Cloning And Expressional Analysis Of Several Fruit Ripening-Related Genes In Momordica Charantia L.

Bitter gourd(Momordica charantia L.)belong to the family Momordica,is an important cucurbit vegetable widely grown in Southern China,India,Philippines,Malaysia,and some subtropical or tropical areas.As a typical climacteric fruit,bitter gourd has a very short period of postharvest.The tribute of fruit ripening of the bitter gourd negatively influences its quality and shelf-life,deprives of the value of its commodities,and brings lots of losses to farmers.According to physiological differences of three genotype of bitter gourd,subgynoecious bitter gourd line '24-K'was identified as accession in this study.A normalized full-length cDNA library was constructed with the bitter gourd fruits during breaker period,by DSN normalization method combined with SMART technique.After massive EST sequencing and function annotation analysis,seven full length cDNA of ripening and softening associated genes were cloned by 3'RACE,and the sequence characteristics were analyzed.Recombinant plasmids containing seven genes combined with GFP were used for subcellular location.Then the expression modes of the seven genes were studied during fruit ripening and softening.Furthermore,the function genes,McACS5 and McACO2,were analyzed using transient expression method,respectively.The promoters of McACS5,McACO2 and McGal were also cloned and analyzed.The results provided further knowledge to understand the molecular mechanisms during fruit ripening and softening in bitter gourd,and this would contribute to the quality genetic improvement.The main results were as follows:1 Variations of major postharvest physiological characters and correlation analysis of different genotypes in bitter gourdThree different genotypes of bitter gourd were used to investigate the effects of the postharvest on the firmness change,respiration rate,ethylene production,and ?-Gal activity during the fruit ripening.The relationship of four physiological indexes was analyzed.The result showed that the fruit firmness continuously declined,the ethylene production characterized by a typical climacteric,the respiration rate model showed firstly increased then decreased during ripening stage of three genotypes.The results indicated that the fruit ripening of bitter gourd had close relation with respiration,ethylene metabolism,firmness of fruit,?-galactosidase activity.Subgynoecious bitter gourd line '24-K' showed the firmness rapidly decreased,and the peak of ethylene-production and respiration-rate were simultaneously appeared,the character of ripening phenotype was easily distinguished.So the line was identified as material in this experiment.the process of fruit ripening of bitter gourd were divided into four stages,such as green-mature stage,breaker stage,turning stage and yellow stage.2 Construction and EST Analysis of the Normalized Full-Length cDNALibrary of Momordica charantia L.at Fruit Ripening stage A normalized full-length cDNA library was constructed at breaker stage,by DSN(duplex-specific nuclease)normalization method combined with SMART(switching mechanism at 5'endof RNA transcript)technique.The Conversion efficiency of unamplified cDNA library was 3,600,000 colonies·?L-1.The average size of inserted cDNAs was more 1.4 kb,and a recombination ratio was about 98%,772 clones were random selected and sequenced,684 high qualities EST were obtained which including 17 contings and 562 singlets.The redundancy was 7.41%.The ratio offull-length cDNAs was 67%.Annotation by the online Blas on NCBI web server revealed that most of predicted function genes were related to catalytic activity,binding,transcription regulator activity,transporter activity and Eenzyme regulator activity.The analysis result indicated that library was efficient and reliable.After further analysis,some new genes related with bitter melon fruit ripening and softening were obtained.3 Isolation of several fruit ripening-related genes by 3'RACE3.1 A full cDNA clone encoding ACS protein,named McACS5(GenBank:JN860423.1),was obtained by 3'RACE technique based on the related EST sequence from the normalized full-length cDNA library of bitter gourd fruit.The cDNA sequence of McACS5 was 1194bp in length with an ORF of 1488bp,encoding a protein of 495amino acids.The protein molecular weight and isoelectric point were predicted to be 55.46 kD and 6.04 respectively.3.2 A full cDNA clone encoding ACO protein,named McAC02(GenBank:JQ613285.1),was cloned from the normalized full-length cDNA library of bitter gourd fruit.The nucleotide sequence of the gene is 1277bp,containing a 1073bp ORF encoded 317 amino acids.The protein molecular weight and isoelectric point were predicted to be 35.85 kD and 5.27 respectively.3.3 A full cDNA clone encoding EIN3/EIL protein,named McEIL2,was obtained from the normalized full-length cDNA library of bitter gourd fruit.The nucleotide sequence of the gene is 2122bp,containing an open reading frame of 1890bp corresponding