Isolation Of Ripening-related Genes From Red-fleshed Papaya Through RNA-seq

Papaya(Carica papaya L.)is one of the most important fruit crops cultivated in tropical and sub-tropical areas.Papaya is a climacteric fruit which undergoes a series of changes from the interior to the outer,such as ethylene production,cell wall and pectin degradation,as well as respiration rate,color and volatile matter changes after harvest.RNA-Seq technology is an important means of plant functional genomics research,which can obtain high-throughput transcript sequence information and expression data quickly.In this paper,papaya fruits was used as experimental materials,the transcriptome of different ripening papaya fruits was analysised,and several fruit ripening-related genes were screened.The major results obtained from the study are summarized as follow:1.Papaya flesh firmness,ethylene production,chlorophyll and carotenoid content,aroma components and content in different ripening stages were determined.In the ripening progress,the color of peel is changing from green to yellow while the color of flesh is changing from white to orange-red;the chlorophyll content in peel gradually decreased,but chlorophyll was almost undetectable in pulp;the content of carotenoids in peel and pulp both increased,the carotenoids content in peel was higher than that in pulp.The color of peel is changing from green to yellow because of fast degradation of chlorophyll and carotenoids appearance.With the ripening of the papaya fruits,pulp firmness decreased rapidly in color break stage,and ethylene production increased gradually,reaching its maximum value in half yellow stage.The aroma components of three papaya cultivars were analyzed by GC-MS with head space solid-phase micro-extractions.A total of 68compounds were detected,including 2 acids,11 ketones,5 aldehydes,6alcohols,22 esters,13 terpenoids,and 9 other compounds.There were significant differences in the relative content and types of these volatile aroma components in three papaya cultivars.2.Based on the Illumina HiSeq~TMM 2500 high-throughput sequencing platform,the transcriptomes sequencing was carried out on six samples,such as F1(the flesh of GS),F2(the flesh of CB),F3(the flesh of HY),P1(the peel of GS),P2(the peel of CB),P3(the peel of HY)in three different maturity stage(GS,green stage;CB,color breake stage;HY,half yellow stage)of‘Daqing No 10'papaya fruits.A total of 27769Unigenes were detected,including 19714 known genes and 1273 new genes.The analysis of differentially expressed genes showed that F1-vs-F2,F2-vs-F3,P1-vs-P2,and P2-vs-P3 had 2663,1739,2317,931up-regulated genes and 451,3601,3749,2767 down-regulated genes,respectively.Metabolic pathway enrichment analysis showed that the pathways of significant enrichment of differentially expressed genes were fatty acid degradation,starch and sucrose metabolism,porphyrin and chlorophyll metabolism,pentose and glucuronic acid conversion.85candidate genes related to fruit ripening and senescence were screened,including 19 cell wall metabolism-related genes,17 fruit color-related genes,18 aroma metabolism-related genes,8 ethylene-related genes,and23 transcription factors.3.An expansion gene CpEXPA2(GeneBank accession No MF662209)associated with papaya softening was cloned.The bioinformatics method was used to analysis the protein encoded by the gene.The open reading frame(ORF)of the gene was 780 bp,encoding259 amino acids,with a typical expansin conserved structure:the N-terminal is rich in 8 cysteine,the C-terminal is rich in 4 tryptophan and1 histidine domain(His-Phe-Asp,HFD).CpEXPA2 protein has high homology with amino acid sequences of mountain papaya,tomato,strawberry,and arabidopsis.The expression level of CpEXPA2 in fruit was higher than that in leaf,flower and stem.And in different ripening stages of papaya fruits,the relative expression of CpEXPA2 in peel was higher than that of pulp.With the increase of ripening and softening degree of papaya fruits,the expression level increased accordingly.