谷子SiHd3a基因的克隆及表达分析

Cloning and Expression Analysis of SiHd3a Gene in Foxtail Millet

分析谷子成花素基因(Hd3a)在不同光周期条件下的表达规律,旨在揭示成花素基因在光周期调控谷子开花过程中扮演的角色。首先利用生物信息学方法获得位于谷子4号染色体的成花素基因序列(Seita.4G067600),命名为SiHd3a,然后根据基因序列设计1对特异引物,提取谷子农家种黄毛谷总RNA,利用RT-PCR技术克隆得到黄毛谷SiHd3a基因cDNA序列,其长度为792 bp,包含一个537 bp的CDS区,编码178个氨基酸;预测SiHd3a蛋白的分子质量为19.74 ku,等电点为6.82,初步判断其为亲水蛋白;蛋白质的二级结构中无规则卷曲所占比例最高(43.26%),其次为延伸链(β-折叠,29.21%)、α螺旋(16.29%)、β转角(11.24%);亚细胞定位分析发现,SiHd3a蛋白定位在细胞质和细胞间质;基于Hd3a蛋白序列进化分析发现,谷子与野生黍、玉米、高粱之间的亲缘关系比较近,聚在一组,而与水稻亲缘关系较远。半定量PCR发现,SiHd3a基因在短日照条件下表现为昼夜节律性表达,在早晨6:00、中午12:00各有1个表达峰,而在长日照条件下基因24 h内表现稳定表达水平。推测谷子SiHd3a基因长、短日照条件下昼夜表达模式的差异是导致其光周期敏感性的可能原因。

Analysis of the expression pattern of florigen gene(Hd3a)under different photoperiod conditions,aims at revealing the role of florigen gene that played in photoperiod-regulated flowering process of foxtail millet.First,a SiHd3a gene sequence(Seita.4G067600,named SiHd3a)on Chr.4 of foxtail millet was obtained by bioinformatics method and according to the transcribed sequence of Seita.4G067600,a pair of specific primers were designed successfully.Then the total RNA of millet land race Huangmaogu was extracted and after reverse transcription the cDNA sequence of SiHd3a gene was obtained by RT-PCR technology.The cDNA sequence was 792 bp which included a 537 bp CDS region that coding 178 amino acids.The molecular weight,isoelectric point of SiHd3a protein were 19.74 ku and 6.82 respectively,which was preliminarily judged as a hydrophilic protein.The proportion of irregular curl was the highest in protein secondary structure(43.26%),following it were extended chain(β-sheet,29.21%),α-helix(16.29%)andβ-turn(11.24%).Subcellular localization analysis showed that SiHd3a protein was located in the cytoplasm and intercellular substance.Phylogenetic analysis based on Hd3a protein sequences showed that there was a close relationship between foxtail millet and crown,maize,sorghum,but a distant relationship between foxtail millet and rice.Semi-quantitative PCR showed that under short-day condition,SiHd3a gene gave a circadian rhythm expression pattern,with two expression peaks at six am,and twelve am respectively.While under long-day condition,SiHd3a gene gave almost stable expression level in 24 hours.It could be speculated that the diurnal expression pattern difference of SiHd3a gene between long-day and short-day might be the cause leading to photoperiod sensitivity of foxtail millet.