Heterologous Expression Of CYP51and The Research Of Molecular Resistance Mechanism To Imazalil From Penicillium Italicum

China is one of the primary citrus-producing countries, so the effective prevention and control of citrus diseases directly affect the economic benefit brought by citrius production. Diseases caused by Penicillium italicum and Penicillium digitatum are the main problems of citrus. For the infectious ability of diseases, it can happen during all the postharvest course of processing, transportation, storage and sales. Blue mold easily occurs in the stage of refrigerated storage. And the incidence of blue mold is higher than green mold caused by Penicillium digitatum. Even when the storage temperature is as low as0℃, the disease can also occur. At low temperature, spores of Penicillium italicum can also spread and directly contact with the healthy citrus, and then make it catch the illness. Therefore, the damage caused by blue mold is more serious than the damage caused by green mold. Sterol14a-demethylase(CYP51)is the most original and common member of the P450superfamily, who participates in the reaction of sterol’s demethylate. All kinds of CYP51have been found in many species. For the key role of Cyp51in sterol’s synthesis process, it is chosed as the target enzyme of herbicides and fungicide.14a-demethylation inhibitors (DMIs) choose CYP51as a target, and the mechanism of sterilizing is to block the synthetic pathways of ergosterol by inhibiting the catalysis of CYP51, ultimately affect the structure and function of pathogenic funguses’ cell membrane. The widespread use of fungicide DMIs directly leads to the resistance to drug of fungus.This article choosed Penicillium italicum as the reseach object. We analyzed the relationship between CYP51of Penicillium italicum and the resistance to drug, and achieved the prokaryotic expression of CYP51gene. The results of the study are as follow:1. We successfully identified42citrus pathogenic fungi strains of Penicillium italicum by isolation and purification. The result of the sensitivity to imazalil of these strains was as follow:most of strains are sensitive type, and only six strains can be treated as resistive type (they are HSPi-3、HSPi-L3、HSPi-N8、HSPi-O12、HSPi-O14、 HSPi-P14).2. This is the first time to obtain the sequences of CYP51B gene and its upstream of Penicillium italicum. The results analyzed by DNAMan and DNAStar software were shown:the size of ORF of PiCYP51B gene was1575bp, which was predicted to encode peptides of525amino acids; PiCYP51B gene contains3introns, and the size were74bp、 51bp and52bp, which were located between247bp and320bp、519bp and569bp、1635bp and1686bp, respectively. The bioinformatics analysis of PiCYP51showed that PiCYP51A and PiCYP51B genes located in two different branches in the phylogeny trees, and they have only57.14%similarity; PdCYP51B and PiCYP51B located in the same branch, and the similarity reached to96%.3. The sequences of PiCYP51A/B genes and its upstream were cloned by using DNA as templates of all resistive type and several sensitive type strains. The results were as follow:there was no199bp insertion in the upstream of PiCYP51A/B genes of all resistive type strains; there was a10bp insertion in the promoter region of all resistive type strains’PiCYP51B gene. And there was no clue to show the relationship between Imazalil resistance mechanism and PiCYP51A/B genes.4. PiCYP51A gene from Penicillium italicum was cloned, and the recombinant expression plasmid (pET28-PiCYP51A) was constructed for heterologous expression. The result of SDS-PAGE showed that nothing was detected, even optimized the express condition; it manifested that the whole ORF of PiCYP51B could not achieve the heterologous expression. PiCYP51A is a trans-membrance protein by the analysis and prediction of transmembrane structure, which contains two transmembrane region. To truncate and predict the N-terminal sequences of44and66amino acid residues, the transmembrane structure disappeared. By gene cloning techniques, the PiCYP51A gene coding N-terminal sequences of44and66amino acid residues were truncated, and the two mutants were named as PiA-44and PiA-66, respectively. Then the recombinant expression plasmids (pET28-PiA-44and pET28-PiA-66) were constructed for heterologous expression.The results of SDS-PAGE showed that the obvious expression bands were detected, and the quantities of recombinant protein expression of pET28-PiA-66were higher. To optimize the express condition, such as time, temperature, IPTG induced concentration; the recombinant protein of pET28-PiA-66still existed in the form of inclusion body.