to 629 amino acids,with a 5'UTR of 174bp,and a 3'UTR of 58bp.The protein molecular weight and isoelectric point were predicted to be 71.58 kD and 5.33 respectively.3.4 The full length cDNA sequence of ethylene response factor gene named McERF1 was obtained by the related EST sequence from the normalized Full-Length cDNA Library of bitter gourd Fruit.Sequence analysis showed that the nucleotide sequence of the gene is 960bp,containing a 690bp open reading frame which encoded 230 amino acids.The protein molecular weight and isoelectric point were predicted to be 24.81 kD and 9.3 respectively.3.5 The full length cDNA sequence of S-adenosylmethionine decarboxylase(SAMDC)gene named McSAMDC(GenBank:KC632099)was cloned from the normalized Full-Length cDNA Library of bitter gourd Fruit.The cDNA sequence is 1900bp in full length with a 501 bp 5'-UTR and a 325bp 3'-UTR.The cDNA sequence consists of three open reading frames(tiny,upstream and main),and the main open reading frame is 1077bp encoding 358 amino acids with a molecular weight of 39.31 kD.3.6 A full cDNA clone encoding lipoxygenase protein,named McLOX2(GenBanK:KC733800.1),was obtained from the normalized full-length cDNA library of bitter gourd fruit.The nucleotide sequence of the gene is 2738bp with an open reading frame encompassed 2538bp encoded 845bp amino acids.3.7 The cDNA sequence of ?-galactosidase gene named McGAL(GenBank:AFD54987.1)was cloned from the normalized Full-Length cDNA Library of bitter gourdfruit.The full length of cDNA sequence was 2261 bp,including a 2187 bp open reading frame which encoded 719 amino acids.4 Bioinformatics analysis of seven fruit ripening-related genes4.1 The McACS5 was an unstable hydrophilic protein with two transmembrane helices.The secondary structure and 3D structure of the protein included a-helices and random coils mainly.Multiple sequence alignment and of phylogenetic analysis showed that the C-terminal region of the protein contained the Ser residue in the'RLSF' motif,followed by a long C-terminal tail with the three conserved Ser residues,and predicted proteins of McACS5 is belong to Typel ACS isozymes.Amino acid sequence analysis revealed that McACS5 shared relatively high identity with McACS1(61.17%),McACS2(46.99%),McACS3(61.77%),McACS4(61.37%).4.2 The McACO2 was a stable hydrophilic protein.The secondary structure included a-helices and random coils mainly.The predicted 3D structure showed that residues H177,D179 and H234 were required to bind iron in the active site.McACO2 protein and other cucurbit plants were located in the same tree by phylogenetic analysis.Amino acid sequence alignment shows that the McACO2 has higher identity with CmACO1,CsACO,CpACO1,and CmACO3,as 88.36%,87.11%,84.95%and 83.91%.4.3 McEIL2 was an unstable hydrophilic protein.The amino acid sequence contained the consensus sequence of one Acidic domain,five basic conserved domains,one aproline-rich region,one nuclear localization signal and the lysine residuewith enzyme activity.4.4 McERF1 was an unstable hydrophilic protein.Amino acid sequence alignment shows the protein contains the typical AP2/ERF domain and two NLSs,which belong to Group ?a protein in the ERF subfamily as typified by the C-terminal ERF-associated Amphiphilic Repression(EAR)motif.In the secondary structure and tertiary structure prediction indicated the AP2/ERF domain was conserved.4.5 McSAMDC has two conserved function domains(proenzyme cleavage site and rapid degradation of SAMDC protein domain).In the secondary structure,random coil,?-helix,extended strand,?-turn were 45.53%,29.33%,19.83%and 5.33%,respectively.Amino acid sequence alignment shows the McSAMDC shows higher identity with AtSAMDC,AISAMDC and BjSAMDC which as 69%,68%and 68%.4.6 The amino acids sequence of McLOX2 shared three conserved region with the other plant by NCBI BLAST.It showed that the gene had high identity with NtLOX1,TomLOXA,and StLOX1.McLOX2 gene belonged to the 9-LOXs subgroup of type I group with phylogenetic tree analyzed.4.7 Sequence analysis showed that McGal contained the putative active site-containing consensus sequence pattern G-G-P-[LIVM]-x-Q-x-E-N-E-[FY]belonging to glycosyl hydrolase family 35 without lectin domain at their C-termini.The McGal exhibited a typical structure with a membrane-spanning domain.In the secondary structure,a-helix,extended strand,?