A method to construct cDNA library of the entomopathogenic fungus, <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>, in the hemolymph of the infected locust |
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Authors: | Cangsang Zhang Yueqing Cao Zhongkang Wang Youping Yin Guoxiong Peng Yuxian Xia |
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Institution: | (1) Genetic Engineering Research Center, School of Bioengineering, Chongqing University, Chongqing, 400030, P. R. China;(2) Present address: College of Bioinformation, Chongqing University of Communications and Posts, Chongqing, 400065, P. R. China |
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Abstract: | A method was developed to construct cDNA library of pathogenic fungus in the blood of the infected insect for cloning the
fungal genes expressed in the host. This method is designed to take advantage of the obvious difference between the cell structures
and components of the pathogen cells and that of the host cells. The host blood cells only have cell membrane, which can be
disrupted by using SDS/proteinase K (PK). The fungal cells grown in the animal blood have cell wall, which can protect the
fungal cell from the disruption of SDS/proteinase K (PK). By this method, the blood cells were disrupted by SDS/proteinase
K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase. Therefore, the fungi grown
in the blood were harvested without any contamination of host RNA and DNA. The pure fungi harvested from the infected blood
can be used for mRNA extraction and cDNA library construction. The purity of the fungal mRNA was confirmed by PCR and RT-PCR
with specific primer pairs for the host and specific primer pairs for the fungus, respectively, and the clones of cDNA library
constructed by using the fungal mRNA was also analyzed. The results showed that there was no detectable contaminated insect
DNA or RNA existing in the fungal mRNA. Randomly selected cDNA clones from cDNA library were sequenced and analyzed against
GenBank using Blastx; no selected sequences had significant similarity with insects’ genes in comparison with the data of
GenBank. The results further confirmed that the method to purify the pathogenic fungus from the host animal is reliable and
the mRNA extracted from the fungus is eligible for cDNA library construction, and other molecular analysis including RT-PCR.
This method may be applied to other pathogenic fungi and their host animals. |
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Keywords: | Pathogenic fungus Insect mRNA extraction cDNA library |
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