Construct Of OsXDH Expression Vector And Identification Of Transgenic Rice

Rice is one of the most important food crops in the world. Half of global population lives on rice. However, world population continues to increase at an explosive rate, our arable land is deterorating. In normal agricultural production, premature senescence of rice leaf has become the important restricted factors influencing the yield and grain quality of rice. Xanthine dehydrogenase (XDH), as a member of the molybdenum enzymes, plays an important role in purine metabolism, catabolizes xanthine and hypoxanthine to ureides, and involved in nitrogen assimilation, hormone homeostasis, reactive oxygen species production, and articipate in the regulation of leaf senescence. In this study, xanthine dehydrogenase gene was isolated from rice leaves, and preliminary studies on the mechanism of delaying the senescence of rice. The results are as followed:(1) Based on the OsXDH structure and XDH homology analysis, the result display that, OsXDH was a single copy gene, composed of14exons and13introns. The full-length of OsXDH cDNA is4485bp, contained an ORF of4110bp, and the5’UTR and3’UTR are130bp and245bp separately. A peptide of1369amino acid residues and150KDa protein coding sequences was deduced according to the ORF, The leader sequence have43amino acid of N terminal. About70%homologous amino acid sequences of OsXDH protein encoding amino acid sequences with castor, Arabidopsis thaliana, multiple alignment of amino acid sequence showed that the XDH relationship of rice and Arabidopsis is very close.(2)Overexpression(OE) and interference(Ri) vector of OsXDH were constructed, and introduced into rice cv. Nipponbare by Agrobacterium mediated transformation, get the expression and the interference transgenic lines, the phenotype of wild type(WT), overexpression and interference lines, chlorophyll content, OsXDH gene and protein expression levels were analyzed. The results showed that the OE plants were better than WT and Ri lines in height, leaves greener, chlorophyll content, the performance of the aging delay. But XDH interference lines showed small in height, yellow leaves, lower chlorophyll content, and premature senescence.(3) To observe the changes of OsXDH mRNA transcript abundance in rice seedling root, stem and,leaf, the method semi quantitative RT-PCR and real-time PCR expression were used. The results showed that the OsXDH gene is constitutively expressed in rice. But the expression of the OsXDH gene in rice at different growth stages have different levels, the amount in seedling stage and tillering stage was lower, the highest in filling stage. The expression of XDH upstream and downstream gene(UO, ALN, AAH) and senescence-associated genes(SGN2, WRKY23, WRKY53, GH27, Pse (t), ACD1, XERO1) was analyzed by real-time PCR. The results showed that UO expression was high and no significantly different in WT and OE, senescence-associated gene expression level increased significantly in Ri. ALN and AAH in Ri with high expression, WT is less, OE is lowest. Compared with WT, senescence associated gene expression levels is increased significantly in OE and decreased in Ri. Constructed a vector of fusion promoter and reporter gene GUS expression, by chemical staining in transgenic plants, are found in GUS gene expression in transgenic plants in root and shoot tip, The root GUS differentially expressed, the root cap has the highest expression level, meristematic zone is less, elongation zone expression is the lowest.(4) The XDH activity of the germplasm at seedling stage in the leaves was determined from Jiangxi, Guizhou and Guangxi local variety. The study found, the expression quantity of GYN was the highest, R3027, R66, R402, JKB, R838, XF70A, R644, CDMG, R4277,42203, HGN, MN, DLM, JKB for the expression of XDH rice varieties less.