| Entomopathogenic fungi are an important class and widely distributed which maintain the number and density of insects in the ecosystems, it plays an important role in nature. Because of the environmentally safe, harmless to humans and animals, entomopathogenic fungi are considered ideal alternative to chemical pesticides, but its long time of lethal, high cost in production, environmentally sensitive and other shortcomings limit the large-scale application. Therefore, the study of pathogenic fungi in pathogenicity, sporulation and anti-stress mechanism is of great significance for biological control.Entomopathogenic fungi are widely distributed in nature, from the Arctic to the tropics, from the highlands to the plains, from swamp to desert, so it is bound to face a variety of environmental stresses such as hypertonic, temperature, UV, oxygen. Alcohol dehydrogenase are widespread in animals, plants, fungi, bacteria, the transcriptome analysis showed Ma ADH1 highly expressed in sporulation, the expression pattern analysis indicates Ma ADH1 highly expressed in sporulation, and low or no expression in other periods. In this study, the Ma ADH1 gene was disrupted by homologous recombination, and the function of Ma ADH1 and the struction and function of Ma ADH1 promoter were analyzed. The main results are as follows:1. Cloning and functional analysis of Ma ADH11.1 Gene cloning, molecular characterizationThe M. acridum ADH1 is 1131 bp and contains one intron. It encodes a predicted protein of 353 amino acids residues, with a deduced molecular weight of 37235 Da and a p I of 7.61. The Signal P 3.0 program(http://www.cbs.dtu.dk/services/Signal P/) revealed no signal sequence suggesting that ADH1 is a cell-bound protein. ADH1 contains typical ethanol dehydrogenase structural domain such as NAD+ binding domain, Zn2+ binding site and ADH1 activity domain(Fig.1 A). Phylogenic analysis indicated that Ma Adh1 protein was closely related to ADH1 from the entomopathogenic fungus M. anisopliae and Beauveria bassiana(Fig. 1B). This protein has 94.05% and 73.16 identity with the ADH1 from M. anisopliae and B. bassiana respectively.1.2 Ma Adh1 disruption and complementationTo analysis the function Ma ADH1, Ma ADH1 was knockdown by homologous recombination and a plasmid for complementation was also generated. The recombinant plasmid was transformed into M. acridum with Agrobacterium tumefaciens. Transformants were screened by PCR and confirmed by Southern blot. As expected, the results were correct.1.3 Expression pattern of Ma ADH1The transcription level of Ma ADH1 were analyzed by semiquantitative reverse transcription polymerase chain reaction and quantitative real-time polymerase chain reaction(q RT-PCR). The results indicated strong expression of Ma ADH1 in conidium, but significant less expression in 14 h mycelium, appressorium and hyphal body.1.4 Ma ADH1 effects the biomass of M. acridum under hypoxic conditionsUnder hypoxic conditions, the biomass of ?Ma ADH1 significantly lower than WT and CP, the biomass is only 50% of WT and CP. Under normal conditions, the biomass of ?Ma ADH1 was no significant difference with WT and CP.1.5 Ma ADH1 regulated conidiation of M.acridum under hypoxic conditionsThe conidiation of M.acridum in the rice and 1/4 SDAY liquid medium were analyzed under hypoxic conditions, the results indicated that the sporulation of ?Ma ADH1 were significantly lower than WT and CP in both solid state fermentation or liquid fermentation. The spores of ?Ma ADH1 were lower in the solid fermentation, which was only the 48% of WT mainly existed in the form of mycelium.1.6 Dissolved oxygen and expression of Ma ADH1 in the liquid cultureThe dissolved oxygen in the liquid culture medium of ?Ma ADH1 was no significant difference compared with WT and CP under hypoxic conditions. The analysis of RT-PCR suggested that the expression of ADH1 in hypoxic conditions was significant higher than the normal conditions.1.7 Enzyme activity of Ma ADH1pThe analysis of enzyme activity of Ma ADH1 p suggested that the activity in the low oxygn dissolved conditions was significant higher than the normal conditions, the result was similar with RT-PCR.1.8 The determination of ethanol and acetaldehyde in the medium and the tolerance assayThe concentration of ethanol and acetaldehyde in the medium were determined at different time, the results suggested ethanol and acetaldehyde content gradually increased, the concentration of ethanol in ?Ma ADH1 was significantly lower than WT and CP, however the concentration of acetaldehyde in ?Ma ADH1 was significantly higher than WT and CP. Tolerance analysis show the tolerance of WT, ?Ma ADH1, and CP to acetaldehyde was significantly lower than ethanol.1.9 The acetaldehyde affected the growth and conidiationTo analyze the effect of acetaldehyde to growth and sporulation in the medium contained different concentrations of acetaldehyde, the results showed that acetaldehyde inhibited the growth and sporulation of M.acridum when the concentration of acetaldehyde is 0.1% and 0.01%(v/v) and the growth and sporulation ?Ma ADH1 strain was significantly lower than WT and CP.2. The structure and functional analysis of PMa ADH12.1 The bioinformation analysis of PMa ADH1A 3246 bp upstream sequence from the start codon(ATG) of the Ma ADH1 gene was retrieved from the genomic DNA sequence of M. acridum. The analysis of bioinformation indicated that the 163 bp upstream sequence from the start codon was transcription start site. PMa ADH1 contain conserved regulatory elements, such as the TATA-box, CAAT-box and CT-rich region and some conserved sequence. Conserved elements and other region had a lot of forward and reverse repeat sequence.2.2 Transformants screeningVarious lengths(3246 bp, 2702 bp, 2248 bp, 2219 bp, 1670 bp, 1138 bp, 521 bp, 226 bp) of DNA fragments of the Ma ADH1 promoter region were amplified using M. acridum genomic DNA and primers using EGFP as a repoter which was inserted into Pk2-sur vector and were transformed into M. acridum mediated by A. tumefaciens.The transformants were obtained and analyzed by PCR and Southeern blot, the results suggested that the copy number of transformants was 1-2 range, but most were one copy.2.3 The deletion analysis of PMa ADH1To analyze the activity of each truncated PMa ADH1, both transcription level and fluorescence intensity of EGFP were determined. Deletions spanning the region from-3246 bp to-2702 bp(Del1) didn't affect the activity of EGFP, however, the deletion spanning the region from-2702 bp to-2248 bp(Del2) and-2219 bp to-1690 bp(Del4) resulted in a 20% and 15%increase in the transcription level of EGFP, respectively, indicating that the negative regulatory element presented in this region. Deletions of the regions upstream of-2219 bp,-1138 bp and-521 bp caused a 15%, 20% and 99.7% decreases the transcription level of PMa ADH1, respectively(Del3, Del5, Del6),indicating that the strong positive cis-acting elements were located in this region. When the region upsream of-226 bp was deleted, there was not any activity of EGFP.2.4 PMa ADH1-Mcl1 significantly increased the virulence of M. acridumMcl1 was special expression with the Del3 promoter. The survival of insect after topical infection application of WT, PADH1-Mcl1 conidia indicated that PADH1-Mcl1 transformants significantly increased the virulence of M. acridum, the LT50 was 4.0±0.2 days, while the LT50 of WT was 5.0±0.3 days.In summary, Ma ADH1 regulated the growth and sporulation of M. acridum by the metabolism of acetaldehyde under hypoxic conditions, Ma ADH1 gene was specific expression during conidium. The structural and functional analysis of Ma ADH1 promoter showed there was an important oxygen pressure responsive element and had the potential to build the engineered strains. |
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