Cloning And Sequencing Of TMV Rehmannia Isolate Movement Protein Gene And Transformation To Tomato

According to published nucleotide sequence of tobacco mosaicvirus(TMV) .movement protein (MP) gene of TMV isolated from Dihuang ( Rehmannia glutinosa) was synthesized by reverse transcripation polymerase chain reaction( RT-PCR) and cloned into pMD-18-T vector to obtain the recombinant plasm id pTMP-D. The results of sequence analysis shown that the ORF of MP gene is 804bp.encode 268 amino acids .Comparing with strain TMV-U1, which shares 78% identity in terms of nucleotide and 80% of deduced amino acids respectively. Because the lower homologous, it is supposed that the Rehmannia isolate maybe a new strain of TMV. The study of biological characteristics and genome of this isolate is in process.A deletion mutant (5-terminal 21bp deleted) was obtained by PCR .The full-length MP gene and the deletion mutant were constructed into expression vector pET30a(+).The two expression vectors (pEMP-F and pEMP-D) containing full-length MP gene and its 5-regional deletion mutant were acquired respectively.With tomato variety ZhongShu 6 as tested material .the preliminary studies on cotyledon differentiation medium and the natural tolerance of Kanamycin and Cephalosporin .It showed that the best optimum medium for callus differentiation and bud regeneration is MS + 6-BA 1.0 mg/L +IAA0.2 mg/L. The appropriate concentration of kanamycin and cephalosporin were 10 mg /L and 400 mg/L in transformation of ZhongShu 6.The full-length MP gene and the 5-regional deletion mutant were cloned into the binary vector pBIN19 with the help of the intermidiate vector pROK219 to obtain the plant expression vector pBMP-F and pBMP-D. The constructs were used for transformation of tomato variety ZhongShu 6. Calluses have obtained through leaf discs transformation. Transformation work is still in processing.