| Nomuraea rileyi, an important entomopathogenic fungus, can infect many lepidoptera insects, especially noctuids, such as Helicoverpa armigera, Spodoptera litura, Tricoplusiani, Anticarsia gammatalis, and Pseudoplusia, can cause the epidemic disease among the pest populations, plays an important role in the biological pest control, and has the potential to be developed into a mycoinsecticide. But mycoinsecticide of Nomuraea rileyi has the disadvanges of slow effect, vulnerable to the environmental factors, short storage time and unstability. To construct a safe and high virulent engineering strain by the gene engineering method is really count now. The target realization of these projects is built on the basis of the work on elucidating fungal infection mechanism during fungi infecting insects and the findings of new efficient insecticidal targets. This study has important significance not only in elucidating the mechanism of N. rileyi resistant to the immune response in the infection process of S. Litura, but alao providing theoretical evidences and technical supports for practical application of target genes.The main purpose of the paper is to elucidate the molecular mechanism of Nomuraea rileyi infection Noctuidae insects and improve Nomuraea rileyi control effect, variety of ways and means were carried out in the research. The results are as follows:(1) Nomuraea rileyi spores were injected into S. litura, and hyphae body of different developmental intervals were isolated from haemolymphe. There were 42 differentially expressed EST frangments coloned in Differential Display, and the EST fragments were between 47 to 542 bp, blastX alignment analysis result shows that these differentially expressed sequences mainly involved in biological process of resistance to insect immune system, antioxidation, escaping insect immune system, electron transportation, cell cycle control, stress response, arsenate reduction, peroxisomal membrane protein, signal transduction, fatty acid desaturation, et al. The RT-qPCR method was used to verify the expression level of the differentially expressed genes at different stages. The results of quantitative PCR showed that the expression of genes was not completely consistent with the expression in the gel bands.(2) Three methods were used in this paper to obtain full-length sequence of target genes: RACE,FPNI-PCR and local BLAST methods(cDNA sequences were searched in the transcriptome sequencing library). The gDNAs were used as the templates in FPNI-PCR protocol, the full length of differentially expressed genes were rapidly amplified,and the upstream and downstream sequences were also amplified using this method. The full length cDNA, gDNA and flanking sequences of SR-RCI, PI-PLC, MCL1, NrFADS2 genes were obtained.In this paper, we mainly studied the up-regulated expression gene NrFADS2, bioinformatics analysis showed that the gene genome sequence contains three introns, open reading frame is composed of 1758 bp,encoding for a protein consisting of 585 amino acids, the protein contains a conserved domain of the membrane-FADs-like superfamily, including the Cyt-b5 domain and the HPGG conserved region, as well as the delta6-FADs-like domain in the middle and C terminal. Quantitative PCR test showed that NrFADS2 gene was induced expressed at different stages of infection.(3) The establishment of genetic transformation system is an important prerequisite for the study of gene function, it is necessary to establish a stable and reliable genetic transformation system to identify the function of candidate genes. In this study, the blastospores were used as the genetic transformation recipient, solved the problem of unsuccessful transformation of N. rileyi mediated by A. tumefaciens, parameters influenced the transformation efficiency were optimized: the optimum hygromycin screening concentration was 450 μg/m L, the optimum inducor concentration of AS was 200 μM. Transformation efficiencies of four Agrobacterium strain AGL-1, LBA4404, GV3101, C58 were compared, result showed that Agrobacterium strain GV3101 had the maximum transformation efficiency. Randomly insertion vectors pPZP-Ptrpc-Hph and pPZ-Hph-DsRED2 were constructed. It was the first time that hygromycin resistance gene and red fluorescent protein gene were transformated into the genome of N.rileyi CQNr01 with blastospores as the recipent mediated by Agrobacterium, and the system of N.rileyi genetic transformation mediated by Agrobacterium was established.(4) The Nomuraea rileyi random insertion mutant library was constructed using Agrobacterium mediated transformation with the random insertion binary vector pPZP-Ptrpc-Hp and pPZP-Ptrpc-Hph-DsRED2, lots of mutants phenotypes were found and both sides of the flanking sequences of the insertion sites were amplified by FPNI-PCR. Because there is no clear genetic background of Nomuraea rileyi, the rich phenotypic changes is much more important for identification of gene function, this method is very useful for Nomuraea rileyi for forward genetics researches.(5) Nomuraea rileyi has no clear background of genomic sequence, the FPNI-PCR was performed to amplify the flanking sequences of the target gene, and the knockout vector was constructed. Mediated by A. tumefaciens, the target gene was knocked out, and the N. rileyi knockout mutation system was established.(6) The gene complementary system is essential for functional identification of target genes. In this paper, the promotor sequence of NrFADS2 was cloned by FPNI-PCR and the promotor and ORF sequence was inserted into the complementary vector. The nourseothricin resistance marker Sat1 gene was used as the marker gene for the phenotype recovery of the genes, and the optimal selection concentration of nourseothricin was 300 μg/m L.(7) The physiological and virulence changes were compared between the wild type, knockout and completary strains. The results showed that knocking out NrFADS2 gene influenced the utilization of fatty acid, decreased the antioxidant capacity of the mutant strain. The cell wall integrity of the knockout strain was partially changed, but the NrFADS2 gene had no direct effect on maintancing the stability of cell osmotic pressure. The ultraviolet resistance ability of NrFADS2 knockout mutant was decreased, the gene may be involved in the repairing of cell membrane light injury. NrFADS2 gene influenced the virulence of N. rileyi to topical infection of Spodoptera litura, but does not affect the ability of proliferation by injection infection, the gene maybe mainly play an important role in the stage of penetrating through host cuticles. |
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