| Protozoa ciliates is a kind of eukaryotic unicellular animal with highly cell differentiation,many species of ciliates will form cyst when the environment changes.These ciliates have become important patterns of animals to explore the regulation mechanism of eukaryotic cell structure and function.Because the formation of ciliates dormancy cyst is a complex process of gene regulation precision,so the corresponding selective expression of genes is responsible for its molecular mechanism of cell morphology and physiological and biochemical changes.Based on previous research,this study use Pseudourostyla cristata which is easy to cultivate and easy to induce to cyst as our experiment material.We systematically compared the related signaling pathways and genes about the encystment with transcriptome(RNA-seq)technology?RNAi technology?qRT PCR technology and bioinformatics.Namely using the transcriptome technology supplemented by a variety of modem molecular biology technology to explore the molecular mechanism of ciliates encystment,and from the overall level to reveal the molecular nature of encystment.The results and conclusions are as follows:(1)Transcriptome library construction and sequencing analysis of cysts cell and vegetable cell of Pseudourostyla cristata.Using the Illumina HiSeqTM4000 sequencing platform on Pseudourostyla cristata to transcriptome sequencing,we received 41,965,669 total unigene,and there were 2,582 differential unigene.Compared with the vegetative cells,cyst cell raised unigene 316,come down unigene 2,266.And there were 36,934,220 unigene got noted,the differential unigene had noted 2,519 GO entries,which compared to vegetative cells,cyst cell raised 430 items,decline 2,317 items.These different entries were mainly related to the entry of cell wall synthesis,intracellular material transportation and the ribosome organelles;these genes were mapped to 284 differential pathways,compared with the vegetative cell,cyst cell raised 73 pathway and fall 279 pathway.Differential signaling pathways were selected as:calcium transport signaling pathway,protein degradation depended ubiquitin signaling pathway and MAPK signaling pathway.We also find out some important differential genes in the pathway,such as in calcium transport signaling pathway,we revealed 24 differentially expressed genes,and compared with vegetative cell,cyst cell had 23 differentially expressed genes downregulated and 1 genes up-regulated;There were 18 differentially expressed genes in protein degradation depended ubiquitin signaling pathway,and compared with vegetative cell,cyst cell had 16 genes downregulated and 2 differentially up-regulated genes;While in MAPK signaling pathway,there were 16 differentially expressed genes come down in cyst cell compared with those in vegetative cell and 1 genes were up-regulated among total 17 differential genes.Most the differentially expressed genes in these three signaling pathways were down,so the signal effects of these three pathways were weakened.Understanding these signaling pathways and its associated genes help us to explore its role in encystment of Pseudourostyla cristata,and this also lay the foundation for studying other ciliates encystment.(2)Hsp70 shRNA construction and the identification of Hsp70 gene interference effect of Pseudourostyla cristataUsing pHBAd-U6-CMV carrier as the basic frame,build three kinds of expression vector of Hsp70 gene interference with RNA interference.By feeding method,we transferred strains containing expression vector into vegetative cells,and measured relative expression of Hsp70 gene,then we checked RNA interference strains silence efficiency of Hsp70 gene interference effect of Pseudourostyla cristata.The results showed that among vegetative cells after Hsp70 shRNA interference,the expression of the Hsp70 gene was knocked down,and Hsp70 gene expression was the most significant in vegetative cells Hsp70 shRNA2 interference.And the relative expression of Hsp70 gene in fifth days decreased most significantly,so we selected vegetative cells Hsp70 shRNA2 interference fifth day as the following experiment materials.It was also found that after 5 days' interference with Hsp70 shRNA2,part of the vegetative cells formed the cyst cell,while those cyst cell were weak.