研究以工业大麻云麻 7 号(Y7)为材料,克隆CsIAA1(Auxin-responsive protein IAA1)基因,通过生物信息学分析CsIAA1序列特征,经RT-qPCR研究CsIAA1组织特异性表达模式.结果表明,CsIAA1基因的CDS全长为621 bp,编码了 206 个氨基酸,含AUX_IAA保守结构域,属于不稳定蛋白.系统进化分析可知,工业大麻CsIAA1与糙叶山黄麻、东山麻等亲缘关系较近.通过烟草叶片瞬时表达和共聚焦试验得知,CsIAA1基因定位在细胞核中.通过RT-qPCR分析发现,在外源IAA处理12h内,CsIAA1在1h时快速响应生长素的处理,表达量最高.在工业大麻雄蕊不同发育阶段中,发育初期表达量比发育期和盛花期要高,且随着发育的进行呈逐渐下降趋势,推测CsIAA1参与调控工业大麻雄蕊发育.该研究从工业大麻中克隆出生长素响应基因CsIAA1并对其进行生信和表达分析,为后续探究工业大麻性别发育机制奠定了研究基础.
Cloning and Expression Analysis of CsIAA1 Gene in Industrial Hemp
In this study,the CsIAA1gene was cloned from the industrial hemp Y7.The sequence characteristics of CsIAA1were analyzed by bioinformatics,and the tissue-specific expression pattern of CsIAA1was studied by RT-qPCR.RT-qPCR was used to analyze the expression level of industrial hemp Y7 at different stages of Male development,and the CsIAA1 gene in different varieties of hemp was cloned and its amino acid sequence difference was analyzed.The results showed that the full-length cD-NA sequence of CsIAA1gene was 621 bp,encoding 206 amino acids,containing AUX_IAA conserved domain,which was an unstable protein.The phylogenetic tree analysis showed that the industrial hemp CsIAA1was closely related to Parasponia andersonii and Trema orientale.Through transient overexpres-sion and confocal experiments in tobacco leaves,it was found that CsIAA1was localized in the nucleus.Through qRT-PCR analysis,it was found that within 12 h of IAA treatment,CsIAA1had the highest ex-pression level of auxin at 0.5 h,indicating that CsIAA1gene could respond to exogenous IAA.In the male of different developmental stages of industrial hemp,the expression level of CsIAA1in the early stage of male development was higher than that in the development stage and the full-bloom stage,and it showed a gradual downward trend.It was speculated that CsIAA1was involved in regulating the male de-velopment of industrial hemp.Its specific function remains to be further studied.In this experiment,the auxin-responsive gene CsIAA1was cloned from industrial cannabis and its function was analyzed,which laid a foundation for the subsequent research on the mechanism of gender development of industrial can-nabis.
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