| Onchidium reevesii is a brackish water amphibian living in tidal flat wetlands.It belongs to Mollusca,Gastropoda,Pulmonata,and Systellommatophora.O.reevesii is a shellfish with high edible and medicinal value.Its living distribution area is common in the high tide zone at the junction of freshwater and seawater in Jiangsu,Shanghai,Zhejiang,Fujian,Guangdong and other places in China.Its temperature adaptability is very strong,its distribution range is wide,and its wild resources are very rich.Observing from the morphological point of view,O.reevesii has not evolved a complete cardiovascular system.There is only a small amount of blood sinuses in the skin tissue,which makes it incapable of obtaining oxygen through skin respiration."Lung sac" is the main organ that the body uses to obtain oxygen.In terms of behavior,O.reevesii also exhibits a long-term and accurate "homecoming behavior".It lives in nests,caves and rock cracks during high tide,and comes out to absorb humus and food such as algae,O.reevesii from different nests rose again at an hour before being able to go home at the same time.It is speculated that it may be due to its inherent position memory ability,which reflects the high accuracy and complexity of this process.The homing behavior is influenced by factors such as the zeitgeber of the external environment(light intensity,sea breeze size,humidity change,and tidal sound waves,etc.)or the precise operation of the rhythmic molecular mechanism in living organisms.The purpose of this study was to construct the first full-length transcriptome data of O.reevesii,and to screen the biorhythmic and environmentally sensitive genes associated with the homing behavior of O.reevesii by RNAseq technology.O.reevesii high-throughput sequencing and biological tidal rhythm studies provide a reference.The main research contents are as follows: 1.Sequencing of full-length transcripts and RNAseq from O.reevesii nerve ring tissueUsing RNAseq Next-generation transcriptome data to correct and de-reduce Pac Bio third-generation sequencing results,the full-length transcriptome data obtained was 346,560,411 bp in size,with a total of 144,372 isoform,and an open reading frame of 43,283 sequences was predicted reading frame(ORF),and analysis predicts that 3,665 translated proteins are belong to secreted protein,with an average sequence length of 2,400 bp,the longest sequence can reach 12,779 bp,the shortest sequence is only 367 bp,the median length of the sequence and N50 lengths are 2,322 bp and 2,719 bp,respectively.The annotated results of the full-length transcript sequence showed that the number of isoforms that could not be annotated was as high as 89,446,accounting for 61.96% of all isoforms.In the results annotated in 5 databases,the predominant number of isoforms was involved in cellular processes,metabolic processes,and signal transduction pathways.NGS sequencing technology is used to select O.reevesii at different tidal time periods as the research object to construct a sequencing library,and the obtained full-length isoform data is used as the reference transcriptome for Map analysis.According to the differential expression analysis,a comparison of transcripts in ebb tide(A)and low tide(B)showed that there were significant differences in 3,986 transcripts,of which 2,145 were up-regulated and 1,841 were down-regulated.Comparing the transcripts in ebb tide(A)and flood tide(C),a total of 3,538 transcripts were significantly different,of which 1,114 transcripts were up-regulated and 2,424 were down-regulated.Comparing the transcripts of low tide(B)and flood tide(C),there were 4,261 transcripts significantly different,of which 1,230 were up-regulated transcripts and 3,031 were down-regulated transcripts.Enrichment analysis of differential genes shows that the number of differentially expressed transcript genes enriched in pathways related to signal transduction is dominant.As the center for the analysis of external environmental signals by the treatment of O.reevesii,transcript expression differences in signal transduction pathways are also in line with general cognition.2.Functional analysis of rhythmic genes in tide perceptionThe rhythmic genes Period and Timeless are genes that are periodically oscillated,and the oscillation period is about 24 hours,and the daily tidal rhythm is about 24.8 hours(two tides),which is also close to 24 hours.The expression of rhythmic genes at different tidal moments was analyzed using real-time PCR.The full isoform sequences of Or TIM1 and Or TIM2 were cloned from the O.reevesii nerve ring tissue.The full-length sequences of Or TIM1 and Or TIM2 genes are 2,781 bp and 4,995 bp.Or TIM1 sequence analysis showed that the length of the ORF box was 1,431 bp,encoding 476 amino acid residues to form a polypeptide chain,and the relative molecular mass of the polypeptide chain encoded by the transcript was 53.426 KDa.The theoretical acid-base equilibrium isoelectricity of the polypeptide chain Point is 4.70,and the highest amino acid in the amino acid chain was Leucine(Leu,11.6%)and serine(Ser,11.6%).Or TIM2 sequence analysis showed that the length of the ORF box was 1,662 bp,encoding 554 amino acid residues to form a polypeptide chain,and the relative molecular mass of the polypeptide chain encoded by the transcript was 62.888 KDa.The theoretical acid-base equilibrium isoelectricity of the polypeptide chain Point is 5.14,and the highest amino acid in the amino acid chain was leucine(Leu,12.3%).In the advanced structure prediction,the TIMless superfamily domain exists in the amino acid sequences