Transcriptome Analysis Of Trichoderma Harzianum Th-33in Chlamydospore Formation

Trichoderma spp. are very important biocontrol fungi. There are a variety of commercializedTrichoderma preparations and most of which are conidium preparations. But conidium preparationshave the disadvantages of short shelf-life and weak resistance to soil fungistasis. Chlamydosporepreparations are significantly better than conidium preparations in these aspects. However, inducingmass production of Trichoderma chlamydospores has much difficulty. So far there is no research reportof mechanism on Trichoderma chlamydospores formation. We have acquired the culture medium andculture control process that can induce mass production of Trichoderma chlamydospores by years ofresearch, which provide the basic technical conditions for studying the molecular mechanism ofchlamydospore formation. In this study we analyzed four different transcripts of T. harzianum Th-33during chlamydospore formation process by transcriptome sequencing, and screened differentiallyexpressed genes associated with chlamydospores formation. This was of great significance forelucidating the Trichoderma chlamydospore formating mechanism and fermentation control. In thisstudy, the main results obtained were as follows:1. We optimized the chlamydospore induction medium obtained by our laboratory. We extractedinducing ingredient from insoluble original medium and got a dissolutive medium that could producemass chlamydospores. At the same time, through systematic observation, we determined four differentstages of T. harzianum Th-33in chlamydospore formation for transcriptome sequcing: pre-sporulation:31h; early sporulation:45h; middle sporulation:54h and late sporulation:68h.2. By transcriptome sequencing four transcripts of T. harzianum Th-33in different periods, we gained15,773unigenes, and11,847unigenes had annotation information through comparison with Uniprot andNCBI nr protein databases, accounting for75.11%of the total unigenes.3. Pairwise comparisons of four samples in different stages, we found that the number of differentiallyexpressed genes between pre-sporulation and middle sporulation was the most, which was1309. Forthese1309genes, there were954up-regulated and355down-regulated expression genes. The numberof differentially expressed genes between middle sporulation and early sporulation was the least, whichwas252. For these252genes,156genes were up-regulated,96genes were down-regulated. Clusteranalysis of differentially expressed genes also showed that the samples between middle sporulation andpre-sporulation were of the biggest differences, and the differences between middle sporulation andearly sporulation were the smallest.4. GO functional enrichment analysis showed that a total of7287unigenes had GO annotation and theywere significantly enriched in50GO terms. Between the two most significantly different samples,pre-sporulation and middle sporulation, there were726differentially expressed genes annoted to GO,including34branches.5. According to the two main components of chlamydospore cell wall, chitin and glucan, we found15chitin-related unigenes,12chitinase genes and3chitin synthase genes, and11chitinase genesdifferentially expressed. The expression of3chitin synthase genes was no differences. Meanwhile, the differentially expressed genes between pre-sporulation and middle sporulation stages contained these11chitinase genes. The number of unigenes associated with dextran was a total of32, including11differentially expressed genes, of which only two were glucan synthase genes.6. KEGG enrichment analysis showed that the four samples were significantly enriched in twometabolic pathway, which were amino sugar and nucleotide sugar metabolism, starch and sugarmetabolism respectively. We also found that these two metabolic pathways contained many substancesthat involved in chitin metabolism pathway reported by others. This implyed that the formation ofTrichoderma chlamydospore was probably linked with chitin metabolism. Thus, we speculated onepossible chitin metabolism pathway in T. harzianum, but the specific genes and metabolic pathwayscorrelated with chlamydospore formation needed further study.7. Meanwhile, we selected seven differentially expressed genes associated with chitin and glucan tovalidate by Real-time PCR. The relative expression trend of three genes in four different periods wasconsistent with the results of transcriptome sequencing. The remaining four gene expression trend inone period was not in line with transcriptome sequencing results. But in view of the overall level, thistranscriptome sequencing results were credible.