Cloning And Expression Analysis Of Peroxidase-related McAPX2 And McPrx And Their Promoters In Momordica Charantia.L

The Bitter gourd(Momordica charantia L.)is a cultivar in the Cucurbitaceae momordica,it was belong to a climbing annual herb,and widely distributed in tropical and subtropical regions.The sustain low temperature in the winter and springr in South China was a major limiting factor affecting the growth,development,yield and quality of Momordica charantia in facility greenhouse.External cold stress could affect balance between production and elimination mechanism of the cellular reactive oxygen species(ROS,Reactive Oxygen Species),resulted in the accumulation of reactive oxygen,and caused oxidative stress to limit plant growth,and even death.The genes related activity oxygen and peroxide were obtained through filter gene library of the Momordica charantia,studding on their regulation mechanism and function could further clear molecular mechanism in stress cold,therefore,this research obtained McAPX2 and McPrx from cDNA library and carried out-works in follow:1.a full APX EST sequences was cloned from cDNA librarys of the Momordica charantia leave by RACE,which named McPrx(Fen bank ID KJ722767.1),bioinformatics analyses shown that McAPX2 length was 1086 bp,open reading frames(ORF,open reading frame)for the 747 bp,encoding 249 amino acids.Analysis online to conservative reign found that the protein encoded by this gene belongs to the POD super-family,which hadh peroxidase activity and heme ligand-binding domain same as conserved domain other plant APX,it was confirmed that McAPX2 as a member of the POD super-family of the Momordica charantia.2.Using a genome walking method,the1267 bp promoter sequences of McAPX2 were obtained and named for pMcAPX2,the analysis results using PLANTCARE software showed that promoter have general features of the cis-acting element,it could combines with RNA or interacts as TATA-box and CAAT-box,they contained also elements that combination with bio stress factors as GA,ABA,CTK,ethylene and pathogens et el,and abiotic stresses as hot stress,and low temperature,and drought,and light.3.The qRT-PCR results indicated that McAPX2 was expressed highly in root,stem and young fruit,which was highest in root and lowest in leave,suggesting the transcript level of McAPX2 was tissue-specific.The mRNA level of McAPX2 was significantly increased until 12 h after cold treatment,The data showed that the transcript level of McAPX2 could be induced by cold treatment,and the induce intensity was closely related to the time course.4.a full Prx EST sequences was cloned from cDNA librarys of the Momordica charantia leave by RACE,which named McPrx(Fen bank ID KJ722767.1),bioinformatics analyses shown that McPrx length was 1068 bp,open reading frames(ORF,open reading frame)for the 972 bp,encoding 324 amino acids,this gene belongs to ClassIII family.5.The qRT-PCR results indicated that McPrx was expressed highly in stem,root,female flowers and so on,which was highest in stem and lowest in female flowers,suggesting the transcript level of McPrx was tissue-specific.The mRNA level of McPrX was significantly increased until 3 h after cold treatment,The data showed that McPrx response to cold treatment.6.Using a genome walking method,the 1237 bp promoter sequences of McPrx were obtained,the analysis results using PLANTCARE software showed that promoter have general features of the cis-acting element,such as CAAT-box and TATA-box,they contained also elements that combination with bio stress factors as GA,ABA,ethylene and pathogens et el,and abiotic stresses as drought,and hot stress,and low temperature,and dark.