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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。
Salt over-sensitive (SOS) signal transduction pathway is a signal pathway that controls ion balance, which can release Na ~ + ion balance at the cellular level to improve the salt tolerance of plants. SOS1, SOS2 and SOS3, SOS3-like calcium binding protein 8 / calcineurin Blike 10 (SCa BP8 / CBL10) contained in the polygenic plant expression vector pSOS, Transformation of multiple salt-tolerant genes of bialaphos resistance (Bar) gene into alfalfa (Medicago sativa) is of great practical significance for enhancing salt tolerance of alfalfa. In this study, the Agrobacterium tumefaciens mediated method was used to transform the cotyledon node of Medicago sativa L. as explants. The optimal screening concentration of herbicide glufosinate was 0.6 mg / L The screening concentration of cefotazime was 300 mg / L. A total of 25 independently transformed plants were obtained by herbicide screening. According to the results of PCR, 6 transgenic plants had PCR amplification bands with a positive rate of 24%. Four positive plants were randomly selected for Southern blotting. Three hybridization signals were found, with 1 ~ 2 copies. RT-PCR analysis showed that the Bar gene (463 bp) and the SOS1 gene (700 bp) corresponding to the expected band size were expressed in the transgenic plants. The results showed that a number of exogenous genes of interest have been integrated into the alfalfa genome and have been expressed at the transcriptional level. After 250 mmol / L NaCl stress, the growth of both transgenic and wild type plants was inhibited to some extent, but the plant height of the transgenic plants was significantly higher than that of the wild type. The leaf area of P1 and P2 strains were significantly higher than that of the wild type Compared with the control, the fresh weight of the three lines was significantly higher than that of the control, and the chlorophyll content of P2 and P4 lines was significantly higher than that of the wild-type control. The results showed that overexpression of SOS pathway multi-gene increased salt tolerance of transgenic alfalfa. In this study, transgenic salt-tolerant multi-gene alfalfa was successfully obtained, which provided a theoretical basis for the selection of new salt-tolerant alfalfa cultivars suitable for salinized soils, salt tolerance identification and salt tolerance mechanism of alfalfa.