Cloning And Preliminary Functional Study Of Glucosinolates Transport Genes BnGTRs In Rapeseed(Brassica Napus L.)

Rapeseed is one of the major oilseed crops in China. Rapeseed meal is a good feed stuffs for livestock. However, the hydrolysis products from glucosinolates in Rapeseed meal have negative effect on livestock. On the other hand, glucosinolates and their hydrolytic products play important role in plant defense system. Various glucosinolates have different effects on resistance to insect and disease. Generally, the reduction of glucosinolate content in rapeseed associates with concomitant decrease of glucosinolate content in leaves, and it will in turn results in susceptibility to insect and disease. The contradiction would influcen the extension of “double low” canola. Therefore, it is important to control glucosinolate distribution in plant including high content in vegetative organs and low content in oilseed. In previous study, At GTRs genes have been identified as glucosinolate transporters in Arabidopsis thaliana. In our research, Bn GTR1 and Bn GTR2 have been isolated in Brassica napus, using GENE RACE technology. Then we explored biological function of Bn GTRs by utilizing ami RNA technology. The main results are as follows:1. The glucosinolates contents in leaves and seed of several varieties with high middle and low glucosinolates content.In leaves, the difference of glucosinolates content is mainly Aliphatic glucosinolates, which is refledted in 2-hydroxy-3-butenyl-GSL and 4-pentenyl-GSL. And no difference was detected in indole glucosinolates.In seeds, the analysis shows the similarity with leaves in glucosinolates content.2. Isolation and architectural feature of Bn GTRsBn GTRs were cloned by the homologous sequence method. Their ORFs are the same length with 1839 bp. They encode 612 amino acids. The structure and functional domain was predicted by SMART and the result showed that Bn GTRs contain a putative transmembrane domain PTR2, which is conserved in At GTRs. Then, we conducted GFP fluorescent subcellular localization. The result confirmed that Bn GTRs are mainly located on the plasma membrane.3. The biological function of Bn GTRsUsing ami RNA technology, we gained T0 transgenic plants. By Real-time PCR, we found the decreased expression of Bn GTR2 in T0 plants. Furthermore, HPLC analysis showed that total content of glucosinolates in the T0 plants has significant reduction than the negative control, especially in aliphatic glucosinolates content. There were no significant differences in the indole glucosinolates content between T0 and untransformed wild type.In summary, Bn GTR1 is not clearly proved to be connected with glucosinolates transport process and Bn GTR2 may be a transporter of glucosinolates.