chi3 Gene Encodes a Chitinase, CHIT30: An Attempt To Locate chi3 in Entomopathogenic Fungi




Pamela Greengarten

Introduction

Entomopathogenic fungi are naturally occurring fungi that act as pathogens towards insects, ticks and other arthropods. Traditionally, tick populations have been regulated through the use of acaricides but due to the evolution of resistance, (Ostfeld et al. 2006, Roush 1993), biological control (biocontrol) is being looked at as a way of controlling pest populations.

Fungal chitinases are enzymes which are required for hyphal growth and are thought to be partially responsible for host penetration (Takaya et al. 1998, Tikhanov et al. 2002). Additionally, chitinases break down chitin microfibrils which make up approximately 30 % of a ticks cuticle. (Silva et al 2005, Tikhanov 2002). In this study, I sought to locate a particular chitinase gene in four entomopathogenic fungi. The chi3 gene encodes the CHIT30 chitinase that has been sequenced and is present in the NCBI database for the species Metarhizium anisopliae (M. anisopliae). This gene has not been characterized in three common entomopathogenic fungi, namely Beauveria bassiana (B. bassiana), Paecilomyces fumosoroseus (P. fumosoroseus) and Paecilomyces farinosus (P. farinosus). I sought to characterize the chi3 gene sequence of these species.

Figures


Figure 1-Figure 1. PCR products using Primer Pair #1 , Lanes 1-5 and Primer Pair # 2, Lanes 6-10. Sizes of the PCR products were determined by using the 100 bp ladder as a comparison. The 170 and 209 bp products were of the expected size. M. anisopliae was the only species that generated product.


Figure 2-Figure 2. PCR products using Primer Ba02. All lanes generated the expected 140 bp size for the microsatellite loci designed for B. bassiana. A 100bp ladder was used to assess the size of the products.


Figure 3-Figure 3. Aligned sequences from NCBI database. A) Sequence alignment for the 170 bp product using Primer Pair #1. The alignment shows 93% identity in the sequence for M.anisopliae for the chi3 gene. B) Sequence alignment for the 210 bp product using Primer Pair #2. The alignment shows 92% identity in the sequence. C) Sequence alignment for the 140 bp product using Primer Pair Ba02. The alignment shows 100% identity to the B. bassiana clone Ba02 microsatellite sequence.


The goal of this project was to locate and characterize the chi3 gene in three species of entomopathogenc fungi, namely B. bassiana, P. fumosoroseus and P. farinosus.

The primers designed for use in this experiment were found to be unable to amplify the gene in B. bassiana, P. fumosoroseus, and P. farinosus. The only amplified DNA fragment came from M. anisopliae, which was the species whose DNA sequence was used to design the primers.

Since the designed primers were unable to amplify the gene in three of the species used for this study, I can not make a definitive conclusion as to the presence or absence of the chi3 gene in these organisms. However, I am able to conclude that either the gene does not exist in B. bassiana, P. fumosoroseus and P. farinosus, or they are not homologous enough to M. anisopliae to enable PCR amplification using the primers generated.

Future studies should be performed to characterize the chi3 gene of these three species.

Full Paper

Acknowledgments

I would like to thank Dr. Amy Tuininga and Dr. Berish Rubin for their gracious input in helping me come up with this project. Thank you to Leleesha Samaraweera, Joe Frezzo, Jinsong Qui and Dr. Sylvia Anderson for your tireless efforts and guidance throughout this project. Thanks to my classmates for their good cheer and humor when things got stressful. Finally, I would like to thank the Biology Department for funding this project.


This document was last modified 05/09/2007.
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