玉米叶序对生候选基因筛选及克隆

Screening and cloning the opposite phyllotaxy genes in maize

根据近等基因系对生叶序玉米H4D与互生叶序玉米H4d的转录组测序数据筛选出的差异基因,结合玉米对生基因OPP-1和OPP-2的SSR分子标记定位,将19个玉米对生叶序差异基因进行了染色体定位。利用比较基因组学和生物信息学分析对染色体定位的差异基因进行了详细分析,发现11个对生叶序差异基因具有同源基因报道,对其进行荧光定量PCR验证后发现基因表达量变化与转录组数据较为一致,其中8个基因在对生中表达量显著提高,3个基因在对生中表达量下降。结合差异基因染色体定位与同源性分析,筛选确定了SSR标记定位之间的5个差异基因作为候选基因。启动子分析表明这些基因启动子区域发现了多个与激素和光信号诱导相关的作用元件。以对生玉米近等基因系H4D和H4d的四叶期茎尖生长点c DNA为模板,对候选基因进行克隆,成功克隆出GRMZM2G077744_T01,GRMZM2G064845_T01和GRMZM2G348959_T01基因。经测序和氨基酸序列比对,发现在H4D和H4d中,GRMZM2G077744_T01基因氨基酸序列同源性为97.30%,GRMZM2G064845_T01同源性为98.85%,GRMZM2G348959_T01同源性为98.50%。氨基酸序列上的差异可能与玉米叶序对生变异具有密切关系。

Opposite phyllotaxy（two opposite leaves at the same stem node） is a new variation type in maize. Opposite phyllotaxy maize has high yield potential and is an important germplasm for plant breeding. It is also an important genetic resource for researching developmental biology. In this study, the transcriptome data of the opposite phyllotaxy maize H4 D and the alternate phyllotaxy maize H4 d were compared. The chromosomal locations of the DEGs were confirmed according to the SSR result of the opposite phyllotaxy-related OPP-1 and OPP-2 genes and 19 putative opposite phyllotaxy-related genes were located in chromosome. Using comparative genomics and bioinformatics analysis, the chromosomal locations of the DEGs were analyzed. The result showed that 11 putative opposite phyllotaxy-related genes have homologous genes in Gen Bank, which were further detected by q RT-PCR. The expression levels of these genes were found to be consistent with the transcriptome data. Of the 11 genes, the expression levels of eight genes significantly increased in opposite phyllotaxy maize, while the expression levels of other 3 genes decreased. Using differential gene chromosomal location and homology analysis, five differential genes between SSR marker positioning were screened as candidate genes. The promoter analyses showed that the promoter regions of these genes have multiple functional elements related to hormone（auxin, gibberellin, etc.） signal recognition and light signal conduction. Using opposite/alternate maize c DNA as templates, three candidate genes, GRMZM2G077744_T01, GRMZM2G064845_T01 and GRMZM2G348959_T01, were successfully cloned. Analysis of the amino acid sequences showed that the similarity of GRMZM2G077744_T01, GRMZM2G064845_T01, and GRMZM2G348959_T01 between H4 D and H4 d is 97.30%, 98.85%, and 98.50%, respectively, indicating that the difference in amino acid sequences may have close relationship with the maize phyllotaxis mutation.