| Microsporidia is a class of obligate intracellular parasitic fungi that are ubiquitous in nature and may infect almost all animals from protozoa to invertebrates and vertebrates including humans.Microsporidia can cause significant economic losses when it infects economic animals,and when infects people with weak or deficient immunity,it will accelerate the death of patients and seriously threaten human health.Microsporidia have unique biological characteristics.The degradation of mitochondria leads to the loss of many typical energy metabolism pathways,and the substances and energy required for intracellular parasitism are highly dependent on the host.Therefore,studying the energy metabolism of microsporidia and the regulation of host energy metabolism will help to elucidate the biochemical adaptation mechanism of microsporidia in host cells.Nosema bombycis(N.bombycis),as the first identified microsporidia,is a representative species of microsporidia.The Pébrine caused by the infection of N.bombycis is one of the most serious diseases in sericulture production.Taking N.bombycis as the research object,this study analyzed the functional characteristics of N.bombycis nucleotide transport protein1 gene(NbNTT2)in the process of microsporidia proliferation,identified its functional characteristics in regulating host energy metabolism,determined its effect on N.bombycis proliferation,and elucidated the mechanism of N.bombycis hijacking host energy metabolism beneficial to its own proliferation and replication,which lays the foundation for the biological research of microsporidia and the prevention and control of microsporidia.The main results are as follows:1.Characterization of Nosema bombycis NbNTT2 geneIn order to investigate the regulatory mechanism of energy metabolism in N.bombycis,the nucleotide transport protein 1 gene NbNTT2 was screened by analyzing the energy-related transport protein genes that are widely involved in the metabolism during their proliferation.To analyze the basic characteristics of NbNTT2 gene,domain prediction and phylogenetic analysis determined that NbNTT2 has a typical TLC domain and transmembrane domain,and is a member of the ADP/ATP transporter protein family.Through indirect immunofluorescence experiments and western blot analysis,it was determined that NbNTT2 is localized on the plasma membrane of the sporoplasm of N.bombycis,with a molecular weight of about 23 k Da.The expression of NbNTT2 gene was characterized by q RT-PCR in silkworm cells and midgut tissues infected with N.bombycis,and it was determined that NbNTT2 gene was expressed during the proliferation of N.bombycis and was up-regulated in the schizophrenia proliferation stage after infection.2.Functional identification of NbNTT2 geneAimed to identify the function of NbNTT2 gene,different concentrations of ATP solution were added to the medium under the condition of overexpression of NbNTT2 gene,and it was found that the intracellular ATP content increased significantly compared with the control group,indicating that NbNTT2 gene has a transport function for ATP;when no ATP was added,the ATP level in the cells overexpressed of NbNTT2 gene also increased significantly,indicating that NbNTT2 gene could promote the production of intracellular ATP.In order to reversely verify the function of NbNTT2 gene,double-stranded interfering RNA of NbNTT2 gene(ds NbNTT2)was designed.The effect of interference was tested by q RT-PCR,and it was determined the transcript level of NbNTT2 gene significantly down-regulated.The function of NbNTT2 gene was verified in reverse by interfering with NbNTT2 after infection with N.bombycis,and a significant decrease in intracellular ATP content was found.Meanwhile,after overexpression of NbNTT2 gene,its function was verified again by incubating cells with different concentrations of antiNbNTT2,and it was found that the intracellular ATP level decreased significantly.The above results confirmed that NbNTT2 gene could promote ATP production in host cells.To further identify the functional key domains of NbNTT2,the ATP binding site of NbNTT2 was analyzed.The NbNTT2 mutants were constructed by amino acid site mutation and functional detection was carried out to determine that phenylalanine at position 27 in NbNTT2 is essential for promoting cellular ATP production and is the key structural domain of its function.3.Effect of NbNTT2 gene on the proliferation of N.bombycisTo determine the effect of NbNTT2 gene on the proliferation of N.bombycis at the cellular level,the NbNTT2 gene was interfered in the cells infected with N.bombycis,and the number of N.bombycis was significantly reduced as determined by indirect immunofluorescence observation and statistical analysis using an antibody to β-tubulin of N.bombycis.The DNA copies of N.bombycis Small Subunit ribosome RNA(NbSSUr RNA)gene in the cells was analysed by q RT-PCR and