Analysis Of Codon Usage Bias And Cloning Of Promoter In Tremella Fuciformis

Tremella fuciformis is a well-known edible and medicinal fungus. It, as a rising bioreactor, is of genetic stability as the same as that of prokaryotes or yeast, in that it is both asexual and sexual reproduction. What's more, eukaryotic genes maybe expressed at a higher level in T. fuciformis than in prokaryotes such as E.coli, because the former possesses eukaryotic post-translational modification and processing system but the latter not. In order to improve the expression level of foreign genes expressed in T. fuciformis, which will provide the theoretical basis for actual production of bioreactor, this study investigates in two main aspects as following: first, analyzed the codon usage bias of T. fuciformis using the advanced bioinformatics tools, which can be the foundation of transformation of heterologous genes codon to enable them adapt to the host environment of T. fuciformis; second, cloned promoters by using thermal asymmetric interlaced PCR (TAIL-PCR.) so that the expression level of foreign genes might be increased in T. fuciformis.1 Construction of cDNA library of yeast-like conidia in T. fuciformisThe total RNA was successfully extracted from yeast-like conidia of T. fuciformis by RNAiso Reagent kit. The mRNA purified by Oligotex mRNA purification kit was reversely transcripted into single cDNA and then was synthesized double strands cDNA using cDNA synthesis kit. After the small cDNA was removed by CHROMA SPIN+TE-1000, the further modified double strands cDNA was ligated to pBluescript II SK (+) vector. Next, the ligation product was electroporated into E.coli. The electroporation condition of DH10B competent cell was:1.5KV of pressure,200? of resistance,25?F of capacitance.16 monoclones picked randomly from the plate were amplified by T7 primer and T3 primer to detect the recombination rate and the size of inserts of the library. The result showed that:the recombination rate of the library was 100%; the average size of inserts was about 1300bp; the cDNA library contained almost 7.5×106 recombination clones. Those above indicated that the standard cDNA library from yeast-like conidia of T. fuciformis was constructed in this study.2 Analysis of codon usage bias of T. fuciformisRelative synonymous codon usage (RSCU) and relative frequency of synonymous codon (RFSC) for eighty CDSs which were obtained from cDNA library of yeast-like conidia in T. fuciformis were analyzed based on the use of sofeware CodonW and programme RUSP respectively. Eighteen high frequency codons were revealed by screening rule of frequency-codons. The DNA G+C content of genes in T. fuciformis was 59.02%, and this content of the third codon position was as high as 69.72%, which demonstrated that genes in T. fuciformis trend to code G or C in the third codon position.Seventeen high expression optimal codons of T. fuciformis were confirmed by analytical method of high expression optimal codon. The relative synonymous codon usage (RSCU) of high expression sample group and low expression sample group were calculated respectively by using software CodonW. The results showed that the degree of codon usage bias in high expression sample group was stronger than that in low expression sample group. It proved that the expression level of gene was one of the important factors to affect the extent of codon usage bias in T. fuciformis.3 Cloning and verification of the promoter of T. fuciformisBased on the results of sequencing and gene expression profile, three high expression genes (test NO. were E02?C02?G06 respectively) were screened as the templates for designing special primers. Promoters were cloned using thermal asymmetric interlaced PCR. Expression vector pBHg-E02, pBHg-C02 and pBHg-G06 carring hygromycin phosphotransferase gene (hpt) as selectable marker gene were constructed and were transformed into competent cells of Agrobactrium tumefaciens by freeze-thaw method. A. tumefaciens carrying plasmids pBHg-E02, pBHg-C02 and pBHg-G06 were cocultured with logarithmic phase yeast-like conidia of T. fuciformis Tr21-01 which was sensitive to hygromycin B and then transformants were selected on selective medium containing 50?g/mL hygromycin B. The resistent colonies of Tr21-01 and A. tumefaciens carrying pBHg-G06 cocultured were observed on SM plate. Fragments of promoter from gene G06 in T. fuciformis and phosphotransferase gene were contained in the genome DNA of recommendation colonies according to the electrophoresis. The results from the investigation suggested that the promoter and hpt successfully inserted the genome. Promoter G06 of T. fuciformis was gained in our study.