| Auricularia auricula-judae is one of the important cultivated edible fungi in China with important nutritive and medicinal value. Under Basidiomycetes, A. auricula-judae belongs to the genus Auricularia, which has a wide distribution. Within the genus Auricularia, all the species havejelly-like and ear-shaped fruiting body lacking the differentiation of pileus, gill and stipe, which can separate this genus from others under Agaricomycetes obviously. At present, no report about the mechanism of fruiting body formation of Auricularia and the differential regulation between Auricularia and other genus was published.In this study, RNA-Seq was used to obtain the transcriptome of A. auricula-judae cultivatedstrain Au916. After sequencing and annotation, two types of important functional genes, glyceraldehyde-3-phosphate dehydrogenase gene and laccase gene, were cloned and analyzed, and the gene sequences were used in phylogenetic analysis of genus Auricularia and basidiomycetes. The main results of this study were as follows:1. Without genomic information of A. auricula-judae, in this study, the transcriptomes of mycelium and fruiting body of strain Au916were sequenced using de novo RNA-Seq, and1G data were obtained from each tissue. After assembling, total length of transcriptomewas19.6M, and the N50of them was808nt. For annotation, the Nr, GO, Swissport, COG and KEGG databases were used. Although the genome of A. delicata, which was together with A. auricula-judae belonging to genus Auricularia, was sequenced, the genome was useless to assemble and annotate the transcriptome of A. auricula-judae.2. The glyceraldehyde-3-phosphate dehydrogenase gene, which can be used in genetic transformation, qRT-PCR and phylogenetic analysis, was selected from the transcriptome based on the annotation. The putative gpd fragment was207bp in length, after amplifying, the full-length cDNA and DNA sequence were sequenced. It was worth to note that two variant allelic gpd gene were present in strain Au916. Sequence analyses showed that the coding regions of allelic genes contained five introns, from which73bp length discrepancies was present. In exons,10SNPs were present. The promoter regions of allelic gene, which contained typical TATA box and GC box in the upstream of the transcription start site, also showed length variation. The analysis of conserved substrate binding site showed that the isolated gpd gene was functional. Direct sequencing of the RT-PCR product and qRT-PCR results indicated that both of the two variant alleles were expressed with no significant difference in mycelium of strain Au916. Intron positions and molecular evolution analyses showed that the phylogenetic relationship between A. auricula-judae and A. delicata, and other basidiomycetes were distant.3. Monokaryon is an important phase of A. auricula-judae life cycle and important for understanding the growth and development of A. auricula-judae. In this paper, based on the variant regions of allelic gpd genes, an InDel molecular marker was developed to identify the protoplast monokaryons of A. auricula-judae. PCR results showed that this marker can obviously separate the dikaryotic and monokaryotic regeneration strains with different genotype.Fluorescence microscopic examination and paired test agreed well with the genotyping by InDel suggesting that the allele’s InDel could provide a quickly and accurately method to identify the monokaryons from protoplast regenerated strains of A. auricula-judae strain Au916.4. Owing to the gene conservation, the gpd gene has been used for interspecific phylogenetic analysis. In this study, the phylogenetic relationship of the genus Auricularia was investigated using the gpd gene and internal transcribed spacer (ITS),18S and28S ribosomal gene sequences with34samples from eight species from Auricularia. The molecular phylogenetic trees indicated that the genus Auricularia is monophyletic, and A. auricula-judae species also is monophyletic. A. auricula-judae was positioned at the most basal node. A. polytricha and A. delicata are found to be polyphyletic. Molecular analyses using the four gene sequences indicated a close relationship of A. peltata species to A. cornea, and A. polytricha to A. reticulate, A. maxima, A. reticulate and A. fuscosuccinea. Gpd gene sequence with intron regions can separate intraspecies strains, and then it can be used for genetic diversity analysis of species and varieties.5. Based on the annotation of transcriptome of strain Au916,11putative laccases coding fragments were isolated from Au916. After assembling the aligning, two pairs of sequences matched each other perfectly suggesting that they might be from the same gene; two pairs of sequences showed six and five nucleotide length discrepancies at introns, and the PCR performed with the specific primers containing the SNP sties indicated each pah-was alleles. Hence, the cloned putative laccase genes were renamed as lcc1, lcc1, Icc3, Icc4, lcc5, lcc6a, lcc6b, lccf and lcc7b. Intron positions and phylogenetic analysis showed the members of laccase gene family in A. auricula-judae were divergent.In the NJ tree, the laccase from genus Auricularia was nested within Ascomycete laccases.The expression patterns of the seven laccase genes were analyzed in different stages: free-living mycelium, sawdust mycelium; sawdust primordium; log primordium (stage I), immature fruiting body (stage II, stage III) and mature fruiting body (stage IV). The results showed that:Icc3mainly involved in vegetative growth; Iccl and Icc5played a role in primordium formation; and Icc5gene played the major role during the maturation of fruiting body from the primordium to mature fruiting body stages. During the development, in addition to the aforementioned three major genes, other genes also expressed with lower level at different stages indicating the some genes of the laccase family were functional redundancy in A. auricula-judae.In conclusion, based on the de novo transcriptome of A. auricula-judae cultivated strain Au916, the gpd and laccase family multigene were isolated. Using qRT-PCR, the expression feature of allelic gpd genes and laccase genes were obtained. The results of intron position analysis of gpd genes, molecular evolution analyses of GPD and laccase amino acid and phylogenetic analysis of genus Auricularia indicated that genus Auricularia had a special taxonomic status under Basidiomycetes; the species within Auricularia were diversity, and among Auricularia, the evolution of A., auricula-judae, which had distant phylogenetic relationship with A. delicata, was earlier. |
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