, IT-227-82, 2736-00 abroad , but lower homologies of 87.2% ,89.2%, 87.6% with LaSota , F48E9 and V4.The above results show PB9601 has obvious regional and genus traits compared with isolated strains LaSota ,F48E9 and V4 .

SRAP markers were employed to analyze the genetic diversity of potato varieties. The results showed that 23 out of the random selected 27 primer pairs were polymorphic among the 44 varieties tested, 104 polymorphism bands were obtained, and the polymorphism ratio of primers was 85.2%; the average polymorphism bands was 4.5 per primer pair. These results demonstrated that SRAP had higher polymorphism in the tested potato varieties. Based on cluster analysis with the SRAP makers, the genetic distance range among the tested 44 varieties was from 0.147 to 0.741. At the genetic distance（D）of 0.67, 44 varieties were clustered into 4 main groups, including one compound group and three discrete ones. The compound group could be subdivided into 7 subgroups. The great genetic diversity of the tested potato varieties was thus confirmed at the DNA level.

The intron 5 of the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene was cloned and its SNP was tested by PCR-SSCP in Wanxi White goose (WW). The carcass trait, meat quality trait and feather-down trait of 116 WWs were mensurated and analyzed by relating with SNP. The results showed that intron 5 sequence length of HMGR gene in goose was 704bp, and the homology between goose and chicken was 44.0%. A and B alleles were tested in the intron 5 by PCR-SSCP, and gene frequencies of A and B were 0.7716 and 0.2284 respectively. Genotype AA was dominant genotype on increasing chest muscle weight, gland stomach weight (P<0.05) and 1000-downs weight (P<0.01), and on decreasing down hold-water percentage (P<0.05). Genotype AB was dominant genotype on decreasing fiber diameter of chest muscle (P<0.01) and on increasing chest muscle 45pH (P<0.05).

The highly-efficient plant binary expression vector PGBIF4ABCVHB was constructed by applying the artificially-modified-synthesized VHb gene and fusedly-insectidal gene(GFMcryIA and CPTI ) , 38 transgenic tobacco plants were obtained by Agrobaterium mediated transformation ,32 of them were indentified that the bivalent genes were inserted into the tobacco genome by PCR amplification detection and Southern blot analysis,western blot analysis proved that VHb gene was expressed in the transgenic plants,Toxicity assay indicated that fused insectidal gene was expressed pesticadal toxin protein .the net weight of transgenic tobacco plants exceeded 8% of that of non- transgenic ones . compared to non- transgenic tobacco plants, the transgenic ones were high-yield varities of insect-resistant plants.

The gene structure of the T.reesei cellobiohydrolase Ⅱ(cbh2) was reconstructed by fusing with additional genes which encode catalytic domains from EGⅣ(cdE ) and CBHⅡ(cdC ), respectively. The target genes were obtained through PCR and the gene cdE was ligated on the 5’ end of cbh2 while cdC was ligated on the 3’ end through gene fusion, generating reconstructed genes cdE –cbh2 and cbh2-cdC, respectively. The reconstructed genes were expressed in Pichia GS115 and results the recombinant strains P.pastoris PEC11 and P.pastoris PCC16 with the most efficient activities. When cultivated in the optimized incubation condition, the CMC activities of the cultivation supernatant of P.pastoris PEC11 and P.pastoris PCC16 attained 3.87U/ml and 7.66U/ml, respectively. The modified CBHⅡwith an additional catalytic domain from itself improved the CMC activity about 2-fold.

In order to study gene expression profile during appressorium developmental process of Magnaphorthe grisea strain Y34 that isolated from the rich area for Asia cultivated rice resources, a cDNA array with 4108 TUTs was used to monitor gene expression patterns during appressorium development of M. grisea. A total of 511 differentially expressed genes were identified from 0, 2, 8, 20, and 30 h time points of the infection structure development. Compared with ungerminated conidia, the number of up-regulated and down-regulated genes was almost consistent at any time-points. More genes were differentially expressed during appressorium maturation than during appressorium induction and formation. Gene expression profiles between 8 and 20 h time-points were similar. By 30 h time-point, all genes that were down-regulated in conidia had changed to up-regulated, and half of the genes showing up-regulated in conidia changed to down-regulated. During 20 to 30 h time-points, genes seemed to be most actively expressed. Therefore, a detailed and comprehensive analysis of the programmed gene expression that occurs during the continuous stages of appressorium development is necessary to better understand the pathogenesis of M. grisea.

