Rhythmic Expression Analysis Of Genes Involved In Circadian Clock Regulation In Red Dragon Fruit(Hylocereus Polyrhizus) By Full-length Transcriptome Sequencing

The circadian clock is an internal invisible“clock”developed by the organism to adapt to the periodic rhythm changes of the living environment.It is closely related to the physiological cycle,metabolic activity,growth and development of organisms.In plants,the plant’s external appearance characteristics adapt to changes in environmental factors through the regulation of gene expression and physiological and biochemical levels,such as flowering and fruiting,stomata opening and closing,hormone response,nutrient absorption and growth rate.At present,researches about plant circadian clocks have been mainly concentrated in model plants such as Arabidopsis,rice,and wheat.They are all model plants of C₃ or C₄photosynthetic metabolism.The plants of crassulacean acid metabolism pathway（CAM）are contrary to these model plants in the regulation of photosynthetic activity and the rhythm of stomata opening and closing,but there are relatively few studies on the biological clock system of CAM plants.In this study,we use the typical CAM plant red pitaya（Hylocereus polyrhizus）as research object to provide important guiding significance for the future research of CAM plant circadian clock system and understand the molecular mechanism of pitaya circadian clock regulation,through exploring the rhythmic expression of genes related to the circadian clock.Since the genome of the red pitaya has not been reported,this study used the third-generation sequencing technology to obtain the full-length transcriptome of the red pitaya,and explored the rhythmicity of the biological clock-related genes.The main research results are as follows:1、Through the use of Pac Bio Sequel sequencing platform single molecule real-time（SMRT）sequencing technology to obtain the red pitaya full-length transcriptome sequence information.The subreads data size is 7.66 G,the total number of sequences is 4,552,474,the average length is 1683 bp,and the N50 length is 2446 bp.The number of CCS sequences was 355,575,and 260,718 FLNC sequences were identified,with an average length of 2398 bp.The number of identical sequences obtained by clustering FLNC reads with similar sequences is 132,836.124,466 SSRs were searched out from 4,552,474unigenes,which were distributed on 79,169 unigenes.2、By sequencing the full-length transcriptome of red pitaya,we obtained the transcript sequence information of the circadian clock-related genes,and based on the gene homology,the BLAST alignment method was used to determine the sequence information of the candidate genes,the selected circadian clock-related genes are as follows:core oscillator genes:CCA1（CIRCADIAN CLOCK ASSOCIATED1）,ELF3（EARLY FLOWERING 3）,GI（GIGANTEA）,LHY（LATE ELONGATED HYPOCOTYL）,LNK1（NIGHT LIGHT-INDUCIBLE AND CLOCK-REGULATED 1）,and TOC1（TIMING OF CAB EXPRESSION 1）;The input pathway:CRY1（CRYPTOCHROME）,ZTL（ZEITLUPE）,PHYA（PHYTOCHROME A）,PHYC（PHYTOCHROME C）,PIF3（PHYTOCHROME INTERACTING FACTORS）;The output pathway:CO（CONSTANS）;And genes related to the CAM pathway:MDH（MALATE DEHYDROGENASE）,ME（MALIC ENZYMES）,PPDK（PYRUVATE PHOSPHATE DIKINASE）and PPCK1（PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE1）.3、The circadian expression patterns of these genes in red pitaya were quantitatively explored by fluorescence in real time,and they were divided into three categories:Hp CCA1,Hp ELF3,Hp LHY,Hp LNK1,Hp GI,Hp PHYA,Hp PHYC,and Hp PIF3,which have expression peaks during the day.Hp TOC1,Hp CO and Hp CRY1 have the highest expression levels at night.The genes Hp MDH,Hp ME1,Hp PPDK and the blue light receptor Hp ZTL involved in the CAM pathway have expression peaks during the day and night.The expression of Hp CCA1,Hp LHY and Hp TOC1 is consistent with the feedback mechanism of arabidopsis.The peak expression of Hp LNK1 appeared at noon,which was different from the time when the peak expression of LNK1 in Arabidopsis thaliana appeared.It may be that Hp LNK1 is involved in regulating the expression of pitaya afternoon genes.The expression of CRY1 can inhibit the degradation of CO protein.The expression curves of Hp CRY1 and Hp CO show the same change trend,and the expression peaks appear at night,but the expression peaks of arabidopsis and soybeans are during the day.This may be related to the flowering characteristics of pitaya.Hp ZTL inhibits the expression of Hp TOC1 under dark conditions,and Hp ELF3 inhibits the expression of Hp GI.Such expression characteristics can be reflected in the gene circadian transcription curve.In addition,the CAM pathway related genes Hp MDH,Hp ME1 and Hp PPDK all showed double peak expression,and the peak during the day was higher than the peak at night,but no related reports were found to describe the double-peak expression pattern of MDH,ME1 and PPDK genes.In summary,this study obtained the full-length transcriptome information of the red pitaya,and designed primers based on the gene homology to amplify the circadian clock-related genes since there is no reference genome,and quantitatively explored the rhythm of the red pitaya’s circadian clock-related information by q PCR.It provides some theoretical references for the future research of CAM plant circadian clock regulation mechanism.