Studies On The Construction Of CHS RNAi Expression Vector And Genetic Transformation Of Petunia Hybrida

RNA interference, as an important gene silencing methods, it has a significantapplication to the research of gene function and genetic melioration. This paper constructthe siRNA (short interference RNA) expression vector of CHS(chalcone synthase) gene ,research the foundation of regeneration system about Petunia Hybrida and inheritabletransformation system by gene gun and Agrobacterium . Then Research the inheritabletransformation of the siRNA expression vector of CHS about Petunia Hybrida .Get theresults as below: 1.Construct two RNAi vectors named as pBI121-CHS to intervene the expression ofgene CHS about Petunia Hybrida .The vector has a reverse repeated sequence as a foreignsegment. The segment should match between the reverse sequences and form a shorthair-pin RNA (shRNA) after the transformation of vector. 2.Found a mature regeneration system of Petunia .Research the inducement effect ofphytohormone ,confirm the optimum culture medium of callus inducement is MS + 2,4-D2.0(mg/L)+ 6-BA0.1(mg/L)+ KT0.1(mg/L)+ NAA0.1(mg/L);the optimum culturemedium of sprout differentiation is MS + 6-BA1.0(mg/L)+ KT0.2(mg/L)+ NAA0.3(mg/L);the optimum culture medium of root is MS + NAA0.4(mg/L)+ IBA1.0(mg/L). 3.Confirm the optimum fastness concentration of Kan about Petunia Hybrida .By thesusceptivity experiment of Petunia Hybrida under the different concentration ofKan ,conclude the optimum concentration of Kan is 80mg/L ,the plant can be choosed bythis concentration. 4.Found the inheritable transformation system of gene gun .Study the effect of targetdistance and bombardment times on transformation. Confirm the optimum bombardmentparameter is that 60mm as target distance and twice as bombardment times. Finally , obtainfour fastness seedling . 5. Found the inheritable transformation system of Agrobacterium . Study the effect ofAgrobacterium concentration and infection period on transformation. Confirm theoptimum concentration is (OD₆₀₀) is 0.5,and the optimum infection period is 5min. Finally ,obtain seven fastness seedling . 6.By the means of PCR and Southern blot, the extrinsic segment be proved to beconformed into the genome of Petunia Hybrida, and obtain 3 transgene plant. 7.By the observation of the flower colour between the transform with untransformplant.We find the colour has a slight change. Therefor, we determine the extrinsic segmentbe transformed into the genome of Petunia Hybrida, and have an interfering effect on theflower colour. To make a summary, this paper take the lead in studying the interference function ofgene CHS by siRNA. By the studying on construction of expression vector,foundation ofregeneration system and transformation methods. Found a mature RNAi inheritabletransformation system .Provide a reference on studying and utilizing of the RNAitechnique in the improvement of plant quality.