it was determined that the proliferation of N.bombycis was significantly inhibited.Further,the relative expression levels of genes related to N.bombycis proliferation were detected after interfering NbNTT2 gene.It was found that NbPTP2 gene,an essential gene for N.bombycis infestation,NbSWP5 gene,an important component of the spore wall,and the continuously expressed NbHSP76 gene were significantly inhibited.These results indicated that the proliferation of N.bombycis was significantly inhibited by interfering with NbNTT2 gene in vitro.In order to determine the effect of NbNTT2 gene on the proliferation of N.bombycis at the individual level,silkworm larvae were injected with ds NbNTT2 and fed on N.bombycis.After confirming that the expression of NbNTT2 could be significantly downregulated in silkworm,the copy number of NbSSUr RNA gene was detected by q RT-PCR to analyze the proliferation of N.bombycis,and it was found that the proliferation of N.bombycis was significantly inhibited.Meanwhile,the midgut tissue of silkworm was observed by pathological section staining,and it was confirmed that the number of N.bombycis was significantly reduced.The above results indicated that the proliferation of N.bombycis was significantly inhibited by interfering with NbNTT2 gene in vivo.4.NbNTT2 gene regulates the metabolic pathway of host hexokinaseTo investigate the regulatory characteristics of NbNTT2 gene on host energy metabolism,intracellular hexokinase(BmHXK)in glycolysis,thiokinase in fatty acid metabolism and cytochrome C oxidase subunit 4(COX 4)in the electron transport chain,which are key enzymes for major ATP production,were examined.The q RT-PCR analysis revealed that the transcript level of BmHXK gene in glycolysis was significantly upregulated,whereas no significant changes were found in thiokinase gene and COX 4 gene after NbNTT2 gene overexpression;while interference with NbNTT2 gene revealed a significant down-regulation in the relative expression level of BmHXK gene.The above results suggest that NbNTT2 gene and BmHXK gene have a regulatory relationship at the transcriptional level and that the effect of NbNTT2 gene on host energy metabolism can be achieved through the regulation of the host glycolytic pathway by BmHXK gene.To further clarify the regulatory mechanism of NbNTT2 gene on host glycolysis,the substrate glucose of BmHXK gene was examined and it was found that intracellular glucose content decreased significantly after overexpression of BmHXK gene and NbNTT2 gene,respectively,indicating that the two may regulate host glycolysis synergistically.Under the condition of overexpression of NbNTT2 gene,the content of glucose decreased more significantly when BmHXK gene was overexpressed;there was a certain degree of reversion when BmHXK gene was knocked down,indicating that NbNTT2 gene could regulate the host glucose level through BmHXK gene.Through the detection of ATP content,it was found that under the knockdown condition of BmHXK gene,the cell’s ability to produce ATP was reduced even when NbNTT2 gene was overexpressed;the ATP level could be recovered to a certain extent after the infection of N.bombycis,indicating that NbNTT2 gene could regulate the host ATP level through BmHXK gene.Based on the mechanism that NbNTT2 gene regulates host energy metabolism through BmHXK gene to facilitate its own proliferation,a transgenic knockout line of BmHXK gene in silkworm was created by transgenic technology.To test the knockout effect of the transgenic lines,the sequence analysis of the target site revealed that the target genes were successfully edited.Statistical analysis of the growth,development and economic traits of the BmHXK gene knockout line silkworm showed that there was no significant difference in the weight of the larvae of the transgenic line at 5th instar 3 days and 5th instar 6 days compared to the control line,and no significant difference in the cocoon volume,cocoon layer weight and cocoon layer rate.In order to analyse the disease resistance of the transgenic line against N.bombycis,the survival rate of BmHXK knockout lines after infection with N.bombycis was analysed and it was determined that the time of death of BmHXK knockout line after infection with N.bombycis was delayed from 5 to 7 days after infection,and the survival rate at day 10 after infection was increased by 175% compared to the control line.The q RT-PCR analysis showed that the DNA copies of N.bombycis in the transgenic line was not significantly different from that of the control line before 48 h of infection,while the DNA copies of N.bombycis in the larvae of the transgenic line was significantly lower than that of the control line from 4 to10 days of infection.The above results indicated that the BmHXK knockout line could delay the death time and improve the survival rate after infection with N.bombycis and was resistant to N.bombycis. |
