The Sequence Analysis And Expression Regulation Of Circadian Clock Frequency Gene (CmFRQ) In Cordyceps Militaris

Blue light and temperature are important environmental factors, which cancontribute to changes of frequency gene in some fungi. In Cordyceps militaris, thecore component of biological oscillator is frequency (CmFRQ) gene that plays a roleto generate and control biological rhythem. The paper is focused on cloning, sequenceanalysis of frequency gene of core component of the clock gene in Cordycepsmilitaris, and it was studied the effect of blue light and temperature on the expressionof CmFRQ gene.We blasted the sequence of frequency gene mRNA of Neurospora crassa inGenBank and found a homologous sequence in a Whole-genome shotgun contigs,Cordyceps militaris CMO1contiq50. By using the ORF finder, the ORF of Cordycepsmilitaris frequency gene was found. According to the ORF sequence which we got,the primers were designed to obtain the CmFRQ-ORF sequence by PCR, sequencingand alignment. It is suggested that CmFRQ-ORF is the open reading frame ofCordyceps militaris. The total length of CmFRQ-ORF sequence is2727bp. Accordingto CmFRQ gene sequence and the3’ RACE method, then3-terminal sequence primerswere designed to obtain a656bp3-terminal partial sequence of CmFRQ mRNA. Itshowed high homology with Cordyceps militaris CM01contig50by alignment.A2727bp ORF of CmFRQ gene encode903amino acids, and it is found acoiled-coil domain and a LA motif RNA-binding domain by sequence analysis.CmFRQ-ORF partial sequence was constructed into pEASY-E1Expression Vector,then both were transformed into BL21(DE3) strain of Escherichia coli. Prokaryoticexpression system of CmFRQ-ORF partial gene was detected and confirmed.The effect of blue light and temperature on the relative expression of CmFRQgene was studied through Real-Time Quantitative PCR. The results suggested thatdifferent blue light time and temperature can cause the changes of relative expressionof CmFRQ, which showed a updulating change and a highest relative expression levelin18℃, respectively.