of Or TIM1 and Or TIM2,and it the evolutionary relationship is closer to the Biomphalaria glabrata and the Aplysia californica.Analysis of Or TIM1,Or TIM2,and Or PER2 genes at different tidal moments in the O.reevesii ring nerve tissue shows that the relative expression changes of the three genes are roughly similar.During the two tidal fluctuations within 24 hours,there were both a time period during which the expression level rose and a time when the expression level was relatively stable,and the time when the relative expression level was stable was mostly at the time when the sampling time was daylight.Among them,it is relatively obvious that the expression levels of the three genes Or TIM1,Or TIM2 and Or PER2 at night are more than those of daylight.It is speculated that the oscillation expression of the three selected rhythmic genes is more closely related to the circadian rhythm.3.Expression analysis of hypoxia-induced pathway during tidal processWhen the tumbling O.reevesii comes out of the flooded state,the oxygen content in the cells of the organism should be relatively low.In this section,the isoform sequences of two genes,Or HIF1a and ORFIH1,have been successfully cloned from the neural ring tissues of O.reevesii.Bioinformatics analysis revealed that the full-length transcript sequences of Or HIF1a and Or FIH1 genes were 4,980 bp and 2,988 bp.Or HIF1a sequence analysis showed that the length of the ORF box was 1,827 bp,encoding 609 amino acid residues to form a polypeptide chain,and the relative molecular mass of the polypeptide chain encoded by the transcript was 67.651 KDa.The theoretical acid-base equilibrium isoelectricity of the polypeptide chain Point is 5.97.For leucine(Leu,9.5%)and proline(Pro,8.7%).Or FIH1 sequence analysis showed that the length of the ORF box was 471 bp,encoding 157 amino acid residues to form a polypeptide chain,and the relative molecular mass of the polypeptide chain encoded by the transcript was 17.813 KDa.The theoretical acid-base equilibrium isoelectricity of the polypeptide chain Point is 8.74.Lysine(Lys,10.2%).In the advanced structure prediction,a typical PAS domain was found in the amino acid chain of the Or HIF1a gene translation.The phylogenetic tree graphical results show that the genetic relationship between the two genes is very close to the Biomphalaria glabrata.The expression trend of Or HIF1a gene was roughly similar in the 14 tidal fluctuation cycles.Generally,the expression level was relatively higher during ebb tide than during low tide and flood tide.Corresponding to this,Or FIH1 gene expression trend is the lowest expression level at ebb tide.However,the sampling rule at 21:20 on May 20 th showed abnormalities,and both genes were highly expressed during this time period.Although it is assumed that the O.reevesii can return home in time and the dissolved oxygen status in the body is still insufficient,there is still insufficient data to support it,but it can provide basic data for future discussion of the adaptive evolution of the O.reevesii in the intertidal flats.5.Expression analysis of low-frequency sensory genes under different sound wave frequenciesThis section mainly focuses on whether low-frequency sensing genes play a role in sensing low-frequency sound waves.The full-length isoform sequences of Or PLC and Or CALMβ genes cloned were 2,578 bp and 1,903 bp.Or PLC sequence analysis showed that the length of the ORF box was 1,881 bp,encoding 627 amino acid residues to form a polypeptide chain,and the relative molecular mass of the polypeptide chain encoded by the transcript was 71.473 KDa.The theoretical acid-base equilibrium isoelectricity of the polypeptide chain Point is 6.52,and the highest amino acid in the amino acid chain was Serine(Ser,9.4%)and leucine(Leu,9.1%).Or CALMβ sequence analysis showed that the ORF box length was 501 bp,encoding 167 amino acid residues to form a polypeptide chain,and the relative molecular mass of the polypeptide chain encoded by the transcript was 18.972 KDa.The theoretical acid-base equilibrium isoelectricity of the polypeptide chain Point is 4.59.Glutamic acid(Glu,12%)and aspartic acid(Asp,10.2%).In the advanced structure prediction,the Or PLC protein chain has the typical PH and EF hand domains of phospholipase C,while the FRQ domain exists in Or CALMβ.The evolutionary relationship between the two genes is closer to Pomacea canaliculata.From the quantitative analysis of phospholipase C,it can be seen that by comparing with the control group,it can be seen that when the stimulating sound wave frequency is 10 Hz to 70 HZ,the expression level of the Or PLC gene is basically stable,and when the frequency is increased to 100 Hz-160 Hz,it can be seen that the activity of Or PLC gene expression is reduced.When the sonic frequency reaches 220 Hz and 280 Hz,the Or PLC gene is highly expressed.Or CALMβ expression analysis chart shows that the expression of Or CALMβ is different during the 24-hour sampling time of the control group.When the acoustic stimulation frequency is 40 Hz-70 Hz,the expression trend of Or CALMβ is very stable.Similar to the Or PLC gene,the expression of Or CALMβ showed a downward trend at 100Hz-160 Hz.When the frequency of sonic stimulation increased to 220 Hz,the expression of Or CALMβ had a sudden increase stage at 0.5 h.Quantitative analysis results support that the O.reevesii can perceive sound wave stimuli in the air,and provide basic data for further confirmation of the adaptive evolution of intertidal organisms such as the O.reevesii. |
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