Screening Of Transcription Factors Involved In Regulation Of Capsaicin Biosynthesis And Functional Identification

Capsaicin is only synthesized in Capsicum plants, and has a wide range of applications of capsaicin. The frame diagram of capsaicin biosynthetic pathways is basically known, but capsaicin biosynthesis regulatory mechanism is still rarely reported. The AP2/ERF transcription factor family and WRKY were screened based on Kim(2014)'s genome data and bioinformatics technology, their functions were identified based on VIGS, q RT-PCR in order to find the difference between varieties with different capsaicin in transcript factors and expect to find out the key genes controlling the higher capsaicin. The results obtained are as follows:(1) Twenty nine transformant tomato plants were obtained Agrobacterium-mediated with CaAT3. Confirmation work is being done.(2) The promoters of the last step structural gene AT3 in the capsaicin synthesis were isolated from the varieties with different capsaicin. There was only one base difference in promoter sequence between “59”(variety with medium capsaicin)and “WL”. Bigger differences were existed between “740”(variety with the highest capsaicin)and “59”(variety with medium capsaicin) or “WL”.There was a base difference in the promoter sequence of p AMT between “59” and “170”(sweet variety without capsaicin).There were 3 bases difference between '59 'and' XN ',2 bases differences between '170' and 'XN', and big difference between'740' and other varieties. In the promoter sequence of COMT, there were 4 base differences between “170” and “59”, “740”. No differences were found between “59” and “740”. In the promoter sequence of KAS, 11 base differences were found between '59' and '170', 21 base differences existed between “59” and “740”, 26 base differences were found between “170”and “740”.(3) The promoters of the structural gene AT3, p AMT, COMT, KAS,PAL were isolated from“59”. Their elements were analyzed. The results showed that all of them contained the core promoter elements and recognition sequence of ERF and WRKY.(4) According tocapsicum genome sequencing results(Kim et al. 2014), through bioinformatic analysis, five possible ERF transcription factors, CaERF1, CaERF2, CaERF3, CaERF4, and CaERF5 were selected. According to the genome sequencing of 'Zunla-1'(Capsicum annuum L.) and wild species 'Chiltepin'(C. annuum var. Glabriusculum, also termed C. annuum var. Aviculare)(Qin et al. 2014), the biological information analysis, CaWRKY1, CaWRKY2, CaWRKY3, CaWRKY4 and CaWRKY5 were selected.(5) Analyzed expression of '740' inbred pepper's five CaERF transcription factor genes and five transcription factor genes of WRKY family, expression levels of CaERF1, CaERF2, CaERF5 in the placenta are higher than pepper flesh, wherein CaERF2 and CaERF5's expression in placenta was significantly higher than the expression of flesh, while CaERF3 and CaERF4's expression in pulp were significantly higher than the placenta. CaWRKY1, CaWRKY2 and CaWRKY5's expression in the placenta are higher than the expression level in the pulp, wherein the expressions of CaWRKY1 and CaWRKY2 in the placenta were significantly different with the pulp. CaWRKY3 and CaWRKY4's expression in the flesh higher than its expression in the placenta, which the expression of CaWRKY3 was significant differences in pepper placenta and the pulp.(6) The tobacco rattle virus(TRV)-based VIGS system was employed to silence above-mentioned 10 transcript factors respectively. To determine the relative transcript levels of selected genes, real-time PCR was performed with specific primers. CaERF1, CaERF2, CaERF4 and CaERF5 genes were silent, expression level of CaERF1 was less than the 1/5 amount of expression of CK, and expression level of CaERF4 and CaERF5 were about a half of CK, the expression of CaERF2 was equal to CK. but compared with PV2(treated with empty vector), CaERF1, CaERF4 and CaERF5's expression were less than PV2, expression of CaERF2 and CaERF3 were higher than PV2.Expression level of structural gene AT3 was significant and lower than CK after the CaERF1, CaERF2 and CaERF3 were silenced.After PV2 treatment, the expression level ofAT3 was insignificant with CK. The expression amount of pAMT was lower than CK after the CaERF1, CaERF2 were silenced. However, it was higher than CK after the CaERF3, CaERF4 and CaERF5 were silenced. the expression level of p AMT after PV2 treatment was insignificant with CK; after VE1, VE2, VE3 treatment, the expression level of structural gene FATA was higher than CK, of which it was the highest after it was treated with VE3.The expression level of structural gene KAS was lower than CK only after the ERF2 was silenced., It were higher than CK when CaERF1, CaERF3 transcript factors were silenced. The silence effect of WRKY family transcript factors was not good in the tobacco rattle virus(TRV)-based VIGS system.(7) Capsaicin was measured after VIGS treatment. Compared with p TRV2, after the pepper plants were treated with VE1, VE2, VE3 and VE5, the capsaicin content decreased significantly, of which it was the lowest after it was treated with VE1 and VE3. After VE4 treated, capsaicin content was higher than p TRV2.And dihydro- capsaicin content was lower than p TRV2, of which it was the lowest after it was treated with VE1.after VE4 treatment dihydrocapsaicin content was higher than p TRV2.(8) The subcellular localization prediction of CaWRKY and CaERF family by the online software Ba Cel Lo showed that the screened genes were localized in the nucleus.(9) Analysis of transcription factor's conserved regions by online software MEME, showed that the transcription factor CaERF4 contained a conserved region, CaERF1, CaERF2, CaERF3, CaERF5 gene sequence contained two conserved regions, but according to the SMART software online analysis, ERF1 and ERF4 had two AP2 conserved domains, and CaERF2, CaERF3 and CaERF5 contained only one AP2 conserved domains. So it indicates ERF1 has two AP2 conserved domains belong to AP2 protein. 5 CaWRKY genes contain two conserved regions and belong to the second category WRKY.(10) Yeast one-hybrid verified CaERF1, CaERF3, CaWRKY1, CaWRKY2 could bind to the promoter of CaAT3.