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WRKY蛋白属于锌指型转录调控因子,能够参与植物多种逆境响应。本研究利用前期野生大豆盐碱胁迫RNA-seq测序数据,从构建的碱胁迫基因调控网络中筛选并克隆到GsWRKY15基因。分析GsWRKY15在碱胁迫下野生大豆根中的表达模式,发现该基因受碱胁迫诱导显著上调表达,且在胁迫后1 h表达量最高。分析GsWRKY15基因在野生大豆各组织中的表达特异性,发现该基因在各组织中均有表达,花中表达量最高。采用根癌农杆菌侵染苜蓿子叶节方法,将GsWRKY15转化肇东苜蓿,获得39株抗性植株。通过PCR、Southern blot和RT-PCR方法分析抗性植株,获得了超量表达GsWRKY15基因的转基因株系并对其进行了耐碱性分析。在150 mmol L–1 Na HCO3处理2周后转基因苜蓿生长状态良好,而非转基因苜蓿出现萎蔫、变黄,甚至死亡;非转基因苜蓿的相对质膜透性和丙二醛含量显著高于转基因苜蓿,而叶绿素含量显著低于转基因苜蓿;同时分析碱胁迫下转基因植株中胁迫相关基因的表达模式,发现H+-Ppase、NADP-ME、KIN1、RD29A基因的表达量高于非转基因苜蓿。结果表明GsWRKY15基因的超量表达能够显著增强苜蓿的耐碱能力。
WRKY protein is a zinc finger transcriptional regulator that can participate in a variety of plant stress response. In this study, we sequenced and sequenced the RNA-seq of wild-type soybean (Glycine max) under salt stress, and then screened the GsWRKY15 gene from the regulatory network of constructed alkali stress genes. The expression pattern of GsWRKY15 in the roots of wild soybean under alkali stress was analyzed. It was found that the expression of GsWRKY15 was significantly up-regulated by alkali stress and reached the highest at 1 h after stress. Analysis of GsWRKY15 gene expression in wild soybean tissue specificity and found that the gene expression in all tissues, the highest expression in the flower. Agrobacterium tumefaciens was used to infect alfalfa cotyledons section, and GsWRKY15 was transformed into Zhaodong alfalfa to obtain 39 resistant plants. The resistant plants were analyzed by PCR, Southern blot and RT-PCR. The transgenic lines that over-expressed GsWRKY15 gene were obtained and analyzed for their alkali resistance. The transgenic alfalfa grew well after two weeks of treatment with 150 mmol L -1 Na HCO 3, but non-transgenic alfalfa appeared wilting, yellowing and even death. The relative plasma membrane permeability and malondialdehyde content of non-transgenic alfalfa were significantly higher than those of transgenic alfalfa , While chlorophyll content was significantly lower than that in transgenic alfalfa. Meanwhile, the expression patterns of stress-related genes in transgenic plants under alkali stress were also analyzed. The results showed that the expression of H + -Ppase, NADP-ME, KIN1 and RD29A genes was higher than that of non-transgenic alfalfa. The results showed that GsWRKY15 gene overexpression can significantly enhance alfalfa’s alkali resistance.