中国农业科学 ›› 2014, Vol. 47 ›› Issue (23): 4573-4581.doi: 10.3864/j.issn.0578-1752.2014.23.003

• 作物遗传育种·种质资源 • 上一篇    下一篇

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

郭鹏1,邢鑫2,张万筠1,姜健1   

  1. 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
  • 收稿日期:2014-04-14 修回日期:2014-07-15 出版日期:2014-12-01 发布日期:2014-12-01
  • 通讯作者: 姜健,Email:jjx@dlnu.edu.cn
  • 作者简介:郭鹏,E-mail:gp@dlnu.edu.cn
  • 基金资助:
    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)

Cloning and Function Analysis of a Salt-Stress-Induced Receptor Like Protein Kinase Gene MsSIK1 from Alfalfa

GUO Peng1, XING Xin2, ZHANG Wan-jun1, JIANG Jian1   

  1. 1College of Environment and Resources, Dalian Nationalities University, Dalian 116600, Liaoning
    2Weihai Agricultural Bureau, Weihai 264200, Shandong
  • Received:2014-04-14 Revised:2014-07-15 Online:2014-12-01 Published:2014-12-01

摘要: 【目的】对紫花苜蓿(Medicago sativa L. cv. Zhongmu-1)stress-induced protein kinase gene 1(MsSIK1)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MsSIK1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MsSIK1的亚细胞定位瞬时表达载体,使用基因枪转化法将MsSIK1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MsSIK1在NaCl、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MsSIK1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NaCl、ABA和干旱处理条件下上调表达,其中在盐处理条件下,MsSIK1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,MsSIK1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,MsSIK1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。MsSIK1GFP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MsSIK1条带明显、亮度高,且T1-10中表达量最高;但在野生型中检测不到该条带,说明外源基因已经整合到拟南芥染色体中并能遗传到子代。成苗期转基因拟南芥盐处理后发T3-2、T3-6、T3-10转基因株系较野生型植株长势好,说明MsSIK1的转入提高了拟南芥的抗盐性。与对照相比,转MsSIK1拟南芥在NaCl处理下,叶绿素含量下降较少,其中,野生型叶绿素含量降低了77%,T3-3降低了53%,T3-6降低了44%,T3-10降低了35%;同样盐胁迫下,3个转基因株系的MDA含量积累较少,其中,野生型MDA的含量是T3-10株系的1.3倍。【结论】MsSIK1作为一个类受体蛋白激酶受多种逆境胁迫诱导,该基因的过量表达提高了拟南芥的抗盐性。

关键词: 紫花苜蓿, MsSIK1, 类受体蛋白激酶, 盐胁迫, 功能研究

Abstract: 【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 MsSIK1 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. MsSIK1 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 NaCl, 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 MsSIK1 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 kinase domain was predicted. The subcellular localization result suggested that MsSIK1 is located in the plasma membrane of onion epidermal cell. Real time-PCR indicated that the mRNA accumulation of MsSIK1 was induced by salt stress, abscisic acid (ABA) and drought. In the salt treatment, expression was induced quickly and the level of the product peaked at 4 h (about 7 times as to the control). In the drought treatment, MsSIK1 transcription was not induced immediately, but reached its maximum at 2 h (about 6 times compared with the control). In the ABA treatment, MsSIK1 transcripts accumulated rapidly and peaked after 3 h (about 6.8 times compared with the control). The RT-PCR identification of transgenic plants indicated that there were 6 lines with obviously band in T1, and highest expression in T1-10, but not detected in the wild type, therefore, it indicated that the foreign gene had been integrated into the chromosome of Arabidopsis thaliana and inherited in the offspring. Compared with the wild type plants, T3-2, T3-6, and T3-10 transgenic lines grew well at seedling stage in salt treatment, indicating that MsSIK1 improved the salt tolerance in Arabidopsis thaliana. Additionally, compared with the control, the content of chlorophyll in transgenic Arabidopsis decreased less in NaCl treatment, among them, the chlorophyll content of wild type decreased by 77%, T3-3 decreased by 53%, T3-6 decreased by 44%, T3-10 decreased by 35%; equally, the content of chlorophyll in transgenic Arabidopsis accumulated less in NaCl treatment, among them, the content of MDA in wild type was 1.3 times as that in T3-10 line. 【Conclusion】MsSIK1 as a receptor like protein kinase was induced by a variety of stress and could increase salt tolerance by overexpression in A. thaliana.

Key words: alfalfa, MsSIK1, receptor like protein kinase, salt stress, functional study