Cloning And Drought Resistance Functional Analysis Of Flavonoid Biosynthesis Pathway Genes BjTT5 And BjTT6 In Brassica Juncea L.

Shanbei yellow mustard is a unique mustard type rapeseed in China.Its erucic acid content is about 25%,and it has strong resistance to barren,split pod and drought.It is not only the main oil crop in northwest China,but also an important germplasm resource for improving the quality of rapeseed.According to our previous fine mapping of the genetic populations constructed by Shanbei yellow mustard and brown mustard,two important genes TT5(CHI)and TT6(F3H)in flavonoid biosynthesis pathway were screened,which may be related to drought resistance.Therefore,in this study,drought resistance of Shanbei yellow mustard and Guanzhong brown mustard at germination and seedling stages was first identified.TT5 and TT6 genes were cloning in the two materials,and the sequence and structural differences of TT5 and TT6 genes were compared and analyzed.Overexpression analysis was performed in Arabidopsis thaliana to verify the drought resistance function of these genes.The specific results were as follows:1.PEG6000 with different mass fractions was used to simulate drought stress at germiantion stage.The results showed that the germination rate and seedling formation rate changed little under 5% and 10% PEG6000 treatment,and the other indexes reached the maximum under 10% PEG treatment.Mild drought stress promoted the elongation of the buds.Seeding germination and root growth were inhibited under 15% PEG treatment.Seedlings could not be formed under 20% PEG treatment,and seeds died under 25% PEG treatment.In the process of drought,there were differences in the degree of change of physiological indexes between yellow and brown mustard.The Guanzhong brown mustard was more sensitive to drought stress,and the drought resistance of yellow mustard was stronger than that of brown mustard.2.The soil culture method was used to identify the drought resistance at seedling stage.The results showed that the leaves of Shanbei yellow mustard began to wilt after 18 days of drought.After 21 days of drought,leaves of Guanzhong brown mustard began to wilt,and the wilting situation was relatively mild.Drought resistance at seedling stage was evaluated by membership function method and the drought resistance of brown mustard was stronger.According to the results of principal component analysis,SOD activity should be considered as the main index of drought resistance identification at seedling stage.3.According to the previous fine positioning results,the homologous genes Bju A044462 and Bju A004576 of TT5 and Bju A035478 and Bju A032280 of TT6 were identified in Brassica juncea.The amino acid sequence alignment,domain prediction,secondary structure prediction and tertiary structure prediction were carried out based on the cloning results of the two plants.The amino acid sequence alignment results showed that the amino acid sequence consistency of Bju A044462 was 43.43% in the two materials.The sequence of amino acids encoded by Bju A004576 was identical.The sequence consistency of Bju A035478 was 99.72%,and there was only one difference in the 254 th amino acid.The sequence consistency of the amino acids encoded by Bju A032280 was 96.86%,with 11 differences.It was found that Bju A044462 and Bju A004576 contain PLN02559 domain,and Bju A035478 and Bju A032280 contain PLN02515 domain.4.The above four gene promoter sequences amplification,Bju A035478 promoter had11 bases difference in the yellow and brown mustard.The analysis of cis-acting elements showed that Bju A035478-Y promoter containede 34 kinds of cis-acting elements,while Bju A035478-B promoter contained 33 kinds,and there were 4 differences between them.In addition to the basic cis-acting elements,both of them contained a number of cis-acting elements associated with stress response,including cis-acting elements ABRE,MYB,and MYC associated with drought and abscisic acid(ABA)responses,as well as TC-rich repeats,which were involved in defense and stress responses.5.Overexpression vectors were constructed to transform tobacco by Agrobacterium tumefaciens and subcellular localization results showed that all the four genes were located in the cell nucleus and cell membrane.The overexpression vectors of candidate genes were transformed into Arabidopsis thaliana,and the overexpression plants of Bju A044462-Y and the T3 generation seeds were obtained successfully.It was found that overexpression of Bju A044462-Y made Arabidopsis thaliana seed coat color lighter.And the drought resistance of Arabidopsis was evaluated,the results showed that overexpression of Bju A044462-Y improved the germination rate and seedling formation rate of Arabidopsis at germiantion stage under drought osmotic stress,shortened the germination and seedling formation time and promoted the root growth.