Functional Analysis Of Rice HL-CMS Candidate Gene Orf216 And Tobacco Glycosyltransferase Gene Sm-Ngt1

Honglian cytoplasmic male sterility (HL-CMS) is one of the three major CMS types in rice (Oryza sativa L.) used in commercial production of hybrid rice. There are two described candidate genes, orfH79 and orf216, involved in HL-CMS, but their sterile-induced mechanisms are still unknown.Glycosyltransferases (GTs; EC 2.4.xy) are involved in the biosynthesis and modification of many substances, and it plays a critical role in the construction of organism cell, cell metabolism and regulation of life activities. In our laboratory, a new tobacco glycosyltransferase gene, sm-Ngt1, has been cloned. It is demonstrated that its expression could be dually induced by methyl jasmonate (MeJA) and salicylic acid (SA), but its physiological function in vivo is still unclear.In this study, the transgenic plants with over expression of orf216 and mx1, and with RNAi of sm-Ngt1 are used, and functional analysis of orf216 and sm-Ngt1 were carried out.1. To achieve a good embryo callus of indica rice YTB for the specific transgenic use in this study, the conditions for embryo callus inducing and subculturing of indica rice YTB were first optimized by analysis and regulation of content of endogenous polyamines. The results showed that the changes in endogenous polyamines content in the embryo callus of YTB could be caused by exogenous putrescine during the process of callus inducing and subculturing. Exogenous putrescine also had a strong impact on the mRNA expression levels of two key polyamine biosynthetic enzyme genes, adc and samdc, respectively. Thereby, it could effectively improve the growing state of YTB callus and enhance the callus embryogenic traits.2. The GTpB/GTpC∷Rf1b5’∷orf216 expression vector for genetic transformation was constructed, and a lot of positive transgenic plants with different degrees of pollen sterility were obtained using the HL-CMS maintenance line YTB as an acceptor. It is proved that overexpression of orf216 gene could cause sterility of HL-CMS maintainance line YTB.3. The CaMV35S∷Rf1b5’∷mxI over expression vector for genetic transformation was constructed and ten positive transgenic plants with parts of fertile pollens and seed-set after self-pollination were obtained using HL-CMS sterile line YTA as an acceptor. It is demonstrated that overexpression of mxI gene could partially restore fertility of HL-CMS sterile line YTA.4. The sm-Ngt1-RNAi vector was constructed by method of Gateway technology and eight survival tobacco W38 transgenic plants were achieved by Agrobacterium-mediated plant transformation. Based on the phenotypes observed in T0 transgenic plants, it is suggested that RNAi of sm-Ngt1 gene could obviously result in shortened internodes, curled leaf edges and retarded growth of tobacco plants.