Functional Verification Of BnaERF5/6 Transcription Factor In Brassica Napus L.and Effect Of 5-hydroxytryptamine(5-HT)on Seedling Under Drought Stress

1.Functional Verification of BnaERF5/6 Transcription Factor in Brassica napus L.The cDNA of rape buds treated with waterlogging for 12 hours was used as the template to clone the genes related to waterlogging tolerance in Brassica napus L.variety ZS9,and the genes were genetically transformed into Arabidopsis by agrobacterium-mediated method.The main results are as the following:Two genes BnaERF5 and BnaERF6 were cloned.Plant over-expression vectors PBI121-BnaERF5and PBI121-BnaERF6 were successfully constructed.Agrobacterium-mediated genetic transformation of Arabidopsis was carried out,through the screening of kanamycin medium for three successive generations.The positive seedling DNA was detected,and the exogenous gene expression was detected by qPCR in pure lines.After the treatment of waterlogging,the leaves of both transgenic and wild-type Arabidopsis lines turned yellow,and the color of some leaves were purple.Some old leaves were dry.After the recovery of 10 days after 7 days of waterlogging,the transgenic plants could flower normally,while the growth of wild-type plants were inhibited,and could not flower normally.Detection of the expression levels of waterlogging resistance related genes ADH1 and PDC1 in Arabidopsis at 0,3,6 and 9 h after waterlogging.The result showed that the expression levels of ADH1gene of transgenic BnaERF5 Arabidopsis seedlings at 0 h and 3 h after waterlogging were higher than those of wild-type Arabidopsis,and the expression levels of ADH1 were lower than those of Wild-Type Arabidopsis at 6 h after waterlogging.When the BnaERF6 transgenic Arabidopsis seedlings were flooding for 0 hour and 3 hours,the expression level of ADH1 gene was higher than that of Wild-Type Arabidopsis.However,when the flooding for 6 hours,the expression level of ADH1 gene was not significantly different from that of Wild-Type Arabidopsis seedlings.When the waterlogging for 9 hours,the expression level of ADH1 gene was lower than that of Wild-Type Arabidopsis seedlings.The PDC1gene of transgenic BnaERF5 Arabidopsis seedlings was increased at 0 h and 3 h after waterlogging compared with that Wild-Type Arabidopsis,and the expression level of PDC1 was lower after 6 h of waterlogging than that of Wild-Type Arabidopsis.PDC1 gene of BnaERF6 transgenic Arabidopsis seedlings was increased at 0 hour and 3 hours after waterlogging compared with that Wild-Type Arabidopsis.However,the expression level of PDC1 gene was not significantly different from that Wild-Type Arabidopsis seedlings at 6 hours after waterlogging,and the expression level of PDC1 was lower than that of Wild-Type Arabidopsis seedlings at 9 hours after waterlogging.Fresh weight and plant height of transgenic Arabidopsis were significantly higher than those of wild type plants after the recovery of 10 days after 7 days of waterlogging.2.Effect of 5-hydroxytryptamine（5-HT）on seedling under drought stress5-hydroxytryptamine（5-HT）is a naturally occurring compound in plants,and this study is to reveal the effects of 5-HT on rape seedlings under drought stress.The effect of exogenous 5-HT on rape（Brassica napus L.）growth under PEG stress were investigated in this experiment,and the traits of seedling growth,active oxygen metabolism and osmotic adjustment substance were evaluated.The results showed that drought stress inhibited the seedling growth,resulted in H₂O₂ and malondialdehyde（MDA）accumulation,increased proline（Pro）and soluble sugar（SS）content,and significantly enhanced the activity of catalase（CAT）,superoxide dismutase（SOD）and ascorbate peroxidase（APX）.Under drought stress condition,exogenous 5-HT could alleviate the inhibition of seedling growth.The activity of antioxidant enzymes（CAT,SOD and APX）was improved significantly by exogenous 5-HT,and the content of H₂O₂ and MDA was decreased.The results also showed that solute accumulation under stress was significantly enhanced with exogenous 5-HT by increasing the content of Pro and SS.This study indicated that exogenous 5-HT can improve H₂O₂ scavenging capacity of rape seedlings by enhancing the activity of antioxidant enzymes and can also alleviate osmotic stress by promoting the accumulation of osmoregulatory substances.Ultimately,exogenous 5-HT improved the biomass of rape seedlings under drought stress,thereby effectively alleviating drought stress in rape seedlings.