紫花苜蓿盐诱导HD-Zip类转录因子MsHB2的克隆及功能分析

doi:10.3864/j.issn.0578-1752.2014.04.002

摘要/Abstract

摘要： 【目的】在已有的一条紫花苜蓿（Medicago sativa L. cv. Zhongmu-1）盐诱导未知基因的EST序列基础上，克隆其（MsHB2）全长序列并分析其功能，以进一步了解其在紫花苜蓿中的耐盐调控机理。【方法】以先前获得的EST序列为基础设计引物，使用RACE法从紫花苜蓿中克隆得到MsHB2的3′和5′末端序列，经DNAMAN软件拼接后得到其全长序列。使用多种生物信息学软件对MsHB2的开放阅读框，编码蛋白等电点、分子量、疏水性、亚细胞定位、进化关系等进行预测分析。构建MsHB2的亚细胞定位瞬时表达载体，使用基因枪转化法将MsHB2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。将生长30 d的中苜一号分别进行300 mmol?L-1 NaCl和0.1 mmol?L-1 ABA胁迫处理，取胁迫处理0、2、4、10、24 h后的根和茎叶组织提取总RNA并进行荧光定量PCR分析。构建了MsHB2的植物超表达载体，转化农杆菌GV3101菌株，使用农杆菌花序侵染法转化拟南芥并在盐胁迫条件下对转基因株系进行相关表型分析。【结果】将RACE法克隆得到MsHB2的3′和5′末端序列拼接后得到1 126 bp的全长序列，序列分析表明，其编码蛋白包含247个氨基酸，具有一个同源异型结构域和一个亮氨酸拉链结构域，与拟南芥HD-Zip第I类转录因子ATHB12具有较高相似性。进化树聚类分析表明MsHB2属于第Ⅰ类同源异型域亮氨酸拉链蛋白。洋葱亚细胞定位分析表明MsHB2定位于细胞核。转录水平表达分析表明MsHB2受300 mmol?L-1 NaCl和0.1 mmol?L-1 ABA胁迫诱导而表达量显著升高。转基因研究表明在NaCl和ABA胁迫下，过量表达MsHB2的转基因拟南芥表现比野生型拟南芥更敏感。【结论】从紫花苜蓿中克隆得到一个定位于细胞核的同源异型域亮氨酸拉链蛋白基因MsHB2，其在紫花苜蓿中受NaCl和ABA胁迫诱导，并在NaCl和ABA胁迫条件下抑制转基因拟南芥的生长。推测MsHB2可能通过ABA信号途径参与紫花苜蓿盐胁迫应激调控，并在盐胁迫等非生物胁迫下对紫花苜蓿的抗逆性起负调控作用。

关键词: 紫花苜蓿 , 同源异型结构域 , 亮氨酸拉链 , 亚细胞定位 , 功能分析 , 转录因子

Abstract: 【Objective】Based on an EST of unknown gene, cloning and function analysis of a salt-induced gene (MsHB2) from alfalfa (Medicago sativa L. cv. Zhongmu-1) were conducted to further research the salt tolerance mechanism in alfalfa. 【Method】The RACE primers were designed according to the known EST sequence. The 3′- and 5′-end of the MsHB2 were amplificated by RACE method. The full length of the gene was assembled by DNAMAN program. The ORF of MsHB2, and the isoelectric point, molecular weight, molecular weight, subcellular localization, phylogenetic tree of the encoding protein were analyzed by some bioinformatics programs. The subcellular localization transient expression vector was constructed and transformed into onion epidermal cell by particle gun. MsHB2 and GFP were expressed in a fusion, which could be used to analyze the subcellular localization by the fluorescence signal. After being treated with 300 mmol•L-1 NaCl or 0.1 mmol•L-1 ABA for 0, 2, 4, 10 and 24 h, total RNA was extracted from root and shoot of 30-day-old Medicago sativa L. cv. Zhongmu-1 to analyze the expression pattern of MsHB2. The overexpression vector of MsHB2 was also contructed and transformed into GV3101 Agrobacterium. The phenotype of transgenic Arabidopsis plants was analyzed under salt and ABA stresses. 【Result】 A full length of 1 126 bp sequence was obtained by assembling 3′- and 5′-end RACE sequence. The sequence analysis result indicated that MsHB2 encoded 247 amino acid and contained a homeobox domain and a leucine zipper domain. MsHB2 had a high similarity with ATHB12. The phylogenetic tree analysis indicated that MsHB2 belonged to the class Ⅰ of plant homeobox domain protein. The subcellular localization result suggested that MsHB2 located in the nucleus of onion epidermal cell. MsHB2 mRNA was induced by NaCl and ABA stresses in alfalfa root and shoot. Its overexpression driven by a constitutive cauliflower mosaic virus-35S promoter in Arabidopsis plants conferred salinity and ABA sensitivity, as compared with WT plants. 【Conclusion】 A homeobox domain and leucine zipper domain protein gene (MsHB2) was cloned from alfalfa, which is induced by NaCl and ABA stresses. MsHB2 also retards the growth of transgenic Arabidopsis after NaCl and ABA stresses. These imply that MsHB2 may affect growth through an ABA dependent regulation pathway. MsHB2 may play a negetive role in salt and some other abiotic stress regulation in alfalfa.

Key words: alfalfa , homeobox domain , leucine zipper , subcellular localization , function analysis , transcription factor

李明娜1, 2, 龙瑞才1, 杨青川1, 2, 沈益新2, 康俊梅1, 张铁军1. 紫花苜蓿盐诱导HD-Zip类转录因子MsHB2的克隆及功能分析[J]. 中国农业科学, 2014, 47(4): 622-632.

LI Ming-Na-1, 2 , LONG Rui-Cai-1, YANG Qing-Chuan-1, 2 , SHEN Yi-Xin-2, KANG Jun-Mei-1, ZHANG Tie-Jun-1. Cloning and Function Analysis of a Salt-Stress-Induced HD-Zip Trascription Factor MsHB2 from Alfalfa[J]. Scientia Agricultura Sinica, 2014, 47(4): 622-632.

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