Identification And Functional Analysis Of CYP51B Promoter From The High-resistance Strain Of Penicillium Italicum In Citrus

Citrus mold caused by Penicillium digitatum and Penicillium italicum is the mainly pathogen to lead the postharvest diseases of citrus fruits,causing economic losses.Sterol 14?-demethylase inhibitors(DMIs)are used to treat this agricultural disease.DMIs are a group of site-specific fungicides that inhibit the activity of CYP51,whereas CYP51 are involved in the process of 14?-demethylation of ergosterol precursors,which regulates cell membrane fluidity and permeability.With the DMIs widely use to prevention and cure the citrus mold results in high resistance of P.italicum to it.The resistance of P.italicum to DMIs is closely related to the overexpression of CYP51,which is controlled by promoter.Therefore,it is significant to study the promoter and its various cis-acting elements.In this paper,we chose P.italicum as the research material to analyse of PiCYP51B promoter structure by means of molecular biology and bioinformatics.The recombinant plasmids were constructed by GFP fusion expression to analyzed function of the promoter.The main results of this study are as follows:1.142 strains of Penicillium in citrus were collected and isolated from Hubei,Chongqing and Nanchang,including 58 strains of P.italicum and 84 strains of P.digitatum.The results showed that 30 strains of P.digitatum are resistant strains,accounting for 36%of the total number of P.digitatum.30 strains of P.italicum are resistant strains,accounting for 52%of the total number of P.italicum.Calculating the EC50 of isolates,the most sensitive one is HSPd-X10 with an EC50 value of 0.081 mg/L;the most resistant one is HSPi-CQ3-2 with an EC50 value of 11.63 mg/L..2.The upstream regulatory sequence of PiCYP51B gene was cloned from P.italicum HSPi-N8.The NNPP software was used to predict the transcription initiation site of PiCYP51B 5'upstream region.The upstream regulatory elements of PiCYP51B gene were analyzed by PlantCARE and PLACE software.3.The full-length(1003 bp)of HSPi-N8 promoter PiCYP51B was cloned and named as PB1.The recombinant plasmid pTFCM-PB1-GFP-TtrpC regulated by HSPi-N8 PiCYP51B full-length promoter was constructed by GFP fusion expression.Meanwhile,12 deleted mutants with different length of PiCYP51B gene promoter deletion mutants were constructed.The recombinant vectors were successfully introduced into the P.italicum genome by Agrobacterium-mediated transformation.The PiCYP51B full-length promoter can drive the expression of GFP.When the PiCYP51B gene promoter with different lengths drives the expression of GFP,the first 7 long fragments(PB2-PB8)can drive the expression of GFP,while the latter 5 fragments(PB9-PB13)cannot drive the expression of GFP.The results showed that the core sequence of PiCYP51B gene promoter was located between PB8(-218 bp)and PB9(-191 bp).At the same time,a strong fluorescence was generated at PB2(-367 bp),whereas fluorescence intensity at PB3(-338 bp)was normal,indicating that there is a key element in the promoter from PB2 to PB3.4.PlantCARE analysis showed that there is a light-responsive element I-box between-210 bp and-201 bp of the promoter sequence,and I-box and GATA-motif are present at the same sequence between-358 bp and-352 bp.The HSPi-N8 PiCYP51B full-length promoter sequence was deleted from the sequence between-210bp and-201 bp and from the sequence between-358 bp and-352 bp respectively,to construct a transformation vectors named DI(deleted I-box)and DG(deleted GATA-motif).Transformants are obtained by Agrobacterium-mediated genetic transformation of P.italicum.Fluorescence detection revealed that no fluorescence was observed in the hyphae when the promoter deleted the sequence between-210 bp and-201 bp(DI).When the promoter lacks a sequence between-358 bp and-352 bp(DG),normal intensity fluorescence is observed in the mycelium,indicating that the I-box and GATA-motif elements in these two regions are sensitive to the PiCYP51B gene promoter.In this paper,a number of Penicillium strains in citrus were collected and isolated.The structure and function of the promoter of P.italicum PiCYP51B gene were studied.This research laid a good foundation for the further study of its target enzyme and also provides a theoretical basis for the research of novel and effective antifungal agents.