农杆菌介导的耐盐多基因转化紫花苜蓿及分子检测

发布时间：2018-02-24 21:01

本文关键词： 盐超敏感信号转导途径(SOS) 苜蓿遗传转化基因组耐盐性　出处：《农业生物技术学报》2017年08期 　论文类型：期刊论文

【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。
[Abstract]:Salt hypersensitive overly sensitive overly signal transduction pathway is a signal pathway to control ion balance, which can expel Na ~-to control ion balance at cell level. In order to improve the salt tolerance of plants, the multiple salt tolerance genes of SOS1, SOS2 and SOS3S3 calcium binding protein 8 / calmodulase B10S3-like calcium binding protein 8 / calcurin Blike 10n SCa BP8 / CBL10) and the gene resistant to dialaphos resistanceBarwere transformed into alfalfa with multiple salt tolerance genes contained in the polygene plant expression vector p SOS. Medicago sativaa has important practical significance in enhancing salt tolerance of alfalfa. Agrobacterium tumefaciens-mediated method was used in this study. The cotyledons of "Jinhuang Queen" alfalfa were used as explants for genetic transformation. The optimum screening concentration of herbicide glufosinate was 0.6 mg / L, and that of cefotazime was 300mg / L. 25 independent transformed plants were obtained by herbicide screening. There were 6 transgenic plants with PCR electrophoresis amplified bands, and the positive rate was 24.The Southern hybridization of 4 positive plants was carried out at random, and 3 of them had hybridization signals, and the copy number was 1 ~ 2. RT-PCR showed that, Bar gene (463 BP) and SOS1 gene (700 BP) were expressed in transgenic plants. The results showed that many foreign target genes had been integrated into alfalfa genome and were expressed at the transcriptional level of .250 mmol/L NaCl. The growth of transgenic and wild-type plants has been inhibited to some extent. However, the plant height of transgenic plants was significantly higher than that of wild type P _ 1 and P _ 2 lines, and the leaf area of the three lines was significantly higher than that of wild type control, and the fresh weight of the three lines was significantly higher than that of control P _ 2 and P _ 4, and the chlorophyll content of the three lines was significantly higher than that of wild type control. The overexpression of multiple genes in the SOS pathway improved the salt-tolerance of transgenic alfalfa. This paper provides a theoretical basis for breeding new salt-tolerant alfalfa varieties suitable for salinized soil cultivation and for the identification of salt-tolerance and salt-tolerance mechanism of alfalfa.
【作者单位】：宁夏大学农学院;
【基金】：宁夏自然科学基金(No.NZ15002)
【分类号】：S541.9

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