The Cloning Of The Genes Associated With Fertility Transformation Of Thermo-sensitive Genic Male Sterile Rice And The Construction Of RNAi Plant Expression Vector

OBJECTIVE: The aim of the research is to isolate the genes associated with fertility transformation of thermo-sensitive genie male sterile rice. METHODS: Two-directional (forward and backward) Suppression Subtractive Hybridization (SSH) was performed on cDNAs of fertile young panicle and sterile young panicle of thermo-sensitive genie male sterile rice (Tb7S), and two Subtracted cDNA libraries were established with the forward and the backward subtracted cDNAs, positive clones from each subtracted cDNA libraries were selected for sequencing and BLAST analysis, the differential expression of 10 clones between fertile young panicle and sterile young panicle of Tb7S were analysed by RT-PCR; RACE technique was used to amplify the 5' cDNA fragment and 3' cDNA fragment of S2; The plant expression vector of pCAMBIA1301-P-A-S was constructed. In pCAMBIA1301-P-A-S, gene-specific sequences in the antisense (A) and sense (s) orientations were linked with a 1300-bp of the GUS gene and controlled by ubiquitin promoter, the 400-bp gene-specific sequences in the antisense (A) and sense (s) orientations were inserted in the Sal IIIBgl II and Pml l/Bst Ell sites, the ubiquitin promoter was inserted in the EcoR HSma I site, the callus from tissue cultured young embryo were used as material in agrobacterium-mediated genetic transformation.RESULT: The experiment of subtractive efficiency indicated that the subtractive efficiency is high, and the plasmid digestion by EcoR I indicated that all the positive clones picked randomly contained cDNA fragments of 200-600bp. The sequenceanalysis demonstrated that picked randomly 74 clones represented 61 different genes, which showed the repeat copies of subtracted population of cDNA fragments were few and the abundance between different genes was normalized. Sequencing and BLAST homology search revealed that 17 clones contain sequences of known gene fragments and 44 possibe novel genes show no sequence homologies with any know sequences in the database. 4 clones of 10 clones picked randomly were identified as differentially expressed genes by RT-PCR, among which 2 clones were expressed specifically in the fertile young panicle of Tb7S and the other 2 clones were expressed specifically in the sterile young panicle of Tb7S. A novel full-length of S2 cDNA containing 2976bp was obtained by RACE technique, which contain the ORF of 2367bp, 5'-UTR of 75bp and 3'-UTR of 534bp. The results of 3DPSSM Protein prediction and protein domain prediction indicated that it might be a regulatory factor involved in signal transduction;. The result of PSORT (Prediction of Protein Localization Sites) indicates that the protein encoded by S2 has a mitochondrial targeting peptide. Southern Blotting showed that the S2 gene is a single cope gene.The plasmid digestion showed that the 400-bp gene-specific sequences in the antisense (A) and sense (s) orientations were inserted in the Sal 11/Bgl II and Pml l/Bst Ell sites, the ubiquitiin promoter was inserted in the EcoR l/Sma I site of the improved pCAMBIA1301. CONCLUSION: (1) All results confirmed the effectiveness and sensitivity of SSH;(2) S2 gene is a single copy gene; (3) S2 gene is expressed specifically in the sterile young panicle of Tb7S ,which suggest that S2 gene might be a novel gene associated with fertility transformation of thermo-sensitive genie male sterile rice;(3) The fact that the protein encoded by S2 cDNA has a mitochondrial targeting peptide suggests that there might be relationship between the two-line rice and three-line rice; (4) The RNAi plant expression vector(pCAMBIA1301A-P-A-S) was constructed successfully. In the pCAMBIA1301A-P-A-S, the gene-specific sequences in the antisense (A) and sense (s) orientations were linked with a fragment of the GUS gene and controlled by the ubiquitin promoter.