CASTOR encoded predictable ion channel protein in Lotus japonicus ,which was the functional gene in the pathway of symbiosis, acted upstream of intracellular calcium spiking, mutants affected in CASTOR gene didn’t form root nodules. To research the proteins interacting with CASTOR, the CASTOR gene was used as the bait protein to mate with the cDNA lib of Lotus japonicus by yeast two-hybrid system. It had 111 positive clones in prescreening, it had 99 blue clones by thorough detection of β- Gal, there may be seven species proteins interacting with CASTOR after sequencing and NCBI blast analysis, then identified the expression of these genes in Lotus japonicus by RT-PCR, it was predicted that CASTOR may form compound with these proteins to thansfer symbiosis signal.

Intergeneric transfer of plasmid vectors pSET152 and pHL212 from Donor E. coli ET12567/pUZ8002 and S17-1 to Streptomyces cinnamonensis were demonstrated and optimized. NsdA gene disruption structure was constructed by PCR-targeting system and then introduced into Streptomyces cinnamonensis BIB2005 through intergeneric conjugal transfer.The disruption of nsdA was confirmed by PCR assays. Compared with the start strain, the yield of monensin of the nsdA mutant BIB309 increased 270 percent in the level of flask.

The gene MalQ was amplified from Streptomyces coelicolor by PCR, then the gene MalQ was cloned into pTrc-CKS and sequenced. MalQ sequence was analyzed by BLAST and it displayed high similarity(97%) to gene MalQ of Streptomyces coelicolor reported in GenBank. The recombined plasmid pTrc-CKS-MalQ was transformated into E.coli Top 10F’and induced by IPTG.. Gene MalQ and CKS fusion protein(molecular weight：about 106kDa) could detected by SDS-PAGE gel electrophoresis. The crude enzyme was demonstrated having 4- -glucanotransferase activity with TLC.

Actinobacillus pleuropneumoniae (APP) is the causative agent of porcine contagious pleuropneumonia which causes great economic losses in the pig industry worldwide. There are at least 15 serotypes of APP so that it is very difficult to diagnose and control this disease. An ApxIV-ELISA which could distinguish the vaccinated pigs from APP-infected ones was established in our previous study based on the fact that the toxin ApxIV expresses only in vivo (in APP-infected pigs), but not in vitro. In this study, we developed this method a differentiating diagnostic kit, whose specificity, sensitivity, repeatability and stability were evaluated and compared with an analogous ELISA kit (CHEKIT-APP-APXIV) from the company IDEXX and the indirect haemagglutination assay (IHA). A total of 1453 clinical sera from China, UK, Denmark, USA, Canada and Australia were screened using our ApxIV-ELISA kit. The results showed that the ApxIV-ELISA kit has very high specificity、 sensitivity and repeatability [the inter- and intrabatch imprecision (CV%) < 15%] and stability (very stable at 4-8 C for 9 months). Compared with CHEKIT-APP-APXIV, the overall agreement rate was up to 91.37%. Our data confirmed that the ApxIV-ELISA kit can be used to distinguish the vaccinated pigs from APP-infected ones.

Abstract: The chromosomal assignment of porcine NDUFS7 gene was carried out in this experiment using radiation hybrid panel. The complete coding sequence of porcine NDUFS7 gene was obtained by the reverse transcriptase-polymerase chain reaction (RT-PCR), and the prediction of its protein structure and potential function was carried out by regarding tools. The spatial and temporal expression profile of NDUFS7 gene was analyzed by semiquantitative-RT-PCR in porcine 11 tissues and skeletal muscle from pig fetuses at 33days post coitus (dpc), 65dpc, 90dpc and grown pigs, respectively. The results showed that NDUFS7 gene was assigned on the SSC２q21-q24 and linked with marker SW395. The coding sequence of NDUFS7 is 648 nt, coding for 215 amino acids. The predicted results indicated that the porcine NDUFS7 gene has a conservative NuoB domain and 3 phosphorylation sites. Meanwhile, the NDUFS7 gene showed a different expressive level in different porcine tissues, while the expressive level of NDUFS7 in prenatal skeletal muscle at 65dpc and 90dpc was relatively higher than that of in skeletal muscle from the fetuses at 33dpc and the grown pigs.

