Cloning Of 42kDa Chitinase Encoding Gene From Trichoderma Viride LTR-2 And Construction Of Transgenic Tomato

As an important economic crop and a biological model in genetics,tomato is of considerable value in science research and is widely studied in plant genetic engineering.Construction of transgenic tomato by inserting foreign resistant genes cloned from other organisms to its genome is an effective method in developing new disease-resistant species to reduce the chemical pesticide application.Chitinase is one of the fungal cell wall degrading enzymes which exist in animals,plant,and microorganisms.Transgenic plants expressing chitinase encoding gene can resist many kinds of pathogens efficiently.However,chitinases coming from different organisms have quite variable effects in resisting pathogens and/or diseases.Thus,to choose the most effective chitinase gene is one of the most important steps in constructing pathogen-resistant transgenic plant.Trichoderma is an important biocontrol fungus which produces various chitinases to degrade the cell wall of the fungal pathogens in the process of hyperparasitization.Among all the chitinases produced by Trichoderma, 42kDa chitinase is considered to be the most effective one in degrading the cell wall of pathogens.Trichoderma viride strain LTR-2 isolated from the rhizosphere of vegetable field is effective in controlling various plant pathogens,and its preparation has been patented in China Bureau for Patent and registered as a new bio-pesticide in China Ministry of Agriculture.One of the important mechanisms of LTR-2 is the production of chitinases. A fragment from Trichoderma viride LTR-2 was successfully cloned by the method of PCR amplification with a pair of primers designed for 42kDa chitinase encoding sequence.The PCR product is 1508 bp and contains an open reading frame of 1459 nucleotides starting with the initiation codon ATG at position 45 and ending with the termination codon TAA at position 1501,and the number of deduction amino acid is about 424.DNA sequencing analysis shows that the sequence has a homology of 99%to those Trichoderma 42kDa chitinase encoding genes published in GenBank,and thus the PCR fragment was verified as the 42kDa chitinase encoding gene and was published in Genebank(GenbankID:EF635427).The fragment was ligated with CaMV35S promoter and 35S-polyA terminator,and then was inserted into the vector of the pCAMBIA1302.Then,the vector of pCHI1302-42 was introduced into Agrobacterium tumefaciens LBA4404 by freezing-thawing method.Leaf disc method was adopted to transfer 42kDa chitinase gene into tomato.Finally,the insertion of the target gene in tomato chromosomal DNA was verified by PCR and Southern blotting methods.