Gene Expression Analysis Of Embryonic Development From Termite Parthenogenetic And Sexual Eggs Based On Single-cell Transcriptome Sequencing

Termite species Reticulitermes aculabialis and R.flaviceps found in China.R.aculabialis have ability to the facultative parthenogenesis,while R.flaviceps lack of parthenogenetic knowledge.Therefore,previous studies have been shown that the parthenogenetic eggs of R.aculabialis can continue the healthy development of the embryo after ovulation.However,the parthenogenetic eggs of R.flaviceps began to develop slowly about 3-5 days after ovulation,and the embryo development was almost wholly arrested about ten days after ovulation.In the current study Illumina HiSeq~TMM 2000 platform used for single-cell transcriptome sequencing analysis of embryo development to day four of R.flaviceps and R.aculabialis parthenogenetic and sexual eggs.By analysing the differential genes of R.flaviceps and R.aculabialis between parthenogenetic and sexual eggs,to find genes that affect R.aculabialis,parthenogenetic eggs can develop normally and R.flaviceps parthenogenetic eggs block their development.Using RT-PCR to study the trends of gene expression levels of these regulatory genes,reveal the reason that affects the parthenogenesis of termites.The conclusion is as follows:(1)A transcriptome assembly consisting of R.flaviceps parthenogenetic and sexual eggs yielded 422,213 unigenes.A total of 123,667 Unigenes were annotated into the database.The Nr database is annotated to 122,100 Unigenes,While the COG database is annotated to 36,805 Unigenes,involving 26 functional categories.The SwissProt database is annotated to 48,416 Unigenes.The KEGG database is annotated to 11,086 Unigenes,involving 43 biological pathways.The Unigene annotated by the Go database includes 54physiological,metabolic functions involving biological processes,cellular components,and molecular features.According to the different significance criterion(Differential gene expression changes more than 2 times and P-value≤0.05),the number of differential genes between R.flaviceps parthenogenetic and sexual eggs was 19,153,the number of up-regulated genes was 484,and the number of down-regulated genes was 18,669.(2)A transcriptome assembly consisting of R.aculabialis parthenogenetic and sexual eggs yielded 324,454 unigenes.A total of 62,253 Unigenes were annotated into the database.The Nr database is annotated to 61,289 Unigenes.The COG database is annotated to23,507 Unigenes,involving 26 functional categories.The SwissProt database is annotated to 24,808 Unigenes.The KEGG database is annotated to 2,354 Unigenes,involving 42biological pathways.The Unigene annotated by the Go database includes 49 physiological,metabolic functions involving biological processes,cellular components,and molecular features.According to the different significance criterion(Differential gene expression changes more than 2 times and P-value≤0.05),the number of differential genes between R.aculabialis parthenogenetic and sexual eggs was 937,the number of up-regulated genes was 710,and the number of down-regulated genes was 277.(3)Analysis of the results of RT-PCR,we found RfFFegg-4,RfFMegg-4,RaFFegg-4 and RaFMegg-4.There was no significant difference in the expression levels of cdk2 between the groups.The expression level of cpeb in RaFFegg-4 was significantly higher than RfFMegg-4(P<0.05),and there was no significant difference between the other groups.In addition to the expression levels of pka,map2k1,mapk1/3,hgk,mkp and pax6 in RaFFegg-4 were significantly higher than RfFFegg-4(P<0.05).The expression levels of pka,map2k1,mapk1/3 and hgk in RfFMegg-4 were considerably higher than RfFFegg-4(P<0.05).There was no significant difference between RfFMegg-4 and RfFFegg-4 about the expression level of pax6 and mkp.It is speculated that genes with significant differences may be involved in the regulation of embryonic development of termite eggs and the occurrence of parthenogenesis.(4)In the five periods of RfFMegg,RaFFegg and RaFMegg embryo development,the cdk2,pka,map2k1,mapk1/3,hgk and mkp reached the maximum in the egg embryo development to the next day and continued to maintain a high level of expression to the fourth day,and the gene expression began to decrease gradually after the fourth day.In the five periods of RfFFegg embryo development,the cdk2,pka,map2k1,mapk1/3,hgk and mkp have reached the maximum level when the egg just produced,or the gene expression reached its maximum when the egg embryo developed to the next day,and the gene expression began to decrease after the second day.It can be speculated that the slow development of R.flaviceps parthenogenetic eggs starting 3-5 days after ovulation may be due to the growth of cdk2,pka,map2k1,mapk1/3,hgk and mkp in embryonic development until the next day,resulting in decreased gene expression.For the study of specific mechanisms of action,we need to conduct more in-depth research.