Functional research on JrbHLHA2 transcription factor targeting chalcone synthase gene JrCHS4 regulating anthocyanin biosynthesis in red walnut

Abstract: 【Objective】Walnut (Juglans regia L.), which ranks first among the four major nut crops, has been widely planted and processed for utilization. Red walnut RW-1 with red leaves, pericarps and seed coats has been researched because of its high contents of anthocyanin. Anthocyanins are important secondary metabolites in plants, which play an important role in avoiding UV damage, attracting insect pollination and resisting low temperature stress. Although the anthocyanin biosynthesis gene has been stud-ied in other plants, the function in walnut is still unclear. Chalcone synthase is the first key enzyme in anthocyanin biosynthesis pathway, which determines the final product of anthocyanin biosynthesis. In this study, the function of JrCHS4 was researched by transient transformation in tobacco leaves.【Methods】The expression patterns of key chalcone synthase genes related to anthocyanin biosynthesis (JrCHS1-JrCHS4) were detected by qRT-PCR. The promoters of JrCHS4 in two different color types of walnuts were cloned by the Phytozome database. The cis-acting elements were predicted by PLACE databases. The promoters were inserted into pCAMBIA1381-GUS vector, and the recombinant vector was transformed into Agrobacterium strain GV3101 for transient expression. The activity of two promoters was detected by GUS histochemical staining and quantitation GUS protein. The regulatory effect of upstream bHLH transcription factors (JrbHLHA1, JrbHLHA2, JrEGL1a and JrEGL1b) on the JrCHS4 promoter was detected by yeast one- hybrid (Y1H) and luciferase assay (LUC). The over-expression vector of JrCHS4 was transient transformed into tobacco leaves, and the changes of leaf color and anthocyanin content were observed.【Results】The expression patterns of four CHSs genes related to anthocyanin biosynthesis were detected by qRT-PCR using different development stages of seed coat. The results showed that at 60th and 120th days after flowering, the expression level of JrCHS4 was significantly higher in the red walnut seed coat than in the normal walnut seed coat and the difference in expression level was the largest, which indicated that JrCHS4 may be the key gene in red walnut anthocyanin biosynthesis. To investigate whether the different expression trends of CHS4 gene in the seed coat development of red walnut RW-1 and normal walnut Zhonglin 1 were related to their promoters, the promoters of JrCHS4 were cloned from the two types of walnuts by the Phytozome database, and 98.50% nucleotide identify were shared. From PLACE database, some elements related to hormone response and stress, like ABRE, MYC, ERE, GARE and MYB1AT, were found in RW-JrCHS4 promoter. Compared with the GW- JrCHS4 promoter, the RW- JrCHS4 promoter lacked one MYB binding site MYB1AT and inserted one bHLH binding site MYCCONSENSUSAT. In order to determine the difference in activity of two JrCHS4 promoters, the promoters were cloned into pCAMBIA1381-GUS vector. After they were transformed into Agrobacterium strain GV3101, the positive clones were transient transformed into tobacco leaves. The result of histochemical assay showed that the negative control (only GUS without the promoter) showed almost no expression, the positive control (35S- GUS) showed a strong expression, and the GUS activity under RW-JrCHS4 was higher than that of GW-JrCHS4. The same results were also gotten by quantitation of GUS protein. The results from both assays showed that compared with the GW- JrCHS4 promoter, the promoter of RW- JrCHS4 showed high activity, which suggested that the different expression patterns of JrCHS4 may be caused by their promoter activities. To screen out the bHLH transcription factors, which were in the upstream of JrCHS4, four bHLHs related to anthocyanin biosynthesis (JrbHLHA1, JrbHLHA2, JrEGL1a and JrEGL1b) were selected out. After cloned into pGADT7 vector, four bHLHs were co-transformed into yeast stain Y1HGold with RWJrCHS4pro- pAbAi. The optimal AbA concentration to inhibit the expression of JrCHS4 promoter was 150 ng · mL- 1 . After they grew on the selected medium, only JrbHLHA2- pGADT7 + RW- JrCHS4propAbAi stain ensured the normal growth, while none of the other combinations could grow, which indicated that JrbHLHA2 could bind to the promoter of JrCHS4. Moreover, the results of LUC assays showed that the activity of RW- JrCHS4 promoter co- transformed with JrbHLHA2 was almost three times more than co-transformed with empty vector. So the results indicated that JrbHLHA2 and JrCHS4 may be the key genes of anthocyanin biosynthesis in red walnut, and JrbHLHA2 was bound the promoter of JrCHS4 to promote the biosynthesis and accumulation of anthocyanin. In order to verity the func-tion of JrCHS4 in anthocyanin biosynthesis, the over-expression vector of JrCHS4 was transformed into tobacco leaves. After they were injected for seven days, the accumulated anthocyanin content of injected JrCHS4 tobacco leaves was higher than the empty vector injected. The results indicated that JrCHS4 promoted the accumulation of anthocyanin.【Conclusion】The JrbHLHA2 transcription factor targeting chalcone synthase gene JrCHS4 is the key factor to regulate the biosynthesis of anthocyanin in red walnut RW-1, which provided important theoretical significance and application value for seed coat color improvement as well as breeding new varieties of red walnut.