Screening And Cloning Of Key Candidate Genes In Opposite Phyllotaxy Maize

Opposite phyllotaxy maize is a new variation type,which characterized is that there are two opposite leavies at the same sote growing on the stem.It contains high yield potential,and is an important genetic resources to research developmental biology, which have important significance in breeding. The current research on molecular mechanism of forming process of opposite phyllotaxis is rarely reported. This study is based on transcriptome sequencing dates of the nearly isogenic opposite phyllotaxy maize (H4D) and alternate phyllotaxy maize (H4d),combining the previous opposite gene mapping of SSR molecular markers to screen for candidate genes of opposite phyllotaxy maize. And we cloned and analyzed these genes. The main findings are as follows:1. Basing on the transcriptome data, the chromosomal locations of the DEGs were confirmed according to the results of SSR molecular markers of opposite phyllotaxy-related genes OPP-1 and OPP-2. A total of 19 putative opposite phyllotaxy-related genes were located. According to comparative genomics and bioinformatics analysis, the chromosomal locations of the DEGs were analyzed in detail,finding that there were 11 putative opposite phyllotaxy-related genes with homologous genes reported2.The 11 putative opposite phyllotaxy-related genes with homologous genes were detected by qRT-PCR.,finding that the changes in these genes expression level are consistent with the datas of transcriptome very well. A total of 8 genes expression level significantly increased in opposite phyllotaxy maize,but only 3 genes decreased.In these genes,GRMZM2G343317_T01、GRMZM5G821024_T02 and GRMZM2G088293_T01 all had a high expression level in opposite phyllotaxy maize,but only GRMZM2G065344 had a high expression level in alternate phyllotaxy maize.3. Difference gene chromosomal location and homology analysis, screening to determine the five differences between SSR marker positioning gene as candidate gene, the promoter analysis showed that these gene promoter regions found multiple and hormone (auxin, gibberellin, etc.) and optical signal induced the role of related components, in plant hormone signal recognition and may play an important role in the process of conduction.4. These candidate genes were cloned from the opposite/alternate maize cDNA,and the GRMZM2G077744_T01、GRMZM2G064845_T01 and GRMZM2G348959_T01 gene successfully were cloned. Sequencing and amino acid sequences showed that between the H4D and H4d the similarity of GRMZM2G077744_T01 is 97.30%, GRMZM2G064845_T01 is 98.85%, GRMZM2G348959_T01 is 98.50%. Amino acid sequence differences may has close relationship with maize phyllotaxis mutate.5. Select GRMZM2G064845_T01 gene prokaryotic expression vector for prokaryotic expression preliminary analysis, we found GRMZM2G064845_T01 not express its activity in E.coliDH5α.Select GRMZM2G064845_T01 gene to construct the overexpression vectors,and then the plasmids were transferred into rice zhonghua 11 by agrobacterium-mediated transformation, obtaining 8 transgenic plants.