Cloning, Characteristics Of Study And Sequence Analysis Of Sucrose And Phosphatidylcholine Transporter Protein Genes In Pear

The sugar of Pear,in the main form of sucrose and sorbitol, enters into the fruit by phloem to be stored and used. The mode of transport as well as its distribution not only plays a role in coordinating the overall growth and development of pear, but also determines the yield and quality of the economic parts of the crop. In the directional source to sink transfer process, sucrose transporter (SUT) plays an important regulating role. Phosphatidylcholine is one of the important components of the biofilm which plays an important role in stabilizing the cell membrane structure. It is transported mainly by phosphatidylcholine transporter protein (PCTP) which has the START domain,participates in intercellular lipid transport, metabolism and cell signaling,and plays an important role in development regulation process and adversity response of higher plants.The understanding and research of the function of these two genes is very limited at home and abroad. SUT2as a membrane protein plays an important part in the Sucrose transport process. PCTP is studied more in yeast and animals, but little in plants. PCTP takes charge of lipid transport, with important influence in the sucrose transport process and the stability of the membrane. On the basis of on the cloning, bioinformatics analysis, expression analysis, subcellular localization of these two genes, this study investigate the function of these two genes and the role they played in the process of sugar and lipid transport in pear. It would lay the theoretical foundation for the promotion of the accumulation and transship of the fruit sugar, and improve the quality of pear fruit. Main results of this study are as follows:1. Take the pear (Pyrusp yrifolia NaKai.’Cuiguan’) as research material. Based on the sequences generated from its transcriptome,primers were designed to in crease SUT2.It was searched by using BLAST against the sequences in GeneBank and found to be81%sequence homologous to strawberry SUT2, belonging to the sucrose-proton co-transporter family. The protein has11transmembrane domains and is designated as PySUT2. Hydrophobicity proteins of PySUT2indicated that it contains higher proportion of hydrophobic amino acids, a requirement for transmembrane domain formation. The analysis of real-time fluorescent quantitative PCR indicated that the gene was expressed all over the plant, with bud tissue having the highest level of expression. The analysis of its expression during the fruit development showed that its expression increases at the end of April, and then decreases to the lowest level at the end of July. Subcellular localization studies with pCXDG vector as probe demonstrated that PySUT2is located on cell membranes. SDS-PAGE analysis detected an induced protein of about70kD in size. Further analysis indicated that the gene may not be directly involved in sucrose transportation, and may function as sucrose sensor on the cytoplasm membrane.2.According to the results of transcriptome sequencing, combined with RACE and Genome walking, the open reading frame of a phosphatidylcholine transfer protein gene (named as PyPCTP1) was cloned from pear (Pryus pyrifolia NaKai.’Housui’). BlAST analysis showed that it was69%identical to the phosphatidylcholine transfer protein in Ricinus communis. START, ZnF TAZ and DCD conserved domains were found in PyPCTP1. Genes encoding amino acid of PyPCTPl bioinformatics analysis found that this protein is a hydrophilic protein, and has a transmembrane region. Then a plant expression vector was constructed by injecting PyPCTP1into a vector containing GFP open reading frame. Onions epidermal subcellular localization showed that PyPCTP1is located on the cell membrane. We compared the PyPCTP1gene expression pattern after IAA, ABA, SA, GA,6-BA and NaCl treatment,and found that compared with CK,the expression level of PyPCTPl increased significantly after beinig treated with SA and GA, and increased gradually after treating with NaCl and IAA. However, PyPCTPl showed no response to ABA and6-BA treatment. It is inferred that PyPCTP1is involved the lipid transporter gene, and may be involved in the pear resistance to stress reaction.