In this research DNA Barcodes was based on the special section of the cytochrome c oxidase 1 (COⅠ)sequence. Three primers were designed in three special sections(Bar1 Bar2) to identify six indigenous chicken breeds. The Bar1 sequence of COⅠ(648 bp : from 712 sites to 1358 sites) was the highest in polymorphism and had 24 SNP in six chicken breeds. The average interspecific divergence was 3.860%, and special sites were found in each chicken breed which was the bases to identifying chicken breeds. The DNA taxonomy of Bar1 used NJ was consistent with morphological taxonomy among the six indigenous chicken breeds. However, the analysis of Bar2 sequences of COⅠindicated that they were lower polymorphism and no special site in most chicken breeds which was not benefit to identify chicken breeds. The DNA taxonomy of Bar2 used NJ was not consistent with morphological taxonomy among the six indigenous chicken breeds. The results illustrated that the DNA barcoding was a rapid and inexpensive method for the identifying breeds, especially when coupled with traditional taxonomic tools in disclosing hidden diversity and molecular phylogeny relationship. It was a better tool for identifying chicken breeds through more improvement.

Abstract: β-1.4-N-acetylgalactosaminyl-transferase(Galnact2) is responsible for the first committed step in the synthesis of mucin-type O-glycans on protein substrates。In this study, primers flanking the galnact2 c.363 C>G mutation were designed according to the known chicken galnact-2 DNA sequence. This polymorphism was genotyped in 276 animals from six chicken breeds including White Recessive Rock, Xinghua, Taihe Silk, Baier Huang, Kangle, Nancheng Black, and in a F2 full sib resource population by crossing White Recessive Rock with Xinghua using PCR-XhoI-RFLP. The results showed that：(1) The genetic polymorphism of the Galnact2 c.363C>G mutation was abundant; (2) This polymorphism was significantly associated with body weight at all ages measured. Individuals with the GG genotype had heavier body weight (BW) at the ages of 1, 21, 28, 35, 49, 56, 63, 77 and 84 days than those with the CC genotype (p<0.01). GC individuals had heavier BW at the ages of 1, 21, 56, 63 days (p<0.01), and 35, 49, 77 and 84 days (p<0.05) than CC individuals. There was no significant difference between GC and GG animals for BW at all ages measured; (3) This polymorphism was not significantly associated with fatness traits including fat pad under the skin, abdominal fat weight, width of fat band, crude fat content in breast muscle and crude fat content in leg muscle.

Polymerase chain reaction (PCR) is perspective for detection of animal derived feedstuffs. In order to avoid safety risks deriving from bovine spongiform encephalopathy and detect fish-derived material efficiently by PCR method, specific primers were designed according to mitochondria 16S rRNA sequence. DNeasy Tissue Kit of Qiagen was applied to extract nucleic acid. Specificity detection results showed primer NC_Fish2480/2501F, NC_Fish2565/2586R had no cross-reaction with bovine, ovine, porcine and chicken derived material. Sensitivity results showed that the detection limitation was 0.1% fish derived material. This study provided a method for PCR detection fish-derived material in ruminant feedstuffs.

