| Three differential expressed cDNA fragments (B1, B2, B3) were obtained by DD-PCR in rice callus mRNA of IR26 (resistance to Xanthomonas oryzae pv. oyzae; Xoo) and jingang30 (susceptible to Xoo) in previous work.. Sequence identity among them were 70-80%. It was confirmed by Northern that their expression were induced by Xoo. BLAST searching showed they are novel genes. It was proposed that the mentioned three cDNA fragments might be different splicing pattern of a same gene, R1X4.This work was designed to study further the splicing pattern of this gene, the relationship among them, expression characteristics and the function.3' RACE and 5' RACE were carried out Result showed that the ORF of RIX4 gene has 1530 bp, and the kozak frame confirm the ORF. Another transcripts, RIX4-4I was cloned by M-EST microarray. Based on 3' RACE, some other transcripts were obtained and sequence analysis were on studing. RIX4 DNA were cloned from rice IR26. Expression of RIX4 gene was analysed using RT-PCR. Expression abundance in IR26 was similar to that in jingang30. But in IR26, the expression was induced by Xoo mightily. RIX4 gene might be related to rice resistance to Xoo.The sequence of RDC4 gene and the transcripts were analysed. Result showed that this gene has six introns and seven exons, and was 266Ibp long. All the intron has the /GT-AG/ consensus characteristics except for the forth intron. Bl, B2, B3 were the different transcripts of the same gene produced by alternative splicing. ORF of the transcript Bl has 1530bp that had all the six introns of this gene excised; B2 was the same with Bl but the first alternative splicing component was cleavaged, and with ORF of 306 bp; B3 was the tanscript has all the two alternative splicing component cleavaged, and with ORF of 222bp. CAAG at me splicing site were the same characteristic of these two alternative splicing components. Transcripts RIX4-4I had five of the six intron excised, and retained the forth intron which has % base pair.Bioinfomatics analysis showed that R1X4 was a soluable protein, pI4.6, localized in nucleus. There were N-glycosylation site, Protein kinase C phosphorylation site, Casein kinase II phosphorylation site, N-myristoylation site, Amidation site, Tyrosine sulfation site, Bipartite nuclear location site, low complexity, Pfam-B2301 and "VQDEMNAQP" four times repeats in mis protein.Search in the internet database showed that Rî–ž4 protein was partial identity with Arabidopsis RAD21 family and has the conservative Pfam-B2301 domain in the end. In addition, RDC4 was localized in nucleu. So, It was proposed that RIX4 gene was one of the RAD21 gene family in rice and play a important role in mitosis and meiosis.Sense and antisense plant expression vector were constructed. Through agrobacteriwn hanefaciens-mediated transformation of japonica rice varieties Zhonghuall, 41 (sense) and 22 (antisense) transgenic plants were obtained, respectively. It was proved that 94% of theregenerated plants showed positive by PCR. The setting percentage of the sense and antisense transgenic plants were different distinctly.RIX4 was mapped on chromosomes 8.The expression profiles, signal transduction, RIX4 gene expression were studied using cDNA microarrary containing 2200 Expression Sequence Tags (ESTs). The leaves of rice near isogenic line (NIL) zhongzaoHR (Resistance) and 9S (Susceptible) were induced with Xanthomeneus oryzae pv. oyzae (Xoo) for 3 hours at 4-leaves stage .315 genes were up-regulated while 176 genes were down-regulated by the induction. Further studies on the 78 induced genes with known functions indicated that the changes of gene expression pattern not only reflect coordinated gene regulation in plant defense but also show the basic model of plant in response to pathogen. Novel genes induced by Xoo were also found. Further studies indicated that ethylene signal pathway may play important role in rice defense response while infected by Xoo. The expression of RIX4 was upregulated slightly in this experiment. |
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