Functional Analysis Of Plant Asparagine Synthetase Gene In Regulation Of Disease Resistance

Plant disease resistance is an extremely complicated biological process. Cloning and further functional analysis of genes involved in regulation of plant resistance will provide great insights into the molecular mechanism of this process. Asparagine synthetases (ASs) (EC 6.3.5.4) are encoded by a small gene family. It is a kind of amino transferase to use ammonia or glutamine as the substrate to biosynthesize the asparagine, existing widely in prokaryotic and eukaryotic organisms. It can enhance translocation of nitrogen from source tissues to stock tissues, adjusting the nitrogen metabolism to participate in regulation of various biological processes. However, the function of plant ASs in regulation of disease resistance remains unclear. This research focuses on cloning of AS genes from tomato and rice, and analyzing the its function in regulation of disease resistance employing virus-induced gene silencing (VIGS) and transgenic (overexpressing and RNAi) technique. Results are summarized as follows:1 Cloning of SlAS and OsAS genes: AS genes were obtained by PCR amplification from cDNA of tomato and rice with Taq enzyme, using four pairs of primers designed according to the published nucleotide sequences. The obtained PCR products were SlAS1 of 1680bp, OsAS1 of 1776bp, OsAS3 of 1815bp and OsAS4 of 1512bp, respectively, which were subcloned into T-vector and verified by DNA sequencing.2 Functional analysis of AS genes in resistance:Role of the AS genes in regulation of plant disease resistance was analyzed employing VIGS and transient overexpression technique. Compared to control plants, those were VIGS-treated for AS genes, respectively, did not show or only showed much less severe Cf-4/Avr4-dependent and Xoo-induced hypersensitive response (HR). Futhermore, compared to control plants, those were transient overexpression-treated for AS genes shown more severe HR symptoms. These results demonstrate that AS genes might be involved in positive regulation of Cf-4/Avr4-dependent HR and nonhost resistance to Xoo.3 Construction of the tomato and rice over-expression and RNAi constructs: Considering that tomato is dicotyledonous while rice is monocotyledonous,35S and Ubi promoters were chosed to control AS gene expression in tomato and rice, respectively. As for vectors, pCHF3 expressing kanamycin-resistance in plant and pC1301-35S expressing hygromycin-resistance in plant were used for over-expressing and RNAi analysis in tomato; while pCZD and pANDA both expressing hygromycin-resistance in plant were used for over-expressing and RNAi analysis in rice. Constructs pCHF3-SlAS1 and pC1301-35S-SlAS1, pCZD-OsAS1,3,4 and pANDA-OsAS1,3,4 and pANDA-OsAS were made for study on overexpression and RNAi in tomato and rice, respectively for a single AS gene or a gene family (OsAS). These binary over-expression and RNAi constructs were transformed into Agrobacterium strain EHA105 by electroporation. Positive transformants were identified through PCR, and were then used for transformation of tomato and rice.4 Rice tranceformation and obtaining of transgenic rice plants:The binary over-expression and RNAi vectors harbouring OsAS gene were transformed into rice by Agrobacterium-mediated method via rice calli. A total 89 hygromycin-resistance plants were obtained, including 28,11 and 14 over-expression lines of OsAS1,3,4,,and 7,12,14 and 2 RNAi lines of OsAS1,3,4 and OsAS, respectively. Most of the generated T1 transgenic plants were positive according to detection analysis on hygromycin resistance tests.5 Effect of OsAS genes on rice agronomic traits: Most of the transgenic plants grown nomallly. The AS-over-expressing plants developed and grown better than AS-RNAi plants, with worst in the AS-family-RNAi plants.