Dr lin who works in the biotechnology research institute has isolated and cloned the structural gene aroAG2 coding for EPSP Synthase sequence from Pseudomonas fluorescens G2. The aroAG2M gene was artificially synthesized according to codon usage preferred by dicotyledonous plants.Impactvector1.4 with the chloroplast signal peptide is one of high performing vectors for expression in plant. The aroAG2M gene was placed under the Rubisco promoter and then a plant expression vector was constructed. Transgenic tobacco were obtained by Agrobacterium-mediated transformation．PCR and Southern analysis of transgenic tabacco plants indicated that the exogenous genes were integrated into the genomes of the transgenic plants,and in transgenic plants herbicide-resistance to glyphosate reaches 4‰.The vector was used for cotton transformation by Pollen Tube Pathway Transgenic Technique. PCR and Southern analysis of transgenic cotton plants showed the target gene was integrated into the genome. To date, T1 generation of transgenic plants have been obtained. Transgenic plants are resistant to glyphosate herbicide in field．

In order to determine a set of SSR core primers fitting for maize hybrids’ purity identification, 420 maize hybrids were analyzed by 10 SSR primers: bnlg439, bnlg125, umc2105, phi072, umc1705, bnlg161, bnlg1792, bnlg162, phi065 and bnlg1450. Primer polymorphism and heterogosite rate were evaluated, which varied much among different primers with PIC value ranged from 0.838 (bnlg1450) to 0.482 (phi072) and heterogosite rate ranged from 0.888 (bnlg161) to 0.431 (bnlg162). There have no obvious relevance between the two parameters. Based the evaluating result, two primers bnlg161 and bnlg1450 were determined as preferred core primers for maize hybrid purity identification; five primers bnlg439、bnlg125、umc2105、umc1705、bnlg1792 were determined as candidate core primers for maize hybrid purity identification; three primers phi072, bnlg162 and phi065 were deleted because of their low polymorphism or low heterozygosis rate. 412 hybrids (accounting for 98％) have heterozygosis band type in at least one primer if the two preferred core primers bnlg161 and bnlg1450 were used,, and if another primer bnlg439 added, 419 hybrids except one hybrid ShennongT11 have heterozygosis band type. It indicated that 2-3 carefully selected core primers could solve most hybrids’ purity identification if only need detect selfed individuals. Specific primers for purity identification were also studied, which could reduce the cost and time of purity identification. There are two sorts of specific primer: (1) within specific range of varieties, the genotype of a primer appeared only in one variety, then the primer could be used as specific primer of the variety; (2) the genotype of a primer had a genotype frequency lower than a specific value, then the primer could be used as specific primer of the varieties with such genotype. 29 varieties have specific primers within the 420 varieties, Both umc1705 and bnlg1450 were used as specific primers of 8 varieties, while phi072 and phi065 were not used as specific primers of any variety. Specific primer have no direct relevance to primer polymorphism and heterozygosis rate, but have some relevance to gene frequency of different alleles in a primer. Primers with complementary band types were further screened, and DNA fingerprint maps for maize purity identification were constructed. Based the above results, we bring forward that the core primers fitting for maize hybrids’ purity identification should have both high polymorphism and high heterogosite rate, and specific primers for a specific maize hybrid should have complementary band type and low genotype frequency.

Polyphenol oxidases (PPOs) present in mature wheat kernerals have been implicated in the undesirable darkening of dough. To accelerate the functional characterization of wheat PPOs, allow the identification of those PPO genes that are primarily involved in food biochemistry, and improve the appearance quality of cereal products of china, basic local alignment search tool (BLAST) searches of expressed sequence tag (EST) databases were performed, and the sequences were aligned using software DNAMAN. Results from this study suggest that the presence of at least 13 PPO sequences in hexapolid wheat fell into two clusters (I、II). Genes in ‘II’ cluster are expressed during kernel development and may therefore influence cereal product quality, and the genes in ‘i’ class of II cluster which can be used in the further molecular marker related research seemed not on the wheat chromosome 2A and 2D. The allelic variations of PPO genes on chromosome 2D (PPO-2Da、b) were richer than PPO genes on chromosome 2A (PPO-2Aa、b) according the sequence alignment between four PPO genes with open reading frame (ORF). 94 single nucleotide polymorphisms (SNP) were detected between PPO-2Da and PPO-2Db and 80 SNPs were found in coding region (coding SNP) while 36 SNPs,which affect the PPO amino acid sequece were non-synonymous cSNPs. To understand the effect of non-synonymous cSNPs in PPO gene on grain PPO activity, primer was designed as STS-H at some non-synonymous cSNPs site. The STS-H can amplify a 460-bp fragment in most cultivars with high PPO activity (b), while no PCR product was detected in most cultivars with low PPO activity (a). A total of 130 wheat cultivars were used to validate the correlation between the polymorphic fragments of STS-H and grain PPO activity. The results showed that PPO alleles ‘a’ and ‘b’ at the STS-H locus gave significantly different effect on grain PPO activity. The cultivars with allele ‘b’ had greater (P<0.01) PPO activity than the cultivars with allele ‘a’. To enhance the selection efficiency of single dominance molecular marker, the multiplex PCR was developed between STS-H and STS01, which is a STS molecular marker for low PPO activity and complementary with STS-H tested in this study.

