杨树儿茶素合成相关基因DFR和LAR的克隆与功能初步分析
发布时间：2018-12-16 11:13
【摘要】：在许多植物中,二氢黄酮醇-4-还原酶(Dihydroflavonol-4-Reductase, DFR)和无色花色素还原酶(Leucoanthocyanidin reductase, LAR)是抗菌物质儿茶素形成步骤中的两个关键酶。但在杨树中,这两种酶是否对儿茶素的合成途径产生影响作用并不清楚。为研究杨树中DFR和LAR基因在欧美杨细菌性溃疡病菌(Lonsdalea quercina subsp. populi)侵染后的表达量变化,探索DFR和LAR与儿茶素合成之间的关系,本研究通过对3个不同抗性品种的杨树在田间进行欧美杨细菌性溃疡病菌的接种,利用荧光定量PCR和高效液相色谱法(HPLC)分析不同杨树品种在接种病原菌后不同的时间段中DFR和LAR的表达量差异和儿茶素的含量变化,并从中林46杨(Populus× euramericana cv.'Zhonglin 46')中克隆出DFR和LAR的ORF序列,构建植物表达载体,转化受体植物。用qRT-PCR检测转基因植株中相应基因的表达量,并用HPLC法检测转基因植株中儿茶素的含量。以期为抗病基因工程育种提供候选基因。研究主要结果如下：对‘中菏1号’、‘107杨’和‘河南毛白杨’三个不同品种的一年生杨树苗在田间进行欧美杨细菌性溃疡病菌的接种,根据对接种后不同时间段发病情况的观察,‘中菏1号’发病最为严重,为高感品种；‘107杨’次之,为感病品种；‘河南毛白杨’不发病,为抗病品种。不同抗性品种杨树的DFR和LAR两个基因对溃疡病菌的侵染产生不同的响应。三个杨树品种中儿茶素的含量在病原菌的胁迫下均发生显著变化,且根据品种间抗病性的不同呈现出差异。儿茶素的上升比例大小为：抗病品种感病品种高感品种。表明儿茶素能够对溃疡病菌做出响应,且儿茶素与杨树的抗病性有关。克隆出DFR基因和LAR基因的ORF序列,构建CaMV 35S启动子调控的反义表达载体anti-pBI121-DFR和正义表达载体pCAMBIA1301-LAR。反义表达载体anti-pBI121-DFR通过农杆菌介导转化84K杨,得到4株完整的转基因植株。转基因株系中儿茶素的含量与野生型植株相比均降低,且差异显著。将正义表达载体pCAMBIA 1301-LAR通过农杆菌介导转化烟草,得到6株完整的转基因烟草。在转基因植株中,LAR基因的表达量均显著上调,且有4株转基因烟草中内源儿茶素的含量大幅度增加。上述研究结果表明,杨树中基因DFR和LAR在受到欧美杨细菌性溃疡病菌侵染后其表达量出现明显变化,中林46杨中DFR基因和LAR基因参与儿茶素的合成,且儿茶素可抑制病原菌的生长。这为通过转基因方法利用儿茶素合成相关基因增强树木抗病性,提供了理论参考。
[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.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.11
本文编号：2382242
[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.
【学位授予单位】：北京林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.11
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1 左涛;杨树儿茶素合成相关基因DFR和LAR的克隆与功能初步分析[D];北京林业大学;2016年
，本文编号：2382242
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