【摘要】：在許多植物中,二氫黃酮醇-4-還原酶(Dihydroflavonol-4-Reductase, DFR)和無色花色素還原酶(Leucoanthocyanidin reductase, LAR)是抗菌物質(zhì)兒茶素形成步驟中的兩個(gè)關(guān)鍵酶。但在楊樹中,這兩種酶是否對(duì)兒茶素的合成途徑產(chǎn)生影響作用并不清楚。為研究楊樹中DFR和LAR基因在歐美楊細(xì)菌性潰瘍病菌(Lonsdalea quercina subsp. populi)侵染后的表達(dá)量變化,探索DFR和LAR與兒茶素合成之間的關(guān)系,本研究通過對(duì)3個(gè)不同抗性品種的楊樹在田間進(jìn)行歐美楊細(xì)菌性潰瘍病菌的接種,利用熒光定量PCR和高效液相色譜法(HPLC)分析不同楊樹品種在接種病原菌后不同的時(shí)間段中DFR和LAR的表達(dá)量差異和兒茶素的含量變化,并從中林46楊(Populus× euramericana cv.'Zhonglin 46')中克隆出DFR和LAR的ORF序列,構(gòu)建植物表達(dá)載體,轉(zhuǎn)化受體植物。用qRT-PCR檢測(cè)轉(zhuǎn)基因植株中相應(yīng)基因的表達(dá)量,并用HPLC法檢測(cè)轉(zhuǎn)基因植株中兒茶素的含量。以期為抗病基因工程育種提供候選基因。研究主要結(jié)果如下：對(duì)‘中菏1號(hào)’、‘107楊’和‘河南毛白楊’三個(gè)不同品種的一年生楊樹苗在田間進(jìn)行歐美楊細(xì)菌性潰瘍病菌的接種,根據(jù)對(duì)接種后不同時(shí)間段發(fā)病情況的觀察,‘中菏1號(hào)’發(fā)病最為嚴(yán)重,為高感品種；‘107楊’次之,為感病品種；‘河南毛白楊’不發(fā)病,為抗病品種。不同抗性品種楊樹的DFR和LAR兩個(gè)基因?qū)儾【那秩井a(chǎn)生不同的響應(yīng)。三個(gè)楊樹品種中兒茶素的含量在病原菌的脅迫下均發(fā)生顯著變化,且根據(jù)品種間抗病性的不同呈現(xiàn)出差異。兒茶素的上升比例大小為：抗病品種感病品種高感品種。表明兒茶素能夠?qū)儾【龀鲰憫?yīng),且兒茶素與楊樹的抗病性有關(guān)�？寺〕鯠FR基因和LAR基因的ORF序列,構(gòu)建CaMV 35S啟動(dòng)子調(diào)控的反義表達(dá)載體anti-pBI121-DFR和正義表達(dá)載體pCAMBIA1301-LAR。反義表達(dá)載體anti-pBI121-DFR通過農(nóng)桿菌介導(dǎo)轉(zhuǎn)化84K楊,得到4株完整的轉(zhuǎn)基因植株。轉(zhuǎn)基因株系中兒茶素的含量與野生型植株相比均降低,且差異顯著。將正義表達(dá)載體pCAMBIA 1301-LAR通過農(nóng)桿菌介導(dǎo)轉(zhuǎn)化煙草,得到6株完整的轉(zhuǎn)基因煙草。在轉(zhuǎn)基因植株中,LAR基因的表達(dá)量均顯著上調(diào),且有4株轉(zhuǎn)基因煙草中內(nèi)源兒茶素的含量大幅度增加。上述研究結(jié)果表明,楊樹中基因DFR和LAR在受到歐美楊細(xì)菌性潰瘍病菌侵染后其表達(dá)量出現(xiàn)明顯變化,中林46楊中DFR基因和LAR基因參與兒茶素的合成,且兒茶素可抑制病原菌的生長(zhǎng)。這為通過轉(zhuǎn)基因方法利用兒茶素合成相關(guān)基因增強(qiáng)樹木抗病性,提供了理論參考。
[Abstract]:In many plants, dihydroflavonol-4-reductase (Dihydroflavonol-4-Reductase, DFR) and colorless anthocyanin reductase (Leucoanthocyanidin reductase, LAR) are two key enzymes in the formation of catechin. But in poplar, it is not clear whether these two enzymes affect catechin synthesis pathway. To study the expression of DFR and LAR genes in poplar (Poplar) by bacterial ulcer pathogen (Lonsdalea quercina subsp.) in Euramerican poplar. To explore the relationship between DFR, LAR and catechin synthesis, three varieties of poplar with different resistance were inoculated in the field by bacterial ulcer pathogen of Euramerican poplar. Fluorescence quantitative PCR and high performance liquid chromatography (HPLC) were used to analyze the difference of DFR and LAR expression and catechin content in different poplar varieties at different time after inoculation. The ORF sequences of DFR and LAR were cloned from Populus 脳 euramericana cv.'Zhonglin 46', and the plant expression vector was constructed to transform the recipient plants. The expression of corresponding genes in transgenic plants was detected by qRT-PCR and the catechin content in transgenic plants was detected by HPLC method. In order to provide candidate genes for genetic engineering breeding of disease resistance. The main results were as follows: the annual poplar seedlings of 'Zhonghe 1', '107 poplar' and 'Henan white poplar' were inoculated in the field by bacterial ulcer bacteria of American and European poplar. According to the observation of the incidence in different time after inoculation, Zhonghe 1 was the most serious and highly susceptible variety. '107 poplar' was the second susceptible variety, and 'Populus tomentosa' had no disease and was resistant to disease. The DFR and LAR genes of poplar cultivars with different resistance had different responses to the infection of ulcers. The catechin content in the three poplar varieties changed significantly under the stress of pathogen, and there were differences according to the disease resistance of the three poplar varieties. The increasing proportion of catechin was: resistant varieties and high susceptible varieties. The results showed that catechin was able to respond to ulcers, and catechin was related to the resistance of poplar. The ORF sequences of DFR gene and LAR gene were cloned, and the antisense expression vector anti-pBI121-DFR and the sense expression vector pCAMBIA1301-LAR. regulated by CaMV 35S promoter were constructed. The antisense expression vector anti-pBI121-DFR was transformed into 84 K poplar by Agrobacterium tumefaciens and 4 intact transgenic plants were obtained. The catechin content in transgenic lines was significantly lower than that in wild type plants. The sense expression vector pCAMBIA 1301-LAR was transformed into tobacco by Agrobacterium tumefaciens. In transgenic plants, the expression of LAR gene was significantly up-regulated, and the content of endogenous catechins in 4 transgenic tobacco plants increased significantly. The results showed that the expression of DFR and LAR in poplar was significantly changed after infection by bacterial ulcer pathogen of American and European poplar. The DFR gene and LAR gene of Zhonglin 46 poplar were involved in the synthesis of catechin. And catechin can inhibit the growth of pathogenic bacteria. This provides a theoretical reference for using catechin biosynthesis genes to enhance tree disease resistance through transgenic methods.
【學(xué)位授予單位】：北京林業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S792.11

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1 左濤;楊樹兒茶素合成相關(guān)基因DFR和LAR的克隆與功能初步分析[D];北京林業(yè)大學(xué);2016年

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本文編號(hào)：2382242