| Brassica juncea is an important vegetable crop in the Yangtze River basin in China.It is a brassica crop of the cruciferous family and is a vegetable crop with important economic value.Studying the related genes that regulate the flowering of Brassica juncea is helpful to the genetic improvement of varieties,and is of great significance to avoid early bolting and increase yield.Preliminary studies have shown that in the model plant Arabidopsis,both WRKY12 and WRKY13 can affect flowering time.WRKY12 promotes flowering,while WRKY13 inhibits flowering in Arabidopsis.However,there is still a lack of in-depth research on their molecular mechanisms affecting flowering time.At the same time,although there have been some studies on the mechanism of flowering integrtors such as SOC1 and FUL,it has not been reported whether they are directly affected by BjuWRKY12 and BjuWRKY13 to regulate flowering time.HISTONE DEACETYLASE 9(HDA9),a member of the deacetylase family,can also participate in flowering regulation.It is also worthy of further study whether BjuHDA9 can coordinate with BjuWRKY12 and BjuWRKY13 to regulate flowering.Therefore,in this experiment,the BjuWRKY12 and BjuWRKY13 genes were cloned,and Agrobacterium-mediated transformation of Brassica juncea was used to obtain transgenic Brassica juncea plants.Subsequently,we observed the flowering phenotype and analyzed the gene function in Brassica juncea.Then,using yeast two-hybrid,Bi FC,yeast one-hybrid and dual luciferase assays,the interactions of both BjuWRKY12 and BjuWRKY13 with BjuHDA9,BjuSOC1 and BjuFUL were studied respectively.The results laid a foundation for the in-depth study of the flowering molecular regulation of BjuWRKY12 and BjuWRKY13 on Brassica juncea.The results are as follows:1.Bioinformatics Analysis and Expression Pattern Research of BjuWRKY12 and BjuWRKY13 GenesAfter searching and comparing,we successfully cloned BjuWRKY12 with a full-length CDS of 660 bp as well as BjuWRKY13 gene with a 633 bp.Among them,BjuWRKY12 encodes 219 amino acids,and the predicted protein molecular weight is24.72 k Da.While BjuWRKY13 encodes 210 amino acids,and the predicted protein molecular weight is 24.22 k Da.Sequence and domain analyses showed that both of them belong to the IIc subfamily of the WRKY family.Subcellular location indicated that they are located in the nucleus.It was found by qRT-PCR that BjuWRKY12 and BjuWRKY13 are expressed in stems,leaves,and flowers.And their expression patterns are similar in various tissues of Brassica juncea.That is,BjuWRKY12 and BjuWRKY13 are both expressed in flowers with the highest expression levels,but low in stems and leaves.It is worth noting that the expression levels of BjuWRKY12 and BjuWRKY13 showed opposite trends with the growth of seedling age.The expression of BjuWRKY12 increased slowly with the increase of seedling age,while the expression of BjuWRKY13 gradually decreased with the increase of seedling age.2.BjuWRKY12 promotes the flowering of Brassica juncea,while BjuWRKY13 inhibits the flowering of Brassica junceaTransgenic Brassica juncea plants were obtained by Agrobacterium-mediated transformation.Compared with wild-type plants,BjuWRKY12 overexpression plants had earlier flowering time,indicating that BjuWRKY12 promoted mustard flowering.However,BjuWRKY13 overexpression plants had delayed flowering time,indicating that BjuWRKY13 inhibited flowering.3.Protein-protein interaction analyses of BjuWRKY12 and BjuWRKY13 with BjuHDA9,BjuSOC1 and BjuFULYeast two-hybrid assays showed that the fused yeast zygotes of BjuWRKY12 with BjuHDA9,BjuSOC1 and BjuFUL did not grow normally in QDO/X/A(SD/-Ade/-His/-Leu/-Trp/X-α-Gal/Ab A).It demonstrated that BjuWRKY12 could not interact with each of them.The yeast zygotes of BjuWRKY13 with BjuSOC1 and BjuFUL did not grow normally on QDO/X/A.It suggested that BjuWRKKY13 could not interact with each of them in yeast.However,the fusion strain of Y187(pGBKT7-BjuWRKY13)and Y2HGold(pGADT7-BjuHDA9)grew normally and turned into blue on QDO/X/A,indicating that BjuWRKY13 was able to interact with BjuHDA9.Bimolecular fluorescence complementation experiments further confirmed that BjuWRKY12 did not interact with BjuHDA9,BjuSOC1 and BjuFUL proteins.BjuWRKY13 was able to interact with BjuHDA9.4.Protein-DNA interaction detection of BjuWRKY12 and BjuWRKY13 with promoters of BjuSOC1 and BjuFULYeast one-hybrid assays showed that the yeast fused strains of pGADT7-BjuWRKY12 and pGADT7-BjuWRKY13 with p Ab Ai-BjuSOC1 and p Ab Ai-BjuFUL grew normally on SD/-Leu/Ab A solid media.The results suggested that both BjuWRKY12 and BjuWRKY13 were able to interact with promoters of BjuSOC1 and BjuFUL.The further studies in the dual-luciferase assays confirmed the above results. |
