Abstract: The 1414bp 5’ flanking sequence of Ha ds10 G1 gene, a late embryogenesis abundant gene of Helianthus annuus L., was cloned by PCR. The similarity compared with the reported sequence was 100%. The promoter was fused to the glucuronidase gene_to construct plant expression vector, which was transferred into tobacco NC89 by Agrobacterium tumefaciens-mediated method. PCR results showed that the promoter had been integrated into genomic DNA of tobacco successfully. GUS activity assays indicated that expression of GUS was active only in transgenic tobacco seeds. However, the GUS activity didn’t exist in the stem and leaves. So, Ha ds10 G1 promoter is seed-specific.

Abstract: Fifty-three strains of Staphylococcus. aureus were isolated and identified from 93 milk samples of dairy cows suffering from mastitis, it’s the main pathogen in the three cow fields in Nanjing region. From the isolates six typical strains were selected to analysis with the biochemical characters and the inherited stability. Furthermore the other pathogenic factors of the six isolates were detected, such as coagulase, hemolysin and antimicrobial resistance factors etc. The crushed bacterial cells of 6 strains and 1 reference strain were processed with SDS-PAGE. The protein maps of the strains are similar except for the higher protein density of TQ-1. Mixture of whole bacterial cells of four S. aureus isolates(XL-1、TQ-1、XG-2 andT Q-2) were chosen to inoculate rabbit, and the hyperimmunized serum was used in the Western-blot assay to analyze the cross protective antigens of the isolates. The result shows the isolate XG-2 has unique band whose molecular mass is about 52KD in the NC membrane. According to the results, 2 S. aureus isolates were selected as the strains for preparing vaccine against bovine mastitis.

An immuno-capture PCR (IC-PCR) method for detection of Acidovorax avenae subsp. citrulli, a pathogen of bacterial fruit blotch of watermelon, has been established by combination of the ISE method with classical PCR and compared with the direct PCR method and growth-checking method. The results showed that all the strains of A. avenae subsp. citrulli tested produced 360bp specific fragments by the IC-PCR and direct PCR method, while others strains of 10 different genera showed negative PCR result. The minimum detection concentration for the IC-PCR and direct PCR method was about 50-102cfu/ml and 104cfu/ml, respectively. The sensitivity of the former is 100 times higher than that of later. Detection of 7 batches of different melon seeds from the markets by IC-PCR showed that 1 cantaloupe variety, 2 honey melon varieties and 2 watermelon varieties of seeds carried the pathogen which almost matched with the result of the growth-checking of the melon seeds. It indicates that the IC-PCR is accurate, sensitive, rapid and low cost.

The cDNA encoding the ubiquitin-53aa extension protein(ubi-53,UBE) was isolated from the fat of Helicoverpa assulta larvae by reverse transcription polymerase chain reaction (RT-PCR). The UBE in H. assulta was 390bp in length, encoded a peptide of 129 amino acid residues, in which there was an ubiquitin fused with a ribosomal L40 protein, the predicted MW was 14.8 kDa. The deduced amino acid sequence has a high identity (90%-98%) with the reported sequence of UBE from other eukaryotic species and 69% with H. armigera single nucleopolyhedrovirus (HaSNPV) ubiquitin. The fragment containing UBE gene was inserted into pGEX-4T-2 expressive vector, and the expression was induced by IPTG in E. coli BL21(DE3). It’s molecular weight was about 40 kDa, by checking with SDS polyacrylamide gel electrophoresis and Western blotting using a mouse monoclonal antibody against GST.

Abstract：Differential Display Reverse Transcript Polymerize Chain Reaction (DDRT—PCR)was applied to study gene expression in submergence-tolerant rice FR13A and submergence sensitive rice IR39595-503-2-1-2 under submergence stress. The results showed that 1428 bands were amplified from 40 pairs of primers in the two varieties, 102 of them appear different obviously between the two varieties and which accounted for 7.1%, 42 of 102 bands which came from submergence-tolerant variety. The northern blot results showed that 7 fragments of the 42 were confirmed to express in submergence tolerant variety FR13A. Those fragments were then cloned into vectors for sequencing．Sequence analysis through Internet Blast searching showed that 4 of them related to response and biochemical changes under water logging, one of them showed high homogeneity to ATP binding protein,3 of them showed high homogeneity to partial sequence of Isocitrate dehydrogenase, NADH2 dehydrogenase and Terminal acetyltransferase .the other 3 fragments ware novel cDNA fragment.

Hybrid soybean is one of the most significant scientific achievements that Chinese scientists are holding the intellectual property. While the invention improved the soybean yield greatly, the difficulty to eliminate the maintain-line seeds from the cytoplasm male sterile line seeds is often a problem limiting the application of the new technology. To solve the problem, we initiated the research. The purpose of the research is to insert a selectable marker gene into the cytoplasmic genome of the male sterile line through chloroplast transformation. Based on the published soybean chloroplast genome sequence (GenBank X07675), two pairs of primers were synthesized to amplify two adjacent soybean plastome sequences allowing its targeted insertion between the trnV gene and the rps12/7 operon of soybean plastid. Within the targeting region, an aadA gene encoding the Aminoglycoside Adenylyltransferase was inserted . The gene was flanked by the rrn promoter and the 3’ sequence of psbA gene. The constructed vector was designated as pJY01. Expression cassettes of bar gene and GFP-GUS fusion gene were then inserted into the pJY01 vector to construct two new vectors: pJY03 and pJY04, which we successfully transformed in the tobacco chloroplast. We detected the expression of the aadA gene by PCR amplification and found that the transformed seedlings conferred green fluorescent activity. This research build a solid base for the future soybean chloroplast transformation.

A complete chitin deacetylase (CDA) cDNA from Mucor racemonus was cloned and sequenced by using RT-PCR and RACE with special conserved primers. The cDNA sequence was submitted to GeneBank (DQ538514). The results showed: (1) The complete gene with length of 1506bp contains 67bp 5’-untranslated region, an open reading frame of 1357bp and 82bp 3’-untranslated region including tailing site AATAAA. The gene encodes a sequence of 448 amino acid residues and consists of core nucleotides encoding a polysaccharide deacetylase domain which covering 32 of the entire sequence. (2) The CDA gene shares high sequence similarity with that of fungi including Rhizopus oryzae (75%), CDA1(58%) and CDA2(56%) of Rhizopus circinans, Mucor rouxii(56%), Gongreonella bulteri(48%), Rhizopus stolonifer(39%), Phycomyces blakesleeanus(39%), CDA1(17%) and CDA2(16%) of Saccharomyces cerevisiae. The corresponding homology of the deduced amino acid sequences was 69%, 57%, 59%, 55%, 47%, 30%, 32%, 18% and 21%, respectively. (3) Phylogenetic analysis according to the deduced amino acid sequences was marched with the classical taxonomic classification of the fungi. (4) The 3-D-structure of this protein was predicted. The protein has a whole CDA functional domain and a polysaccharide deacetylase domain.