白桦BpBEE基因的功能研究

发布时间：2018-05-24 09:38

  本文选题：白桦 + BpBEE2　； 参考：《东北林业大学》2016年硕士论文

【摘要】：Brassinolide Enhanced Expression(BEE)基因编码植物所特有的bHLH转录因子,是调控油菜素内酯信号转导的重要元件。本实验通过构建白桦BpBEE2基因的植物过表达载体和抑制载体,进行白桦的遗传转化,对获得的转基因株系进行生长量测定和盐、旱胁迫,验证白桦BpBEE2基因所发挥的作用。主要研究结果如下：1.根据白桦转录组数据,获得了3条白桦BpBEE基因,对3条白桦BpBEE基因进行生物信息学分析,结果表明,3条白桦BpBEE基因均具有典型的bHLH结构域,基因序列特性非常相似,且均有5个内含子与6个外显子。2.通过实时荧光定量PCR的方法,对3条白桦BpBEE基因的表达模式进行分析,结果表明,这3条基因在叶片,雌花,雄花,茎和顶芽中均有表达,叶中BpBEE基因表达量明显高于其他部位。在生长旺盛时期,叶和顶芽中BpBEE基因均呈现上调表达,表明BpBEE基因可能参与到白桦的生长过程。BR激素处理之后,BpBEE1在早期的芽和木质部中呈显著上调；BpBEE2在早期的木质部和韧皮部显著上调外,其他处理时间也呈现不同程度的上调。表明3个白桦BpBEE基因参与BR激素应答,且BpBEE1和BpBEE2呈现早期应答响应。3.应用Gateway技术构建白桦BpBEE2基因的植物过表达载体和酶切连接方法构建的白桦BpBEE2基因的植物抑制载体,采用根瘤农杆菌介导的合子胚法进行白桦的遗传转化,通过PCR鉴定转基因植株,结果表明,白桦BpBEE2基因已经成功整合到了植物基因组上,获得了1个超表达株系和5个抑制表达株系。4.对获得的转基因植株进行生长量测定。结果表明：超表达株系的生长量显著高于野生型,而抑制表达株系则明显低于野生型。表明超表达BpBEE2基因能提高植株的生长量。5.抗盐和抗旱的逆境胁迫分析,分别选取0.3%和0.6% NaCl;5%和10%PEG的胁迫处理,进行鲜重和生根率统计。结果表明：转基因株系并没有显著的提高抗盐旱性。6.克隆BpBEE1基因的启动子,通过原件预测,发现：该启动子含有很多与激素相关的元件；转基因拟南芥株系进行不同生长时期的组织特异性染色和激素处理后的GUS活性检测和染色,结果表明：转基因株系中GUS染色在叶脉处染色更重,MeJA处理后的GUS酶活性较未处理的明显提高,IAA和ABA处理后明显降低,与GUS染色结果相符。
[Abstract]:Brassinolide Enhanced expression bee (Bee) gene is a unique bHLH transcription factor in plants, which is an important element in the regulation of rapesinolide signal transduction. In this experiment, the plant overexpression vector and inhibition vector of Bai Hua BpBEE2 gene were constructed, and the genetic transformation of Bai Hua was carried out, and the growth of the transgenic lines was measured, salt and drought stress were used to verify the role of Bai Hua BpBEE2 gene. The main results are as follows: 1. Three Bai Hua BpBEE genes were obtained from Bai Hua transcriptome data, and three Bai Hua BpBEE genes were analyzed by bioinformatics. The results showed that all three Bai Hua BpBEE genes had typical bHLH domains, and the gene sequence characteristics were very similar. There were 5 introns and 6 exons. The expression patterns of three Bai Hua BpBEE genes were analyzed by real-time fluorescence quantitative PCR. The results showed that the three genes were expressed in leaves, female flowers, male flowers, stems and terminal buds. The expression of BpBEE gene in leaves was significantly higher than that in other parts. During the period of vigorous growth, the expression of BpBEE gene was up-regulated in both leaves and terminal buds. It was suggested that BpBEE gene might be involved in the growth process of Bai Hua. Br hormone treatment showed that BpBEE1 was upregulated significantly in early bud and xylem, while BpBEE2 was upregulated in early xylem and phloem, and other treatment time was also up-regulated in different degree. The results indicated that three Bai Hua BpBEE genes were involved in Br hormone response, and BpBEE1 and BpBEE2 showed early response. 3. The plant overexpression vector of Bai Hua BpBEE2 gene and the plant inhibition vector of Bai Hua BpBEE2 gene constructed by restriction endonuclease ligation were constructed by Gateway technique. The Bai Hua genetic transformation was carried out by zygotic embryo mediated by Agrobacterium tumefaciens (Agrobacterium tumefaciens). The results of identification of transgenic plants by PCR showed that Bai Hua BpBEE2 gene had been successfully integrated into the plant genome, and one super-expression strain and five suppressive expression lines .4were obtained. The growth of transgenic plants was measured. The results showed that the growth of overexpression lines was significantly higher than that of wild type, while the growth of suppressive expression lines was significantly lower than that of wild type. The results showed that overexpression of BpBEE2 gene could increase plant growth. The stress treatments of 0.3% and 0.6% NaClO 5% and 10%PEG were used to calculate the fresh weight and rooting rate respectively. The results showed that the transgenic lines did not significantly improve salt drought tolerance. 6. The promoter of BpBEE1 gene was cloned. By original prediction, it was found that the promoter contained a lot of hormone related elements, and the transgenic Arabidopsis thaliana lines were tested for tissue specific staining and GUS activity after hormone treatment at different growth stages. The results showed that the GUS enzyme activity of the transgenic lines treated with GUS at the leaf vein was significantly higher than that of the untreated ones, which was consistent with the results of GUS staining.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.153

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