Sequence Analysis And Blue Light-influenced Expression Of CmWC-1Ma CmWC7-2Gene From Fungus Cordyceps Militaeis

Cordyceps militaris is an important medicinal and edible fungi, which containsmany physiologically-active components such as Cordyceps Polysaccharide,Cordycepin and so on. As an environmental factor which can affect light receptor offungi, blue light can control growth and physiological processes of many organisms.This study intended to know the induction of blue light to the functional genes inCordyceps militaris through the molecular biology research. This study can alsoprovide support for further exploring resources of the precious functional genes inCordyceps militaris. The main results were displayed as follows:(1) We got the3’ end partial sequence of the CmWC-1and CmWC-2gene Whichmay be the potential light receptor through the3’ RACE method. Sequencing resultsshowed that the length of3’ end partial sequence of the CmWC-1gene is1020bp, the394-452bp is introns. The length of3’ end partial sequence of the CmWC-2gene is1009bp, the288-353bp is introns.(2) We got the open reading frame sequence of CmWC-1and CmWC-2genethrough PCR. The length of them were2823bp and1575bp respectively. Sequencinganalysis showed that the length of the amino acid sequence translated by the ORFsequence of CmWC-1gene was940aa. It might be the Zinc finger domain-containingprotein(GATA-type), which including three PAS domains(a specialized class of PASdomain known as a LOV domain) and a Zinc finger domain. The length of the aminoacid sequence translated by the ORF sequence of CmWC-2gene was525aa. It mightbe the Cutinase palindrome-binding protein, which including a PAS domain.(3) According to the ORF sequence which we got, we designed the primers andgot the functional domain fragment of CmWC-1and CmWC-2gene ORF throughPCR. Target fragments combined with pEASY-E1expression vector weretransformed in Trans-T1cells to amplify. The positive recombinant-clone wasconfirmed and propagated. The plasmids which extracted were transformed inBL21(DE3) cells to amplify. The positive recombinant-clone was confirmed throughcolony PCR and sequencing analysis. Through these steps, We constructedprokaryotic expression vector of ORF functional domain of CmWC-1and CmWC-2 gene. The functional domain sequence of CmWC-1gene began to express from the844th nucleotide of its ORF sequence. The full length of the expression sequencewas1266bp, expressed433amino acids totally. The functional domain sequence ofCmWC-2gene began to express from the286th nucleotide of its ORF sequence. Thefull length of the expression sequence was636bp, expressed212amino acidstotally.(4) We intended to study the change of the expression levels of CmWC-1andCmWC-2gene induced by blue light through Real-Time Quantitative PCR. Resultsshowed that CmWC-1, CmWC-2gene had a certain amount of expression with theinduction of blue light or not. When cultural conditions was always dark, the relativeexpression level of CmWC-1gene was the highest(0.44). When Blue light existed, apeak value of the relative expression level appeared at16hours(0.37). UnlikeCmWC-1gene, a peak value of the relative expression level of the CmWC-2geneappeared at16hours(3.28). The expression level in other conditions was low. Thisshowed that direct exposure to blue light can cause the changes of expression levelof CmWC-1and CmWC-2gene.