大叶落地生根类糖原合成激酶KdSK基因的克隆与表达分析

摘要

类糖原合成酶激酶(SKs)属于丝氨酸/苏氨酸类蛋白激酶,在植物器官发育、激素信号传导过程中十分重要,并参与生物胁迫、非生物胁迫的应答过程.大叶落地生根中的胎生苗发育过程,同时具备胚胎发生和器官发生的特征,是研究无性生殖的理想模型.为了更好地理解大叶落地生根中胎生苗发育的分子机制,该研究利用RACE-PCR技术,从大叶落地生根中克隆了1个新的基因KdSK.该基因具有423个氨基酸残基,分子量为47.79 kD,等电点为8.37,其开放阅读框长为1 272 bp.其蛋白与黄瓜的同源性最高,属于植物类GSK3/shaggy蛋白激酶家族的第Ⅳ类,与苜蓿(MSK4)蛋白在进化关系上最近,且与拟南芥(AtSK4-1、AtKSK4-2)聚为一枝.保守域结构分析表明,KdSK蛋白具有明显的蛋白激酶的结构域,包括蛋白激酶的ATP结构域和丝氨酸/苏氨酸蛋白激酶活化结构域.实时荧光定量PCR分析表明,该蛋白基因在大叶落地生根的根中表达量最高,且受渗透胁迫(甘露醇)的诱导上调表达.该研究首次从大叶落地生根中克隆出KdSK基因,该研究结果为进一步研究该基因的功能打下了基础.%Shaggy-like protein kinase(SKs) plays numerous roles during the plant development, such as organ development, phytohormone conduction, abiotic stress and biotic stress.Kalanchoe daigremontiana is an attractive model system for the study of the molecular mechanisms of somatic embryogenesis and organogenesis competence because of its ability to form embryos and to acquire organogenic competence through plantlets.To better understand the molecular mechanisms of plantlet formation involved in K.daigremontiana, a shaggy-like protein kinase, KdSK, was identified using rapid amplification of cDNA end (RACE) PCR.KdSK gene consists of an ORF of 1 272 bp that was predicted to encode a 483 amino acid residue-long protein of 47.79 kD with an isoelectric point of 8.37.The sequence analysis of the KdSK revealed homology to Cucumis sativus shaggy-related protein kinase kappa.Phylogenetic analysis showed that KdSK gene was closely related to Medicage sativa protein(MSK4) and formed a subgroup with Arabidopsis thaliana(AtSK4-1, AtKSK4-2), all clustered to Clade Ⅳof GSK3/shaggy kinases family.Conservation domain structure analysis indicated that KdSK protein had typical structure of the protein kinase domain, including ATP domain and Serine/Threonine protein kinases active-site signature.Real-time PCR analysis revealed that KdSK transcript was expressed the highest in root and upregulated under osmotic stress.This study characterized the novel KdSK gene from Kalanchoe daigremontiana for the first time and the results will be useful for further functional determination of the gene.