Cloning And Functional Characterization Of DFR And LAR Associated With Catechins Synthesis In Poplar

In many plants, Dihydroflavonol-4-Reductase(DFR) and Leucoanthocyanidin Reductase(LAR) are two key enzymes in antibacterial material catechin biosynthesis. However, whether the two enzymes have effects on synthesis way of catechin is not clear in poplar. In order to study the differential expression of DFR gene and LAR gene related to the way of catechin synthesis after inoculated with Lonsdalea quercina subsp. populi, and to explore the relationship between two genes and catechins synthesis, quantitative real-time PCR and high performance liquid chromatography were used to detect the expression of DFR、LAR, and content of catechin in three different resistant varieties of poplar inoculated with Lonsdalea quercina subsp. populi. The ORF sequences of DFR and LAR from Populus x euramericana were cloned and plant expression vectors were constructed, and transformed them into plants. Quantitative real-time PCR was used to detect expression of genes in transgenic plants, and high performance liquid chromatography was used to detect the contents of catechins in transgenic plants. The study may provide candidate genes for disease resistance genetic engineering breeding. The main results are as follows:Three varieties of one year old poplar seedling(P. deltoids cl.Zhonghe-1’、Populus ×euramericanacv.’74/76’、Populus tomentosa cv.’henan’) were inoculated with canker pathogen in the field. Based on disease incidence after inoculation in different time, "Zhonghe 1", seriously diseased, was highly susceptible variety, and Populus ×euramericana cv.’74/76’ also diseased, but was not serious, Populus tomentosa cv.’henan’ was resistant. The DFR and LAR genes had differential expression and the content of catechin had significant changes under the stress of the pathogen in different varieties of poplar. The rising ratio of catechins in three poplars were in high resistant variety> in medium resistance varity> in susceptible variety. The results showed that catechin can respond to canker pathogen infection, and be connect with the disease resistance of poplar.ORF sequences of DFR and LAR genes were cloned, and antisense expression vector anti-pBI121-DFR and sense expression vector pCAMBIA 1301-LAR were constructed by CaMV 35S promoter regulation. Antisense expression vector anti-pBI121-DFR was transformed into Populus alba×Populus glandulosa using Agrobacterium tumefaciens mediated method. Four complete transgenic plants were obtained, and the content of catechin in transgenic plants were significantly lower than that of wild type plants. Sense expression vector pCAMBIA1301-LAR was transformed into tobacco using Agrobacterium tumefaciens mediated method. Six complete transgenic plants were obtained. The expression of LAR gene in transgenic plants was significantly higher, and the endogenous content of catechins were significantly increased in four transgenic plants.The above research results show that the expression of DFR and LAR genes in poplar were significantly changed after infected with canker pathogen, and the DFR and LAR genes were involved in the synthesis of catechins in Populus xeuramericana, and the growth of pathogen can be inhibited by catechin in vitro. This will provide the theoretical reference for enhancing tree disease resistance through using the genes related to catechin synthesis by transgenic method.