Construction And Preliminary Analysis Of A Full-length CDNA Library From Mycobiont Of Endocarpon Pusillum Hedwing (Lichen-forming Ascomycota)

Lichens, as terrestrial fungal-algal organisms, are widely distributed with wide ecological amplitude ranging from cold to hot deserts and other extreme habitats. Lichens are principal biological agents and pioneers in the rock-weathering and pedogenic processes. In order to carry out a desert biocarpet engineering to cover the sandy desert with drought-resistant transgenic plants, a study to screen drought-resistant genes from the arid desert lichen is being made. The fungal partner or mycobiont of lichens was isolated from the desert lichen Endocarpon pusillum Hedwing collected from the Shapotou Region of Ningxi. The research reads as follows:(1) The first full-length cDNA library for lichenized fungi was constructed from cultured mycobiont of the arid desert lichen E. pusillum. The titer of the primary library was 1.5×106 cfu/mL, and the rate of insection was 95.5%. These results indicated that the library was constructed successfully. Using this cDNA library, 111 genes are identified from lichenized fungi for the first time in this study. BLAST analysis revealed that 42 CDSs showed high homology to known fungal genes (identities >75%), 58 CDSs had some homology, and there are 11 genes producing no hits, indicating that these genes share no homology to known fungal genes. It suggests that these 11 genes maybe are unique genes in lichenized fungi, which should play important roles in lichen symbiosis.(2) The absolute quantitative PCR method for genome sizing was verified with genomes of known size. We applied this method to size the genome of the mycobiont from E. pusillum. 32 primer pairs from 15 identified genes were designed to be applied in qPCR. Finally, ten primer pairs with the similar and reasonable amplification efficiency were screened to determine the genome size of the mycobiont from E. pusillum. Statistical analysis showed that there was no significant difference between the results calculated by these ten genes. The average size of the genome of the mycobiont is about 39.13Mb.(3) The copy number of rRNA gene repeat units was determined by relative quantification PCR. Three genes (SCD, ATA and RPB11a) whose amplification efficiency are nearly the same and approach to the theoretic calculated value (E theoretic = 2) were chosen as the single-copy genes according to the size of genome and the BLAST analysis in other fungi genomes. The results indicate that the copy number of rRNA gene repeat units is 43.(4) Construct a recombinant vector containing the cDNA which ecodes Tropomyosin-1 and transfect it in E. coli BL21. Induced by IPTG, the recombinant protein was purified with Ni2+ affinity chromatography.The above results will facilitate research on genes involved in stress-resistance in lichenized fungi, and our knowledge on genes in lichens. The result of the size of genome and the copy number of rRNA gene will facilitate the study about sequencing the genome of this mycobiont in assembly and annotation. The work will span the gap in our knowledge of molecular understanding of lichens.