The Identification Of The ABC Transporter In Calonectria Pseudoreteaudii And The Preliminary Study Of MDR Gene

Calonectria leaf blight is one of the most serious diseases in the Eucalyptus plantations,which seriously threatens the development of Eucalyptus industry.This disease mainly caused by Calonectria spp..Among them,C.pseudoreteaudii YA51 is the most pathogenic strain,which distributed most widely and be found earliest in Fujian Province.Eucalyptus are rich in secondary metabolites,it can be used to against the pests and plant pathogen.At the same time,the content of polyphenols and flavonoids,the activity level and isoenzyme bands of Polyphenol oxidase in the Eucalyptus leaves were positively correlated with the resistance of Eucalyptus.ATP-binding cassette transporters is one of the largest superfamily in the known transporter family.Lots of pathogenic fungi ABC transporters have been proved to be able to regulate pathogenic processes or achieve resistance to fungicides by transporting exogenous chemicals.Therefore,in order to clarify the relationship between the ABC transporter of C.pseudoreteaudii and the molecular mechanism of pathogenicity or drug-resistance,we identified and classified the ABC transporters of C.pseudoreteaudii by bioinformatics methods,and elucidated the role of ABC transporters played in transporting exogenous sources.The main results are as follows:(1)67 ABC transporters were identified from the genome of C.pseudoreteaudii YA51 by bioinformatics tools,all of them are belong to 7 subfamilies,namely ABCA,ABCB,ABCC,ABCD,ABCE,ABCF and ABCG,respectively.The ABCG subfamily is the largest ABC subfamily of C.pseudoreteaudii,which accounting for about 37.3%.The length of ABC transporter of C.pseudoreteaudii is divided into full-length,3/4-size and half-size,and the number of transmembrane helix in TMD domain varies from 0 to 8.The subcellular localization prediction results showed that 76.11%was located on the plasma membrane and the rest of them were located on the intimal system.According to the TCDB database comparison results,the ABC transporter of C.pseudoreteaudii can also be dvided into PFF,MDR,STE,MPE,HMT,DCT,P-FAT,U-ABC1,DRUG RA and PDR,respectively.(2)The results of comparative analysis of ABC transporters were carried out between 8 model fungal species and C.pseudoreteaudii showed that the number of ABC transporter in Ustilago maydis,Saccharomyces cerevisiae,Sclerotinia sclerotiorum,Botrytis cinerea,Neurospora crassa,Magnaporthe oryzae,Colletotrichum graminicola and Fusarium graminearum were 33,25,36,48,31,44,51,and 58 respectively.The number of ABC transporters in C.pseudoreteaudii was significantly higher than other species.Except of S.cerevisiae,the other 8 species contain 1 to 2 ABCA transporters.The number of ABCD and ABCE are the same for each strain.Except that S.cerevisiae has 5 ABCF transporter,the C.pseudoreteaudii,C.graminicola,B.cinerea,F.graminearum and M.oryzae have 3 ABCF transporter,the U.maydis,S.sclerotiorum and N.crassa only have 2 ABCF transporter.Among 9 strains,the subfamilies of ABC transporter,ABCC,ABCB and ABCG,which involved in the transport of exogenous chemicals were significantly higher than other subfamilies.And the number of these 3 subfamilies of C.pseudoreteaudii was higher than other plant pathogenic fungi.(3)The results of digital gene expression profile of C.pseudoreteaudii infected Eucalyptus 48 h showed that 22 ABC gene were up-regulated and 15 ABC gene were down-regulated.Phylogeny and transcriptional analysis results showed that CpABCB7,CpABCC10,CpABCCll,CpABCG8,CpABCG14,CpABCG15,CpABCG18,CpABCG21,CpABCG22 and CpABCG23 may play an important role in the stage of C.pseudoreteaudii infected 48 h.(4)The sequence characteristics and physicochemical properties of MDR transporter were analyzed by bioinformatics software.The secondary structure prediction results showed that the secondary structure of MDR transporters are consist of α-helix,radandom coile,and β-sheet.Proportion of them are α-fold>β-turn>random coil,respectively.We also used Model-Swiss to predict the 3D structure of MDR protein,and the predicted results were consistent with the prediction results of the secondary structure,which are typical MDR protein structure.(5)QPCR was used to detect the expression of 12 MDR genes in conidiation,germination,post inoculation 24 h,post inoculation 48 h,post inoculation 72 h and the condition of C.pseudoreteaudii induced by H2O2,NaCl,KCl,LiCl,Chlorothalonil,cycloheximide,matrine,gramine and miconazole.The results showed that CpABCB10 may be involved in the regulation of spore germination,CpABCB4 and CpABCB9 genes may be involved in the regulation of metal ion transport,and CpABCB7 and CpABCB8 genes are involved in the regulation of the drug-resistance mechanism of C.pseudoreteaudii.In addition,the CpABCB7 gene can also participate in the whole pathogenic process.The remaining MDR genes play a supporting or negative regulatory role.Based on the whole genome of C.pseudoreteaudii,we identified the ABC transporter,and its domain,subcellular localization,transmembrane helix were analyzed in this study.Then the evolution relationship of the ABC transporters among 9 species and the transcriptional expression analysis of ABC gene were carried out.At the same time,we also studied the expression of MDR gene under different conditions,and analyzed its physicochemical properties,secondary structure and 3D structure.This result can construct a foundation for the research ofpathogenicity and multidrug-resistance mechanism of C.pseudoreteaudii.