本文選題：啟動(dòng)子 + 酪氨酸酶基因�。� 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：甜菜素是一種水溶性含氮色素,在多數(shù)石竹目植物中替代花青素賦予植物以艷麗的顏色。它的生物合成涉及到三步關(guān)鍵酶促反應(yīng)：羥基化L-酪氨酸形成L-DOPA.氧化L-DOPA生成cyclo-DOPA、轉(zhuǎn)化L-DOPA為甜菜醛氨酸(生色基團(tuán))。甜菜醛氨酸與氨基酸或胺“自發(fā)”縮合形成甜菜黃素,但與cyclo-DOPA"自發(fā)”縮合形成紅色的甜菜苷配基。多年來學(xué)術(shù)界推測PPO型酪氨酸酶參與了前兩步反應(yīng),但缺乏分子生物學(xué)證據(jù)。本實(shí)驗(yàn)室從紅柄甜菜葉中純化到了酪氨酸酶并克隆了基因BycTYR,為了研究該基因在甜菜素合成中的作用,本研究在克隆與解析其啟動(dòng)子的同時(shí),還共表達(dá)了BvcTYR與介導(dǎo)第三步的DOD。主要結(jié)果如下：從紅柄甜菜葉中克隆到了BvcTYR編碼區(qū)的5’端側(cè)翼區(qū)域,長度為1670 bp,預(yù)測結(jié)果顯示它在3’端含有轉(zhuǎn)錄起始位點(diǎn)(TSS)、TATA-box、CAAT-box等調(diào)控元件,具有TATA-box型啟動(dòng)子的特征,將其命名為BvcPPOP。用BvcPPOP驅(qū)動(dòng)GUS基因經(jīng)農(nóng)桿菌介導(dǎo)導(dǎo)入花青素植物擬南芥。GUS染色和定量分析結(jié)果顯示,3vcPPOP::GUS在T3代轉(zhuǎn)基因擬南芥的營養(yǎng)器官特異性表達(dá),特別是在根和葉柄中優(yōu)勢表達(dá),而在角果、種子、花序及花序葉中的表達(dá)不明顯。它的表達(dá)強(qiáng)度受發(fā)育階段的影響,但表達(dá)模式幾乎不變。BvcPPOP::GUS的表達(dá)受非生物脅迫的影響,SA、MeJA能上調(diào)其表達(dá),而NaCl、ABA.高pH值(pH8.0)、GA和甘露醇能下調(diào)其表達(dá)。為了探尋控制營養(yǎng)器官特異性表達(dá)的元件和獲取核心啟動(dòng)子,對BvcPPOP進(jìn)行了5’端刪除,獲得了4個(gè)不同長度的短截片段(F2-F5)。與BvcPPOP相比,4個(gè)短截片段驅(qū)動(dòng)GUS基因在T3代擬南芥中的表達(dá)強(qiáng)度變化較大,特別是從F3開始,表達(dá)強(qiáng)度急劇減弱,但是其表達(dá)模式卻未見明顯變化。最短的短截片段(247 bp,F5)仍保留了在根和葉柄中優(yōu)勢表達(dá)的模式。在本生煙中瞬時(shí)共表達(dá)了BvcTYR與多種生物來源的DOD,結(jié)果顯示BvcTYR與BvcDODAl共表達(dá)的本生煙葉片在藍(lán)光下具有最強(qiáng)的甜菜黃素特征熒光信號。穩(wěn)定表達(dá)BvcTYR和BvcTYR+BvcDODA1的普通煙草的葉盤均能催化酪氨酸/酪胺生成紅/黃色物質(zhì)。該紅色物質(zhì)在470 nm左右有一個(gè)吸收峰,可能是Dopa-quinone。這些結(jié)果為酪氨酸酶參與甜菜素生物合成的第一步酶促反應(yīng)提供了分子生物學(xué)支持,也為在營養(yǎng)器官中表達(dá)目標(biāo)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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2
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本文編號：2112899