Abstract: Resveratrol is a member of stilbenoids with the capcity of disease-resistantance for plants and health benefit for human. Carrot is one of the most important vegetables rich in β-carotene. To further promote carrot’s heath care value by expression of foreign resveratrol, we conducted the genetic transformation of RS to carrot. the cDNA of grape resveratrol synthase gene was isolated from a cDNA library of grape and its codon for amino acids was optimized for efficient expression in carrot. With a cDNA of RS, the transformation vecter was constructed by inserting the cDNA into a transformation vector pCB, whose HPT was eliminated. pCB and pCB-RS-hyg- were co-transformed into carrot via a efficiently Agrobacterium mediaed transformation method and transgenic plants were acquired. By screening in the T0, T1 and T2 generation of transgenic plants, 2 plants with high content of resveratrol were found, Which laid a very important base for further use in health care food exploitation and study of resveratrol metabolic pathway.

Ageratina adenophora (Crofton weed) is one of the most widespread invasive species in China. Its genetic diversity and population structure in China was analyzed by amplified fragment length polymorphism (AFLP) in this study. Three primer pairs were selected for the analysis and 490 bands were produced from 62 A. adenophora populations of 5 major geographic areas in China, of which 328 bands showed polymorphic. Diversity levels within populations were relatively high (mean expectedheterozygosity = 0.171, mean Shannon index = 0.268), and that between populations was low (Fst = 0.287). At the regional level, the AMOVA indicated that about 70% variations in the data set were from genotypic variations within populations, 8.04% variations due to regional differences, and the remaining 21.71% due to differences among populations within the provincial regions. Cluster analysis based on the UPGMA method grouped the majority of the 62 A. adenophora populations into four main clusters, corresponding with their geographic regions. Elevation was approved by Sigmaplot 2000 analysis to be the main environmental factor affecting the genetic diversity of A. adenophora. Mantel test indicated that there was a low correlation between geographic and genetic distances among populations (r =0.34). It is concluded that A. adenophora spread mainly by wind or water and its genetic diversity level in newly invaded area was lower than that in former invaded areas.

A gene encoding β-endoglucanase (EG) from Vibrio fischeri EM17 was amplified by PCR and inserted into E.coli expression plasmid pGEX-6p-1.The recombinant plasmid was transformed into E.coli BL21 and induced to express. Sequecing analysis showed that the sequence of β-endoglucanase gene fragment exhibited 97.1％ homology with Vibrio fischeri ES114’s ,while it was very low compared with other organisms’ .The optimal enzyme reaction temperature is 60℃, and the optimal pH is 9.0. The result of the effect of ion on enzyme activity showed that the enzyme activity relied on metal ion, Mg2+and Ca2+ showed a stimulating effect while Mn2+ and Pb2+ showed an strong inhibitory effect.

Avermectin production of Streptomyces avermitilis could be stimulated by increasing the dosage of orfX gene. Integrative expression vector pWJ827 containing ermEp* and construction gene of orfX, was constructed based on pIJ8600. The recombinant strain BIB0827 was obtained by introducing pWJ827 into the industrial strain BIB9903 through intergeneric conjugal transfer. HPLC assays showed the avermectin production of BIB0827 reached 3350μg/mL, 1.5-fold increasing when compared with BIB9903. The characteristic of over producing strain BIB0827 was stable after ten rounds of non-selective growth, which could be applied in the industrial production of avermectin.

