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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。
According to the near-isogenic lines, the differential genes screened from the transcriptome data of leaf-opening maize H4D and inter-leaf-derived maize H4d, and combining with the SSR markers of maize OPP-1 and OPP-2, 19 maize Chromosome mapping of differentially expressed leaf sequences. Using comparative genomics and bioinformatics analysis to analyze the differences of chromosomal location genes, we found that 11 of them have homologous genes of leaf-differentiating genes, and they were verified by real-time quantitative PCR and found that the changes of gene expression and transcription The data of the two groups were more consistent. Among them, the expression of 8 genes was significantly increased in the offspring, while the expression of 3 genes in the offspring was decreased. Based on the chromosomal location and homology analysis of the differentially expressed genes, five differential genes were screened and identified as candidate genes. Promoter analysis showed that several promoter elements related to the induction of hormones and light signals were found in the promoter region of these genes. The cDNAs of GRMZM2G077744_T01, GRMZM2G064845_T01 and GRMZM2G348959_T01 were successfully cloned using c DNA of four-leaf stage shoot tip growth of the near-isogenic lines H4D and H4d as the template. The results of sequencing and amino acid sequence alignment showed that the amino acid sequence homology of GRMZM2G077744_T01 was 97.30% in H4D and H4d, 98.85% in GRMZM2G064845_T01, and 98.50% in GRMZM2G348959_T01. Amino acid sequence differences may be closely related to maize leaf sequence variation.