用基因工程技术创造低酚棉材料

发布时间：2018-07-29 17:37

【摘要】：棉酚与其它保护萜类都是次生代谢物,在植物对害虫的自我保护系统中增加植物的抵抗能力。(+)-δ-杜松烯合酶基因与棉酚合成过程有密切的关系。本课题分别构建(+)-δ-杜松烯合酶基因的两个亚组CAD1-c、CAD1-a目标序列的干涉载体并进行了遗传转化。其中,CAD1-a也构建了含有种子特异启动子的干涉载体,以期望得到种子中没有棉酚,而植株中含有棉酚的棉花材料。随后,用qRT-PCR技术验证转基因材料中目标基因和棉酚合成相关基因的转录水平。获得以下实验结果:(1)通过RT-PCR分析杜松烯合酶基因与棉酚合成相关基因的表达模式得出,FPS、P450、dHG-6-OMT在根和子叶中高度表达;CAD1-c在棉花茎、叶、萼片和果皮中有所表达;CAD1-a在这些组织中均未表达。(2)分析转基因棉花材料CAD基因表达水平发现,当干涉掉CAD1-c基因后,CAD1-a基因上调表达;干涉掉CAD1-a基因,CAD1-c基因上调表达,说明基因CAD1-a和基因CAD1-c在功能上可能有一定的互补作用。同时也发现把CAD1-a基因干涉很彻底时,CAD1-c基因的表达量也会很低,说明CAD1-a基因在一定程度上可以调控CAD1-c基因的表达。(3)CAD1-c基因参与的信号传导机制可能属于PTI途径,而CAD1-a基因参与的信号传导机制可能属于ETI途径。JA可能通过抑制WRKY1转录因子的表达来调控CAD1-a基因的表达。CAD1-c基因可能影响植物激素信号ABA的传导路径,并参与了植物激素信号传导和抗病原菌等过程。(4)分析转基因材料的转录组数据发现,编号为A-2(CAD1-a)的株系有122个基因上调表达,58个基因下调表达;C-15(CAD1-c)的株系有421个基因上调表达,115个基因下调表达。其中453个基因在CAD1-c中发生变化,97个在CAD1-a中发生变化,有83个基因在两个材料中都发生变化。(5)通过对转基因材料的棉酚测定发现,CAD1-a和CAD1-c基因的干涉系棉酚都有所降低,CAD1-a的效果更好。种子特异启动子转基因后代棉籽中棉酚的含量显著降低,如果棉籽直接用于养殖业和食品加工行业等领域,可以带来巨大的经济价值。
[Abstract]:Gossypol and other protective terpenoids are secondary metabolites, increasing the resistance of plants to the self protection system of pests. (+) - Delta Du synthase gene is closely related to the synthesis process of gossypol. Two subgroups of (+) - Delta - Du - Du synthase gene, CAD1-c, interference carrier of CAD1-a target sequence, are constructed. Genetic transformation was performed. Among them, CAD1-a also constructed an interference carrier containing seed specific promoters to expect to get no gossypol in the seed and cotton with cotton in the plant. Then, qRT-PCR technique was used to verify the transcriptional level of the target gene and the gossypol synthesis related basis in the transgenic materials. The following experimental results were obtained: (1) RT-PCR analysis of the expression patterns of the genes associated with duropase and gossypol synthesis showed that FPS, P450, dHG-6-OMT were highly expressed in the roots and cotyledon, and CAD1-c was expressed in the stems, leaves, sepals and pericarp of cotton, and CAD1-a was not expressed in these tissues. (2) the expression level of CAD gene in transgenic cotton material was found to be detected when interfering with CAD1 After the -c gene, the CAD1-a gene is up-regulated, interfering with the CAD1-a gene and the CAD1-c gene up-regulated, indicating that the gene CAD1-a and the gene CAD1-c may have some complementary functions. At the same time, when the interference of the CAD1-a gene is very thorough, the expression of the CAD1-c gene is also very low, indicating that the CAD1-a gene can regulate CAD1- to a certain extent. The expression of C gene. (3) the signal transduction mechanism of CAD1-c gene involvement may belong to the PTI pathway, and the signal transduction mechanism of the CAD1-a gene may belong to the ETI pathway.JA may regulate the expression of the CAD1-a gene by inhibiting the expression of the WRKY1 transcription factor, and the.CAD1-c gene may affect the conduction path of the plant hormone signal ABA and participate in the transplantation. (4) analysis of the transcriptional data of the transgenic materials found that there were 122 up-regulated and 58 down-regulated genes of A-2 (CAD1-a), and 421 C-15 (CAD1-c) genes were up-regulated and 115 genes were down regulated. 453 genes were changed in CAD1-c and 97. Change in CAD1-a, 83 genes were changed in two materials. (5) through the determination of gossypol in transgenic materials, it was found that gossypol in CAD1-a and CAD1-c genes decreased, and the effect of CAD1-a was better. The content of gossypol in seed specific promoter was significantly reduced, if cotton seeds were used directly for breeding. The fields of colonization and food processing can bring huge economic value.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562

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