A Full-length cry1Ia gene fragment, which obtained by PCR amplification with a pair of primers designed according to cryIa-type gene sequences and DNA from Bacillus thuringiensi Btc008 as template, was introduced into expression vector pET-21b and transformed into E. coli BL21(DE3). Molecular weight of the induced express product was 81kD.The encoded protein was composed of 719 amino acid residues and the predicted MW is 81.2kD. The amino acid sequence of the Cry1Ia was very different from those of 12 known Cry1Ia-type proteins. This gene with accession number AF373207 was designated as cry1Ia8 by International Bt Insecticidal Gene Nomenclature Committee. The bioassay results indicated that the Cry1Ia toxin protein showed distinctly insecticidal activity against Ostrinia furnacalis and Plutella xylostella with LC50 of 0.268 µg/g and 2.227 µg/ml respectively, while it also had insecticidal activity against Leguminivora glycinivorella，but no activity against Pyrrhalta aenescens. The novel cry1Ia8 gene will be new resource for the construction of genetically engineered bacterium and transgenic plant for biocontrol of insect pests. It also is available for screening gene stacks to delay the resistance produce of the pests.

Targeting-induced local lesions in genomes (TILLING), as an important research technology in reverse genetics has been widely applied to study in molecular biology of many model organisms. CELⅠ is the key enzyme of this technology. In this study, we extracted the endonuclease CELⅠ with high activity from celery by (NH4)2SO4 deposition, affinity chromatography, anion and cation chromatography. Using heteroduplex DNAs as the substrates, we further investigated the various activities of CELⅠ under different conditions, the various combinations of different purified levels, digestion temperatures, treatment time, presence or absence of Taq DNA polymerases. The results indicated that the CELⅠin our test is much purified, the optimum temperature is between 40℃ and 50℃, well special digestion for mismatch bases in a long time, and that the activity of CELⅠ would be higher when Taq enzyme is present in the reaction system. In conclusion, our results will provide technology supports for application of TILLING in silkworm with large scale and low cost.

Screening microsatellite marker from ESTs database (10691 item) of common carp, we have found 127 microsatellite sequences which occupied 1.19% of ESTs database. Besides these markers, we have isolated 20 microsatellite sequences from constructing cDNA library for common carp. These microsatellite sequences allowed us to design 68 pairs of primers with Primer Premier 5.0. 27 primer pairs obtained expected product, and 20 primers showed polymorphism among populations in synthesized 40 primers. Using the 20 polymorphic loci, we analyzed common carp genetic structure of two geography populations, Yangtze River and Heilongjiang River. The results showed that the average effect alleles numbers were 3.9135 and 3.4820 respectively; the average observed heterozygosity was 0.6067 and 0.5667; the average expected heterozygosity was 0.6737 and 0.6194; the average polymorphism information content was 0.6308 and 0.5714. Population structure of Yangtze River is better than that of Heilongjiang River. Hardy-Weinberg deviate index of the two populations were small, indicating that the populations was equilibrium. Amplified results in HLJE1 and HLJE10 were very different between Yangtze River and Heilongjiang River, and polymorphism in Yangtze River was much higher than in Heilongjiang River. The two loci may be used for common carp population identification of Yangtze River and Heilongjiang River.

Several Phytophthora infestans-susceptible mutants had been obtained based on the different reactions of Arabidopsis activated tagging mutants after being inoculated by the fungus, and flanking sequence of Arabidopsis T-DNA insertion had also been obtained by TAIL-PCR technology and genomic DNA from disease-susceptible mutants as template. Some disease resistant-related genes had been found using NCBI sequence homology analysis and AD1,AD2 and AD4 were the most efficient arbitary primers. Two mutants had been found with T-DNA insertion through sequencing and analyzing eight specific TAIL-PCR products.

