Abstract: 【Objective】Walnut (Juglans regia L.), which ranked first among the four major nuts, had been widely planted and processed for utilization. Red walnut RW-1 with red leaves, pericarps and seed coats were researched because of the high contents of anthocyanin. Anthocyanins are important secondary metabolites in plants, which played an important role in avoiding UV damage, attracting insect pollination, and resisting low temperature stress. Although the anthocyanin biosynthesis gene had been studied in other plants, the function in walnut were still unclear. Chalcone synthase is the first key enzyme in anthocyanin biosynthesis pathway, which determined the final product of anthocyanin biosynthesis. In this study, the function of JrCHS4 was researched by transient transformed 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 color 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 were 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 were detected by yeast one-hybrid (Y1H) and luciferase assay (LUC). The over-expression vector of JrCHS4 was transient transformed into tobacco leaves, 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 60 and 120 days after flowering, only 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, 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 walnuts by the Phytozome database, and 98.50% nucleotide identify were shared. From PLACE database, some elements related to hormone response and stress-related, like ABRE, MYC, ERE, GARE and MYB1AT, were found in RW-JrCHS4 promoter. Compared with the GW-JrCHS4 promoter, the RW-JrCHS4 promoter lacks one MYB binding site MYB1AT and inserts 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 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, and the positive control (35S-GUS) showed a strong expression, the GUS activity under RW-JrCHS4 was higher than that of GW-JrCHS4. The same results were also gotten by quantitation of GUS activity. The results from both the assays showed that compared with the GW-JrCHS4 promoter, the promoter of RW-JrCHS4 showed high activity, which suggests that the different expression patterns of JrCHS4 may be caused by their promoter activities. To screen for the bHLH transcription factors which were 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 RW-JrCHS4pro-pAbAi. The optimal AbA concentration to inhibit the expression of JrCHS4 promoter was 150 ng·mL-1. After grown on the selected medium, only JrbHLHA2-pGADT7+RW-JrCHS4pro-pAbAi stain ensured the normal growth, while none of the other combinations grown indicated that JrbHLHA2 could bind to the promoter of JrCHS4. Following, the results of LUC assays showed that the activity of RW-JrCHS4 promoter co-transformed with JrbHLHA2 was almost three times 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 bound the promoter of JrCHS4 to promote the biosynthesis and accumulation of anthocyanin. In order to verity the function of JrCHS4 in anthocyanin biosynthesis, the over-expression vector of JrCHS4 was transformed into tobacco leaves. After injected seven days, the accumulated anthocyanin of injected JrCHS4 tobacco leaves were 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 from 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.
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