The Study Of Relationship Between AP2/ERF Transcription Factor And Gene Of Capsaicinoid Synthetase Pun1

Capsicum(Capsicum ssp.)is a popular choice of vegetables and spices,with a distinctive pungent flavor,which is a substance called Capsicum.At the same time,the capsaicin substance is an important secondary metabolite,which has the role of anti-cancer,analgesic and weight loss.The current cultivation of pepper(Capsicum annuum),capsaicin content of it is low,so how to promote the synthesis of capsaicin and accumulation,by for the extraction of capsaicin to provide quality raw materials become an important research subject.The capsaicin genome has been sequenced and the capsaicin pathway has been clear.Pun1 is key genes in the capsaicin synthesis pathway,it decided to spicy fruit whether and how many levels of capsaicin,therefore,to study the gene expression regulation and to analyze molecular mechanism of capsaicin biosynthesis regulation,provide important theoretical basis for pepper germplasm genetic improvement.In this paper,the relationship between the AP2 / ERF family transcription factor and the key gene Pun1 of capsaicin biosynthesis is discussed,and the main results are as follows:1.Cloning and bioinformatics analysis of ERF/JERF transcription factorCloning two ERF transcription factors gene ERF and JERF in pepper,Genbank login number are KF060657 and KF169943.The full-length of ERF gene is 950 bp, encoding 264 amino acids,has one AP2 domain which containing 58 amino acids in the sequence of 533-706 position;the full-length of JERF gene is1219 bp,encoding145 amino acids,has one AP2 domain which containing 58 amino acid in the sequence of 736-912 position.Using Infusion technology,the ERF and JERF sequences were connected to the vector p GAD424,and the recombinant vectors are p GAD424-ERF and p GAD424-JERF.2.Gene expressions of different Pepper Cultivars at different developmental stages in placentaUsing real-time quantitative PCR technology to analysis the expression of ERF,JERF gene and Pun1 gene in different pepper cultivars at different developmental stages in placenta.The results showed that the expression change trends of these three genes in the three varieties of peppers were basically the same,which indicated that ERF and JERF could promote the expression of Pun1.3.Cloning and bioinformatics analysis of Pun1 promoterPrimers were designed to clone the promoter sequence of Pun1 gene,the full-length of promoter sequence of Pun1 gene is 1535 bp,which contains G-BOX,TAAT-BOX and other related boxes.Infusion technology was used to construct recombinant vector of Pun1 gene promoter and p Lac Zi which is p Lac Zi-Pun1 Pro.4.Verification of interaction between ERF,JERF transcription factors and Pun1By using yeast one hybrid technology,transform AD target vector p Lac Zi-Pun1 Pro,BD reporter vector p GAD424-ERF/JERF into yeast strain YM4271,use FTG-cry2 as positive control group which is preserved in laboratory,use with LEU single missing,LEU/URA double missing boards to screen cultures.The results show that the interaction between transcription factor ERF and Pun1 is weak,JERF transcription factor and Pun1 is strong.