The Regulation Of Cmfrq、Cmwc-1and Cmwc-2 Genes Expression, Preparation Of Their Antibodies And Detection Of The Protein In Cordyceps Militaris

All organisms have their own circadian clocks which can provide circadian rhythm. Circadian clock protein FREQUENCY(FRQ), blue light receptors White Colar-1(WC-1) and White Colar-2(WC-2) are three important components of the core circadian oscillator in clock system of fungi, and they play important roles in the circadian negative feedback regulation system. The paper used Cordyceps militaris F411 as the experimental material. The regulation of clock gene Cmfrq(Cordyceps militaris frq), blue light receptors genes Cmwc-1(Cordyceps militaris wc-1)and Cmwc-2(Cordyceps militaris wc-2)expression in Cordyceps militaris was analyzed, and the antibodies to Cm FRQ(Cordyceps militaris FRQ), CmWC-1(Cordyceps militaris WC-1) and CmWC-2(Cordyceps militaris WC-2)were prepared. Then all antibodies were used to detect expression of the protein in Cordyceps militaris by Western blotting technolgy.To investigate and the regulation of Cmfrq, Cmwc-1 and Cmwc-2 expression, this study designed different culture conditions for Cordyceps militaris, and detected expression of Cmfrq, Cmwc-1 and Cmwc-2 under different conditions by real-time PCR.The results showed that expression of Cmfrq, Cmwc-1 and Cmwc-2 were different under different conditions. The relative transcription level of Cmfrq was the highest,Cmwc-2 was second,Cmwc-1 was the lowest.When Cordyceps militaris was changed from dark conditions to light conditions,the relative transcription of Cmfrq in mycelium was increased, which was same to Neurospore crassa. The imnormal fruiting body had a higer relative transcription of Cmfrq than normal fruiting body. The diference between Cmwc-1 and Cmwc-2 suggested that the structure of WCC in Cordyceps militarys may be diferent to Neurospore crassa. The relative transcription of Cmwc-1 mainly focused on lower part of fruiting body, but the relative transcription of Cmwc-2 mainly focused on upper part of fruiting body, which implied that there may be more CmWC-1/CmWC-2 complexes in the middle of fruiting body, and it may be related to phototropism and extension of fruiting body.Through bioinformatics analysis, primers were designed for cloning of the partial sequence of Cmfrq(1561-2727 bp), the partial sequence of Cmwc-1(1549-2772 bp) and the partial sequence of Cmwc-2(394-1179 bp). The three sequences and plasmid pET-41a(+) were connected, then transformed into BL21(DE3) competent cells, which expressed fusion protein with IPTG inducing.The expressed fusion protein molecular weight size are 74 kDa, 76 kDa and 64 kDa, respectively. The fusion proteins after purified were used as antigens for the preparation of antibodies to anti-CmFRQ, anti-CmWC-1 and anti-CmWC-2. The antibodies to CmFRQ protein were prepared by another method. The antigenic determinant short peptides of chemical synthesis(FRQ1, FRQ2 and FRQ3) were coupled to protein carriers, and the coupled products were used as antigens for the preparation of antibodies anti-FRQ1, anti-FRQ2 and anti-FRQ3.Total protein in mycelium and fruit body of Cordyceps militaris F411 were extracted, and all antibodies were used for Western blotting analysis. The results showed that anti-FRQ1 and anti-FRQ3 cannot detect specific hybridization band from the protein of mycelium and fruit body of Cordyceps militaris, and only anti-FRQ2 could probe specific 50 kDa(Cm FRQ50) and 31 kDa(Cm FRQ31) protein. There were another two hybridization bands between 40 kDa and 50 kDa in fruiting body.Furthermore, the relative content of CmFRQ50 and CmFRQ31 could change in diferent growth stages, which showed that the existence forms of the clock protein CmFRQ could alter with the change of the biological characters in Cordyceps militaris. CmFRQ100 could be probed in the mycelium but not in the fruiting body of Cordyceps militaris by anti-CmFRQ. There were diferent forms of CmWC-1 in mycelium and fruiting body of Cordyceps militaris: two forms of CmWC-1, CmWC1-42 and CmWC1-48, were in mycelium, however, only the form of CmWC1-42 is in fruiting body. It was likely that CmWC1-48 contains a LOV domin. There was the same form of CmWC-2, CmWC2-50, in mycelium and fruiting body of C. militaris F411.This study not only demonstrate the regulation relationship of Cmfrq,Cmwc-1 and Cmwc-2 expression, but also analy the forms of CmFRQ, and Cm WC-1 and CmWC-2 in Cordyceps militaris.This study will promote the understanding of biological clock system in Cordyceps militaris.