紫花苜蓿bZIP转录因子MsbZIP1的克隆、表达特性和遗传转化分析

Cloning,Expression Characteristics and Genetic Transformation Analysis of A bZIP Transcription Factor MsbZIP1 in Alfalfa

bZIP(Basic leucine zipper)是植物中一类非常重要的转录因子,参与植物从生长发育到抗性调控的多个生物学过程。为了了解紫花苜蓿(Medicago sativa)中bZIP转录因子的特性,解析紫花苜蓿MsbZIP1基因的生物学功能,从而阐述MsbZIP1基因响应紫花苜蓿抗逆调控机制,本研究利用RACE技术从紫花苜蓿中获得1个MsbZIP1基因的全长cDNA,该序列全长1176 bp,编码361个氨基酸,预测分子量为42.3 kD,等电点为6.5。分析发现,该蛋白含有bZIP家族典型的BRLZ碱性结构域和亮氨酸拉链,属于bZIP家族蛋白。进化树分析表明,该蛋白属于bZIP转录因子C亚族,与拟南芥(Arabidopsis thaliana)的AtbZIP63具有很高的同源性,推测可能具有与该类蛋白相似的功能。qRT-PCR分析表明,MsbZIP1基因对干旱、高盐、高温、低温,以及脱落酸(Abscisic acid,ABA)和生长素(Auxin,IAA)处理都有不同程度的响应,推测该基因可能参与调控紫花苜蓿多种非生物胁迫。本试验通过构建表达载体PCAMBIA3301-MsbZIP1,以农杆菌介导的花序浸染法转化拟南芥,经后代筛选、扩繁和分子检测,得到7株超表达的转基因拟南芥。本研究第一次分离了紫花苜蓿C亚族bZIP转录因子,初步确定紫花苜蓿MsbZIP1基因响应多种逆境胁迫的反应,并获得了阳性转基因材料,并为进一步探索该类转录因子在紫花苜蓿抗逆性调控中的作用奠定了基础。

The bZIP(Basic leucine zipper)genes is a type of very important transcription factors in plants.It is involved in many biological processes from growth and development to stress resistance regulation in plants.In order to understand the characteristics of bZIP transcription factors in alfalfa(Medicago sativa),to analyze the biological function of alfalfa MsbZIP1 gene,and to explain the mechanism of MsbZIP1 gene response to alfalfa stress resistance,the full-length cDNA of a MsbZIP1 gene was isolated from alfalfa in this research.Sequence analysis showed that the full-length of this gene is 1176 bp,encoding a protein of 361 amino acids with a predicted molecular weight of 42.3 kD and an isoelectric point of 6.5.Smart analysis showed that MsbZIP1 contains a typical BRLZ motif(Basic region domain and leucine zipper domain)of the bZIP family.Phylogenetic tree analysis revealed that MsbZIP1 belongs to C subfamily of the bZIP family and is related closely to Aribidopsis bZIP63.qRT-PCR analysis indicated that MsbZIP1 gene responded to various abiotic stresses(dehydration,salinity,high temperature,low temperature and two kinds of hormones:ABA and Auxin),indicating that it may played an important role in the adaptation to abiotic stress in alfalfa.In this test,an over expression vector pCAMBIA3301-MsbZIP1 was then constructed and transformed into Arabidopsis thaliana by Agrobacterium-mediated floral dip method.Transgenic Arabidopsis plants over expressing MsbZIP1 was obtained by screening and molecular identification.In conclusion,this study isolated the C subfamily bZIP TF in forage legume alfalfa for the first time,preliminarily determined the response of alfalfa MsbZIP1 gene to multiple abiotic stress and successfully obtained the transgenic plants.The results lay a foundation for further exploring the role of this type of transcription factor in the regulation of alfalfa′s stress resistance.