红柄甜菜酪氨酸酶基因及其启动子的功能解析

发布时间：2018-07-10 09:12

  本文选题：启动子 + 酪氨酸酶基因　； 参考：《中国农业大学》2016年博士论文

【摘要】：甜菜素是一种水溶性含氮色素,在多数石竹目植物中替代花青素赋予植物以艳丽的颜色。它的生物合成涉及到三步关键酶促反应：羟基化L-酪氨酸形成L-DOPA.氧化L-DOPA生成cyclo-DOPA、转化L-DOPA为甜菜醛氨酸(生色基团)。甜菜醛氨酸与氨基酸或胺“自发”缩合形成甜菜黄素,但与cyclo-DOPA"自发”缩合形成红色的甜菜苷配基。多年来学术界推测PPO型酪氨酸酶参与了前两步反应,但缺乏分子生物学证据。本实验室从红柄甜菜叶中纯化到了酪氨酸酶并克隆了基因BycTYR,为了研究该基因在甜菜素合成中的作用,本研究在克隆与解析其启动子的同时,还共表达了BvcTYR与介导第三步的DOD。主要结果如下：从红柄甜菜叶中克隆到了BvcTYR编码区的5’端侧翼区域,长度为1670 bp,预测结果显示它在3’端含有转录起始位点(TSS)、TATA-box、CAAT-box等调控元件,具有TATA-box型启动子的特征,将其命名为BvcPPOP。用BvcPPOP驱动GUS基因经农杆菌介导导入花青素植物拟南芥。GUS染色和定量分析结果显示,3vcPPOP::GUS在T3代转基因拟南芥的营养器官特异性表达,特别是在根和叶柄中优势表达,而在角果、种子、花序及花序叶中的表达不明显。它的表达强度受发育阶段的影响,但表达模式几乎不变。BvcPPOP::GUS的表达受非生物胁迫的影响,SA、MeJA能上调其表达,而NaCl、ABA.高pH值(pH8.0)、GA和甘露醇能下调其表达。为了探寻控制营养器官特异性表达的元件和获取核心启动子,对BvcPPOP进行了5’端删除,获得了4个不同长度的短截片段(F2-F5)。与BvcPPOP相比,4个短截片段驱动GUS基因在T3代拟南芥中的表达强度变化较大,特别是从F3开始,表达强度急剧减弱,但是其表达模式却未见明显变化。最短的短截片段(247 bp,F5)仍保留了在根和叶柄中优势表达的模式。在本生烟中瞬时共表达了BvcTYR与多种生物来源的DOD,结果显示BvcTYR与BvcDODAl共表达的本生烟叶片在蓝光下具有最强的甜菜黄素特征荧光信号。稳定表达BvcTYR和BvcTYR+BvcDODA1的普通烟草的叶盘均能催化酪氨酸/酪胺生成红/黄色物质。该红色物质在470 nm左右有一个吸收峰,可能是Dopa-quinone。这些结果为酪氨酸酶参与甜菜素生物合成的第一步酶促反应提供了分子生物学支持,也为在营养器官中表达目标基因提供了有用的工具。
[Abstract]:Betaine is a water-soluble nitrogen-containing pigment that replaces anthocyanins in most carnation plants and gives them brilliant colors. Its biosynthesis involves three key enzymatic reactions: hydroxylation of L-tyrosine to L-DOPA. Cyclo-DOPA was synthesized by oxidation of L-DOPA and transformed into betaine aldehydic acid (chromophore group). Betaine was "spontaneously" condensed with amino acids or amines to form betaine, but cyclo-DOPA "spontaneously" condensed to form red betaine ligand. For many years, it has been speculated that PPO tyrosinase was involved in the first two reactions, but there was no evidence of molecular biology. Tyrosinase was purified from sugarbeet leaves and the gene BycTYR was cloned. In order to study the role of BycTYR in the biosynthesis of betaine, BvcTYR and DODmediated the third step were co-expressed while its promoter was cloned and analyzed. The main results are as follows: the 5'flanking region of the coding region of BvcTYR was cloned from the leaves of beet with a length of 1670 BP. The predicted results show that it contains regulatory elements such as TATA-box CA-box at the 3'end, and has the characteristics of TATA-box promoter. It was named BvcPPOP. The results of staining and quantitative analysis of Gus gene mediated by Agrobacterium tumefaciens in Arabidopsis thaliana, Arabidopsis thaliana (Arabidopsis thaliana) introduced by BvcPPOP, showed that 3vcPPOP: Gus was specifically expressed in vegetative organs of transgenic Arabidopsis thaliana at generation T3, especially in root and petiole, but in pod. The expression of seed, inflorescence and inflorescence leaves was not obvious. Its expression intensity was affected by the developmental stage, but the expression pattern was almost unchanged. The expression of BvcPPOP: Gus was affected by abiotic stress. High pH (pH 8.0) GA and mannitol could down-regulate its expression. In order to search for elements to control the specific expression of vegetative organs and obtain core promoters, BvcPPOP was deleted at 5'end and four truncated fragments (F2-F5) with different lengths were obtained. Compared with BvcPPOP, the expression intensity of Gus gene driven by four truncated fragments in T3 generation Arabidopsis thaliana changed greatly, especially since F3, the expression intensity decreased sharply, but the expression pattern of Gus gene did not change obviously. The shortest truncated fragment (247bpF5) still retained the dominant expression pattern in root and petiole. BvcTYR and several biological sources of DOD were coexpressed in BvcTYR and BvcDODAl. The results showed that BvcTYR and BvcDODAl co-expressed BvcTYR and BvcDODAl had the strongest beetroflavin characteristic fluorescence signal under blue light. Both BvcTYR and BvcTYR BvcDODA1 can catalyze tyrosine / tyramine to produce red / yellow substance. The red substance has an absorption peak at about 470 nm, possibly Dopa-quinone. These results provide molecular biology support for tyrosinase to participate in the first step enzymatic reaction of betaine biosynthesis and a useful tool for the expression of target genes in vegetative organs.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2
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本文编号：2112899