紫花苜蓿MsCIPK8基因的克隆与表达分析

Cloning and Expression Analysis of MsCIPK8 in Alfalfa

CIPK是植物中一类丝氨酸/苏氨酸蛋白激酶,在植物响应逆境胁迫中发挥着重要的作用。本研究根据盐碱胁迫下紫花苜蓿(Medicago sativa L.)转录组数据设计引物,通过RT-PCR克隆获得紫花苜蓿MsCIPK8基因,该基因CDS全长1341bp,编码446个氨基酸,编码蛋白相对分子质量50.73 kD,等电点6.72,具有CIPKS家族蛋白所特有的N端激酶域和C端NAF/FISL结构域。生物信息学分析结果显示,MsCIPK8为可溶性蛋白,二级结构多为无规矩卷曲;系统进化分析表明,紫花苜蓿MsCIPK8与蒺藜苜蓿(Medicago truncatula Gaertn.)MtCIPK8亲缘关系最近。两个蛋白序列比对发现存在4个差异位点,其中3个在保守结构域内。MsCIPK8在低温、干旱、盐和盐碱胁迫下表达量均受到诱导上调表达。低温胁迫下,MsCIPK8在根和叶中的表达量分别在12 h和3 h达到峰值;盐胁迫下,MsCIPK8在根中的表达量12 h达到峰值;盐碱胁迫下,根和叶中MsCIPK8的表达量在12 h后持续高表达;干旱胁迫下,MsCIPK8在根和叶中的表达量在12 h均达到峰值。上述结果表明MsCIPK8参与紫花苜蓿对干旱、低温、盐和盐碱等非生物胁迫的应答。

CIPK(CBL-interacting protein kinase)is a serine/threonine protein kinase which plays an important role in response to stress in plants.In this study,MsCIPK8 was obtained by using RT-PCR based on transcriptional analysis of alfalfa in response to saline-alkaline stress.Sequence analysis of MsCIPK8 showed a 1341 bp CDS encoding 446 amino acids with a relative molecular mass of 50.73 kD and an isoelectric point of 6.72.MsC IPK8 performed an N-terminal kinase domain and a C-terminal NAF/FISL domain as a CIPKS family protein.Bioinformatics analysis indicated that MsCIPK8 is a soluble protein with a high proportion of random coil in secondary structure.Phylogenetic analysis showed that MsCIPK8 was most closely related to MtCIPK8(Medicago truncatula Gaertn.).Four difference sites were found in the two protein sequence alignment,of which 3 were in the conserved domain.MsCIPK8 had induced high expression in response to low temperature,drought,salt and salt-alkali.The expression of MsCIPK8 in roots and leaves had the highest transcript abundance at 12 h and 3 h under low temperature stress respectively.Under saline stress,the highest expression level of MsCIPK8 in roots was detected at 12 h.Under saline-alkaline stress,the expression of MsCIPK8 in roots and leaves remained high after 12 h.Under drought stress,the expression of MsCIPK8 in roots and leaves reached the highest expression level at 12 h.These results indicated that the expression of MsCIPK8 might be related to the resistance of alfalfa to abiotic stresses such as drought,low temperature,salt and salt-alkali.