The Full-length CDNA Sequence Cloning And Expression Analysis Of Clock Genes In Gampsocleis Gratiosa

Circadian rhythm is the most common and deeply studied biological rhythm.Several clock genes have been identified in Drosophila melanogaster,as a model organism.It has been found that the transcription-translation feedback loop composed of these genes and their expression products constitutes the core molecular mechanism of the clock.There are few studies on insect biological rhythm other than D.melanogaster.As a common singing pet,Gampsocleis gratiosa has obvious rhythm in its singing,with a peak in the morning and evening.The thorough study on the regulation of song rhythm of G.gratiosa is helpful to understand the song mechanism and has important scientific significance.Based on the data of adult transcriptome of G.gratiosa,four clock genes including period(per),timeless(tim),cryptochrome(cry),clockwork orange(cwo)were studied in three aspects.Firstly,the full-length cDNA sequences of clock genes of G.gratiosa were obtained by RT-PCR and RACE,and their sequences were analyzed by bioinformatics.Then,the real-time fluorescence quantitative PCR technique was used to study the relative expression levels of four clock genes in different tissues of adult and head at different developmental stages of G.gratiosa.Finally,after a week domestication of three photoperiod conditions(natural photoperiod,L:D=12:12 normal photoperiod and D:L=12:12 inverted photoperiod),the head and testis of male adult of G.gratiosa were dissected at 0:00,6:00,12:00 and 18:00 for quantitative detection of relative expression levels.The results are as follows:1.The complete open reading frames of G.gratiosa clock genes per,tim,cry and cwo were obtained which were named Ggper,Ggtim,Ggcry and Ggcwo,respectively.The length of ORF of four clock genes were 3576 bp,2622 bp,1941 bp and 1773 bp,encoding 1191,873,646 and 590 amino acids,respectively.The predicted molecular weights of proteins were 131.15 kDa,99.53 kDa,74.23 kDa and 65.08 kDa,respectively.The theoretical isoelectric point were 6.09,6.02,7.57 and 6.41,respectively.The sequence alignment showed that the amino acid sequences of four clock genes were highly consistent with those of other insect clock genes,all of which were more than 60%.Structural domain analysis revealed that four clock genes had conserved functional domains: GgPER(PAS,PAC,Period_C,NLS and CLD),GgTIM(TIMELESS,NLS,PER-1 and PER-2),GgCRY(DNA-photolyase and FAD-binding-7),GgCWO(HLH and ORANGE).The phylogenetic analysis were basically consistent with the results of the sequences alignment.These characteristics accorded with the characteristics of clock gene protein,which indicated that the four cloned genes belong to the clock gene of G.gratiosa.2.The temporal and spatial expression patterns of clock genes Ggper,Ggtim,Ggcry and Ggcwo in G.gratiosa were analyzed.The four clock genes expressed in all developmental stages and had significant differences(P<0.05).The expression levels in nymph stage were higher than in adult stage.These differences suggested that clock genes may play an important role in the molting process of nymph stage.The four clock genes expressed in different tissues of G.gratiosa.The expression levels of clock genes in different tissues was significantly different(P<0.05),and the expression patterns of the same gene were basically identical between male and female adults.It was speculated that these four clock genes play the same role in adults of G.gratiosa.3.Under natural photoperiod conditions,four clock genes expressed in circadian rhythm in the head of male adults.In testis,Ggper,Ggtim and Ggcry showed obvious circadian rhythm expression,while Ggcwo showed no obvious circadian rhythm.After changing the photoperiod,four clock genes showed obvious circadian rhythm expression in the head and testis of male adults,and Ggcry was significantly affected by photoperiod.In addition,under natural photoperiod,Ggper and Ggtim expressed in the same trend in the head and testis of male adults;in the head of male adults,Ggcwo showed circadian rhythm which was basically consistent with the fluctuation trend of Ggper and Ggtim,but had no circadian rhythm in the testis.Combining the sequences of four clock genes and their expression patterns,we preliminarily detected that GgPER and GgTIM could combine to form heterodimers and enter the nucleus to play a role in transcription repression;GgCRY might have dual effects of light signal transduction and transcription repression;GgCWO could synergize with GgPER and GgTIM to express rhythmically in the head and exert transcriptional repression.This study not only provided abundant data resources for exploring the song rhythm of G.gratiosa,but also laid a foundation for futher research on clock genes.