The Construction Of Pepper Transformming System And Its Application In The Transformation Of RNAi Vectors Of Several Candidate Genes

Pepper (Capsicum annuum) is a very important solanaceae vegetable crop which is widely cultivated all over the world. The genetic improvement and utilization of good cultivars of pepper is the most economic way to ensure quantity, quality and efficiency in its production. Contrast to conventional breeding, gene engineering improve crop traits more effectively by transferring genes among different plants and among plants, animals, as well as microbes, which is based on the establishment of transformation system. A lot of attention had been paid to the establishment of genetic transformation of pepper in the past decades, but the efficiency of current used transformation system is not high enough for practical use, and most of them showed an obvious genotype-dependence. In this study, we try to establish a high efficient in vitro culture regeneration system and Agrobacterium-mediated transformation procedure for pepper, and vectors of several candidate genes of pepper were transformed into the genome of pepper by this system. The main findings are as follows:1 The foundation of pepper in vitro culture regeneration system: The most suitable explant for in vitro culture was 14d seedling age of aseptic pepper's shoot-tip and the cotyledon; To set up pepper cotyledon and shoot-tip explants for the induction directly of multiple buds system. In differentiation medium, add the BA and NAA conducive to the induction of multiple buds, but high concentration of NAA was easy to produce high root. Add the PJ that OD600 value of 1.1 4ml / L in favor of peppers differentiation. In this study, the most suitable medium for the induction of pepper is MS + BA 5mg / L + NAA 0.1 mg / L + AgNO3 4mg / L + PJ 4ml / L; In different genotypes of pepper cotyledon and shoot-tip induced rate of multiple buds have significant differences; Add 0.5mg / L GA3 in medium conducive to multiple buds elongation, but higher concentration of it will easily induce deformed buds; Through the filter we determined that the optimum medium for rooting pepper is 1 / 2 MS + NAA 0.1mg / L + IAA 0.1mg / L;2 The foundation and application of pepper Agrobacterium-mediated transformation procedure: The experiments of the genetic transformation of pepper show that: Capsicum's Kanamycin lethal concentration can be to 70mg / L; The best conditions for pepper cotyledon genetic transformation is: pepper cotyledon must be pre-culture about 2d, then pick the explants infected by immersion 8 min in a suspension of Agrobacterium which was add AS 200μmol/L that the value of OD600 was about 0.6. After 2d co-culture we wash the bacteria and switch to the filter-culture; We determined that the rooting medium should be added cephamycin to inhibit Agrobacterium growth; This study was obtained a small number of transgenic plants and has been transferred to the target gene by PCR verification. Applies the regeneration system and the transformation procedure to transform the vector of CabZip2,CaLRR,CaP, etc. The vector of CabZip2 has obtained eight clones, the vector of CaLRR has obtained fifteen clones, and the vector of CaP has obtained nineteen clones. The transformation efficiency of this vector were 1.67%,3.13%,2.35. The proportion of PCR-positive transgenic plants in the resistance regenerated plants were 25%,33%,21.05%.The results showed that the efficiency of the regeneration and genetic transformation system was high enough for the application in gene engineering as well as functional genomics study of pepper, and the transgenic plants acquired in this experiment laid an important foundation for further analysis of the candidate genes.