| Plant ATP-binding cassette(ABC)transporter play an important role in the metabolites,excretion and transport accumulation of secondary metabolites.In addition,it plays an important role in the detoxification and resistance response of exotoxins.However,the current researches on plant ABC transporters are mostly focused on model plants.Understanding the molecular regulation mechanism of grapefruit ABC transporters to reveal the secondary metabolites of pomelo,Plant defense response mechanism and improvement of pomelo varieties are of great significance.In this study,based on the RNA-seq database of' Hong rou mi you',the bioinformatics analysis method was used to discover and classify the ABC gene in pomelo grapefruit,which was characterized by real-time fluorescence quantitative PCR,variety-specific analysis,tissue specificity analysis and glyphosate stress response analysis,and part of the genes subcellular localization analysis.The results are as follows:1.Based on the RNA-seq database of ' Hong rou mi you',50 pomelo ABC genes were segregated and their subfamilies were classified,2 ABCA genes,10 ABCB genes,9 ABCC genes,2 ABCD genes,22 ABCG genes,1 ABCE gene,3 ABCF genes and 1 ABCI gene were obtained.In this study,we mainly studied the ABCB,ABCC and ABCG subfamily genes that are currently more studied in the plant kingdom,that is,41 pomelo ABC genes.Build a phylogenetic tree and name them.2.The bioinformatics analysis of the predicted protein of 41 CmABCs in pomelo was carried out.The results showed that the length coding region of CmABCs of 1821-5553 bp encoding a polypeptide of 606-1923 amino acids with an estimated molecular mass of 64.32513?213.34760 kDa.The theoretical isoelectric point of which were 5.72-9.40.There were 13 unstable proteins and 28 stable proteins.There were 4 hydrophilic proteins and 37 hydrophobic proteins.The other 40 CmABCs were non-secreted proteins except for CmABCG24.CmABCs With 4?17 transmembrane structures;CmABCB18 located in the cell membrane and cytoplasm,CmABCG29 located in the cell membrane and chloroplast,CmABCC2 and CmABCC12 located in the vacuole,the remaining 37 CmABCs were localized in the cell membrane;41 CmABCs have different kinase potential Phosphorylation site.3.41 CmABCs were analyzed by fluorescence quantitative PCR before maturation of'Bai rou mi you'?'Hong mian mi you'?'Hong rou mi you' and 'San hong mi you',and 5 genes that were obviously increased in all four varieties were screened out:CmABCB18,CmABCCIO,CmABCG2,CmABCG14-2,CmABCG31,suggesting that they may participate in the accumulation of secondary metabolites,excretion and transport.The characteristics of these five genes were analyzed by fluorescence quantitative PCR after the middle and late columnar cells of 143?213 days after 'Hong rou mi you' flower were found.There was no significant change in the relative expression of CmABC10 and CmABCG31.The expression of CmABCB18 The mesangial column in 143?173 days distal columnar cells in the expression was significantly higher than the mesial column,the rest of the period was no difference;CmABCG2,CmABCG14-2 in mesanglial cells in the relative expression of the overall upward trend,However,CmABCG2 was faster in the mesial columnar cells than in the columnar cells,and there was no difference in CmABCG14-2 between the mesangial columnar cells.The subcellular localization of CmABCB18,CmABCG2 and CmABCG14-2 showed that they were all located in the cell membrane,suggesting that CmABCB18 may be involved in carotenoid transport,CmABCG2 involved in lignin synthesis,and CmABCG14-2 may be involved in the growth and development of pomelo fruit.4.The tissue-specific and variety-specific analysis of CmABCB18,CmABCG2 and CmABCG14-2 was carried out using'Bai rou mi you','Hong mian mi you','Hong rou mi you' and 'San hong mi you' fruit albedos,segment membranes,juice sac handles and juice sacs as samples.The expression level of CmABCB18 was higher than that of 'Bai rou mi you' in the segment membranes,juice sac handles and juice sacs of 'Hong mian mi you','Hong rou mi you' and 'San hong mi you' after 95-165 d from flowering.The expression levels of CmABCG2 were low and almost unchanged in the four parts of the four varieties 95-135 d after flowering,and they were different in different varieties and different parts on 165 d.CmABCG14-2 was expressed in the sponge layers of four varieties.The amount was significantly higher than the rest.5.22 CmABCGs were classified,of which 13 belonged to PDR type.The nomenclature and bioinformatics analysis of 11 pomelo PDR genes were carried out.Homology analysis showed that the amino acid sequence encoded by CmPDRll-2 has the highest homology(69.25%)with the AtPDRll gene that transports paraquat herbicides in Arabidopsis,The ORF of PDR gene was named as CmPDR11-2,which was a full-length of 4371 bp encoding a 1456 amino acids long,the molecular weight of 165.13803kDa,the protein is a stable hydrophilic protein,no signal peptide,with 12 transmembrane structure located in the cell membrane was cloned by using RT-PCR technique.The results of real-time fluorescence quantitative PCR analysis showed that under the condition of glyphosate herbicide stress,the relative expression of CmPDR11-2 in the leaves treated with glyphosate for 1-15 days showed an overall upward trend,both of which were higher than control,and the reaction was rapid and durable.This indicated that the expression of CmPDR11-2 gene was related to 'Hong rou mi you'glyphosate tolerance... |
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