Study On Characterization Of Brassica Napus IMyAP Gene In Plant Defense Responses

In cruciferous plants, Myrosinase-associated protein is a important member in the glucosinolate myrosinase system, with myrosinase and myrosinase-binding protein formate complex. It could hydrolysis glucosinolate to various secondary metabolites. There are two different forms of Myrosinase-associated protein in the Brassica napus, one is woulding-induced Myrosinase-associated named iMyAP (inducible MyAP, iMyAP), mainly expression in the vegetative organs; the other specifically expressed in seeds, named seed-specific Myrosinase-associated protein (Seed-MyAP). In Arabidopsis thaliana, MyAP locates in the tonoplast, as a cellular transport protein, plays an important role to transport system of endometrium, MyAP also inhibits the nitrile and promotes the isothiocyanates formation in the process of hydrolysis glucosinolates. In Brassica napus, iMyAP is present in the vegetative organs as a induced proteins.the iMyAP gene was induced expression by the woulding,MeJA,JA and ABA, suggesting that iMyAP gene may be associated with plant defense responses, and our preliminary study found that both high temperature and insect feeding can induce iMyAP gene expression. We study on Characterization of Brassica napus iMyAP gene in plant defense responses by molecular biology and genetic engineering methods, obtained the following conclusion:1.Construction iMyAP RNAi gene interference vector (pYF280) and over-expression vector (pHB-1152). Handle cotyledons as explants, Agrobacterium mediated transformation of Brassica napus Xiangyou 15, from 2654 explants, obtained 242 resistant seedlings and 1 genetic stability transfer iMyAP RNAi interference vector transgenic plant by PCR detection. Conversion efficiency is 0.04%.2.With a semi-quantitative RT-PCR to analysis iMyAP genes expressied in different parts of Brassica napus Xiangyou 15, iMyAP gene is high expression in rapeseed silique, stems and leaves followed, flowers and roots is low expression.3. High temperature, low temperature, high salt, drought, pests, insect feeding, woulding and methyl jasmonic acid treatment 4h, the semi-quantitative RT-PCR analysis iMyAP genes expression in Brassica napus leaves. On the wound and JA treatment, iMyAP gene expression is in higher levels in Brassica napus leaves, followed by high temperature and pests;iMyAP gene is low expression by low temperature,high salt and drought treatment. Suggesting iMyAP gene is closely related to pests and heat stress. 4. By semi-quantitative RT-PCR detection, On the woulding treatments, the expression of iMyAP gene in transgenic plants leaves was inhibited. In seed of DAP 18 and DAP35, iMyAP gene expression have also been inhibited. Suggesting that iMyAP RNAi vector fragments was inserted into the genome DNA of Brassica napus inhibited iMyAP gene expression.5. JA treatment 4h,Analysised JA pathway marker genes PR4, VSP2 expression. the results show that:the JA pathway marker genes (PR4, VSP2, etc.) are higher expression in wild-type than the transgenic plants. Expression of iMyAP gene is inhibited make the expression of PR4, VSP2 also be inhibited in JA's treatments. Suggesting that iMyAP gene is a positive regulators to JA pathway marker genes PR4, VSP2 and transgenic plant was JA-insensitive.6. Woulding treatment 4h, Analysised JA pathway marker genes expression, the results show that:JA pathway woulding marker gene VSP2 and LOX1 are higher expression in the transgenic plants than the wild-type. whereas the other JA marker genes such as PR4 and PDF 1.2 gene in transgenic plants expression is lower than the wild type. expression of iMyAP gene was inhibited make expression of JA pathway marker genes VSP2 and LOX1 increased. Suggesting that iMyAP gene is a negative regulators to JA pathway woulding marker genes VSP2 and LOX1.