The Gene Cloning And Functional Analysis Of Nrlac1 Gene In Nomuraea Rileyi Nr01-ym

As an entomopathogenic fungus,Nomuraea rileyi can naturally infect a variety of noctuidae pests such as Spodoptera litura.Compared with chemical insecticides,biopesticides are eco-friendly,free of contamination,circularly infectable and not easy to cause drug resitance.As a pesticidal fungus,conidium is the effective form of N.rileyi.Due to long sporulation cycle and strict sporulation condition,N.rileyi coindium is not the ideal product for commercial use.Therefore,it is necessary to study the sporulation characteristics and stress resistance mechanism of N.rileyi.A spore color mutant strain caused by T-DNA accidentally insertion was discovered in the early stage of the study.The intrinsic color of N.rileyi conidia is dark green other than the yellowish color of the mutant.In this study,subsequent experiments were conducted on the mutant.In order to find the disrupted gene or random insertion site,the mutant strain was subjected to genome-wide sequencing analysis and found that the T-DNA insertion site is the upstream promoter site of a laccase gene Nrlac1.To further validate the results of genome-wide sequencing,the Nrlac1 knockout and complemented strains were constructed,and the Nrlac1 gene function was further studied.The main conclusions are as follows:?1?Nrlac1 gene knockout and complementary strain constructionAccording to the whole genome sequence of N.rileyi,Nrlac1 knockout and complementary vector were constructed by the sequences of about 2,000 bp upstream and downstream of the lac1 gene with appropriate cleavage site.The Agrobacterium containing the successfully constructed knockout and recovery vector plasmids was respectively co-cultured with the wild type and Nrlac1 deletion stain blastospores.The mutant strain?Nrlac1 and the complementary strain CP was reconfirmed by Southern blotting experiment.?2?The phenotype of?Nrlac1 strainThe conidia of?Nrlac1 strain are yellow,the colony of?Nrlac1 is compacts,while the conidia of the wild-type strain are green,the spores are fluffy and dry.The spreading ability of spore by wind decreased so much with losing green pigment,which affects the characteristics of spores that can be circulated in the wild.?3?The capacity of?Nrlac1 was decreased to resist ultraviolet and damp-heat stressThe conidia germination rate of mutant was significantly lower than wild type under the same intensity of ultraviolet irradiation and heat-moisture treatment?HMT?,while the deletion of Nrlac1 gene.Compared with wild type strains,the expression level of UV repair-related genes was significantly lower in mutant strains,while treated with the same ultraviolet intensity.All these showed that the conidial pigments of N.rileyi were beneficial to the resistance of spores to extreme environmental changes and could provide survival advantages.?4?The pigmentation effect of?Nrlac1Quantitative analysis after extracting conidial melanin showed that WT melanin content was much higher than?Nrlac1,which proved that melanin was an important component of spore pigments of N.rileyi.At the same time,Nrlac1 participated in the synthesis of conidial pigment or melanin.It was found that the pigments of both microsclerotia were red after extracting the pigments of WT and?Nrlac1,which indicated that conidial pigments and MS pigments of N.rileyi could be two relatively independent synthetic pathways.?5?The effect of Nrlac1 gene deletion on virulenceThe virulence of?Nrlac1 were tested by surface drip and in vivo injection with Spodoptera litura larvae.The results showed,compared with wild type strains,LT₅₀ of?Nrlac1 was delayed around 1d by drip treatment,while it has no significant difference under injection.Nrlac1 deletion reduced the virulence of the mutant strain to a small extent.