The RACE amplifying and sequence analyzing of secreted aspartic proteinase gene SA76 of Trichoderma harzianum

Abstract

Total RNA was isolated from mycelium of T. harzianum by Total RNA extraction kit, and two clear bands of rRNA (28S and 18S) were observed in agarose electrophoresis. By joining the 3′end sequence with the known SA76 EST from cDNA library of T. harzianum, a full-length cDNA sequence of 2019bp was obtained, whose open reading frame contained 1593bp, a stop codon TAA, a 5′untranslated region (5′UTR) of 266bp, a 3′untranslated region (3′UTR) of 201bp, and poly (A) 29 encoded a protein of 530 amino acids, had a signal peptide. T. harzianum shared 53% identity of secreted aspartic proteinase gene with G. zeae, 37% with N. crassa and 36% with C. globosum. The full-length cDNA sequence of secreted aspartic proteinase gene from T. harzianum was cloned for the first time by using BD SMART RACE technique, which provides a foundation to obtain and validate functional genes of T. harzianum.

Keywords

BD SMART RACE / T. harzianum / secreted aspartic proteinase gene

References

[1] Chenchik A., Moqadam F., Siebert P. Marathon cDNA amplification: A new method for cloning full-length cDNAs Clontechniques, 1995, 1: 5-8.

[2] Chenchik, A., Moqadam, F., Siebert, P. 1996. A new method for full-length cDNA cloning by PCR. In: Krieg, P. A (Ed), A Laboratory Guide to RNA: Isolation, Analysis, and Synthesis. Wiley-Liss, Inc., p273–321.

[3] Geremia R.A., Goldman G.H., Jacobs D.A. et al. Molecular characterization of the proteinase-encoding gene prb1, related to mycoparasitism by Trichoderma harzianum Mol. Microbiol., 1993, 8: 603-613.

[4] Harman G.E., Björkman T. Potential and existing uses of Trichoderma and Gliocladium for plant disease control and plant growth enhancement Enzymes, Biological Control and Commercial Application, 1998, 2: 229-265.

[5] Liu P., Yang Q. Identification of genes with a biocontrol function in Trichoderma harzianum mycelium using the expressed sequence tag approach Research in Microbiology, 2005, 15: 416-423.

[6] Louie K., Patricia A.C. Characterization of a cDNA encoding a subtilisin-like serine protease (NC-p65) of Neospora caninum Molecular and Biochemical Parasitology, 1999, 103: 211-223.

[7] Manczinger L., Polner G. Cluster analysis of carbon source utilization patterns of Trichoderma isolates Syst. Appl. Microbiol., 1985, 9: 214-217.

[8] Nathalie P., Stephanie G., Christine R. et al. AspS encoding an unusual aspartyl protease from Sclerotinia sclerotiorum is expressed during phytopathogenesis FEMS Microbiology Letters, 2001, 194: 27-32.

[9] Schaefer B.C. Revolutions in rapid amplification of cDNA ends: new strategies for polymerase chain reaction cloning of full-length cDNA ends Analytical Biochemistry, 1995, 227: 255-273.

[10] Zheng Y., James R.C. cDNA cloning by amplification of circularized first strand cDNAs reveals non-IRE-regulated iron-responsive mRNAs Biochemical and Biophysical Research Communications, 2000, 275: 223-227.