The Study Of Hlstidine Kinase Gene (CmHK) And Kexin Gene (CmKecxin) In Cordyceps Militaris

Cordyceps militaris is a traditional medicinal and edible fungi in China, which isrich in physiologically-active components such as cordycepin, cordyceps acid.Modern pharmacology study shows that Cordyceps militaris has the function ofanti-tumor, hypoglycemic, antibacterial, anti-inflammatory, anti-oxidation andregulate endocrine significantly. Additionally Cordyceps militaris has the function ofresisting hepatic fibrosis and improving immunity. Because its active components andpharmacological effects is similar to Cordyceps sinensis, Cordyceps militaris has highresearch value.The thesis intends to conduct in-depth research based on original research ofhistidine kinase gene(CmHK) and Kexin gene(CmKexin) in Cordyceps militaris. Thefollowing results demonstrate the findings:(1)We got the3’end partial sequence of the CmHK and CmKexin gene through the3’RACE method. Through sequencing, the length of3’end partial sequence of theCmHK gene is985bp, the length of3’end partial sequence of the CmKexin gene is725bp.(2)According to the reported sequence of Cordyceps militaris and the3’endsequence we got, we designed primers and got the open reading frame sequence ofCmHK and CmKexin gene through PCR, sequencing and alignment. The total lengthof the ORF sequence were2928bp and2559bp respectively. The length of the aminoacid sequence which translated by the ORF of CmHK gene was976aa. Sequenceanalysis showed that it contained: four HAMP domains, a HisKA domain, aHATPase_c domain and a REC domain. The length of the amino acid sequencewchich translated by the ORF of CmKexin gene was853aa. Sequence analysisshowed that it contained: a Peptidase S8family domain in Protein convertases and aProtein convertase P-domain.(3)Based on the ORF sequence of CmHK gene we got, we designed the primers,then got the function domain fragment of CmHK gene ORF sequence through PCR.Target fragments combined with pEASY-E1expression vector were transformed inTrans-T1cells to amplify. The positive recombinant-clone was confirmed and propagated. The plasmids which extracted were transformed in BL21(DE3) cells toamplify. The positive recombinant-clone was confirmed through colony PCR.Through these steps, we constructed prokaryotic expression vector of ORF functiondomain of CmHK gene.(4)We intended to study the change of the expression levels of CmHK andCmKexin gene induced by blue light through Real-Time Quantitative PCR. Reslutsdemonstrated that the expression of the CmHK gene had a significantly decrease at thebeginning of the blue light induced. After that, it increased a lot. A peak value of theexpression level appeared at16hours, then it dropped quickly. The expression of theCmKexin gene was stable in the48hours through blue light induced, then it riserapidly. A peak value of the expression level appeared at50hours, then it fell fast.Finally, it kept stable.