GmHMADP参与高温高湿下大豆种子活力形成及铜镉胁迫响应的研究

doi:10.3864/j.issn.0578-1752.2018.14.002

Abstract

Abstract: 【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 35S::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 qRT-PCR. Simultaneously, pBI121 with XbaⅠand BamHⅠ 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 168h,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 168h. In addition, GmHMADP were significantly (P＜0.01) increased by Cu²⁺ and Cd²⁺ 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 Cu²⁺ and 200 μmol·L^-1 Cd²⁺ 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.

Key words: soybean, GmHMADP, high temperature and high humidity stress, copper, cadmium, seed vigor

ZHU YaJing, ZHOU YaLi, LIU SuShuang, WEI JiaPing, LIU XiaoLin, SHEN YingZi, ZHAO HaiHong, MA Hao. GmHMADP Involved in Seed Vigor Formation of Soybean Under High Temperature and Humidity Stress and its Study Responsive to Copper and Cadmium Stress[J].Scientia Agricultura Sinica, 2018, 51(14): 2642-2654.

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