Knock-Out Of RAC Gene In Trichoderma Harzianum

Trichoderma harzianum is an important biocontrol fungus which has the ability to control multiple plant pathogens such as Rhizoctonia solani, Sclerotium rolfsii, Fusarium oxysoprum, Phytophthora nicotine and so on. In the process of T.harzianum against plant pathogens, physical or chemical signals will be generated by the pathogens. T.harzianum has the corresponding receptors to recognize these signals, then the receptors will be activated. Activated receptors initiate multiple signaling pathways in the T.harzianum cell, so a series of cell functions will change. The small GTPase coupled receptor mediated signal transduction is an important signal transduction pathway. The small GTPase RAC protein is a member of Rho family. The function of rac genes in the mammalian cell has been researched more in-depth but relatively less study in filamentous fungi.This study use of PCR methods successfully coined the DNA sequence of rac gene encoding DNA sequence and its upstream and downstream DNA sequence in T.harzianum .The total length of this sequence is 2741bp. The length of upstream DNA sequence, rac gene sequence and downstream DNA sequence are 782bp, 990bp and 969bp. By comparing the rac gene encoding DNA sequence and cDNA sequence we found that the rac gene contains 5 introns which started with GT and ended with AG. Comparing the rac gene DNA sequence with the genome of Trichoderma reesei , Trichoderma atroviride and Trichoderma virens, we found the rac gene DNA sequence have the highest DNA sequence similarity with a part of DNA sequence of T.virens genome.According the 2741bp rac gene DNA sequence we choosing the appropriate restriction enzyme cutting site to construct two plasmid vectors pPZPTtk8.10::rac::hyg1 and pPZPTtk8.10::rac::hyg2 to knockout the rac gene in T.harzianum . Double enzyme digest and plasmid DNA sequence all proved the two plasmids were successful constructed. The two plasmids were transformed into T.harzianum by Agrobacterium tumefaciens-mediated. Through positive selection and negative selection we have successfully get the T.harzianum transformations with the rac gene knockdout.