Construction Of Mutation Library By REMI And Cloning Of Hog1 Gene Form Corynespora Cassiicola Cc01 Of Hevea Brasiliensis

Natural rubber is one of the four strategic goods and materials in the world,it plays an important role in our country’s national security and national economy.The Corynespora Leaf Fall Disease (CLFD) of Hevea brasiliensis, caused by Corynespora cassiicola, has recently made one of the threat, after the South American Leaf Blight(SALB), to natural rubber production in South Asia, southeast Asia and central Africa. Researching about molecular pathogenesis of C. cassiicola were rarely reported in the world.In this study, an insertion mutant library of C.cassiicola Cc01 was generated using the approach of Restriction Enzyme Mediated Integration (REMI), mutant library evaluation,molecular analysis, phenotypes and screening pathogenicity defects mutants were done meawhile. C.cassiicola strains were collected widely and a strong virulent strain Cc01 was screened, whole-genome sequencing were done in cooperate with Beijing BGI Science and Technology co., LTD,and according to the method of homology clone, hog1 gene was obtained from Corynespora cassiicola Cc01, a preliminary analysis to its function was done then. For the following findings:An insertion mutant library of C.cassiicola Cc01 was generated using the approach of Restriction Enzyme Mediated Integration (REMI). In the process of the library construction, pUCATPH, a transformation vector was linearized respectively with KpnⅠ, HindⅢ, SacⅠ and SmaⅠ and used to transform Cc01. The yield of protoplast at highest level when mycelium cultivated to 20 h and snail enzyme (40 mg/mL), digestion (20 mg/mL) enzyme in mixed solution. The optimal amount of enzyme were 45 U,30 U,15 U and 45 U respectively with KpnⅠ, HindⅢ, SacⅠ and SmaⅠ. The transformation generated 2168,978,442 and 407 transformants respectively by the four individual enzyme-mediated insertion in the condition of 2 μg plasmid in each tube system. Twenty transformants were randomly picked up for PCR detection, the results were all positive transformation. Ten transformants were picked for Southern blot analysis,the result showed that single copy insert accounted for about 90%, double copies of the inserted accounted for about 10%, copy number greater than two had not appeared.High-throughput Illumina sequencing technology was used for paired-end sequencing of Corynespora cassiicola Cc01 strain,500bp and 6000 bp library were obtained. According to the results of data assembly, the size of genome was 44.23 M, GC% was 52.48%, total length of it was 42,372,289 bp containing 376 scaffords. Based on k-mer analysis coverage of genome was 95.80% and per 15-mer desired depth was 23, the total number of k-mer was 1,017,290,102.Mutants were screened for defects in pathogenicity with a detached and living copper brown leaf assay,34 mutants showing reproducible pathogenicity defects were obtained from 3995 transformants.Through the phenotypic screening for 12 colony morphology and 14 conidium morphology variation of mutants were obtained. Genomic sequences flanking were recovered from 10 mutants by thermal asymmetric interlaced PCR, local blast sequence alignment showed that three were inserted into the conjecture ORF among five assumption genes(S1187,S269 and S1202), the inserted gene from mutant S1187 might be a pathogenic protein gene MoCrc1,the other two(K689, S1319) were integrated in the upstream of ORF.Full-length hog1 gene which was names CChogl was obtained from Corynespora cassiicola Cc01 using homology clone(Accession number KC262647). Sequence analysis extrapolated that the open reading frames of this gene was 915 bp, contains 6 introns, and encodes a putative protein of 304-bp-amino-acid. Phylogenetic clustering suggested that the amino acid sequence of CCHOG1 showed more than 94% similarity to MAPK HOG1 of Pyrenophora tritici, HOGl-like protein kinase of Alternaria alternata, MAP kinase C1K1 of Cochliobolus lunatus. Clustal X analysis suggested that CCHOG1 had a close relationship with MAPK HOG1, meanwhile together as one with HOGland CLK1 which regulated hypertonic stress, oxidative stress and pathogenicity, thus predicted the function of CCHOG1 was similar with HOGland CLK1 probably.