Cloning Of Cowpea Tryptin Inhibitor Gene And Construction Of Plant Expression Vector

In this paper the total genomic DNAs were extracted from the cowpea young leaf tissue by the phenol/chloroform/isoamyl alcohol/RNAaseA method.Using the DNAs as template and 27bp long oligonucleotide with BamI-1l in 5 terminus as primer which confers gene sequence of the cowpea Bowman-B irk type trypsin inhibitor known and ATG codon, the cowpea tryptin inhibitor (CPu) gene was amplified by polymerase chain reaction(PCR).The length of the double stranded DNA amplified was about 340bp.The 340bp BamHl restriction fragment was cohensive end ligated into the BamHI site of the plamid vector pGEM3zf(+).The resultant recombinant molecules were used to transform DHS a strain of Escherichia coli. for blue/white clones screening. Rapid electrophoresis and restriction endonuclease(BamHl) digestionanalysis showed that the-most of the recombinant plamids from the white clones contained insertions with about 340bp length.The clones with a DNA insert in either orientation were indentified by restriction mapping(PstI,EcoRI).The result showed that the CPu gene inserted was positive orientation ligated into plamid pGEM3zf(+). The cloned CPu DNA fragment also was verified by PCR and DNA sequencing analyses.The insertion contained the 240bp coding sequence for the mature inhibitor and a 81 bp leader sequence with three in-phase ATG-methionine codons.Only one nucleotide in the leader sequence was different from that of CPTI cDNA reported by Hilder .That was TTG codon replaced by CTG.But it was the same to Liu Chunming and Liu Yuan.Both the nucleotide encode Leu.The cloned CPu DNA respectively shared 99.8% and 100% homology with published CPTI cDNA nucleotide sequence.Either by DNA-PCR method or by RNA cDNA-PCR method,it was the same sequence.Deleting a 2Oaa leader sequence by SmaI-NcoI digestion, blunting the cohensive end by' Klenow polymerase and ligating by' T4DNA ligase. the 280bp long SacI-Xbal restriction fragment containing the 8Oaa coding sequence for the mature protein inhibitor and a 7aa leader sequence with one in-phase ATG-methionine codon in the upstream of the coding region was inserted into plant expression vector pCAMBIA 1301 digested with the two same restriction endonuclease. Thus the plant expression vector --pCACL-has been constructed. Introducing CPu gene into many important crop plants should be technologically feasible, and expression of CPTI gene in the transgenic plants resulted in enhancement of insect resistance.This paper also discussed the practical values and future prospects about insect resistance transgenic plants.