| Metarhizium anisopliae, a kind of entomopathogenic fungus widely applied in domestic and abroad, has an important role in biological control of insects. Compared with chemical insecticides,mycoinsecticides are not infective or toxic to human and do not pollute environment, but slow speed of kill is perceived as a potential drawback to the use of fungi against locusts. Studies on mechanisms of fungal pathogenesis and host defense may suggest strategies for the development of more efficient anti-locust mycoinsecticides. After the fungi breakthrough insect epidermis barrier invading host heamlymph, entomopathogenic fungi mainly grows in the blood cavity multiplies by form of insect mycelium etc. in the host in vivo haemolymph until led to the host died. Most of entomopathogenic fungi can penetrate the insect cuticle rapidly, and generally they can invade into host body about 24 hours, but it need a week or even long time to kill insect. So it has the vital significance to study host lethal mechanism after entomopathogenic fungi invade into host body. It is main research work on pathogenic mechanism to explore gene expression after entomopathogenic fungi invade into insect body. It is also significantly important to study the role of entomopathogenic fungi in pathogenic mechanism to directly obtain entomopathogenic fungal bodies from infected insects in order to isolate and clone expressive gene of Metarhizium anisopliae after their invading into insect body. In this paper, orthogonal design was adopted to optimize the factors for disruption of locust haemocytes and degradation of their DNA and RNA from the mycosed locust by Metarhizium anisopliae. The optimum conditions for separation and purification of the fungal cells from mycosed locust were achieved. In the optimum conditions,fungal cells and mycelia were separated and purified from the haemolymph of locust infected by Metarhizium anisopliae without any contamination of the host DNA and RNA tested by PCR and RT-PCR method using a locust specific primer pair. This lays foundation for separating and cloning the fungal genes expressed in the haemolymph of locust after penetration. The main results were as follows: 1. The sensitivity of locust specific primer 1, 2 and Metarhizium anisopliae specific primer 3, 4 was analyzed. 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 primer 3, 4. 2. Orthogonal design was adopted to optimize the factors for disruption of locust haemocytes and degradation of their DNA and RNA from the mycosed locust by Metarhizium anisopliae. The degradation method of the Metarhizium infected host haemocytes, DNA and RNA was as follow:the final concentration of proteinase K in the reaction is 1mg/ml at 26℃for 60min;concentration of deoxyribonuclease I and ribonuclease A is 5U and 0.5mg/ml respectively at 30℃for 35min. 3. According to the optimal enzyme digestion condition, we dealed with infected locust haemolymph, and extracted Metarhizium anisopliae DNA using traditional method of genome DNA extraction after digestion finished. Then we carried out PCR and RT-PCR to validate using locust specific primer and Metarhizium anisopliae specific primer. The result showed that there were no locust DNA and RNA contaminated in Metarhizium anisopliae DNA obtained from infected locust haemolymph. |
