Changes Of Flesh Color, Cloning And Expression Of Anthocyanin Biosynthesis Related Genes In Red-Flesh Kiwifruit

In order to clear color variation during the fruit development of red-fleshed kiwifruit, further find out the key genes in the anthocyanin biosynthesis and then clarify the expression patterns of anthocyanin biosynthesis related genes in the coloring of the fruit, the inner and outer pericarp of’HY-09’, a mutants of’Hongyang’(Actinidia chinesis cv ’Hongyang’) were used as the tested materials. The dynamic changes of pigments, sugar in the the various periods after full bloom of mutant in and changes of’HY-09’fruit were detected, and the relationship between anthocyanin and color of the’HY-09’fruit was analyzed. A MYB transcription factor was cloned from’HY-09’fruit, and the expressions of MYB and other structural genes relate to anthocyanin biosynthesis in the inner and outer pericarp of the same fruit from different periods were analyzed by qPCR. The differentially expressions of those genes from the inner, outer pericarp and different periods were further analyzed in two-way hierarchical clustering method by using Permut Matrix software. The main results are showed as follow:1. There was clearly difference in pigment composition and content between the inner and outer pericarp of’HY-09’fruit. Although chloroplast pigments exist in inner and outer pericarp, but anthocyanin only exists in inner pericarp, none detected in outer pericarp. From the point of chlorophyll, the contents in the inner and outer pericarp presented downward trend with fruit development, significantly decreased in 37-47 day after full bloom, DAFB period. But the outer pericarp was always higher than the inner pericarp, and which shown an obvious fluctuations during descent. At the same time, the h°value (chromaticity angle) of outer pericairp had declined from 37 DAFB to 132 DAFB (harvest), which was slowly closer to 90 with fruit development. While at the part of anthocyanin, there was no anthocyanin accumulation before 37-47 DAFB in the inner pericarp, anthocyanin began to appear at 67 DAFB and its content fast accumulated along with the fruit development, until reached the peak at 122 DAFB (4.32 mg/100 g. FW), but slightly declined at 132 DAFB (harvest). While a* and h° values change rules were consistent with the variation in content of anthocyanin. Dynamic change of soluble sugar were more consistent in inner and outer pericarp, which were slowly upward trend in the early fruit development, and the fast increased at 106 DAFB, but the significant differences of the two was that the soluble sugar contents of inner pericarp were always higher than the out pericarp after 106 DAFB. While there was few difference between the two tissues in carotenoids in the entire development period of’HY-09’.2. A transcription factor AcMYBl were cloned from the inner pericarp of’HY-09’ mature fruit (132 DAFB), the full-length of AcMYB1 is 896 bp, there is a long for 783 bp open reading frame encoding 260 amino acids and having R2R3 domain. The comparison of AcMYB1 with other R2R3-MYBs showed that, AcMYBl shared about 30% similarity with other species of anthocyanin synthesis transcription factors such as petunia AN2 MYB and Arabidopsis AtMYB90, and also low similar with AcMYB in kiwifruit. While shared high similarity (51.77%) with a negative regulation transcription factors AtMYB4 in Arabidopsis, but further analysis found that the amino acid structure of AcMYB1 didn’t have the pdLNLD/ELXiG/s structure in AtMYB4.3. The expression of AcMYB1 and other structural genes in the inner and outer pericarp were analyzed, the results showed that:the expression level of AcMYB1 in the inner pericarp was always higher than the outer pericarp, the transcription levels of AcMYB1 and anthocyanin content were positively correlated. Which can be speculated that AcMYB1 may be a new MYBs transcription factors played a regulatory role in the anthocyanin biosynthesis of red-fleshed kiwifruit. And 8 structural genes relate to anthocyanin biosynthesis are expressed both in the inner and outer pericarp, but on the whole, the inner pericarp expression lever was higher than outer pericarp, especially F3H2 and F3GT1. At any time, the inner pericarp expression levers of that were significantly higher than outer pericarp, and kept a high expression level in anthocyanin accumulation stages. F3GT1 showed higher transcription level after at 67and 122 DAFB than any stages, the two stages just coincided with the period of anthocyanin the beginning accumulation and the highest content. In addition, the hierarchical clustering analysis revealed the related gene expression patterns, AcMYB1 and structural genes F3GT1, F3H2 clustered together with, clearly separated from other genes. It showed that AcMYB1, F3H2 and F3GT1 played a very important role in anthocyanin biosynthesis in the red-fleshed kiwifruit. And these results may provide a good foundation to clarify the molecular mechanism of anthocyanin formed in the red-fleshed kiwifruit.