Fine Mapping And Cloning Of Rice EUI1 Gene Controlling Uppermost Internode Elongation

The rice mutant gene eui(elongated-uppermost-internode) has an important role in hybrid rice seed production, for the CMS line with euieui gene has a better panicle exsertion and the restore lines with it has a taller plant height than the control, euil mutant has the characters of the longer upper-internode, more sensitive to exogenous GA3 and more GA1 content in young panicle than that of control. Isolation of EUI1 gene will make us understand the molecular mechanism of the rice stem elongation, the plant hormones biosynthesis, metabolization and regulation. Some former studies had tagged euil gene on the long arm of the 5th chromosome, and the closer linkage maker of it was AC9 located on the BAC clone AC 108876. In this study, we had fine-mapped EUI1 gene and isolated the candidate gene of it. The main results were here:A F2 population of the cross Xieqingzao eB1 (ssp. indica) and Zhonghua15 (ssp. japonica) was developed to fine map EUI1 gene, and 1100 F2 plants with mutant phenotype were selected to analyze the molecular markers linked to EUI1 gene. For fine mapping EUllgene, a series of SSR markers were designed according to the rice genome database. With these SSR markers and STS markers on the long arm of the 5th chromosome, a high-resolution genetic map of EUI1 gene containing 14 markers was built. Two SSR markers AC40 and AC46 closestly linked to EUI1 gene were obtained, the genetic distance of them to EUI1 gene was 0.55cM and 0.09cM, respectively. AC40 and AC46 were located on the two overlapped BAC clones AC 13 7619 and AC 108289, a 152kb region containing EUI1 gene was defined with the two markers. And a co-segregating marker AC41 with EUI1 in the 152kb interval was identified from the 1100 F2 mutant plants.Analysed the 152kb sequence, 35 putative genes were predicted. 4 out of them were homologous to known protein, others were the ORF of hypotheticalprotein. The co-segregating marker AC41 was located on intron of putative cytochrome P450-related gene. Comprehensived the morphological, physilogical and biochemical characters of eui1 mutant, and functions of plant cytochrome P450, the putative cytochrome P450-related gene was predicted as EUI1 candidate gene. PCR amplification and sequencing of the exons of the mutants and their corresponding wild types were made, the results showed that exon2 of the mutants Xieqingzao eBl and Pei'ai 64eSl respectively deleted 8 nucleotidesGGGAGATC ttc and 2 nucleotides gtg AT atcgc. A/GATCt and gAT/atc were thecleavage site of the restrict enzyme Bg1 II and EcoR V, respectively. PCR product of exon2 of Xieqingzao eBl was not digested by Bgl II, while PCR product of Xieqingzao B could be digested into two fragmants. The same result was occurred between Pei'ai64 eS and Pei'ai64 S. PCR product of exon2 of Pei'ai64 eS was not digested by EcoR V, while that of Pei'ai64 S could be digested into two fragments. The above results indicated that the putative cytochrome P450 containing co-segregating marker AC41 was the candidate gene of EUI1Integrated the sequenced exons of the wild types and the sequence of the full-insert cDNA clone AK109526 of Nipponbare, the cDNA sequences of EUI1 of the wild types XieQingZao B and Pei'ai 64S were predicted.The EUI1 candidate gene consisted of two exons and one intron, the longest ORF of it contained 1734bp, and it encoded 577 amino acids. The BLASTN analysis of EUI1 candidate gene with rice genome database showed that the copy number of it was only one. The homologous analysis of the candidate gene indicated that the biological function of it was unknown, and it was belonged to cytochrome CYP714D1.7 members of the annotated rice cytochrome CYP714 family were all mapped on rice chromosome by silico mapping. And the polygenetic tree of rice CYP714 family member was built by using DNAman software.