Constructing The RNAi Vector Of Pepper POD Gene And Studying On Genetic Transformation

Hot pepper is one of the most widest cultivated vegetables in the world． The sharptaste of hot pepper is owned to capsaicinoids．Nowadays， Scientists has found manykinds of capsaicinoids from hot pepper. Capsaicinoids is made of vanillyl amine andfatty acids chain，and the R-of fatty acids chain contribute to the difference betweenthem． Capsaicinoids in hot pepper is capsaicin, which is accounted of46%～77%.The content of other capsaicinoids is very few. Capsaicin as a plants’natural activecomponent has inestimable application. It has been applied in many areas，such asmedicine、agriculture、military affairs、Food.Therefore, the requirement of capsaicinis to grow with each passing day. In hot pepper, capsaicin’accumulation is under thecontrol of biosynthesis and degradation pathway. Scientists can make degradationpathway weaken to get the high accumulation of capsaicin. Peroxidase, which is one ofthe enzymes in degradation pathway, can effectively cause capsaicin’s oxidativedegradation.In my study, to get the high accumulation of capsaicin，I make peroxidasepost-transcriptional gene silencing by microRNAi.Besides, transformation of PODgene regulated by fruit-specific promoter can make gene silencing only betide in fruitof pepper.I design two pairs of PCR specific primers according to the peroxidase’sconservative sequence which is got by using BLASTN tool, and the PCR productsrespectively are called sense fragment and antisense fragment, which are thecomplementary region of hairpin structure. Cloning the intron fragment of pepper’sKAS gene (ketone acyl carrier protein synthesis enzyme), as the loop region ofhairpin structure. Cloning the tomato fruit-specific promoter named2A12. Then,fourfragments are respectively connected to the expression vector3301.Finally,Agrobacterium makes POD gene insert pepper genome and I can get the transgenicpepper plants which can product capsaicin in high-yield by tissue culture in vitro. Main results of this study are as follows:1、POD gene was cloned successfully. Peroxidase gene was made of three exons.The length of open reading frame was999bp. The protein productinon was made of332amino acids.2、The RNAi vector was builded successfully. The connection sequence of thefour fragments on the basis vectors, from upstream to downstream, was:2A12promoter, justice fragments、 intron fragments and antisense fragment.3、Capsicum genetic transformation system was optimizedinduced and screened medium1：MB5+6-BA（5.0mg/L）+IAA（0.5mg/L）+AgNO3（4.0mg/L）+KL（200mg/L）;induced and screened medium2：MB5+6-BA（5.0mg/L）+IAA （0.5mg/L）+AgNO3（4.0mg/L）+PPT （1mg/L）+KL（200mg/L）;elongated medium：MB5+6-BA（2.0mg/L）+IAA（0.2mg/L）+AgNO3（4.0mg/L）+PPT（1mg/L）+KL（200mg/L）+GA3（1mg/L）.4、I got14regenerated plants by tissue culture and obtained a transgenic pepperplants by PCR.