紫花苜蓿盐诱导类受体蛋白激酶基因MsSIK1的克隆及功能分析  

Cloning and Function Analysis of a Salt-Stress-Induced Receptor Like Protein Kinase Gene Ms SIK1 from Alfalfa

作　　者：郭鹏[1] 邢鑫 张万筠[1] 姜健[1] 

机构地区：[1]大连民族学院环境与资源学院,辽宁大连116600 [2]威海市农业局,山东威海264200

出　　处：《中国农业科学》2014年第23期4573-4581,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31170168);辽宁省科技计划(2011209001);新疆科技支疆项目(201191126);辽宁省高等学校优秀科技人才项目(LR2013055)

摘　　要：【目的】对紫花苜蓿(Medicago sativa L.cv.Zhongmu-1)stress-induced protein kinase gene 1(Ms SIK1)进行克隆与表达研究,了解该基因的分子机制及其应用。【方法】以紫花苜蓿叶片总RNA为模板,根据同源克隆设计简并引物,利用RT-PCR结合RACE技术,获得Ms SIK1的编码序列。利用同源性比对进行序列分析。通过SMART网站(http://smart.embl-heidelberg.de/)模拟该基因的蛋白结构。构建Ms SIK1的亚细胞定位瞬时表达载体,使用基因枪转化法将Ms SIK1与GFP在洋葱表皮细胞中融合瞬时表达并观察其亚细胞定位荧光信号。通过Real time-PCR分析Ms SIK1在Na Cl、ABA和干旱处理条件下的表达特征。利用农杆菌侵染方法获得转基因拟南芥植株,通过RT-PCR对转基因植株进行表达鉴定,获得转基因植株后,利用转基因株系进行盐处理进而对成苗期转基因拟南芥性状鉴定。在盐胁迫处理下,测定野生型与转基因株系的叶绿素含量、MDA含量进而验证该基因的抗盐功能。【结果】获得Ms SIK1编码序列2 478 bp,编码825个氨基酸。该蛋白C端与多种植物激酶具有相当高的同源性,模拟蛋白结构发现该基因具有类受体蛋白激酶高度保守的丝氨酸/苏氨酸结构域、跨膜结构域和富含亮氨酸重复序列的膜外结构域。Real time-PCR分析表明该基因在Na Cl、ABA和干旱处理条件下上调表达,其中在盐处理条件下,Ms SIK1表达先升高后降低,在处理4 h时达到最大值(约为对照值的7倍)。在干旱胁迫处理时,Ms SIK1受诱导表达增强明显,当处理2 h时表达量达到最大值(约为对照值的6倍);ABA处理时,Ms SIK1被诱导表达明显,当处理3 h时表达量达到最大值,约为对照值的6.8倍。Ms SIK1与GFP融合瞬时表达的洋葱表皮细胞中的荧光信号主要集中于质膜附近,转化空载体的洋葱表皮细胞中的荧光信号分布于细胞各个部位。转基因植株的RT-PCR鉴定表明,T1代6个株系中所得到的Ms SIK【 Objective 】 The aim of this study is to clone stress-induced protein kinase gene 1（SIK1） gene, to analyze its molecular mechanisms and promote their applications in breeding. 【Method】 The total RNA from the leaves of alfalfa was used as the template to design the degenerate primers based on homology cloning strategy, and then the full-length ORF sequence of Ms SIK1 was obtained through a combined reverse transcription-PCR（RT-PCR）. Sequence analysis was performed by using homology comparision, the SMART website（http：//smart.embl-heidelberg.de/） was used to simulate the protein structure. The subcellular localization transient expression vector was constructed and transformed into onion epidermal cell by particle gun.Ms SIK1 and GFP were expressed in a fusion, which could be used to analyze the subcellular localization by the fluorescence signal.Real time-PCR was used to investigate the expression pattern under Na Cl, ABA and drought stress, transgenic Arabidopsis plants was obtained by Agrobacterium. Expression identification of transgenic plants was performed by RT-PCR, after the transgenic plants were obtained, T3-2, T3-6, and T3-10 transgenic lines were used for character identification of transgenic Arabidopsis under salt stress at seedling stage. Chlorophyll content and MDA content were investigated for functional verification of salt resistance between the wild type and T3-2,T3-6, T3-10 transgenic lines.【Result】The results indicated that ORF of Ms SIK1 was 2 478 bp and contained a single open reading frame of 825 amino acid residues. The protein C terminal has high homology with a variety of plant kinase, the simulation of protein structure indicated that the protein encodes a putative leucine-rich repeat receptor-like kinase with various domains. In the N-terminal region, a putative extracellular domain containing 10 amino acid leucine-rich repeats（LRR） was identified, and a transmembrane domain was identified. In the C-terminal cytoplasmic region, a serine/threonine protein

关 键 词：紫花苜蓿 MsSIK1 类受体蛋白激酶 盐胁迫 功能研究 

分 类 号：S541.9[农业科学—作物学]