Studies On The Mechanism Of The Missing Sexual Fruiting Body Of Isaria Cicadae

Isaria cicadae commonly known as Isaria cicadae Miquel,is a kind of precious medicinal fungi which has the bioactive effect of improving the eyesight,antipyretic,detoxification and anticonvulsant,and also a very broad spectrum of biocontrol fungi which has the advantages of the high safety,high sporulation and strong ability of infection.However,the I.cicadae only has the asexual reproduction,it's fruiting body was not found.Nowadays the study on the I.cicadae is mainly concentrated in the analysis of the effective components and pharmacological research,while the research on the genetics of I.cicadae is almost none,so reasons for the lack of sexual fruiting body of I.cicada is not reasonably explained yet.In this study,the full-length cDNA of MAT1-1-1 and MAT1-2-1 were cloned from I.cicadae using RACE technology,and the characteristics of the genes were analyzed.Furthermore,multiple alignments of the amino acid sequence were acted,thereby PCR primer sets for the mating-type genes MAT1-1-1 and MAT1-2-1 were designed based on the distinctive amino acid sequences of the alpha and HMG boxes,respectively.Moreover,the mating type of strains from Anhui province were detected,finally,Barrage tests and the artificial induction of the fruiting body have been used to assess vegetative compatibility between different mating-type isolates.The main results are as follows:1.The full-length cDNA of MAT1-1-1 and MAT1-2-1 were cloned from RCEF6213 and RCEF6212 using RACE technology,respectively.The MAT1-1-1 full-length cDNA sequence with 1895 bp was deduced by sequence alignment and comparison with the 5' and3' fragments.Except for 5'-UTR(untranslated regions)with 80 bp and 3'-UTR with 81 bp,the full-length cDNA of MAT1-1-1 possessed a 1734 bp open reading frame(ORF).The sequence bioinformatics analysis shows that the deduced protein had 577 amino acids with calculated pI value of 5.877 and a molecular weight of 128 kDa.The CCD program of NCBI predicted that the deduced MAT-1-1-1 protein contain a MAT alpha1-box domain.The MAT1-2-1 with a whole sequence of 1290 bp,showed that it encompassed an ORF of771 bp encoding 256 amino acids.The deduced protein had a molecular mass of 43.9 kDa with a calculated pI of 9.830.This sequence contained a high-mobility group(HMG)DNA binding domain.Phylogenetic analysis based on the nucleotide sequences of MAT1-1-1and MAT1-2-1 from fungi showed that I.cicadae and I.tenuipes form a natural taxonomic group with the homology of 88% and 89% respectively.2.The amino acid sequence alignment of mating type protein domains with other four ascomycetes showed that the genes had conserved amino acid identity and partial amino acid similarity.Then,PCR primer sets(MAT1-1F/MAT1-1R and MAT1-2F/MAT1-2R)were designed based on the distinctive amino acid sequences of the alpha and HMG boxes.PCR verification showed that the two pairs of primers can amplify the expected MAT1-1-1and MAT1-2-1 fragments,and with no amplification results of mating type gene from the remaining four ascomycetes.3.The mating type composition and proportion of the I.cicada strains from Anhui province was studied.The result showed that 14 MAT1-1 strains were identified while the others were MAT1-2 strains,which indicated there is no strains possessing two mating type genes,and were supposed to be heterothallic.Moreover,the mating type proportion in the region is relatively balanced,and no deletion or a dominant mating type in the distribution.So from the perspective of mating type,we can speculate I.cicada has the possibility of sexual reproduction in this region.4.Vegetative compatibility between different mating-type isolates was assess.Barrage tests showed that all the I.cicada isolates were incompatible no matter between the same or complementary mating type isolates.The morphology of the incompatibility reaction is divided into 2 forms: in form?,there is almost no hypha growth in the contact region.In form ?,there is a dense rumble strip composed of interweaved hyphae.In the artificial induction of the fruiting body,the yield of mycelium,sporulation and coremium were inhibited in the paired isolates,which corresponded with the morphology of the vegetative incompatibility.5.Comprehensive analysis showed that the vegetative incompatibility between complementary mating type strains may be the reasons for the lack of sexual fruiting body of I.cicada.