摘要
【目的】高温高湿、重金属等非生物胁迫是制约大豆生产,影响种子发育和品质的重要因素。通过对大豆GmHMADP的研究,揭示其在重金属镉、铜和高温高湿胁迫下的表达水平以及功能,以期为培育高活力的逆境耐受性大豆新品种提供理论依据和实践基础。【方法】利用Primer Premier 5.0软件设计引物,以宁镇1号和湘豆3号2个品种叶片的cDNA为模板,分离大豆GmHMADP全长cDNA序列。通过NCBI网站BLAST对GmHMADP同源性氨基酸序列进行搜索,并用DNAMAN软件进行氨基酸序列比对。同时,以XhoⅠ和SalⅠ为酶切位点构建35S::GmHMADP-GFP融合表达载体,分析其在烟草叶肉细胞的瞬时表达,进行亚细胞定位。利用实时荧光定量PCR技术对GmHMADP的组织特异性表达及其在高温高湿和CuSO_4、CdCl_2处理下的表达进行分析。以XbaⅠ和Bam HⅠ为酶切位点,利用p BI121载体,构建融合表达载体,将GmHMADP在野生型拟南芥中过表达,获得3个纯合的T3转基因株系,对高温高湿胁迫下拟南芥的种子活力以及过表达拟南芥植株对铜、镉胁迫的耐受性进行分析。【结果】获得大豆GmHMADP全长cDNA序列,该基因包含1个996 bp的完整开放阅读框,具有4个外显子和3个内含子。多序列比对结果表明,GmHMADP氨基酸序列含有2个金属离子结合位点特征序列CXXC。亚细胞定位结果表明GmHMADP蛋白定位于细胞膜与细胞核上。组织特异性分析结果表明,GmHMADP在宁镇1号和湘豆3号2个品种的发育和成熟中的种子中表达量较高。与相应的对照组相比,在宁镇1号种子中,高温高湿胁迫48、96和168 h后,GmHMADP的表达量显著(P<0.01)升高;在抗性品种湘豆3号中,该基因在处理24、48、96和168 h时表达量显著(P<0.01)升高;湘豆3号中,GmHMADP的表达量在不同浓度Cu^(2+)、Cd^(2+)的诱导下均显著(P<0.01)升高;而宁镇1号中,GmHMADP的表达量仅在50μmol·L^(-1) Cu^(2+)和200μmol·L~(^(-1)) Cd^(2+)处理下显著(P<0.01)升高。GmHMADP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;经Cu、Cd胁迫后,转基因拟南芥株系的根长和干重均显著(P<0.05)高于野生型植株。【结论】GmHMADP参与高温高湿胁迫下种子活力的形成并响应Cu、Cd胁迫,在拟南芥中过表达GmHMADP可提高拟南芥的种子活力以及对Cu、Cd胁迫的耐受性。
【Objective】Abiotic stress such as high temperature and humidity(HTH), heavy metal stress are important factors that restrict soybean production and affect seed development and quality. In this study, the expression level and function of GmHMADP gene under heavy metal cadmium, copper and the HTH stress were analyzed, so as to provide a theoretical and practical basis for breeding new soybean resistant cultivars.【Method】Primer Premier 5.0 was used to design primers, and the full length cDNA sequence of GmHMADP was isolated by using the cDNA of leaves of cv. Ningzhen No.1 and Xiangdou No.3 as template. The homologous amino acid sequences of GmHMADP were searched by BLAST of NCBI website, and the amino acid sequence alignment were carried out by DNAMAN. Via constructing 35 S::GmHMADP-GFP fusion expression vector with XhoⅠand SalⅠrestriction sites, subcellular localization of GmHMADP was studied by transient expression analysis of tobacco mesophyll cells. In addition, the tissue-specific expression of GmHMADP gene and the expression pattern of GmHMADP gene under HTH, and CuSO4 and CdCl2 treatment were analyzed, respectively, by q RT-PCR. Simultaneously, p BI121 with XbaⅠand Bam HⅠ restriction enzymes were used to construct fusion expression vectors. The transgenic Arabidopsis plants with over-expressing GmHMADP gene were generated, three transgenic lines were obtained and three independent homozygous T3 transgenic lines were used for analysis.【Result】The full-length cDNA sequence of GmHMADP gene was isolated, which contained a complete open reading frame of 996 bp. Besides, the GmHMADP gene contained 4 exons and 3 introns. Amino acid sequence analysis showed that GmHMADP contains two heavy metal binding core motif(CXXC). The subcellular localization result showed that GmHMADP protein was located on cell membrane and nucleus. The results of tissue-specific analysis showed that GmHMADP gene had the higher expression levels in developing and mature seeds of both the cultivars. Compared to the corresponding controls, the expression levels of GmHMADP gene were significantly(P〈0.01) increased in seeds of cv. Ningzhen No. 1 at the HTH stress time points 48, 96, and 168 h,and the expression level of GmHMADP gene were significantly(P〈0.01) increased in seeds of cv. Xiangdou No. 3 at the HTH stress time points 24, 48, 96, and 168 h. In addition, GmHMADP were significantly(P〈0.01) increased by Cu^2+ and Cd^2+ treatment with different concentrations in seedling roots of cv. Xiangdou No. 3, while in that of cv. Ningzhen No. 1, the expression level of GmHMADP gene wre significantly(P〈0.01) increased only under 50 μmol·L^-1 Cu^2+ and 200 μmol·L^-1 Cd^2+ treatment. The germination potential, germination rate, seed vigor of overexpressed Arabidopsis thaliana were significantly(P〈0.01) higher than that of the wild type plants under HTH stress. The root length and dry weight of transgenic Arabidopsis were significantly(P〈0.05) higher than that of the wild type undercopper and cadmium stress.【Conclusion】 The GmHMADP was involved in the formation of seed vigor under HTH and responded to copper and cadmium stress. Moreover, overexpression of GmHMADP in Arabidopsis could enhance the seed vigor and the tolerance to copper and cadmium stress.
作者
朱雅婧
周亚丽
刘骕骦
魏家萍
刘小林
沈英姿
赵海红
麻浩
ZHU YaJing;ZHOU YaLi;LIU SuShuang;WEI JiaPing;LIU XiaoLin;SHEN YingZi;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14期2642-2654,共13页
Scientia Agricultura Sinica
基金
国家自然科学基金(31371711)
关键词
大豆
GmHMADP
高温高湿
CU
CD
种子活力
soybean
GmHMADP
high temperature and high humidity stress
copper
cadmium
seed vigor