【作者】 张敏；

【导师】 邢连喜；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2019， 硕士

【摘要】 尖唇散白蚁Reticulitermes aculabialis是我国已知的白蚁中唯一具有兼性孤雌生殖能力的白蚁种类,黄胸散白蚁R.flaviceps则不具有孤雌生殖能力。前期研究显示,尖唇散白蚁孤雌卵在排卵之后仍然可以继续进行胚胎的正常发育,而黄胸散白蚁孤雌卵则在排卵之后的3-5天左右开始出现发育缓慢现象,在第10天左右发育几乎完全停滞。本研究利用Illumina HiSeq~TMM 2000平台对胚胎发育至第4天的黄胸散白蚁孤雌卵、黄胸散白蚁两性卵、尖唇散白蚁孤雌卵和尖唇散白蚁两性卵进行单细胞转录组测序分析。通过对尖唇散白蚁孤雌卵和两性卵以及黄胸散白蚁孤雌卵和两性卵之间的差异基因进行分析,找到影响尖唇散白蚁孤雌卵可以正常发育而黄胸散白蚁孤雌卵出现发育阻滞的基因,对这些调控基因利用实时荧光定量PCR技术进行基因表达水平的研究,同时分析其基因表达量的变化趋势,揭示影响白蚁孤雌生殖发生的原因。研究结果如下:(1)黄胸散白蚁孤雌卵和两性卵构成的转录组组装获得422213条unigenes。有123667条Unigenes被注释到数据库中,Nr数据库注释到122100条Unigenes;COG数据库注释到36805条Unigenes,涉及26个功能分类;Swissprot数据库注释到的Unigenes为48416条;KEGG数据库注释到11086条Unigenes,涉及43条生物通路;Go数据库注释到的Unigene分别涉及生物学过程、细胞成分及分子功能相关的共54种生理代谢功能。按照差异显著性标准(差异基因表达变化2倍以上且pvalue≤0.05)进行筛选,黄胸散白蚁孤雌卵和两性卵之间的差异基因个数为19153个,上调基因有484个,下调基因有18669个。(2)尖唇散白蚁孤雌卵和两性卵构成的转录组组装获得324454条unigenes。有62253条Unigenes被注释到数据库中,Nr数据库注释到61289条Unigenes;COG数据库注释到23507条Unigenes,涉及26个功能分类;Swissprot数据库注释到24808条Unigenes;KEGG数据库注释到的Unigenes为2354条,涉及42条生物通路;Go数据库注释到的Unigene分别涉及生物学过程、细胞成分及分子功能相关的共49种生理代谢功能。按照差异显著性标准(差异基因表达变化2倍以上且pvalue≤0.05)进行筛选,尖唇散白蚁孤雌卵和两性卵之间的差异基因个数为937个,上调基因有710个,下调基因有277个。(3)对实时荧光定量PCR的结果进行分析,我们发现在胚胎发育至第四天的黄胸散白蚁孤雌卵(RfFFegg-4)、黄胸散白蚁两性卵(RfFMegg-4)、尖唇散白蚁孤雌卵(RaFFegg-4)和尖唇散白蚁两性卵(RaFMegg-4)中。cdk2在各组间的表达量均无显著性差异。cpeb除了在RaFFegg-4中的表达量水平显著高于在RfFMegg-4中外(P<0.05),其余各组之间亦均无显著性差异。pka、map2k1、mapk1/3、hgk、mkp和pax6在RaFFegg-4中的表达水平显著高于RfFFegg-4(P<0.05)。pka、map2k1、mapk1/3和hgk在RfFMegg-4中的表达水平显著高于RfFFegg-4(P<0.05)。pax6和mkp在RfFMegg-4和RfFFegg-4之间无显著性差异。由此推测表达显著性差异的基因可能参与调控了白蚁卵的胚胎发育以及孤雌生殖的发生。(4)在RfFMegg、RaFFegg和RaFMegg胚胎发育的5个时期中,cdk2、pka、map2k1、mapk1/3、hgk和mkp在卵胚胎发育至第二天时基因表达量达到最大并继续保持较高水平的表达量,或者在卵胚胎发育至第四天时基因表达量达到最大,在第四天后基因表达量都开始逐渐下降。在RfFFegg胚胎发育的5个时期中,cdk2、pka、map2k1、mapk1/3、hgk和mkp在卵刚刚产出的时候基因表达量就已经达到最大或者在卵胚胎发育至第二天时基因表达量达到最大,在第二天后基因表达量都开始下降。由此可以推测,黄胸散白蚁孤雌卵在排卵后的3-5天开始出现发育迟缓的现象有可能就是因为cdk2、pka、map2k1、mapk1/3、hgk和mkp在黄胸散白蚁孤雌卵胚胎发育至第二天后,基因表达量下降而导致的。对于其具体的作用机制,有待进行更深入的研究。更多还原

【Abstract】 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.更多还原