A Method To Obtain Pure Fungus Metarhizium Anisopliae And Construct Fungal CDNA Library From Infected Insect Haemocoel

So far, it is lack of effective method to study the realtime fungal gene expressions which are important to elucidate fungal infection mechanism during fungi infecting insects. In this paper, using Metarhizium anisopliae and Locusta migratoria as research material, we create a new method to obtain fungal bodies from host in vivo and construct fungal cDNA library.We designed the method of isolating fungal bodies from the locusts on the base of the different cell structure between fungus and animal. The outer part of the fungal cell is the cell wall, which is mainly composed of polysaccharide, compared to that of an animal cell, which is membrane. So, during the process of enzyme digestion, the cell wall of fungus can protect its cell from proteinase K digesting while proteinase K can digest any protein remaining from the host eukaryotic cell.Under the experemental conditions, the blood cells had been disrupted by SDS/proteinase K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase, so the fungus grown in the blood were harvested without any contamination of host RNA and DNA. The pure fungus harvested from the infected blood can be used for mRNA extraction and cDNA library construction. 90 randomly selected cDNA clones from cDNA library were sequenced and analyzed against GenBank using BlastX in order to comfirm the method.Two specific primer pairs, which were used as a monitor system to comfirm our experimental result, designed according to fungal tubulin gene and locust histone gene respectively in this study. Primer pair H1and H2 was used to test whether locust DNA existed in the third supernatant, which was washed by MilliQ H₂O after enzymolysis was completed. Before cDNA construction, it was also used to comfirm whether the extracted fungal mRNA was contaminated by locust DNA or mRNA, while the primer pair tu1and tu2 was used to test and confirm whether the extracted mRNA was fungal mRNA.The main results were as follows:1. The sensitivity of locust specific primer 1, 2 and Metarhizium anisopliae specific primer 3, 4 was desiged according to locust histone gene and fungal tubulin gene and analyzed by PCR for their specificity and sensitivity. The results showed that 0.1pg locust DNA can be detected by using locust specific primer 1, 2 and 1pg Metarhizium anisopliae can be detected by using Metarhizium anisopliae specific...