The Knockout And Analysis The Function Of Scytalone Dehydratase In Metarhizium Robertsii

How to control agricultural pests is still a puzzle for people to solve.Currently,more and more people are concerned microbial pesticides,because it use biology to control biology and also has the advantages of safe,non-polluting and so on.In the field of microbial pesticides,insect pathogenic fungi was treated as insecticide fungi and play an important role in the pests control,however the research of insect pathogenic fungi has become a hotspot increasingly.As a model creature of entomogenous fungi,Metarhizium robertsii has played an important role in studying genetic regulatory mechanism,pathogenesis and biological control of entomogenous fungi.DHN melanin synthesis pathway where scytalone dehydratase(SCD)gene is a key gene exists in most fungi,it is closely related to the pathogenicity of fungi and also helps fungi to resist the unfavorable environment.In the current studies,it indicated that wild-type Metarhizium robertsii lacked the machinery for DHN melanin synthesis and the pathway of pigment synthesis in Metarhizium robertsii still needs a further study.In this paper,the SCD gene of Metarhizium robertsii was determined from NCBI and suitable primers were designed according to the sequences of its upstream and downstream.Recombinant plasmid was successfully constructed by digestion and connection.Then recombinant plasmid was transformed into Agrobacterium tumefaciens,so that knockout strain and the complemented strain were obtained through transformation in fungi mediated by Agrobacterium tumefaciens.The differences between knockout strain and wild-type strain were analyzed to predict the function of SCD gene.The main results are as follows:(1)The SCD gene sequence of Metarhizium robertsii was obtained by NCBI,and its upstream and downstream gene sequences were constructed in the same way.The recombinant plasmids were constructed and sequenced.(2)Recombinant plasmid was transformed into Agrobacterium tumefaciens,so that knockout strain and the complemented strain were obtained through transformation in fungi mediated by Agrobacterium tumefaciens.(3)The biological characteristics of wild-type strain,knockout strain and complemented strain were studied.The differences among knockout strain,wild-type strain and complemented strain were analyzed to predict the function of SCD gene.1)Compared with wild-type strain,the colony of knockout strain was smooth and regular on the edge and the hyphaes were presented radial while the size and color of the colony did not change.Especially,the color of the colony on the PDA medium is still dark green,which proved that wild-type Metarhizium robertsii lacked the machinery for DHN melanin synthesis again.2)The wild-type strain,knockout strain and complemented strain were respectively inoculated to PDA,SDAY,1/4 SDAY medium,and the diameter of growth was measured.Compared with wild-type strain,the growth rate of knockout strain on PDA,SDAY,1/4 SDAY medium did not changed.3)The wild-type strain,knockout strain and complemented strain were respectively inoculated on PDA medium,the number of spores was calculated respectively after 12 days of incubation.Compared with wild-type strain,the knockout strain had a decreased sporulation.4)The wild-type strain,knockout strain and complemented strain were respectively inoculated on SDS+PDA,NaCl+PDA,menadione+PDA,congo-red+PDA,carbendazim+PDA,H2O2+PDA medium,the growth of colonies were observed and recorded.The results show that SCD gene has no influence with the anti-fungicide ability,antioxidant ability and anti-hypertonic ability of Metarhizium robertsii,also it does not participate in the synthesis of cell wall.5)The spores of wild-type strain,knockout strain and complemented strain were irradiated under UV lamp,then cultivated with germination solution.After 26 h,germinations were observed.Compared with wild-type strain,the knockout strain showed a significant decrease trend in UV tolerance.6)The corn borers were infected in the suspension of the wild-type strain,knockout strain and the complemented strain,then fed at 25 to observe the infection.Compared with ?wild-type strain,the ability of knockout strain to infect corn borers was not changed.The results showed that the SCD gene was related to pigment in Metarhizium robertsii,which provided that whid-type Metarhizium robertsii lacked the machinery for DHN melanin sythesis.At the same time,the function of SCD gene in Metarhizium robertsii was analyzed,which provided a theoretical basis for the better application of Metarhizium robertsii in biological control and the future study of pigment synthesis pathway of Metarhizium robertsii.