摘要
【目的】大豆(Glycine max (L.) Merr.)种子从发育成熟期(R6或R7期)开始具有活力,在此时期容易受到高温高湿胁迫,导致种子活力下降。通过对Gm LEA的研究,揭示其高温高湿胁迫下的表达水平以及功能,为一步深入研究Gm LEA在参与大豆种子活力形成及响应非生物胁迫等方面奠定基础。【方法】利用Primer Premier 5.0软件设计引物,以宁镇1号和湘豆3号叶片的cDNA为模板,分离大豆GmLEA的cDNA序列全长。通过NCBI网站BLAST对GmLEA同源性氨基酸序列进行搜索,使用MEGA 6.0和DNAMAN多重比对蛋白质序列,并采用MEGA 6.0的N-J算法构建进化树。通过酵母双杂交试验验证GmLEA与GmCDPKSK5在酵母体内的互作。构建亚细胞定位和双分子荧光互补(bimolecular fluorescence complementation,BiFC)载体,通过基因枪介导法转化烟草叶片分析GmLEA与GmCDPKSK5在烟草叶片细胞内的互作及其编码蛋白的亚细胞定位。利用实时荧光定量PCR技术(qRT-PCR)对GmLEA的组织特异性表达及其在高温高湿胁迫下的表达进行分析。构建pBI121融合表达载体,通过农杆菌介导法获得3个纯合的T3代过表达拟南芥株系,对高温高湿胁迫下拟南芥的种子活力进行分析。【结果】获得大豆GmLEA的cDNA序列全长,该基因包含一个长1 377 bp的开放阅读框(ORF);且该基因所编码的蛋白定位在烟草叶片细胞的细胞膜上。酵母双杂交试验与双分子荧光互补(BiFC)试验结果表明,GmLEA与GmCDPKSK5分别在酵母体内与烟草叶片细胞的细胞膜上存在特异性互作。组织特异性分析结果表明,GmLEA主要在宁镇1号和湘豆3号2个品种发育和成熟的种子中表达。在湘豆3号种子发育过程中Gm LEA的表达量呈先上升后下降的趋势,在开花后50 d达最大值,在宁镇1号种子发育过程中该基因的表达量呈上升的趋势,在开花后60 d达到最大值。高温高湿胁迫后,与对照相比,Gm LEA在湘豆3号中96 h时下调表达,其余时间点均上调表达,在宁镇1号中,仅24 h时下调表达。GmLEA过表达拟南芥株系经高温高湿胁迫后的发芽势、发芽率及种子活力均显著(P<0.01)高于野生型植株。【结论】GmLEA参与高温高湿胁迫下种子活力的形成,与GmCDPKSK5存在特异性互作,二者可能共同参与高温高湿胁迫下种子活力的形成。
【Objective】 Soybean(Glycine max(L.) Merr.) seed generally form vitality from their physiological maturity period(R6 or R7 period). However, the seed is susceptible to high temperature and humidity(HTH) stress during this period. This will lead to a decline in seed vigor. The results will lay a foundation for further studying the mechanism of seed vigor formation under abiotic stress. 【Method】 Primer Premier 5.0 was used to design primers, and the full length cDNA sequence of Gm LEA was isolated by using the cDNA of leaves of cv. Ningzhen No.1 and Xiangdou No.3 as template. The homologous amino acid sequence of GmLEA was searched by BLAST at NCBI, the protein sequences were multiple aligned using MEGA 6.0 and DNAMAN, and the phylogenetic tree was constructed using the N-J algorithm of MEGA 6.0. Yeast two-hybrid experiments were performed to verify the interaction of GmLEA and GmCDPKSK5 in yeast. A subcellular localization and bimolecular fluorescent complementation(BiFC) vector were constructed, The interaction between GmLEA and GmCDPKSK5 in tobacco leaf cells and the subcellular localization of the encoded protein were analyzed by gene-gun-mediated transformation of tobacco leaves. In addition, the tissue-specific expression of GmLEA gene and the expression pattern of GmLEA gene under HTH were analyzed by qRT-PCR, respectively. The pBI121 fusion expression vector was constructed and three homozygous overexpressed Arabidopsis lines were obtained through Agrobacterium-mediated method, and three independent homozygous T3 transgenic lines were used for analysis. 【Result】 The cDNA sequence of GmLEA gene contains a 1 377 bp open reading frame(ORF), and the subcellular localization result showed that the encoded protein was located on the cell membrane. The results of yeast two-hybrid rotation verification showed that GmLEA could interact with GmCDPKSK5 in yeast. In addition, bimolecular fluorescence complementation(BiFC) experiment showed that GmLEA could interact with GmCDPKSK5 on cell membrane of tobacco leaf cells. The results of tissue-specific analysis showed that GmLEA gene had higher expression levels in developing and mature seeds of both cultivars. The expression level of GmLEA was increased first and then decreased during the development of cv. Xiangdou No. 3 seeds. During the process of seed development of cv. Ningzhen No.1, the level of GmLEA expression was on the rise and reached the highest at 60 days after flowering. After high temperature and high humidity(HTH) stress, the expression of GmLEA was decreased at 96 h in cv. Xiangdou No. 3, and the other time points were increased. However, the expression was decreased at 24 h in cv. Ningzhen No.1. The germination potential, germination rate and seed vigor of GmLEA gene in transgenic Arabidopsis thaliana were significantly(P<0.01) higher than those of the wild type plants under HTH stress. 【Conclusion】 The GmLEA is involved in the formation of seed vigor under HTH stress,and has specific interaction with GmCDPKSK5, It is speculated that they may participate in the formation of seed vigor under HTH stress.
作者
周亚丽
朱雅婧
赵飞云
王爽
刘骕骦
郭凌凯
赵海红
麻浩
ZHOU YaLi;ZHU YaJing;ZHAO FeiYun;WANG Shuang;LIU SuShuang;GUO LingKai;ZHAO HaiHong;MA Hao(National Key Laboratory of Crop Genetics and Germplasm Enhancement,Nanjing Agricultural University,Nanjing 210095)
出处
《中国农业科学》
CAS
CSCD
北大核心
2018年第23期4397-4408,共12页
Scientia Agricultura Sinica
基金
国家自然科学基金(31671772
31371711)
科技部国家重点研发计划(2018YFD0100905)
