Cloning And Expression Analysis Of Blue Light Receptors PoWC-1 And PoWC-2 From Pleurotus Ostreatus

Pleurotus ostreatus is one of the most widely cultivated and consumed edible fungi in China,which is favored by people because of its high protein,low fat and low price.Light is one of the important environmental factors that affects the growth and development of P.ostreatus.However,there are few studies on its molecular mechanism,especially the blue light receptors associated with primordium formation and the development of fruiting body.First of all,we obtained the PAS domain of blue light receptors WC-1 and WC-2,which has been reported in Schizophyllum commune,then we compared the JGI PC9 and PC15 transcriptome database,and gained the homologous sequences of Pleurotus ostreatus,By designing primers based on the obtained sequence,we obtained the full-length CDS of PoWC-1 and PoWC-2.The genetic structure and function through online websites and databases;The ORF of PoWC-1 and PoWC-2 was verified through PEASY-Blunt E1 prokaryotic expression system;The interaction between Po.WC-1 and Po.WC-2 was identified through the yeast two-hybrid technique;The transcriptional level of PoWC-1 and PoWC-2 were analyzed by fluorescence quantification;The over-expression and antisense vectors of PoWC-1 and PoWC-2 were constructed,and transformed to the P.ostreatus by agrobacterium-mediated method.The conclusions of this paper are as follows:1.We gained the full-length CDS of PoWC-1 and PoWC-2 by PCR using P.ostreatus c DNA as template.The CDS length of the PoWC-1 gene was 1,956 bp,encoded 651 aa.The results of bioinformatics analysis showed that the nucleotide sequence of PoWC-1 was 100% identity with the database.It had three typical PAS domains and one Zn F domain.The CDS length of the PoWC-2 gene was 2,316 bp,which encoded 771 aa.The nucleotide sequence of PoWC-2 is 99% identity with the database.it had one typical PAS domain and one Zn F domain.2.We constructed the prokaryotic expression vector E1-WC1 and E1-WC2,and induced them to express.The results of SDS-PAGE showed that the molecular weight of PoWC-1 was about 73 k Da,and PoWC-2 was about 84 k Da,which was consistent with the expectation.3.The interaction between PoWC-1 and PoWC-2 was investigated through the yeast two-hybrid technique,and the experimental results showed that PoWC-1 could form a complex structure with PoWC-2.4.The expression levels of PoWC-1 and PoWC-2 were analyzed under different light time in the dikaryotic mycelia through RT-PCR;The expression levels of PoWC-1 and PoWC-2 were analyzed in different parts of mature fruiting body or in different periods.The results showed that the expression of PoWC-1 and PoWC-2 increased in a short time,and the final expression level tended to be stable with the increase of light time.For different parts of mature fruiting body,the expression levels of the pileus PoWC-1 and PoWC-2 were the highest,followed by the stipe and the gills;For different periods,the expression quantity of the primordium period was higher,than the mature fruiting-body period.5.The over-expression vectors p Po-GPD-WC1+ and p Po-GPD-WC2+,and antisense vectors p Po-GPD-WC1-and p Po-GPD-WC2-,were constructed and transformed to P.ostreatus through GV3101 mediation.The next verification work is ongoing.It will lay the foundation for the subsequent verification of the functions of PoWC-1 and PoWC-2.