杨树LAR基因的克隆及表达对儿茶素合成的影响  

Cloning of Poplar Leucoanthocyanidin reductase gene(LAR) and transgenic analysis on catechin synthesis in tobacco

作　　者：左涛[1,2,3] 包海[2,3] 陈慧[2,3] 苏衍修 常聚普[4] 郭利民[4] 贺伟[1] 王延伟[2,3] 

机构地区：[1]北京林业大学林木有害生物防治北京市重点实验室,北京100083 [2]北京林业大学北京林业大学林木育种国家工程实验室,北京100083 [3]北京林业大学林木花卉遗传育种教育部重点实验室,北京100083 [4]濮阳市林业科学院,河南濮阳457000

出　　处：《中南林业科技大学学报》2016年第12期121-128,共8页Journal of Central South University of Forestry & Technology

基　　金：国家林业公益性行业科研专项(201104054);国家自然科学基金(31470668;31200511;J1103516)

摘　　要：无色花色素还原酶基因(Leucoanthocyanidin reductase,LAR)是类黄酮生物合成途径中的一个重要基因,为了验证杨树中LAR的功能,明确LAR基因对抗病物质儿茶素合成的影响,本实验以接种后第6 d的一年生‘中林46’杨树树干的树皮为材料,利用RT-PCR技术克隆了LAR基因的OFR序列,并构建LAR的过表达载体p CAMBIA1301-LAR。采用农杆菌介导的叶盘法转化烟草,获得了转LAR基因的烟草6株。用q RT-PCR检测转基因烟草的表达量,结果显示,在转基因植株中,LAR基因的表达量均显著上调。用高效液相色谱法检测转基因植株中的儿茶素含量,结果显示,6株转LAR的烟草植株中,有4株内源儿茶素含量显著升高。以上结果表明,中林46杨LAR是杨树类黄酮生物合成途径中的一个基因,该基因与儿茶素的合成有关。Leucoanthocyanidin reductase gene （LAR） is one of important genes in flavonoids biosynthesis pathway. This investigation was conducted in order to verify function of LAR, and the influence on the synthetsis of disease-resistant substances catechins. Total RNA was isolated from the infected bark of Populus X euramericana cv. ‘Zhonglin 46＇ 6 days after inoculation with Lonsdalea quercina subsp, populi. A specific/MR fragment of about 1000 bp was amplified by RT-PCR and sequenced, and then successfully constructed into an expression vector with sense-orientation driven by CaMV 35S promoter （pCAMBIA1301-/MR）. Subsequently, Pcambial301- /MR was transformed into tobacco using Agrobacterium tumefaciens mediated method. Six transgenic lines was finally obtained and confirmed by PCR. qRT-PCR was further used to detect the expression of LAR in transgenic tobacco, which showed that LAR gene was significantly upregulated in transgenic lines compared to the wild type. High performance liquid chromatography （HPLC） was further used to detect the content of catechin in transgenic plants and wild type plants respectively. The results showed that the content of catechin in 4 transgenic plants were increased, however there were no catechin detected in 2 transgenic plants. The study shown that /MR ofPopulus×euramericana cv. ‘Zhonglin 46＇ is one of genes involved in flavonoids biosynthesis pathway and is associated with the synthesis of catechin, which provides insights to the molecular mechanism of LAR controlling catechin synthesis and the utilization of resistance genes in the improvement of plant disease resistance.

关 键 词：类黄酮 无色花色素还原酶基因 过表达 儿茶素 

分 类 号：S792.11[农业科学—林木遗传育种] S718.46[农业科学—林学]