(3)Comparative analysis of the effect of silencing Hsp70 gene on the formation of the cyst of Pseudourostyla cristata based on the transcriptome analysisVegetative cells treated with Hsp70 shRNA2 interference for 5 days was experimental group,and Vegetative cells treated with no interference was control group.After comparative analyzing them,we obtained unigene 33,354,and differential unigene 2,001.Compared to the control group,the experimental group increased unigene 1,022 and down unigene 979.There were 20,562 unigene got noted,and the differential unigene had noted 2,081 GO entries,which compared to the control group,the experimental group raised 1,026 items and come down 1,484 items.These differential entries such as combined with DNA,the size of repair,and formation of nuclear related items increased,while protein translation,ribosomal subunits formation,and ribosomal structure related entries decreased.These genes were mapped to 267 differential pathways,and compared to the control group,the experimental group increased 185 pathway and down 254 pathway.Selecting some significant differential signaling pathway such as:JNK signaling pathway,autophagy signaling pathway and endocytic signaling pathway.We found that 12 differential genes in JNK signaling pathway,and compared with the control group,the relative genes expressions of 12 different genes in the experimental group were all raised.Changes in these differential genes leads to the enhancement of JNK signaling pathway in the experimental group;and there were 36 differential genes in autophagy signaling pathway.Compared with the control group,the experimental group relative genes expressions of 17 differential genes were up-regulated and 19 differential genes relative expressions were reduction.Changes in these differential genes caused the change of autophagy pathway in the experimental group;and there were 10 differential genes in endocytic signaling pathway.Compared with the control group,the experimental group relative genes expressions of 3 differential genes were up-regulated and 7 differential genes relative expressions were reduction.Changes of these differential genes lead to changes in endocytosis signaling pathway in the experimental group.(4)Comparative transcriptome to analysis the differences between the vegetative cells after Hsp70 shRNA2 interference and cyst cells after Hsp70 shRNA2 interferenceFor the further research on the effect of Hsp70 gene on Pseudourostyla cristata cyst formation,we use comparative transcriptome to analysis the differences between the vegetative cells after Hsp70 shRNA2 interference which used as the experimental group and cyst cells after Hsp70 shRNA2 interference which used as the control group.Thus,we found unigene 35,068,and got differential unigene 1,730.Compared to the control group,the experimental group increased unigene 1,188 and down unigene 542.There were 22,374 unigene got noted,and the differential unigene had noted to 1,665 GO entries,which compared to the control group,the experimental group raised 1,302 items and come down 624 items.These differential entries such as protein translation,plasmodesmata,and other related items were suppressed,whereas the entries associated with chromosome condensation,transcription,and ribosome structure were enhanced.These genes were mapped to 260 differential pathways,and compared to the control group,the experimental group increased 241 pathway and down 165 pathway.Selecting some significant differential signaling pathway such as:P53 signaling pathway,protein degradation and digestion signaling pathway and NF-kappa B signaling pathway.We found that 8 differential genes in P53 signaling pathway,and compared with the control group,the relative genes expressions of 8 different genes in the experimental group were all raised.Changes in these differential genes leads to the enhancement of P53 signaling pathway in the experimental group;and there were 4 differential genes in protein degradation and digestion signaling pathway.Compared with the control group,the experimental group relative genes expressions of 1 differential genes were up-regulated and 3 differential genes relative expressions were reduction.Changes in these differential genes caused the change of protein degradation and digestion pathway in the experimental group;and there were 4 differential genes in NF-kappa B signaling pathway.Compared with the control group,the experimental group relative genes expressions of 4 differential genes were all up-regulated.Changes of these differential genes lead to enhancement in NF-kappa B signaling pathway in the experimental group.Those related genes and pathways may affect the viability of the cyst cell of Pseudourostyla cristata after Hsp70 shRNA2 interference.Using Q-PCR to verify the transcriptome sequencing data,the results showed a high degree of consistencyThis study from comparative transcriptome is to explore Pseudourostyla cristata multiple signaling pathways and related genes related to dormancy,and to provide important information to reveal the mechanism of the formation of cysts.The results can also clarify the interaction between eukaryotic cell and environment and reveal the mechanism of stress resistance in eukaryotic cells. |
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