Construction and transient expression of vacuolar invertase gene antisense expression vector in tomato leaves

Total RNA was isolated from tomato (cv. Zhongshu No. 4) leaves by using the specific primers designed according to the sequence of vacuolar invertase gene in GenBank. A fragment with the length of 668 bp was amplified by reverse transcription and polymerase chain reaction and cloned into pMD18-Tsimple vector. Sequence analysis showed the amplified fragment was the TIV1 fragment of the vacuolar invertase gene. The fragment was cut by two restriction enzymes SalⅠand BamHⅠ, and inserted into plant expression vector pBinAR between CaMV 35S promoter and OCS terminator to form antisense plant expression vector pBinAR-aTIV1. The pBinAR-aTIV1 was identified by PCR amplification with TIV1 gene primers, and the direction of connection was identified by double restricted enzymes digestion. The result showed that the expression vector was successfully transferred into agrobacteriumtum EHA105 with method of colony PCR. The activity of vacuolar invertase was decreased by transient expression of antisense fragment in tomato leaves.