-turn and random coil were 19.89%,26.98%,6.54%and 46.59%,respectively.Amino acid sequence alignment indicated McGal had higher identity with Cicer arietimum,Medicago trumcatula,Vigna radiate,Glycina max and Lupimus angustifolius which as 73%,73%,73%,72%and 71%.5 Subcellular localization of seven fruit ripening-related genes The subcellular localization result showed that McACS5 protein localized in mitochondria,by Protoplast transformation method using Arabidopsis and tobacco transient expression way.The Subcellular localization revealed the McACO2 was located in whole cell by tobacco transient expression method.The Subcellular localization analysis showed that McEIL2 protein was located in the nucleus of epidermal cell.The Subcellular localization analysis showed that the protein showed that McERF1 protein was located in the nucleus of onion epidermal cell.Subcellular localization analysis showed McSAMDC focused mainly in the cytoplasm of onion epidermal cell.The subcelluar localization in the epidermal cells of onion showed that McLOX2 was a cytoplasm protein.The subcellular localization revealed that McGal was located in mitochondrial inner membrane of tobacco leaf cell.6 Expressional analysis of seven fruit ripening-related genes6.1 Real-time PCR results showed that McACSl and McACS5 have the highest expression at green mature stage,along with stable expression at breaker stage and turning stage which predicted that they probably involved in system ethylene I biosynthesis and system ethylene II biosynthesis.McACS3,McACS4 only had high expression at green mature stage which predicted that they probably involved in system ethylene I biosynthesis.McACS2 was regulated by fruit development.6.2 Real-time PCR results showed that McAC02 expressed from highly to lowly in the fruit development,the highest in at green mature stage,and then down regulated gradually with the lowest at turning stage.The result predicted that McACO2 was regulated by fruit development and probably involved in system ethylene I biosynthesis.6.3 Fluorescence quantitative PCR analysis showed that the gene highly expressed in the development,with the lowest level at the green mature stage and the highest at breaker stage then down-regulated gradually.It was predicted that McEIL2 may play an important role in fruit ripening and softening stage in bitter gourd.6.4 Fluorescence quantitative PCR analysis showed that the transcripts of McERF1 accumulated the most highly in at the fruit enlargement initial stage following with medium expression at the fruit enlargement middle stage,and then rapidly decreased thereafter,which indicate that McERF1 might play a repression role in ethylene biosynthesis at t fruit development stage.6.5 Fluorescent quantitative PCR analysis shows McSAMDC expressed the highest levels at fruit enlargement stage then rapidly decreased thereafter,the levels continuously increased from the green mature stage to yellow stage.McSAMDC involved into regulate ethylene I biosynthesis.6.6 Real-time quantitative PCR analysis showed that McLOX2 gene scarcely expressed at the development stage following with abundantly expressed in the green-ripening stage,and then went down slightly.The expression level of McLOX2 gradually increased during the fruit ripening,and up to peak at the yellow stage.McLOX2 might involve in system ethylene II biosynthesis and accelerate fruit ripening.6.7 Fluorescent quantitative PCR analysis displayed the McGal gene was expressed the highest levels at the mature green stage and decreased thereafter,which means that gene might be related to early ripening stage.7 Clone and expression analysis of the McACS5,McACO2 and McGal three promoters7.1 McACS5 promoter with sequence a length of 635bp was cloned by genomic walking method which has besides containing some cis-action elements involved in auxin response,MeJA responsiveness,SA responsiveness and ABA responsiveness.The activity of GUS was declined by the hormonal treatment(JA,ABA,SA and ETH),using GUS histochemical staining and quantitative analysis.7.2 McACO2 promoter with a sequence length of 558bp was cloned which has basic elements besides containing some cis-action elements involved in MeJA responsiveness,ARE,Pathogen-and NaCl-induced expression.Using GUS histochemical staining and quantitative analysis proved the activityof GUS was increased by the hormonal treatments(JA,ABA,ETH,and SA).7.3 McGal promoter with sequence length of 1124bp was isolated.Some cis-action elements involved in defense and stress responsiveness were obtained in this promoter.the GUS activity was increased by the hormonal treatments(JA,ABA,ETH,and SA)using GUS histochemical staining and quantitative analysis.Three promoters may be inducible promoters.8 Transient expression of McACS5,McAC028.1 Transient expression of McACS5 showed the expression level of McSAMDC was inhibited,but and increased the expression level of other six genes including McACS3?McACS4?McAC01?McEIL2?McLOX2,and McGAL were increased in the leaves of bitter gourd.The result showed that the five genes of ACS were Synergistic effect in system ethylene I biosynthesis.8.2 Transient expression of McAC02 could inhibit the expression of McSAMDC,McACO1,McEIL2,McERF1,McLOX2,and increased the expression of McGal.9.Deleted expression analysis of the promoter McAC02McAC02 was deleted from its 5' end to form promoter fragments with 4 different lengths.The result showed that MeJA responsive cis-acting element might locate within-558bp to-493bp,the ethylene responsive cis-acting element might locate withinThe possible cis-acting elements of the promoter responding to MeJA induction and SA induction were located between-498bp to-438bp.