Genetic Transformation And Molecular Analysis Of Transgenic Stem Mustard Plants Coexpressing Coat Protein Genes Of TuMV And CMV

A transformation system of TuMV CP and CMV CP gene mediated by Agrobacterium tumefaciens was established in stem mustard (Brassica juncea var. tsatsa). Cotyledons of stem mustard were used as the explant, and transformants were successfully obtained in the present experiment. This study provided theoretical and technical basis for obtaining virus-resistant plants by modern molecular biotechnology and plant genetic engineering, and technical platform for gene transformation in mustard and Cruciferae as well. The resultes are as follows: 1. Construction of plant bivalent expression vector of TuMV CP and CMV CP genesThe specific primers were designed and synthesized according to the published sequence of TuMV CP and CMV CP gene, TuMV CP and CMV CP gene fragmentes were obtained by PCR amplification using pMD-TuMV CP and pUC-CMV CP as templates, and the TuMV CP amplified fragment was cloned into pCambial301 vector to produce recombinant clone pCam-TuMV CP, and CMV CP amplified fragment was cloned into pBI121 vector to produce recombinant clone pBI-CMVCP.containing CMV CP gene fragment was obtained by digesting the pBI-CMV CP vector with EcoRI and PstI,we could get Plant Bivalent Expression Vector of TuMV CP and CMV CP gene through containing CMV CP gene fragmentinserted into pCam-TuMV CP. pCam-TuMV CP-CMV CP was transformed into A. tumefaciens EHA105 by freezing and thawing method.The positive clones confirmed by PCR and digestion were used for transformation of stem mustard (Brassica juncea var. tsatsd).2. Establish of transformation system using cotyledon as explantThe effect of different concentration of antibiotics on regeneration of cotyledonswas studied. Meanwhile, the effect of pre-culture time(ld, 2d? 3d, 4d),co-culturetime(ld> 2d, 3d. 4d) and on transformation efficiency of cotyledon were investigated in the present study. The best transformation frequency was found in 2-day pre-cultured explants and 10-15 min infection after 2-day co-culture through 12.5mg/l Hgy and 500 mg/1 Carb differentiation medium at 28 °C.3. Molecular identification of transformed shootsBy PCR and RT-PCR analysis, we found that TuMV CP and CMV CP gene were inserted into the stem mustard genome and expressed successfully...