Cloning And Prokaryotic Expression Of Sucrose Transporter Gene From Prunus Persica(L.) Batsch

China is the country of origin of the peach in the world. Peach plays a veryimportant role in people’s life and the horticulture industry. In recent years, with thedevelopment of production and the improvement of people’s living standards. Varietyis richer, better quality of peach production become one very important problem ofdeciduous fruit trees in the north of China. All the fruit quality and yield largelydetermine fruit contained within the amount and type of carbohydrates.Yield andquality of peach fruit are highly dependent on the sugar levels of loading andunloading. Sucrose is assimilation substance from source to sink the main form oflong distance transport. The loading, unloading and distribution of sucrose mainlydepends on the membrane of sucrose transporters. At the moment, about whetherthere is a functional sucrose transporters in peach mediating the transmembranetransport of sugar is not clear. Especially, sink organs of carbohydrates from thephloem unloading and metabolic pathways and molecular mechanism is still unclear.This paper first reported a new sucrose transporters gene from peach. The geneexpression pattern was analyzed and function and of the gene.Construction ofprokaryotic expression vector, induced fusion expression.Laid the foundation forfurther research on the gene. The main result of this paper is as follows:(1)The organization of total RNA extraction. The total RNA of peach leaveswere extracted by the improved Trizol method; the total RNA of peach fruit wasextracted by the CTAB method; the total RNA of peach branches phloem, petal, sepal,pistil, stamen tissue was extracted by the SDS-phenol method. RNA with goodintegrity and high purity were extracted from all samples,28S and18S band withoutdegradation were separated clearly by gel analysis. Therefore, can satisfy therequirement of experiment.(2)Cloning and sequence analysis of sucrose transporter gene of peach fruit. Thecloned peach SUT gene contains a complete open reading frame (ORF), and itssequence length is1500bp, and the coding protein include499amino acid residues,With12transmembrane region. The central cytoplasmic loop is composed of41amino acids. The molecular weight is53.48kD and the isoelectric point is9.23,belong to the basic protein and does not include the signal peptide. The deduced amino acid sequence analysis shows: the gene contains a conservative sucrosetransporters functional domains,belong to a member of major facilitator superfamilyand GPH family sucrose/H+symporter. It has the highest amino acid sequencehomology of Malus domestica and Pyrus×bretschneideri, all for87%. Nucleotidesequence analysis revealed that the similarity with apple up to90%was the highest.Its similarity with strawberry, soybean, kiwi, cucumber were85%,76%,76%and75%respectively. Analysis of phylogenetic tree clustering, the results show that:Malus domestica and Pyrus×bretschneideri clustering on the same branch, but theyencode the amino acid sequence of evolutionary distance are not synchronized.PpSUT1and their closest genetic relatives. Therefore, predict the function of peachPpSUT1similar to apple MdSUT1function. With the evolution of the peony distancefar away, so MFS family genes encoding amino acid sequence may exist in theprocess of evolution of species diversity.(3)Space and time expression of sucrose transporters of peach. The expressioncharacteristic of PpSUT1was investigated at transcriptional level by Real-TimePCR.The results showed that PpSUT1transcripts were present not only in peachleaves and fruit, but aslo in petal, sepal, pistil, stamen, branches phloem, whereasmRNA levels were different and changed with grow period. Peach fruit developmentprocess of the change of the sucrose content and expression of sucrose transporterswere positively correlated.(4)Construction of prokaryotic expression vector and identification. The centralcytoplasmic loop of sucrose transporters cloned into the prokaryotic expression vectorpMAL-c2x. The bacterial liquid PCR identification, double enzyme identification,Sequencing identification.We determine the position of the peach SUT gene insertionSize are correct. Confirmed successfully constructed prokaryotic expression vector.(5)pMAL-c2x-PpSUT1expressed in escherichia coli. A prokaryotic expressionvector pMAL-c2x-PpSUT1was constructed according to the fragment of centralcytoplasm loop and transformed into E.coli BL21. When induced with0.3mmol·L-1IPTG, by13%SDS-polyacrylamidedel gel electrophoresis. A fusion protein was seenabout47.0kD, which laid the foundation for next step antibody preparation.