[Cloning and DNA sequencing of the gene encoding trichosanthin]

A gene encoding Trichosanthin (TCS) was amplified by means of DNA polymerase chain reaction (PCR) and cloned from the genomic DNA of Trichosanthes kirilowii. Its DNA sequence has been determined. Nucleotide sequence analysis indicates that the cloned TCS gene includes 5'-flanking region and the encoding sequence for TCS preproprotein. Comparison of its encoding sequence with the published nucleotide sequences of three TCS genes demonstrates that they have the sequence homologies of 99.20%, 98.74% and 98.64% respectively. Comparing its deduced amino acid sequence with four reported amino acid sequences of TCS demonstrates that they have the sequence homologies of 98.62%, 97.62%, 97.41% and 98.38% respectively. A new TATA-like box sequence was found in the 5'-flanking region of cloned TCS gene. In order to further study the structure, expression, regulating mechanism of TCS gene and the structure-functional relationship of TCS, a series of mutants of TCS gene has been constructed. The expression of these mutants in bacteria and transgenic plant has being studied.