Rice Chloroplast Form Rnai Carrier Construction Of The Related Gene And Genetic Transformation Research

Rice (Oryza Sativa) is one of the main staples for human. The chloroplast is an important photosynthesis place for rice plant. Thus, the chloroplast development is one of the key factors for the yield of rice. However, the molecular regulation mechanism of chloroplast development in rice is largely unknown. In this study, we report that the gene OsDCL1encodes a protein sharing a conservative functional domain in the C-terminus of DCL (Defective Chloroplasts and leaves) reported in tomato and Arabidopsis. Previous studies reported that the content and distribution of auxin affect on rice morphogenesis significantly. As one of the major natural active form of auxin, indole-3-acetic acid (IAA) is able to be biosynthesized through two pathways which are dependent or independent on tryptophan. It was reported that indole-3-glycerol phosphate synthase (IGS) played important role for Arabidopsis development. We found three rice IGSs those are homologous to IGS, encoding proteins predicted to be the precursors of chloroplast biogenesis in Arabidopsis. To study the molecular regulating mechanisms of these genes during the development of chloroplast in rice, we applied RNA interference approach to suppress the expression of these specific genes without changing the genetic background of rice. Main results obtained are listed as follows:I, Based on the published sequences of OsDCL1and OsIGSs on the Gramene website, primers were designed to amplify corresponding target fragments to build the RNAi expression vectors of OsDCL1and OsIGSs:pCAMBIA301-OsDCI1-RNAi, pANDA-Os/GS1-RNAi, pANDA-Os1GS2-RNAi, pANDA-OsIGS3-RNAi and pANDA-OsIGS123-RNAi, respectively.II, Using Agrobacterium-mediated transformation approach, we introduced OsDCL1-RNAi into Kasanasa to repress the expression of the gene OsDCL1and OsIGS1-RNAi, OsIGS2-RNAi, OsIGS3-RNAi and OsIGS123-RNAi, respectively, into Kitaake to repress the expression of their respective genes. After Hygromycin resistance selection and calli differentiation, their respective transgenic plants were obtained.III, After PCR-based genotyping of hygrymicine gene which is surposedly co-integrated with the RNAi-fragment into the receptive rice, we obtained eight positive RNAi transgenic lines of OsDCLl, nine positive RNAi transgenic lines of OsIGSl, two positive RNAi transgenic lines of OsIGS2, three positive RNAi transgenic lines of OsIGS3and two positive RNAi lines of OsIGSl23, respectively.IV, By observing the phenotype of the positive transgenic plants (transgenic generation0, TO), we found there was chlorisis at the tip of leaves in the positive RNAi transgenic lines of OsDCLl, and the whole leaves became yellow in positive RNAi lines transformed with OsIGS1-RNAi. The results of qPCR showed that the expression of OsDCLl decreased significantly in the positive RNAi trangenic lines (TO) compared with wild type (Kasanasa).V, By analyzing the leaf yellow phenotype and genotype of T1plants derived from the positive OsDCL1-KNAi transgenic lines, we found that the leaf yellow phenotype of the T1plants is co-segregated with the genotype of OsDCL1-RNAi. Thus, these results suggest that the gene OsDCLl plays a key role in chloroplast biogenesis during the plant development of rice.