Three apple samples were collected from Xinjiang province with rugose symptoms. The low molecular weight RNAs from three samples were characterized by Return-PAGE. The results showed that two of samples were infected by viroid. Then, it was confirmed to be ASSVd by reverse transcription-polymerase chain reaction (RT-PCR) using two pairs of primers designed according to the reported sequence of ASSVd in GenBank, the amplified products were cloned into pGEM-T and sequenced. Two sequences variants were registered in GenBank DQ362906, DQ362907, The result shows that ASSVd is a quisispecies which contain many very similar but different variants with high sequence homology.

In order to cloning the tandem inhibin gene, the two different primers were desigened according to the Genbank sequence. p1INH and p2INH were Tandem through XbaⅠ. Doubled gene encoding porcine inhibinα(1～32) was cloned and recombined with pcDNA3.1 expression vector to construct inhibin gene vaccine pcINH. The recombinant plasmid pcINH was expressed in E. coli. DH5α.The expression protein had inhibin immunization activity and has no homology with other protein in the body.

In this study，ISSR conditions on pine caterpillars were optimizatied.The factors which affected the ISSR results of pine caterpillars were studied.The results showed that 2.5mmol.L-1 Mg2+，0.8μmolL-1 primer, 0.20mmol. L-1dNTP，1.25ＵTag polymerase，15～30 ng template DNA and the optimal annealing temperature of primers of 45.9～52.4℃ in 25μL ISSR reaction system were the best. Though above PCR system，ISSR fragments of 14 populations from different district of 4 species were obtained and detected.Polymorphism between different populations was abundantly detected by 1.5％ arognose which showed this system was suitable and stable.

To insight into the characteristic of its endosperm-specific promoting regulation activity, –2510- -1bp sequence of the wheat pbf (prolamin-box binding factor) promoter was cloned by Polymerase Chain Reaction (PCR) amplification and a series of pbf deletion fragments were fused with the β-glucuronidase (GUS) reporter gene (UidA) and 3΄ non-coding region of the nopaline synthase gene (nos) in pCAMBIA1381z vector. And these fusions of pbf deletion had been transformed into wheat endosperm in vitro and GUS transient expressions regulated by pbf promoter deletions had been assayed subsequently. Results of GUS transient assay in wheat endosperm and Loss of function displayed that –2510- -1bp pbf sequence could show promoting activity in endosperm, however, 5’ and 3’ deletions of –2510- -670bp, –1288- -1bp sequence resulted in undetectable promoter activity; inner deletion of –2160- -689bp evidently decreased the GUS expression activity. Compared with levels of GUS expression of the Cauliflower Mosaic Virus (CaMV) 35S promoter used as a control, the full sequence of pbf promoter had lower activity. In this article, the number and sorts of main motifs in pbf promoter sequence had been described. It was suggested that the presence of a Skn-1-like motif (G/ATCAT) and a –300bp element (TGHAAARK) in the promoter sequence might be important and essential for expressing pbf promoter activity and specificity. Effects of wheat endosperm developing state and GUS-transient assay system on promoter activity assay had been discussed also.

Tail sequence is a primer sequence which is not belong to maize genome and can be widely applied in maize SSR primers. A lot of common maize SSR primers can be used in fluorescence detection system just by labeling 5` end of the tail sequence with fluorescence, then high-through and low cost detection could be achieved at the same time. A method of designing tail sequence was discussed, including design principle, design process, universality and PCR conditions.

After the accomplishment of the sequence of multiple plant genomes a major effort was started to elucidate the biological function of all open reading frames of these organisms by targeted gene replacement. At the same time, gene targeting which can avoid the deficiency of plant gene transform is a powerful tool for plant genetic modification. Although it is very widely used in yeast and mouse embryonic stem cell, gene targeting is inefficient in plant because of low homologous recombination frequency. Recently, zinc finger nuclease becomes a focus in bioscience because it can stimulate high efficient homologous recombination. Chimeric oligonucleotides, which can stimulate single base replacement, can be a useful tool for plant breeding. The finding that modulating the function of some plant genes can produce plants with a hyper-recombination phenotype bring a hope to the final use of gene targeting technique. It can be forecasted that the set up of efficient gene targeting technique can make an evolutionary progress in both molecular biology and plant breeding science.