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紫花苜蓿MsCIPK8基因的克隆与表达分析
李亚坤, 陈乃钰, 杨晓雪, 安逸民, 陈秀秀, 郭长虹
0
(黑龙江省分子细胞遗传与遗传育种重点实验室/哈尔滨师范大学生命科学与技术学院)
摘要:
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。本文根据盐碱胁迫下紫花苜蓿(Medicago sativa)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 1 341 bp,编码 446 个氨基酸,编码蛋白相对分子质量 50.73 kDa,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)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。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。
关键词:  紫花苜蓿  CIPK8基因  非生物胁迫  基因克隆
DOI:10.13430/j.cnki.jpgr.20190528002
投稿时间:2019-05-28修订日期:2019-12-27
基金项目:国家自然科学基金(No.31770575;31470571);国家重点研发计划项目(No.2017YFD0101303);黑龙江省重大攻关项目(No.GA18B104)和国家转 基因生物新品种培育重大专项(No.2016ZX08004-002)资助
Cloning and Expression Analysis of MsCIPK8 in Alfalfa
LI Ya-kun, CHEN Nai-yu, YANG Xiao-xue, AN Yi-min, CHEN Xiu-xiu, GUO Chang-hong
(Key Laboratory of Molecular Cytogenetics and Genetic Breeding of Heilongjiang Province / College of Life Science and Technology, Harbin Normal University)
Abstract:
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 kDa and an isoelectric point of 6.72. MsCIPK8 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).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.
Key words:  alfalfa  CIPK8  abiotic stress  gene clone

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