农杆菌介导的耐盐多基因转化紫花苜蓿及分子检测  

Alfalfa(Medicago sativa) Transformation with Salt-tolerant Muti-Gene Mediated by Agrobacterium tumefaciens and Molecular Identification

作　　者：王静[1] 麻冬梅[1] 许兴[1] 

机构地区：[1]宁夏大学农学院,银川750021

出　　处：《农业生物技术学报》2017年第8期1255-1264,共10页Journal of Agricultural Biotechnology

基　　金：宁夏自然科学基金(No.NZ15002)

摘　　要：盐超敏感(salt overly sensitive,SOS)信号转导途径是一个控制离子平衡的信号通路,可以在细胞水平上排出Na^+控制离子平衡,从而提高植物的耐盐性。将多基因植物表达载体p SOS中包含的SOS1、SOS2和SOS3、SOS3类钙结合蛋白8/钙调磷酸酶B10(SOS3-like calcium binding protein 8/calcineurin Blike 10,SCa BP8/CBL10)及抗双丙氨膦(bialaphos resistance,Bar)基因多个耐盐基因转化苜蓿(Medicago sativa),对于增强苜蓿的耐盐性具有重要的现实意义。本研究采用农杆菌(Agrobacterium tumefaciens)介导法,以"金皇后"苜蓿的子叶节为外植体进行遗传转化,除草剂草丁膦(glufosinate)最佳筛选浓度确定为0.6 mg/L,除菌剂头孢霉素(cefotazime)筛选浓度确定为300 mg/L,经除草剂筛选共获得了25株独立来源的转化植株。经PCR分子检测,有6株转基因植株具有PCR电泳扩增条带,阳性率为24%。随机将4株阳性植株进行Southern杂交,有3株出现杂交信号,拷贝数为1~2个。RT-PCR检测表明,与预期的条带大小相符合的Bar基因(463 bp)和SOS1基因(700 bp)在转基因植株中表达。结果表明,多个外源目的基因已经整合到苜蓿基因组中并且在转录水平上已表达。250 mmol/L NaCl胁迫处理后,转基因和野生型植株生长都受到了一定程度上的抑制,但转基因植株的株高均明显高于野生型,P1和P2株系叶面积均明显高于野生型对照,3个株系的鲜重均明显高于对照,P2和P4株系叶绿素含量明显高于野生型对照。说明SOS途径多基因的超表达提高了转基因苜蓿的耐盐性。本研究成功获得了转耐盐多基因苜蓿,为选育适合盐渍化土壤种植的耐盐苜蓿新品种,及苜蓿耐盐鉴定和耐盐机理等研究提供了理论依据。The salt overly sensitive （SOS） signal-transduction pathway is a control ion balance signal pathway. This pathway plays a key role in discharge Na^＋ and ion homeostasis on cellular lever and improves plant salt resistance. It has some realistic significance on enhanced salt tolerance in alfalfa （Medicago sativa）, which transformed with muti-gene mediated by Arabidopsis thaliana SOS1, SOS2, SOS3, SOS3-like calcium binding protein 8 （SCaBP8） and bialaphos resistance （Bar） 5 genes of polygenic plant expression vector pSOS. The Agrobacterium-mediated transformation method was used, ＂Golden Empress＂ cotyledonary nodes of alfalfa were selected as explants. The best concentration of glufosinate herbicide was identified as 0.6 mg/L,the best concentration of bacteriostat cefotazime was identified as 300 mg/L. 25 independent sources of transformed plants were obtained through screening after the herbicides selection. These plants applied PCR amplification and molecular detection, which showed that there were 6 transformants with the special bands. The positive rate was 24%. Four of positive transgenic plants were randomly selected Southern hybridization identification, and the results showed that there were 3 hybridization signal, and copy number was 1 or 2. The result of RT-PCR analysis demonstrated that the same as expected electrophoretic bands size of Bar gene （463 bp） and SOS1 gene （700 bp） expressed in transgenic plants. It was proved initially that multiple exogenous salt- tolerant genes have been integrated into the transgenic plant genome and expressed at the transcriptional level. After treated with 250 mmol/L NaC1 stress, the growth of wild type plants and transgenic plants were inhibited, but the plant height of the transgenic plants were significantly higher than the wild type, the leaf area of the transgenic P1 strain and P2 strain were obviously higher than the control group, the fresh weight of 3 strains were significantly higher than controls, leave chlorophy]I content of the

关 键 词：盐超敏感信号转导途径(SOS) 苜蓿 遗传转化 基因组 耐盐性 

分 类 号：S541.9[农业科学—作物学]