| Capsaicin is a kind of secondary metabolites restrictedly biosynthesis in chili pepper(Capsicum spp.). It is an important part of the fruit quality. A high pungency variety “shuanshuanla” is famous for it's high concentration of capsaicin. However, it's fruiting rate is low, growth period is long, It is not a good variety used for the capsaicin extraction. So the research on the molecular biology of biosynthesis of capsaicin is very important, for us to find out the high hot gene or its regulation mechanism and make it satisfied with production requirements. At present,the pathway of capsaicin biosynthesis has elucidated, however, whether these strucrure genes and corresponding promotes are differences among the varieties with different pungency degree has yet to figure out. And the regulation mechanism of transcription factor was still largely unknow. AT3 only express in chili pepper, Capsaicin is biosynthesized in the placenta, All of this illustrates the importance of the AT3 and the biosynthesis approach is regulated by the transcription factor. In this study 3 varities with different pungency degree were used as materials to clone some important structure gene and expression quantity was analysed. We find out the important AT3 structural genes and analyzed it's promoter activity and it's transcription factor. Our results are as follows:1. Because capsaicinoid is only biosynthesized in pepper placenta, so we extract the RNA of placenta in variety “740”, “59”, “170” and cloned the 9 important structure genes of capsaicin synthesis. Comparative analysis was made using bioinformatics methods, and q RT-PCR was carried out in pericarp and placenta. The expression of AMT and AT3 varied in pericarp and placenta, There was a big missing of AT3 in variety”170” and no initiation codon. It imply that AMT and AT3 play great roles in the synthesis of capsaicin.2. Virus-induced gene silencing(VIGS) system were applied for AT3 and AMT silence. The expression level significant decreased in the placenta of silence plants 16 days later. The capsaicin content of ATM has fallen down 25%, but AT3 changed little 45 days later.3. There was a MYB binding site in the promoter of AT3 in variety”740”. Five expression vectors were constructed with 5' end missing and transferred into chili placenta protoplast using PEG method, Through the fluorescence microscope observation, 747 bp active region was found, then construct 5 intercalary deletion expression vector were constructed. Using the same method, they were transferred into the placenta protoplast and observed. Finally we made sure the 237 bp active region. There are 2 MYB blinding sites in this region.4. Based on the capsicum genome sequences, using the placental c DNA of “740” as a template, Cc MYB86,Cc MYB2,Cc MYB48,Cc MYB59 complete CDS were cloned in “740”. Except Cc MYB86,the expression levels of Cc MYB2,Cc MYB48 and Cc MYB59 in the placenta, were significantly higher than in the pericarp.5. Sequence analysis showed that Cc MYB86,Cc MYB2,Cc MYB48 and Cc MYB59 have conservative, containing conserved R2 and R3 MYB domain, belong to the R2R3 MYB transcription factor family. And the results of the subcellular localization showed that all of them were located within the nucleus.6. For further function identification of Cc MYB86,Cc MYB2,Cc MYB48 and Cc MYB59, virus-induced gene silencing(VIGS) system were applied for gene silence. Results showed that Cc MYB86,Cc MYB2,Cc MYB59 expression level significant decreased in the placenta of silence plants, the expression quantity of BCAT,COMT,AMT,AT3 has also Significant changes. The high performance liquid chromatography(HPLC) were applied to determin capsaicin content in silence plants. The capsaicin content was also reduced. This shows that all of this three transcription factors play a role in the regulation of capsaicin synthesis and the BCAT,COMT,AMT,AT3 are regulated by this three transcription factor. |
PDF Full Text Request