紫花苜蓿MsLEA4-4基因的克隆、表达分析及遗传转化  

Cloning and expression analysis of MsLEA4-4 from Medicago sativa

作　　者：贾会丽[1] 王学敏[2] 郭继承 高洪文[2] 吴欣明[1] 刘建宁[1] 石永红[1] 董宽虎[4] 王运琦[1] 

机构地区：[1]山西省农业科学院畜牧兽医研究所,山西太原030032 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]北京绿京华园林工程有限公司,北京102209 [4]山西农业大学动物科技学院,山西太谷030801

出　　处：《草业学报》2017年第12期108-116,共9页Acta Prataculturae Sinica

基　　金：现代农业产业技术体系专项资金(CARS-35-25);物种资源保护费(2130135);山西省农业科学院畜牧兽医研究所项目(xms201506)资助

摘　　要：胚胎晚期富集蛋白(LEA)广泛参与植物对多种逆境胁迫的反应。本研究利用同源克隆的方法,从紫花苜蓿中克隆了一个LEA4类基因的开放阅读框(ORF),命名为Ms LEA4-4。该基因编码512个氨基酸,结构分析显示MsLEA4-4包含5个重复的由11个氨基酸TAQAAKEKTQQ组成的序列特征。利用实时荧光定量PCR检测了Ms LEA4-4在不同逆境下的表达量,结果显示,该基因受干旱、NaCl、Cu^(2+)、Zn^(2+)和外源ABA诱导表达上调,其中Na Cl胁迫2h、Cu2+和Zn2+胁迫8 h,Ms LEA4-4基因表达量最高;冷胁迫和干旱胁迫下,该基因的表达量随处理时间的延长呈逐渐上升趋势,表明该基因可能参与了紫花苜蓿的抗逆性调控。构建植物超表达载体p CAMBIA3301-Ms LEA4-4,采用农杆菌介导法侵染拟南芥花序,通过草铵膦(PPT)筛选和分子检测,7株抗性苗呈阳性,表明目的基因已成功导入拟南芥基因组中。本研究为进一步探索Ms LEA4-4基因在紫花苜蓿抗逆性调控中的作用奠定了基础。Late embryogenesis abundant（ LEA） proteins are widely involved in many adverse situations for plants. In this study,we isolated a LEA4 gene from alfalfa by homology cloning strategy,termed Ms LEA4-4. The Ms LEA4-4 encoding revealed 512 amino acids and structure analysis showed that the gene contains eleven repeating TAQAAKEKTQQ amino acids sequences. Quantitative real-time PCR was used to detect Ms LEA4-4 expression quantities in adverse situations. Results revealed that the expression of Ms LEA4-4 was up-regulated by drought（ PEG）,salinity（ Na Cl）,Cu^2＋,Zn^2＋ and ABA stress. Gene expression was highest at 2 h and 8 h under the stress of Na Cl,Cu^2＋ and Zn^2＋. Under chilling and drought stress,the gene showed a rising trend. These results indicate that the Ms LEA4-4 protein may be involved in the regulation of environment stress responses in alfalfa. The plant expression vector p CAMBIA3301-Ms LEA4-4 was constructed and transgenic plants were obtained through infecting inflorescences of Arabidopsis by Agrobacterium tumefaciens-mediated transformation. The vector sequence was tested by antibiotic（ PPT） and PCR in the genome of transgenic plants and indicated that the target gene had been transferred. This study provides basic information for further study of the Ms LEA4-4 function in stress-tolerance regulation in alfalfa.

关 键 词：紫花苜蓿 胚胎晚期富集蛋白(LEA) 逆境胁迫 遗传转化 

分 类 号：S541.9[农业科学—作物学]