棉花盐胁迫应答基因克隆与功能验证

发布时间：2018-05-02 03:21

  本文选题：陆地棉 + 盐胁迫应答　； 参考：《中国农业大学》2016年博士论文

【摘要】：棉花是盐碱地先锋作物。研究耐盐机理、提高棉花耐盐性具有重要理论与实践意义。基于实验室前期研究工作,从盐胁迫下陆地棉根系抑制性杂交差减文库筛选得到5个候选基因GhSnRK2.6, GhCIPK6, GhCPK5、GhC2H2和GhWRKY41。通过克隆全长基因,构建植物表达载体,分别转化野生型拟南芥及棉花,分析其功能；通过构建瞬时表达载体,进行亚细胞定位；通过调查转基因材料耐盐相关性状,分析这5个盐胁迫应答基因的功能；筛选耐盐品系,为棉花耐盐育种提供材料来源。(1)克隆了盐胁迫应答基因GhSnRK2.6, GenBank登录号为JN872373, ORF全长1,086bp,编码361个氨基酸。该基因包含9个外显子,8个内含子。构建瞬时表达载体p35S-GhSnRK2.6:GFP,基因枪法转化洋葱表皮细胞进行瞬时表达分析,该蛋白定位于细胞核。构建植物表达载体p2301M-GhSnRK2.6,分别转化野生型拟南芥和棉花,分析转基因材料耐盐性。结果GhSnRK2.6过表达拟南芥苗期及成熟期的耐盐性提高；转基因棉花后代材料盐胁迫下发芽率显著高于受体株系,耐盐性提高；另外,盐胁迫下转基因棉花的产量性状和纤维品质也优于受体对照。(2)构建瞬时表达载体p35S-GhCIPK6:GFP,基因枪轰击法转化洋葱表皮细胞进行瞬时表达分析,证明GhCIPK6定位于细胞质。BiFC分析确定GhCIPK6与GhCBLl和GhCBL8均存在互作关系,GhCBLl-GhCIPK6复合体定位于细胞核,GhCBL8-GhCIPK6定位于细胞核及细胞膜。将植物表达载体pBI-GhCIPK6转化棉花,分析耐盐性。证明盐胁迫下,GhCIPK6通过参与不同调控路径,调控细胞K+平衡,维持细胞膜稳定性,降低盐胁迫对质膜的伤害,保证植株正常生长,提高转基因棉花后代的耐盐性。(3)构建瞬时表达载体进行亚细胞定位,结果转录因子GhC2H2定位于细胞核。将前期构建的植物表达载体pBI-GhC2H2转化棉花,分析其耐盐性。结果证明过表达GhC2H2可提高转基因株系的衣分和单铃重,促进纤维增长,保证转基因棉花产量,提高转基因棉花后代的耐盐性。(4)构建瞬时表达载体进行亚细胞定位,结果GhCPK5定位于细胞核。将前期构建的植物表达载体pBI-GhCPK5转化棉花,分析相关性状,结果在盐胁迫下,GhCPK5转基因株系的单铃重和衣分高于受体,纤维长度增长,马克隆值下降,断裂比强度提高,纤维品质优于受体对照。(5)构建瞬时表达载体进行亚细胞定位,结果转录因子GhWRKY41定位于细胞核。将前期构建的植物表达载体p2301M-GhWRKY41转化棉花并分析耐盐性。结果表明,过表达GhWRKY41可提高转基因棉花胁迫下发芽率,盐胁迫下转基因株系的铃重和衣分变化不大,但对纤维品质存在不利影响。
[Abstract]:Cotton is a pioneer crop of saline - alkali land . It is of great theoretical and practical significance to study the salt tolerance mechanism and improve the salt tolerance of cotton . Based on the research work in the early stage of the laboratory , five candidate genes GhSnRK2 . 6 , GhCIP6 , GhCP5 , GhC2H2 and GhWRKY41 were isolated from the root system of upland cotton under salt stress .
carrying out sub - cell positioning by constructing a transient expression vector ;
By investigating the salt tolerance properties of transgenic materials , the function of these five salt stress response genes was analyzed .
The expression vector p35S - GhSnR2.6 : GFP was used to transform wild - type Arabidopsis thaliana and cotton to analyze the salt tolerance of transgenic materials .
Under salt stress , the germination rate of transgenic cotton progeny was higher than that of receptor strains , and the salt tolerance was improved .
The results showed that GhCBL8 - GhCBL1 and GhCBL8 could improve the salt tolerance of transgenic cotton . The results showed that GhCBL8 - GhCBL1 was located in the nucleus . The results showed that GhCBL8 - GhCBL8 was located in nucleus .

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562
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本文编号：1832165