The Construction Of Pepper (Capsicum Annuum L) Transformation System And Its Application In The Transformation Of NAC1,CDPK,CIPK Candidate Genes

Pepper (Capsicum annuum L.) is an important vegetable crop, genetic transformation is required not only for its gene engineering but also for its functional genomics study. However, high efficient genetic transformation system of pepper is corrently unavailable, and the transformation system established so far is obviously genotype dependent. so it is ugent to establish a high efficient and genotype independent transformation system for practical use in gene engineering and functional genomic study in pepper. In this study, a high efficient Agrobacterium—mediated transformation system of pepper was established with 9 pepper which was further used for the transformation of 3 candidate genes for over expression and RNAi analysis. The main results are as followings:1. Optimization of regeneration system of pepperThe regenerating system of pepper was optimizated using cotyledon with handle from seedlings of 12-16 days after germination as explants and bud induce medium (MB+ 5mg/ L 6-BA+ 0.1mg/ L IAA+ 4mg/ L AgNO₃+ 30g/ L sucrose +7g/ L agar) , bud elongation medium(MB+ 3 mg/L 6-BA+1 mg/L IAA+1.5mg/LGA₃+4mg/LAgNO₃+30g/ L sucrose+7g/ L agar) and rootage medium(1/2MB+0.2 mg/ L IAA+ 0.1mg/ LNAA+ 30g/ L sucrose+7g/ L agar.2. Establishment of efficient genetic transformation system of pepperWe adopt different concentration of Kan in different growth stage of explants. 75 mg/ L in stage of bud inducement, 50 mg/ L Kan for cotyledon and 75mg/ L Kan for hypocotyl in stage of bud elongation, 40 mg/ L for rootage. Carbenicillin of 500 mg/L restrained the growth of Agrobacterium efficiently. Cefotaxime of 200 mg/ L was benefit for root inducement. The explant was pre-cultured for 3 days, co-cultured for 2 days and infected by Agrobacterium(OD600=0.6)for 6-8min which is optimum for transformation. Translation rate of Flamingo-bill, cotyledon with handle and cotyledon explant is 33%, 23.6%, 21.1%。3. Application of the transformation system and detection of transgenic pepper plantsOver expression vectors or RNAi vectors of 3 candidate genes of pepper, i.e. NAC1(nascent-associated complex),CIPK (calcium sensor-interacting protein kinase ) and CDPK(Calcium-dependent and calmodulin-independent protein kinase ) were transferred into the genome of pepper via the transformation system we established, 55 regenerating Kan resistance plants of CaNAC1, CaCDPK, CaCIPK were acquired respectively. Among the regenerating Kan resistance plants, 16 were PCR positive with the specific primers of CaNAC1, CaCDPK, CaCIPK repectively.