Molecular Characterization Of RIX4, A Rice Homologue Of Yeast RAD21

The differential expressed cDNA fragments were obtained by DD-PCR from IR26 callus induced by Xanthomonas oryzae pv. oyzae;(Xoo) in previous work.. Blastn searching showed it was a novel gene then named RIX4. This work was designed to study further the splicing pattern of this gene, the relationship between them, expression characteristics and the function.The full-length ORF sequence of RIX4 gene was 2,187 bp long, encoding a 728 amino acid residues presumed protein, with MW 80.35KD, pI 4.65. The genomic DNA of RIX4 gene has about 5.3 kb. Comparison analysis of the sequences between the genomic DNA and cDNA showed that this gene has 11 exons and 10 introns, and all of the 10 introns have the /GT-AG/ consensus characteristics.The RIX4 protein has an entire N-terminal domain Pfam04825 and a C-terminal domain Pfam04824 which are conserved in previously identified RAD21/REC8 proteins. It was evidenced that RIX4 protein belongs to RAD21/REC8 cohesion protein. The secondary structure prediction of RIX4 was conducted on websites. The result showed that RIX4 consisted of 21.98% alpha helix, 7.01% extended strand and 71.02% random coil. The random coil.as the most abundant structure, penetrated through the RIX4 protein, while the alpha helix and extended strands relatively gathered to form three obvious helix/strand groups.Another novel transcript RIX4-5 was obtained from young panicles in both sterile lines and maintainer lines when the distance between the pulvinus of the flag leaf and the pulvinus of the second leaf was zero. Analysis of the sequences and splicing patterns of all the 5 RIX4 transcripts was conducted. The results showed that the alternative splicing patterns in exon 6 and 7 are identical with the typical pattern, namely, alternative splicing on the different 5'or 3' site. But the alternative splicing patterns in exon 6 and 9 are novel. The study showed that RIX4-1 transcript was produced in leaves inoculation with Magneporthe grisea(M. grisea) and RJX4-4 transcript was predominant in callus inoculation with Xoo.The fragment encoding the polypeptide of RIX4 protein N termius was cloned into pET-28a and expressed in the strain BL21 of E.coli. After induction by IPTG at 37℃,the expressed protein was purified by Ni~2+-NTA agrose column. The antiserum was raised by immunizing New Zealand rabbit and used to analysize the content of RIX4 protein in plants. The full length protein can be detected in the IR26 callus induced by pathogen Xoo. during 0~60 min, and the content increased with induction time. The RIX4 protein can also be detected in the youngpanicles of both sterile lines and maintainer lines when the distance between the pulvinus of the flag leaf and the pulvinus of the second leaf was zero, and the content in maintainer lines was higher than in the former.But protein encoded by other transcripts was not detected in all the situations above. In the leaves of rice near isogenic line (NIL) H7R and H7S induced by M. grisea isolate ZB15 (2001-046) and the leaves of varieties Jaxing 02-03 and Zhongzao 27 by isolate 01-14, the content of RIX4 full-length protein varied along with the induction time, but the variation was not identical. Furthermore, both full length protein and protein encoded by RIX4-4 transcript can be detected in all leaves induced by M. grisea. Thus, our result evidenced that the RIX4 gene had different transcripts in the protein level.RNAi expression vectors were constructed and transformed into Zhonghua 11 through shotgun. 9 (pCAMBIA-HANNIBAL-iUX^l)) and 12 (pCAMBIA-HANNIBAL-iUX4(397)) transgenic plants were obtained, respectively. Identification of resistance of sense and antisense transgenic plants to Xoo showed that the RIX4 gene conferred rice more effective resistance to the pathogen.Peiai 64S is a photoperiod- and thermo- sensitive genie male sterile (PTSGMS) line of rice, which is male sterile under long day/high temperature and partial fertile under short day/low temperature. A cDNA array representing 3,328 unique rice genes was used to profile the gene expression patterns in the young panicles of Peiai 64S under these two conditions. The statistical data showed that up to 14.60% of genes exhibited up- or down-regulated expressions in the plants under long day/high temperature compared with plants under short day/low temperature, but the expression abudance of RIX4 gene was not differential. Only four genes were up-regulated while 482 genes down-regulated. Real-time PCR with all the up-regulated and 9 randomly selected down-regulated genes confirmed the differential expressions detected by the array, indicating that the constructed cDNA array is reliable. These differently expressed genes participated in almost all cell biological responses. Analysis of up- and down-regulated genes revealed distinctive changes between the mRNA abundances of MMK1 and MMK.2, both of which are analogs of MAPK, and significant down-regulation of several transcription factors. It is proposed that changes occurred in the MAPK signal transduction pathway might disturb the transcription control necessary for morphogenesis of pollens and corresponding physiological functions.