Cloning Of HL-CMS Associated Candidate Gene Orf216 In Rice And Functional Analysis Of Rice Blast Resistance Gene Pi36

Rice (Oryza sativa L.) is one of the most important crops for human consumption. A variety of hybrid rice cultivars derived from cytoplasmic male sterility (CMS) have largely increased rice yield. However, at present, the increase of human population and decrease of plantation result in the problem of food supplies worldwide. Furthermore, biostress from pathogeny and pest, such as rice blast, continues to be a potentially devastating disease of rice, affecting yield and decreasing its quality. Thus, increasing the yields in unit area by genetic improvement is the main approach to solve the food supply problem.HL-CMS/Rf system is one type of CMS systems of rice. The chimeric mitochondrial genome-associated candidate gene for HL-CMS, named as HL-sp1, was previously identified. In order to isolate the gene, analysis of HL-sp1 flanking sequence by TAIL-PCR and genetic transformation, mediated through Agrobacterium tumefaciems EHA105, were carried out. At the same time, to understand the molecular mechanism of Pi36 gene against rice blast, the technology of RNAi and subcellular localization were used to verify its function for rice blast resistance. The main results were as follows:1. Analysis of HL-sp1 flanking sequences and gene predictionA 6740 bp HL-sp1 sequence, extended 2009 bp and 2555 bp from the 5'- and 3'-end of the original HL-sp1 fragment, respectively, was obtained through the single primer PCR and improved TAIL-PCR. The 6740 bp sequence was then analyzed by Blast and alignment. In HL-sp1, there was an uncompleted mitochondrial atp6 gene with deletion of 1772 bp of at the 5'end; There were only a little homologous at the 5'end about 1700 bp of HL-sp1 with mitochondrial DNA (mtDNA) of Indica rice cultivar 9311, and a partial homologous with the mtDNA of maize T-CMS and C-CMS; The 2555 bp flanking sequence of 3'-end is highly homologous to 9311 mtDNA.Two gene prediction systems, RiceGAAS (http://ricegaas.dna.affrc.go.jp) and Softberry FGENESH (http://www.softberry.com), were used to predict if there are any candidate genes in the 6740 bp HL-sp1. The results showed that there were two ORFs in HL-sp1 sequence. The first one, composed of 4 exons with 171 bp, coded an unknown protein. And another one was a chimeric gene, named as orf216, tentatively, composed of 651 bp without intron, coding an unknown protein with 216 amino acid residues. 2. Analysis of the cytoplasm specificity and expression of candidate gene orf216 The DNAs isolated from the sterile lines, maintainer lines, restorer lines and the hybrid of HL-CMS/Rf, BT-CMS/Rf and WA-CMS/Rf systems were used for analyzing cytoplasm specificity with the special primer from the candidate gene orf216. The result showed that it existed only in YTA and HL-2 with HL-CMS cytoplasm. RT-PCR analysis suggested that the orf216 be constitutively expressed.3. Genetic transformation of HL-CMS associated candidate gene orf216To dissect the function of orf216 in HL-CMS, a fusion protein expression vector was constructed, using the binary vector pCAMBIA1305.1 to combine orf216 with a mitochondrial orientated signal peptide sequence from BT-CMS fertility restorer gene Rf1b. Genetic transformation was carried out by the Agrobacterium-mediated transformation system, using the maintainer line YTB as the acceptor, which is homokaryon with HL-CMS line YTA, and transgenic plants were obtained. The T0 generation was confirmed by PCR, using the selective markers from Hpt gene as well as candidate gene. The results showed that the foreign fragment carried by the construct had been integrated into YTB genome.4. Construction of Pi36 RNAi recombined vector and its genetic transformationTo further identify the function of the resistance gene Pi36, the fragments from 5'-end, 3'-end and NBS region of Pi36 were cloned by using gene-silencing vector pCAMBIA1300RS, which were named as pI1, pI2 and pI3, respectively. The confirmed constructs were subsequently transformed into the donor cultivar Q61 of Pi36 by Agrobacterium-mediated transformation system. And transgenic plants corresponding to the candidate constructs pI1, pI2 and pI3 were obtained, respectively, and confirmed by PCR analysis with the selection marker gene Hpt. Expression of Pi36 in transgenic plants was estimated through real-time quantitative PCR. The results showed that the expression of Pi36 in the selected transgenic plants was decreased, especially in pI1 transgenic plants.5. Construction of Pi36 subcellular localization vector and genetic transformationTo better know the function and molecular mechanism of host-pathogen interaction, N-terminal sequence of Pi36 protein was fused with GFP, and the subcellular localization vector was constructed by utilizing pCAMBIA1302. The transient expression analysis with onion cuticle system showed that the fused protein localized in cell nucleus, which was consistent with the observation in the root tip of stable hereditary transgenic plants obtained by Agrobacterium-mediated transformation system.