Analysis Of Differentially Expressed Genes Of Rhizoctonia Solani During The Sclertium Formation

Rhizoctonia solani, a group of important plant pathogenic fungi, can cause serious disease in a variety of crops, such as rice and wheat.Reports had been made about the mechanism of formation and development of the sclertium of Rhizoctonia solani, but the main focus of these reports was on nutrition and environmental factors. Despite some useful research, there is still lack of the knowledge on the molecular mechanism of sclerotium formation of Rhizoctonia solani, the sclertium formation-related genes are still yet to be found.In order to clarify the molecular mechanism of the sclertium formation, a cDNA library of specifically expressed genes during the sclertiumformation was constructed by using Suppression Subtractive Hybridization (SSH) combined with Virtual Northern Blot technique. Total RNA and mRNA were respectly isolated and purified from the right growth period of the material. The hypha mRNA of Rhizoctonia solaniGD-118, caused rice sheath blight, was used as the driver, and the sclertium mRNA was used as the tester. After digested by Rsa-â… , driver and tester cDNA were ligated with different Adaptors, two steps of hybridization and two round of PCR were followed to normalize and enrich differently expressed genes. Then the SSH product was ligated with T-vector and transformed into E.coli competent cell DH5a, and the recombinant clones were screened by white-blue plaque selection to construct subtracted cDNA library of specifically expressed genes during the formation of sclertium.150 clones were randomly picked from the library and identified by PCR, results showed that 121 differentially expressed cDNA were inserted success fullyand the size of the inserts was mainly between 200 to 500bp. Then these positive clones were selectedand sequenced. The sequence information was analyzed with BLAST program, and differentially expressed ESTs were obtained. The effectiveness of the subtractive hybridization was verified by Virtual Northern Blot of randomly selectedclones used hypha,sclertium cDNA sequence as probes.Finally,28 specific sclertium formation-related gene fragments were obtained after sequencing and homo logy-blasting, whichwere mainly related to stress response, transcription reguLation, signal transduction, metabolismin the pathogen. And 12 out of 28 did not get right match in GeneBank, which may be newly found sclertium formation-related genes. The information generated in this study provides scientific clues to understand the mechanism of the formation of sclertium of Rhizoctonia solani.