小麦TaCO9基因的克隆及功能分析

Cloning and Functional Analysis of TaCO9 Gene in Wheat(Triticum aestivum)

为了明确TaCO9-1A基因在小麦生长发育中的具体调控机理,该研究以同源克隆的方法成功获得了大麦(Hordeum vulgare)光周期基因HvCO9在小麦(Triticum aestivum)中的直系同源基因TaCO9,并对其进行生物信息学分析、亚细胞定位、转录激活以及表达模式分析;利用农杆菌侵染法转化拟南芥,并对过表达株系进行表型分析。结果表明:(1)TaCO9包含2个外显子和1个内含子,CDS区全长876 bp,编码291个氨基酸,蛋白序列含有特有的CCT结构域,且在不同物种间高度保守。(2)生物信息学分析表明,TaCO9基因编码的蛋白质分子量约为30.7 kD,等电点为6.24;TaCO9的启动子区含有光响应、激素应答和胁迫应答等多种顺式作用元件。(3)亚细胞定位和转录活性分析表明,TaCO9主要定位于细胞核中,且具有转录激活活性。(4)qRT-PCR结果表明,TaCO9基因在各个组织中均有表达,叶片中的表达量最高;在光照14 h条件下TaCO9的表达量显著高于光照10 h和12 h条件下的表达量,且TaCO9在大粒小麦‘西农817’子房与籽粒中的表达量显著高于‘中国春’。(5)经潮霉素筛选成功获得3个转基因拟南芥株系;进一步功能鉴定结果表明,过表达转TaCO9基因拟南芥植株的开花期迟于野生型(对照)3~4 d,但角果和籽粒较野生型大。

In order to clarify the specific regulation mechanism of TaCO9-1A gene in wheat growth and development,this study successfully obtained the orthologous gene TaCO9 of the barley(Hordeum vulgare)photoperiod gene HvCO9 in wheat(Triticum aestivum)by homologous cloning.Bioinformatic analysis,subcellular localization,transcriptional activation,and expression pattern analysis were performed.Arabidopsis was transformed by Agrobacterium infection,and phenotypic analysis was performed on the over-expressing lines.The results showed that:(1)TaCO9 contained 2 exons and 1 intron,the whole length of CDS was 876 bp,291 amino acids were encoded,and the protein sequence contained a unique CCT domain,which was highly conserved among different species.(2)Bioinformatic analysis showed that the molecular weight of the protein encoded by this gene was about 30.7 kD and the pI was 6.24.The prokaryotic expression results showed that the molecular weight of the protein was basically consistent with the predicted results under IPTG induction;The promoter region of TaCO9 contained many cis-acting elements such as light response,hormone response and stress response.(3)Subcellular localization and transcriptional activity analysis showed that TaCO9 was mainly located in the nucleus,and TaCO9 had transcriptional activation activity.(4)The results of qRT-PCR showed that TaCO9 gene was expressed in all tissues,with the highest expression in leaves;TaCO9 expression level under 14 h light was significantly higher than that under 10 and 12 h light;and TaCO9 expression level in ovary and grain of‘Xinong817’was significantly higher than that under‘Chinese Spring’.(5)Three transgenic lines were obtained by hygromycin screening;Functional identification showed that the flowering period of transgenic Arabidopsis plants with overexpression of TaCO9 was later than that of wild-type control plants(3-4 d),and the seeds and siliques were larger.