Screening Of CDNA Library Of Crassostrea Gigas And Prokaryotic Expression And Enzymatic Properties Of N-acetylneuraminate Lyase

The access to biological material of reference species,which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects,are often difficult to provide for further studies or third parties due to the consumptive nature of the samples.Although now widely distributed over the Pacific coasts of Asia,Australia and North America,individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries.In this article,we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries.These libraries were then compared to the publicly available oyster genome,and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I(COX1)and NADH Dehydrogenase(ND).The suitability of the generated cDNA library is also demonstrated by cloning of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase(UGD)and UDP-xylose synthase(UXS),which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.Sialic acid is a monosaccharide that appears at the end of glycoside compounds and is involved in the recognition of cells and cells,and between cells and molecules.Its synthesis and decomposition have always been the focus of research.N-acetylneuraminic lyase(NPL)can catalyze the cleavage of N-acetylneuraminic acid(Neu5Ac)into N-acetyl-mannosamine(ManNAc)and pyruvate.It is one of the key enzymes in the sialic acid cycle.This kind of enzyme generally appears in organisms that need sialic acid.Several sialidase have been found in the oyster's hepatopancreas,but there have been no reports of NPL.Therefore,we plan to clone N-acetylneuraminic lyase gene from Pacific oysters for prokaryotic expression,infer its function by comparing with NPL from human.1.Screening of cDNA library of Crassostrea gigasSelect oyster varieties with regional differences:Dandong in Liaoning,Qingdao in Shandong,Lianyungang in Jiangsu,Ningbo in Zhejiang,Dongshan Island in Fujian,Chaozhou in Guangdong.The TRIZOL method was used to extract RNA from oysters,and reverse transcription was used to generate cDNA.Design primers to clone COXI,ND,UXS,UGE and UGD genes.The COXI and ND sequencing results show that except for the Zhejiang samples,all samples have high homology.The samples from Liaoning and Jiangsu have similarities of 100%with ND and COXI.These two libraries can be used to do further experiment.Then the nuclear genes UGE,UGD and UUX were used to test the selection results.The results showed that except for the samples from Zhejiang Province,the similarity was above 97.5%,which was the same as the previous results.So,cDNA libraries from Liaoning and Jiangsu can be used to do further experiment.cDNA library from Jiangsu was used as a template to successfully clone the galactosekinase and N-acetylgalactosinekinase of Pacific oysters,and N-acetylgalactosinekinase show activity to N-acetylglucosamine,N-acetyl-D-mannosamine,N-acetylgalactosamine.2.Cloning and Expression of NPL in Oyster and transformation of E.coli BL21Using the NPL from Escherichia coli(EcNPL)gene as a reference,four suspected NPL sequences in Pacific oysters were retrieved from the NCBI.cDNA library from Jiangsu was used as a template to clone CgNPL-2,CgNPL-3,and CgNPL-4.The sequencing results showed that the cloned genes had only a few amino acid mutation points,but these three genes were expressed when expressed in E.coli BL21.Is an inclusion body protein.CgNPL-1 cannot be obtained by cloning and sent to company for synthesis.The enzyme expressed in E.coli BL21 is soluble,and the effect after Ni2+-NTA affinity chromatography column is not good,containing a large amount Miscellaneous protein.When the enzyme activity was initially measured,the activity of CgNPL-1 on(Neu5Ac was unstable and the efficiency was low.It is speculated that it may be the NPL expressed by E.coli itself,which affects the results of activity verification.Therefore,CRISPR/Cas9 knockout technology was used to knock out the NPL gene in E.coli,and the PCR method and the Neu5Ac induction method were tested to prove that the E.coli NPL gene has been successfully knocked out.And gene knockout did not affect the expression ability of BL21 heterologous protein3.Activity testing of recombinant N-acetylneuraminate lyaseNPL from Human(HsNPL)was synthesized by the company;compared with the amino acid sequence of EcNPL,Lys174 of CgNPL-1 may be enzymatic active site,which was mutated to alanine by PCR.The HsNPL,CgNPL-1,and mutated CgNPL-1 plasmids were introduced into E.coli BL21 with NPL gene knocked out.Ni2+-NTA affinity chromatography column removed most of the heterogeneous proteins.Neu5Ac,KDO,KDN and its analogues are used as substrates to test the activity of the three enzymes.The results show that unlike HsNPL,CgNPL-1 has no activity on Neu5Ac,but has activity on KDO,KDN,and KDN analogs.The mutant CgNPL-1 activity disappeared,indicating that Lys174 is its active center.