Cloning,Subcellular Localization And VIGS Vector Construction Of Aldo-keto Reductase Gene BclAKR4C10 From Flowering Chinese Cabbage

Anthracnose is one of the major diseases in flowering Chinese cabbage（Brassica campestris L.ssp.chinensis var.utilis Tsen et Lee）.The infection of anthracnose will cause“pitting”on the leaves,which will affect the appearance quality of the flowering Chinese cabbage,and continually have enormous economic loss.Frequent investigation and testing showed that Colletotrichum higginsiamum was the primary pathogen causing anthracnose in flowering Chinese cabbage in the major producing areas of Guangdong Province.Plant Aldo-keto reductase gene is an vital gene type of plant stress resistance pathway.In this study,RT-PCR technique was used to clone and obtain a BclAKR4C10 gene of aldehyde ketone reductase from flowering Chinese cabbage leaves,and the BclAKR4C10 gene sequence and biological characteristics were analyzed.Besides,subcellular localization technique of the BclAKR4C10 gene,fluorescence quantitative expression analysis of the infection of anthracnose on the flowering Chinese cabbage at 0 h,6 h,12 h,24 h and 48 h,and VIGS vector construction were also applied in the study.All the methods in this study helped elucidate the BclAKR4C10 gene and the molecular basis of resistance to anthracnose in flowering Chinese cabbage,which provides a new biological approach for accurate control of anthracnose in flowering Chinese cabbage.The main results are as follows:（1）BclAKR4C10 gene:open reading frame 948 bp,encoding 315 amino acids,with（α/β）₈ barrel structure and three variable loop structures.（2）The protein encoded by BclAKR4C10 gene:35.290 k Da relative molecular weight and 5.77 theoretical isoelectric points.The result of signal peptide prediction indicated that the protein had no signal peptide cleavage site and was non-secreted protein.The average hydrophilic/hydrophobic index of BclAKR4C10 protein was-0.248,thus the protein was hydrophilic.Transmembrane region analysis predicted that the protein had no transmembrane region.Phosphorylation site prediction results showed that the protein had18 Ser phosphorylation sites,10 Thr phosphorylation sites,and 2 Tyr phosphorylation sites.Secondary structure prediction showed that the proteinα-helix accounted for 40.63%,extended chain 14.60%,β-turn 6.67%,and irregular curl 38.10%.（3）Subcellular localization of BclAKR4C10 gene in flowering Chinese cabbage:the BclAKR4C10 gene was constructed into the p BI121-e GFP-HA vector.PCR detection and sequencing verification showed that PBI121-BclAKR4C10-e GFP-HA vector was successfully constructed.The subcellular localization analysis showed that empty vector was expressed in the cell membrane and nucleus,and BclAKR4C10 protein is distributed in the nucleus and cell membrane after production.The subcellular localization of BclAKR4C10 gene allows us to understand the expression position of BclAKR4C10 gene in the anti-anthracnose process of flowering Chinese cabbage at the cellular level,providing a theoretical basis for further study on the anti-anthracnose mechanism of flowering Chinese cabbage.（4）Quantitative fluorescence expression analysis of BclAKR4C10 gene in flowering Chinese cabbage:according to the quantitative fluorescence expression analysis at 0 h,6 h,12 h,24 h and 48 h on the leaves of flowering Chinese cabbage inoculated or nor inoculated with anthracnose,the relative expression of BclAKR4C10 gene in leaves of flowering Chinese cabbage was significantly different at different infection stages of anthracnose.The expression level of BclAKR4C10 was always low in the control group（without inoculation）.The expression level of BclAKR4C10 was significantly higher than that in other periods after 24 h of anthracnose infection,about three times higher than that in the control group.The expression level of BclAKR4C10 decreased after 48h of infection,but still higher than the expression level at 0 h～12 h.By analyzing the differences in the expression of BclAKR4C10 gene in different stages of anthracnose infection,the close relationship between BclAKR4C10 gene and resistance to anthracnose of flowering Chinese cabbage was clarified.The study provided an excellent theoretical basis for the expression of the resistance gene and the mechanisms of resistance to anthracnose in flowering Chinese cabbage.（5）VIGS vector construction of BclAKR4C10 gene in flowering Chinese cabbage:the BclAKR4C10 gene was constructed into the p TRV2 vector,and the p TRV2-BclAKR4C10vector was confirmed to be successfully constructed after PCR detection and sequencing verification.Then the recombinants were transfected into plant by vacuum-infiltrating,after a period of time,the BclAKR4C10 gene expression of the infected plants and control plants were analyzed.The results showed that the expression level of BclAKR4C10 in infected plants was significantly lower than that in control plants,which indicated that the VIGS system of flowering Chinese cabbage had been established.It provided the experimental material for further experiments.