中国农业科学 ›› 2019, Vol. 52 ›› Issue (2): 201-214.doi: 10.3864/j.issn.0578-1752.2019.02.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

紫花苜蓿MsGAI的克隆、表达及遗传转化

张涵,王学敏,刘希强,马琳,温红雨,王赞()   

  1. 中国农业科学院北京畜牧兽医研究所,北京100193
  • 收稿日期:2018-07-18 接受日期:2018-09-07 出版日期:2019-01-16 发布日期:2019-01-21
  • 通讯作者: 王赞 E-mail:wangzan@caas.cn
  • 作者简介:张涵,E-mail: 793607453@qq.com
  • 基金资助:
    国家自然科学基金(31761143013);国家自然科学基金(31272495);国家现代农业产业技术体系(CARS34)

Cloning Expression Analysis and Transformation of MsGAI Gene from Medicago sativa L

ZHANG Han,WANG XueMin,LIU XiQiang,MA Lin,WEN HongYu,WANG Zan()   

  1. Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193
  • Received:2018-07-18 Accepted:2018-09-07 Online:2019-01-16 Published:2019-01-21
  • Contact: Zan WANG E-mail:wangzan@caas.cn

摘要:

【目的】 DELLA蛋白属于GRAS家族,是赤霉素信号转导途径中重要的转录因子,负向调节GA转导途径。克隆获得紫花苜蓿GAI,分析其基因生物信息学特征并预测蛋白结构域。明确紫花苜蓿GAI组织表达特征及不同处理下的表达模式,构建该基因超表达载体并转入紫花苜蓿,以探究DELLA蛋白基因在紫花苜蓿赤霉素(GA)信号转导途径及胁迫条件下的作用机理。【方法】 利用同源克隆的方法,从紫花苜蓿中克隆得到MsGAI。利用生物信息学方法分析该基因的序列特征,使用MEGA7.0对MsGAI蛋白序列及同源序列进行多序列比对,构建同源物种间的系统发育树。利用实时荧光定量PCR检测紫花苜蓿各组织GAI表达量以及在PEG、NaCl、GA、ABA和黑暗处理下,GAI的表达变化。同时对转基因GAI株系表达水平进行分析,选择表达量高、中、低株系(L5、L8、L11)分别进行PEG和NaCl处理,分析GAI的表达变化。以pBI121为基础载体,采用双酶切-连接的方法构建植物超表达载体35S:MsGAI-gus。将重组载体转入农杆菌GV3101菌株中,以紫花苜蓿叶片为外植体,采用农杆菌介导的愈伤组织转化法转化紫花苜蓿,经PCR检测和GUS组织化学染色,得到转基因阳性苗。【结果】 该基因序列包含有一个1 818 bp的开放阅读框,编码605个氨基酸。生物信息学分析结果显示,MsGAI蛋白具有GRAS家族的典型结构域和保守区,其中包含N端保守结构域DELLA和TVHYNP,C端保守结构域SAW。多序列比对及系统进化树分析表明,该序列与其他物种的DELLA蛋白序列相似度均高达80%以上,将其命名为MsGAI。该基因与蒺藜苜蓿GAI亲缘关系最近,其次与鹰嘴豆、红三叶等双子叶豆科植物亲缘关系较近,与大麦等单子叶植物较远。实时荧光定量PCR分析表明,MsGAI在紫花苜蓿各组织中均有表达,根中的表达量最高。经PEG、NaCl、GA以及ABA处理后,均有明显响应;黑暗处理显著抑制MsGAI的表达。转基因株系经PEG、NaCl处理后,GAI表达量均上调。对构建完善的35S:MsGAI-gus植物超表达载体进行双酶切检测,琼脂糖凝胶电泳显示,条带大小与预期一致。对转基因植株进行GUS组织染色验证,结果表明,阳性植株呈现蓝色,对照组为白色。对超表达载体携带的MsGAIGUS序列进行PCR检测均呈阳性。【结论】 紫花苜蓿DELLA蛋白基因的克隆和超表达载体构建成功,MsGAI对逆境胁迫有响应。

关键词: 紫花苜蓿, DELLA蛋白, 赤霉素, 表达分析, 同源克隆, 遗传转化

Abstract:

【Objective】 DELLA, one of GRAS family proteins, is an important transcription factor, which negatively regulates the gibberellin (GA) signal pathway. In this study, the Medicago sativa L. DELLA gene MsGAI was cloned, and its secondary structure was also predicted through bioinformatics. To illustrate the molecular function of MsGAI in M. sativa L. gibberellin (GA) signal transduction pathway, we clarify the spatio-temporal expression pattern of MsGAI and its response to different treatments; and the transgenic alfalfa over-expressing MsGAI were obtained by Agrobacterium-mediated transformation to explore gene function as well.【Method】 MsGAI was cloned by homologous cloning and sequence characteristics of MsGAI were analyzed by online bioinformatics tools. The phylogenetic tree of MsGAI and its homologous genes from other species was constructed by MEGA 7.0. The real-time quantitative PCR (qRT-PCR) was used to detect the spatio-temporal expression pattern of GAI and its response to abiotic including PEG, NaCl, GA, ABA and dark. The plant overexpression vector 35S:MsGAI-gus was transferred into Agrobacterium GV3101 strain, and transgenic alfalfa were obtained by Agrobacterium-mediated callus transformation. The transgenic positive seedlings were verified by PCR and GUS histochemical staining. Three transgenic line (L5, L8, L11) were selected to analysis MsGAI expression under PEG, and NaCl treatment.【Result】 Sequence analysis showed that 605 amino acids constitute MsGAI and it contains the conserved DELLA and VHYNP sequences in N-terminal regulatory region, and SAW in C-terminal. Multiple sequence alignment and phylogenetic tree analysis showed that the sequence similarity with other DELLA protein was as high as 80%, and it has the closest relationship with M. truncatula GAI, followed by the dicotyledonous such as Cicer arietinum and Trifolium pratense, and is far away from monocotyledon such as Hordeum vulgare. Real-Time PCR analysis showed that MsGAI had the highest expression in roots and after PEG, NaCl, GA and ABA treatment, the response was obvious while dark treatment significantly inhibited the expression of MsGAI. The GUS tissue staining showed that the positive plants were blue and the control group was white and the expression of MsGAI was upregulated in positive plants under PEG and NaCl treatments.【Conclusion】 The cloning and overexpression vector of DELLA protein in alfalfa was successfully constructed and the MsGAI could respond to stress treatments.

Key words: Medicago sativa L., DELLA protein, GA, expression analysis, homologous cloning, transformation