Cloning And Analysis Of The Pyruvate Kinase Gene And Splicing Factor Of Nosema Bombycis

Microsporidia is a single-celled eukaryotic organism with wide parasitic range.N.bombycis is the pathogens of microsporidian of main infectious disease in silkworm,causing huge economic losses to sericulture.There is no tricarboxylic acid cycle in N.bombycis,but it has the glycolysis pathway.There are three key reactions in the glycolysis process: the conversion of glucose to 6-phosphate glucose under the action of hexokinase,the conversion of fructose 6-phosphate to fructose 1,6-diphosphate under the action of fructokinase phosphate,and the conversion of phosphoenolpyruvate to propionate under the action of pyruvate kinase,these three steps are irreversible reactions.Splicing factor is an important element of RNA editing in eukaryotic organisms.After splicing factor splicing,many functional mRNA with encoded information can be generated,which is essential for the most basic life activities of organisms,and is also an indispensable step for the correct expression of corresponding proteins in eukaryotic genes.In this paper,some studies were carried out on the genes and proteins of pyruvate kinase and splicing factor of N.bombycis,and the amino acid encoded by the two genes,and the following results were obtained:(1)It was found by searched that pyruvate kinase of N.bombycis has two subtypes,each of which has 3 copies.Phylogenetic tree and multiple sequence alignment results indicated that two copies of the M2 subtype of NbPK(EOB11679.1 and EOB11685.1)have higher similarity and may have similar functions;one of the two copies(NbPK-2,GenBank accession number: KB909845.1)and the amino acid sequence(NbPK-2,GenBank accession number: EOB11679.1)were selected for studied.Its bioinformatics analysis results show: the result of secondary structure prediction showed that NbPK-2 contained 38% helix,21% extended strand,40% random coil;and only 5% sequences of NbPK-2 are disorder,while the other sequences are ordered;the result of prediction of signal peptide showed that there was no signal peptide in the sequence.NbPK-2(KB909845.1)was successfully cloned.The length of the gene sequence is 1 359 bp,it contains a complete open reading frame and encodes 452 amino acids.The results of q-PCR showed that the relative expression level of NbPK-2 was the highest at the 2 hours after infected silkworm;and the relative expression of NbPK-2 was relatively high from then on until 24 hours;from 48 hours,the relative expression level of NbPK-2 showed a downward trend and reached the lowest value at 144 hours;at 168 hours,the relative expression of NbPK-2 increased,higher than 144 hours;after infected,the relative expression of NbPK-2 was higher than 144 hours.The trend of quantity arrival is higher in the early stage and lower in the later stage.(2)After searched,it was found that there are two copies of the splicing factor of N.bombycis(EOB15199.1 and EOB11739.1).Its bioinformatics analysis results show: one of the two copies of the splicing factor of N.bombycis(NbSF-1,GenBank accession number: KB908915.1)and its amino acid sequence(NbSF-1,GenBank accession number: EOB15199.1)were selected for studied.Its bioinformatics analysis results show: the similarity between NbSF-1 and other protein sequences is low,not exceeding 60%.The composition of NbSF-1 sequence structure is relatively simple,with only about 6% helix and 6% extension stand,and others are random coil,and there was no signal peptide in the sequence;83% sequence of NbSF-1 sequences are disordered;bioinformatics analysis shows that NbSF-1 contains 71 phosphorylation sites,22 N-glycosylation sites,20 O-glycosylation sites;and predicted result of subcellular localization suggest that it may be localized in the cytoplasm and nucleus.NbSF-1(KB908915.1)was successfully cloned,the length of the gene sequence is 1 449 bp,it contains a complete open reading frame and encodes 482 amino acids.The results of q-PCR showed that the relative expression level of NbSF-1 was increased from 2 h to 24 h after infected silkworm,and decreased from 24 h,but remained relatively stable after 48 h of infection;after infected,the relative expression level of NbSF-1 showed an upward trend in the early stage and a relatively flat trend in the later stage,it suggested that NbSF-1 might be involved in the whole life cycle of microsporidia.(3)NbSF-1 protein was successfully expressed by constructed recombinant expression vector.After optimizing the induction conditions,when the final concentration of IPTG was 0.5 mM,a relatively large amount of target protein could be obtained after 4 hours induction at 37 C.The purity of the purified target protein solution was significantly higher than that of the unpurified protein solution,the purification was successful;the purified NbSF-1 protein was used to immunize rabbits for many times,and the polyclonal antibody was successfully prepared..(4)The results of indirect immunofluorescence showed that NbSF-1 was present in the dormant N.bombycis,and ejected to the spores with the germination of the microsporidia;after the germination,fluorescence was observed in sporoplasm out of spores,and there is no fluorescence in the microcapsules of N.bombycis.It is proved that NbSF-1 is located in the cytoplasm of N.bombycis,and there is no NbSF-1 on the spore wall,nucleus and polar tube.