Activity Detection Of Cordyceps Militaris Lectin Expressed By Procaryotic Cells And Preliminary Study Of Nem1 Gene

Cordyceps militaris plays an important part in anti-fatigue,antitumor,anti-aging,and obviously enhance the nonspecific immune system.It is widely used in our country as food or medicinal fungi.So the study of active ingredients in C.militaris and the related gene will help to develop its pharmacological function better.It will provide scientific basis for its biosynthesis.Lectins are a kind of non-immunogenic proteins or glycoproteins which selectively bind carbohydrates.It can agglutinate cells or glycoconjugate to precipitation.The research reports are very few about lectins in C.militaris at home and abroad,and the previous studies were limited to directly purification from the fruiting body and biological activity detection.But there's no research about lectin gene in C.militaris.In the previous study,through the C.militaris genome data mining,a Jacalin lectin gene encoding lectin-ccm4 was find.In this research,the gene sequence were analysed by bioinformatics,and it was heterologously expressed in Escherichia.coli.The biological activity of product was examined.The lectin-ccm4 gene expression level in different growth periods of C.militaris was tested by RT-PCR.The research results shown that the lectin-ccm4 protein is different from each of the reported lectin.It a new one in C.militaris.This suggests that there may be a variety of lectin in C.militaris.SDS-PAGE was performed to detect target protein.It suggests that lectin-ccm4 was successfully expressed in prokaryotic cells.Molecular weight was in accordance with prediction.Target prodtein is soluble,so it can be directly used to detect biological activities.Different lectins have varies specificity recognition sites to agglutinate kinds of cells.The protein of the JRL-ccm4 could inhibit growth of Staphylococcus aureus but not the E.coli.The protein of the lectin-ccm4 could partly inhibit cervical cancer HeLa cell proliferation;When the protein concentration was 1.8mg/ml,the inhibition ratio achieved was 11.7%.And it can influence the shape of the tumor cells seriously.The lectin-ccm4 had the highest amount of gene expression in primordium,which is 3.49 times that of mycelium period and 1.46 times in the period of the fruiting body.It suggest that the gene may be related to fruit body development.The results provide the foundation work for understanding the function or utilization of C.militaris lectin from molecular level.This study was to reveal the function of Nem1 gene in C.militaris,constructed strains of ?Nem1 gene deletion strains and complemented strain containing different mating type gene.We explore the function of Nem1 gene by observing the variation of lipid metabolism,the influence of agronomic traits in the process of cultivation,as well as the influence of microstructure and infect virulence during the process of hypha growth.Considering Nem1 is a key gene of lipid metabolic pathway,this study using qPCR to quantitative genes related to autophagy.As a result,we constructed C.militaris strains of ?Nem1 gene deletion(1?Nem1,2?Nem1)and complemented(Comp1,Comp2).Through the subcellular localization prediction and conversion of GFP fusion protein,we think Nem1 protein are more likely to be distributed in cell nuclear membrane and cytoplasmic membrane.Growth rate of was severely inhibit.Growth rate of 1?Nem1 and 2?Nem1 were reduced by 41.2% and61.76% respectively.After cultivation Nem1 mutant strain can not form sporocarp.This resulte suggests that Nem1 can seriously affect the growth of C.militaris mycelium and differentiation of fruit body.At the same time,Nem1 gene influences conidium and spore germination process.The experiment of inducting appressorium formation using polystyrene hard hydrophobic surface reveals that the wild strains and Nem1 knockout strains can form appressorium normally after 24 h.Add to the result of qPCR about chitinase and protease gene,it demonstrates that Nem1 gene does not affect the ability to infect the host for C.militaris.Though fluorescent dye we found that the nuclear shape of ?Nem1 mutant strain was abnormal.Which can show Nem1 protein plays an important role in the nucleus formation.Lipid decreased significantly in ?Nem1 mutant strains.Nem1 gene can influence lipid synthesis.Gene expression analysis found that Atg13,Atg22-2,Atg28,which related to autophagy in ?Nem1 mutant strains are lower expressed.It can suggest that Nem1 gene influences the level of autophagy.There is close relationship between autophagy and lipid metabolism in C.militaris.