Cloning Of MAT Genes And Fertility Analysis Of The Bisexual Strain Of Setosphaeria Turcica

Northern Leaf Blight of Corn is an important corn leaf diseases which infected corn plants mainly through conidia.The fungus can also proceed sexual reproduction.Sexual spores and other sexual structures were formed through the combination of organs or cells differentiated from vegetative,those had become another major infection sources.At the same time,the genetic materials were produced through the hybrid which enhanced the pathogenicity and adaptability.Exserohilum turcicum is heterothallic fungi,including two mating types,namely,mating type A and mating type a.At present,sexual state has not yet been found in the natural world,but perithecium,ascus and ascospores could be induced with different mating type strains through artificial culture.The gender mating type strains of Exserohilum turcicum were found in our laboratory at the first time,namely,mating type Aa.Those could produce the hybrids cultured with the A mating type strain or a mating type strain.As we all know,sexual hybridization is one of the main path which lead to fungal variantion.Gender of mating type can increase the risk of sexual hybridization and enhance the mutation rate.Debuchy et al studied that the sexual reproduction of fungal controlled by mating type genes(MAT) In this study,the isolates 08-06(mating type A),08-07(mating type a) and 03-16(mating type Aa) of Setosphaeria turcica were used as researching materials.The genome DNA of these isolates were extracted using CTAB method and the fragment of the MAT1 gene was cloned using degenerated primers with the candidate gene strategy.A fragment of 1000 base pairs in length was cloned from isolate 08-06.The whole length of MAT1 gene was obtained by genome walking strategy.Meanwhile,1382 bp was cloned in the 5' end and 776 bp was obtained in the 3' end of MAT1 gene.The totle RNA was extracted and the first strain of cDNA was synthesized,further more,the cDNA of MAT1 gene was cloned.Both sequences of DNA and cDNA sequences were Alignment with the software of DNAMAN,the result showed that the whole length of cDNA was 1 140 bp, and there was a 51 bp intron.Bioinformatic analysis indicated that there was a complete ORF in MAT1 gene which encoded 379 amino acid residues and molecular weight 38.3 kD protein,143 hydrophobic residues and 236 hydrophilic residues.In addition,a complete DNA binding protein with alpha box was existed which belong to DNA binding protein families.With the same method,the whole length of MAT2 gene was from the strain of 08-07.Meanwhile,1765 bp was cloned in the 5' end and 658 bp was obtained in the 3' end of MAT2 gene.the cDNA of MAT2 gene was also cloned.The DNA sequences and the cDNA sequences were alignment with the software of DNAMAN and the result indicated that the whole length of cDNA was 1038 bp,and there was a 55 bp intron.Bioinformatic analysis demonstrated that there was a complete ORF in MAT2 gene which encode 345 amino acid residues and molecular weight 41.8 kD protein,112 hydrophobic residues and 233 hydrophilic residues.In addition,a complete DNA binding protein with HMG-box was existed which belong to DNA binding protein families too.The result of Southern-blot showed that MAT1 and MAT2 were both single copy in the genome of S.turcica.The special primers were designed based on the MAT1 and MAT2 genes and the mating type genes from a part of strains of S.turcica was cloned.It made we know that the MAT1 gene was cloned from mating type A strains and MAT2 gene was cloned from mating type a strains,strangely,the MAT1 and MAT2gene were both cloned from mating type Aa strains.So Molecular biology methods of identificating mating type of S.turcica was established which is more Accurate,easier and time-saving.The strain 03-16 could selfed and produced Perithecium by sexual induced,showed that this strain has the Self-incompatibility.The MAT1 and MAT2 genes were both cloned from 03-16 strain,which suggested that the formation of gender strains with genetic material foundation,which,to some extent,support the assumption of that the heterothallic fungi to the homothallism evolution.The mating type of S.turcica tending to be sexual extended the selectivity and the race of sexual hybridization.The pathogenicity was enhanced or new physiolo- gical races were generated through sexual hybridization.Appearance of bisexual strains extended the probability of sexual hybridization,and variation.Therefore, the research of the characteristics and the genetic background of the bisexual strain has great significance for the study and the Biological Control of S.turcica and also Laied the foundation for discussion the formation and the control mechanism of sexual reproduction of the Plant pathogenic fungi.