百子莲生长素受体基因TIR1的全长cDNA克隆及功能分析

Cloning and Functional Analysis of Full-Length cDNA of the Auxin Receptor Gene TIR1 from Agapanthus praecox ssp.orientalis

采用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法获得百子莲生长素受体TIR1基因cDNA全长序列,对该序列进行生物信息学分析的结果显示,TIR1基因的mRNA序列全长为2 235bp;5’非编码区296bp,3’非编码区172bp;该基因的ORF全长为1 764bp,编码一个含有588个氨基酸的蛋白。百子莲TIR1推导蛋白序列与葡萄(Vitis vinifera)、二穗短柄草(Brachypodium distachyon)、高粱(Sorghum bicolor),玉米(Zea mays)等植物均具有较高的同源性,同源性最高的是葡萄,可达78%。系统进化树表明,百子莲与葡萄距离最近。由α-螺旋,随机卷曲和延伸链组成一个磁状结构,为亲水蛋白,疏水性值为-0.092,在百子莲体细胞胚胎发生过程中,实时荧光定量PCR结果表明TIR1基因表达量在愈伤组织中含量最低,在胚性愈伤组织和体细胞胚胎中含量均有所升高,而在体胚幼苗中表达量最高,表明该基因介导的生长素信号在体细胞胚胎发生过程中发挥着非常重要的作用。

The RNA was extracted from young leaves of Agapanthus praecox ssp.orientalis and the full length sequence of TIR1 cDNA was cloned by RACE technique.Bioinformatics analysis showed that the full length mRNA was 2 235 bp,including 296 bp of 5＇untranslated region and 172 bp of 3＇untranslated region.It had an open reading frame（ORF）of 1 764 bp,encoding aprotein of 588 amino acids.The deduced protein sequence was highly homologous with some plant species,such as Vitis vinifera,Brachypodium distachyon,Sorghum bicolor and Zea mays.The highest homology of was V.vinifera which reached 78%.Phylogenetic tree revealed that the evolutionary distance between A.praecox ssp.orientalis and V.vinifera was the nearest.The real-time quantitative PCR results showed that the expression of TIR1 in different stages of the somatic embryogenesis was totally different.The expression level in callus was thelowest and continuously elevated in embryonic callus and somatic embryos,while the highest in somatic seedlings,which indicated that TIR1 perhaps played a crucial role in somatic embryogenesis of A.praecox ssp.orientalis.