Cloning And Drought Resistance Function Analysis Of Plant Trehalose Synthesis Pathway Gene TPPB

Brassica napus L.belongs to Cruciferae,Brassica.Brassica napus L.is an important oil crop in China,but it is also a plant with large water demand and poor drought tolerance.Seasonal drought occurred frequently during the growth cycle of Brassica napus L.,which often led to uneven seedling emergence,slow growth and even caused the decreased yield.Therefore,it is of great significance to cultivate drought-resistant Brassica napus L.through genetic engineering to reduce the impact of drought on its yield.Trehalose,as an important cell osmotic protectant,plays an important role in abiotic stress,including drought,salinity and freezing stress.Previous studies have found that overexpression of the rice trehalose synthesis pathway gene TPP1 could improve plant drought resistance and significantly increase the yield of transgenic maize,which imply the possible role of TPP family genes responding to drought stress.As a model plant of cruciferae,Arabidopsis thaliana can provide candidate genes for the exploration of drought resistant genes in Brassica napus L.The research on the trehalose-synthesis genes in Arabidopsis thaliana response to drought stress can help to reveal the molecular mechanism of trehalose synthesis genes responding to drought stress and its dynamic regulation mechanism in Brassica napus L.Here,we found that the loss function of AtTPPB gene,a member of trehalose-6-phosphate phosphatase gene(TPP)family,causes drought-sensitive phenotypes,while the overexpression of AtTPPB gene could significantly enhance drought tolerance in Arabidopsis.Yeast one-hybrid assay showed that DREB1 A could directly bind to the DRE/CRT element located in the AtTPPB promoter region.It indicated that DREB1 A may regulate the expression of AtTPPB gene.In order to explore the molecular mechanism of AtTPPB gene response to drought stress,we further performed RNAseq data analysis of wild type,mutant and overexpressing homozygous lines.GO enrichment showed that the differential genes related to oxidative stress were significantly enriched under drought stress conditions.It indicated that AtTPPB may function in the regulation of the level of reactive oxygen species under drought conditions.Further DAB staining results showed that drought-induced reactive oxygen species accumulated significantly in the tppb mutant compared to the wild type,but decreased in the AtTPPB overexpressing plants.It indicated that AtTPPB could protect cell membrane stability by scavenging reactive oxygen species.In addition,DAB staining results from DREB1 A relative lines showed that the level of ROS in dreb1 a mutant was significantly higher than wild type,while that of DREB1 A overexpressing plants was significantly reduced.It indicated that TPPB and DREB1 A have the same regulation of droughtreactive ROS levels in Arabidopsis.Therefore,our results reveal that DREB1 A probly activates the AtTPPB gene and cause it function in antioxidation to protect plants from injury by keep the balance of reactive oxygen species in plants,then positively regulates drought stress response.It helps to facilitate the uncovering of the molecular mechanism of AtTPPB regulating drought stress.Based on the exploration of the drought phenotype in the early stage of Arabidopsis,we genetically transformed the AtTPPB gene overexpression construct into the Brassica napus L.to obtain the transgenic drought-resistant rapeseed.It is very important for Brassica napus L.in improving its yield and quality under drought conditions,expanding its planting range,and drought resistance researching.