Cloning Of Rhol Gene From Cryptococcus Laurentii And Its Expression In Saccharomyces Cerevisiae

The Cell wall integrity signaling pathway serves as a highly conserved mechanism of cell wall synthesis among the fungus. This pathway plays an important role in formation of a bud, cell division, detecting and responding to the cell wall stress. Rhol protein, as a key factor, transmits wall stress signals from the Cell-surface sensors to activate MAPK cascade, which mobilizes a physiologic process related to cell wall synthesis. Cryptococcus laurentii has been widely reported to control the postharvest fruit disease as a biocontrol yeast, while its physiological and biochemical mechanisms are not yet clear because of lacking of genome sequences. In this research, we explored the transcriptome of Cryptococcus laurentii to ensure rhol gene sequence and investigated the expression of rhol gene in Saccharomyces cerevisiae. Besides, the role of rhol gene enhancing the stress resistance and biocontrol efficacy in antagonistic yeast was also studied. The main results are follow:The analysis of rhol gene sequence and protein structure of Cryptococcus laurentii. Transcriptome analysis was used to obtain 597 bp cDNA sequence of rhol gene. Cryptococcus laurentii rhol gene comprises of 7 exons and 6 introns with total gene length of 966 bp, which is similar with the gene structure of Cryptococcus neoformans. The prediction of Rho1 protein shows that the size of Rholp is 22 kDa and it consists of 199 amino acids. Meanwhile, the typical GTP-bound domain is essential to its fuction and displays interation with GEFs. Rholp has the high similarity with Rho family protein three dimensional structure, including 6 a-Helix,5 β-Strand and 5 Loop structure. Hypothesis that Rholp of Cryptococcus laurentii has the homogeneous molecular biological function as the Rho family of Saccharomyces cerevisiae was obtained.Expression of rhol gene from Cryptococcus laurentii in Saccharomyces cerevisiae. The total cDNA sequence of rhol gene was cloned by PCR method where specific primers were used. Clonexpress recombinant technology was used to insert target gene to expression vector pYES6/CT. The recombinant expression plasmid was transformed into yeast competent cell by the method of electroporation. The minimum concentration of 150 μg/ml blasticidin in culture medium is necessary to identify the transformed yeast strain.The evalution of stress resistance and biocontrol efficy of recombinant yeast. The recombinant yeast strain SC-rhol existed increment in the tolenrance to the thermal stress and hyperosmolality. Moreover, recombinant yeast SC-rhol showed more effective in inhibition of the penidllium expansum in pear fruit and reduced the disease incidence by 33% and 25% under stress, respectively. Our findings had suggested that the proposed mechanism was involved in the over-expression of rhol gene improving the efficiency of CWI signal pathway transcription.