杨树二氢黄酮醇-4-还原酶基因(DFR)的克隆及反义表达对儿茶素合成的影响

为了验证二氢黄酮醇-4-还原酶基因(DFR)在杨树上的功能,明确DFR对抗病物质儿茶素合成的影响,利用抗病基因防治树木溃疡病提供候选基因。以接种欧美杨细菌性溃疡病菌后6 d的一年生中林46杨树苗干的树皮为材料,利用RT-PCR技术克隆DFR基因的ORF序列,并构建DFR的反义表达载体anti-p BI121-DFR。采用农杆菌介导叶盘法转化84K杨,获得了转反义DFR基因的84K杨4株。用高效液相色谱法检测转基因植株叶片中的儿茶素质量分数,结果显示,4株转反义DFR株其内源儿茶素质量分数分别为0.97、2.4、1.6和0.87 ng/g,与84K野生型植株中儿茶素质量分数(8.9 ng/g)相比显著降低。上述结果表明DFR基因参与了杨树类黄酮生物合成途径,该基因与儿茶素的合成有关。

Cloning Dihydroflavonol-4-Reductase Gene (DFR) of Poplar and Its Antisense Expression Effects on Catechin Synthesis

The experiment was conducted to verify the function of Dihydro flavonol-4-reductase gene ( DFR) , and analyze the in-fluence on the synthesis of disease-resistant substances catechins for the use of resistance genes resistant canker providing candidate genes.Total RNA was isolated from the infected bark of Populus×euramericana‘Zhonglin 46’ after inoculating 6 days with Lonsdalea quercina subsp.populi.A specific DFR fragment of the expected size was amplified by RT-PCR and sequenced, and then successfully constructed into an expression vector with antisense-orientation driven by CaMV 35S pro-moter ( anti-pBI121-DFR ) .Subsequently, anti-pBI121-DFR was transformed into Populus alba ×P.glandulosa using Agrobacterium tumefaciens mediated method.Four transgenic lines was finally obtained and confirmed by PCR.High per-formance liquid chromatography (HPLC) was further used to detect catechin in transgenic plants and wild type plants.The contents of catechin in transgenic plants were 0.97, 2.4, 1.6, and 0.87 ng/g, respectively, significantly lower than that of wild type plants (8.9 ng/g).Therefore, DFR of Populus×euramericana‘Zhonglin 46’ was involved in flavonoids biosyn-thesis pathway, and associated with the synthesis of catechin.