Investigation On Major Latex Protein423 (MdMLP423)from Apple In Response To Abiotic Stress

As one of economic crops,apple is cultivated widely in China and often suffers from abiotic stresses such as drought,high salt and low temperature,which greatly affects growth and yields.So,using modern biotechnology to study the mechanism of stress resistance and identification of novel genes related to stresses is quite important.In the previous study of apple transcriptome in this laboratory,a gene was found significantly up-regulated under abiotic stress,which encodes a kind of major latex protein?MdMLP423?.In this investigation,recombinant technique was used to study the function of anti-stresses and the crucial amino acid residues of MdMLP423 in E.coli.The anti-stresses role of MdMLP423 was also studied in plants by transgenic procedure in Arabidopsis thaliana.The main results obtained are as follows:?1?Bioinformatics analysis of MdMLP423.In the previous study of the apple transcriptome in this laboratory,the expression of one gene was found to up-regulated about 7times under salt stress.By blasting in Gen Bank of NCBI,the registration number of this gene is LOC103448744,which encoded a protein of major latex protein?MdMLP423?with 152amino acid residues and was located on chromosome 11 of apple.The MdMLP423 was a water-soluble cytoplasmic protein with no signal peptide and transmembrane domain by analyzing of SignaIP and TMHMM softwares.A long alpha helix??3?at carboxyl end is surrounded by senven of?sheets,forming a helix-grip fold,based on the three dimensional structural model of Arabidopsis MLP?AtMLP28?.In the spatial of Md MLP423,there is a hydrophobic groove composed of random loop,which may play its biological functions by binding with ligands.The whole structure of MdMLP423 is quite similar to that of other MLP members.?2?Recombinant expression of MdMLP423 enhanced the ability of prokaryotic cells to resist stress.The encoding gene of MdMLP423 was cloned and constructed onto the expression plasmid of pET30,then transformed into E.coli cell,transforming empty plasmid was as the control.On the solid medium containing NaCl,mannitol or KCl,the colony number of E.coli cells with recombinant MdMLP423 was significantly more than that of the control group in solid medium.The growth rate of E.coli with MdMLP423 was also much higher than that of the control in liquid medium containing salt or mannitol,much earlier to logarithmic growth period than that of the control.The results indicated that MdMLP423significantly improved the ability of anti-stresses in the prokaryotic cells.?3?Gly-rich loop was closely related to anti-stress function of MdMLP423.In the three dimensional structure of MLP members,a hydrophobic groove,formed by the linker between the first?helix and the second?sheet,contained a conservative Gly-rich loop,Gly-xxxxx-Gly,which is predicted to have functions in MLPs.There are 4 Gly residues at the corresponding sequence of MdMLP423.Each of four Gly was muted to Ala by using site-directed mutagenesis to investigate the role of Gly-rich loop.Compared to the wild type of MdMLP423,mutations on the conservative positions,MdMLP423_G47A and MdMLP423_G53A led to MdMLP423 almost lost the anti-stress function,the colony numbers being similar to that with empty plasmid on the stress medium.However,when mutations were on the non-conservative positions(MdMLP423_G46A and MdMLP423_G48A),little difference with MdMLP423 was observed.The results indicated that Gly47 and Gly53 were essential for anti-stress function of MdMLP423.?4?Expression pattern of MdMLP423 was analyzed.The encoding sequence of MdMLP423 was fused with GFP in the pROKII plasmid and transformed into tobacco leaves.MdMLP423 was located in cytoplasm and nucleus by checking the green fluorescence signal.The signal GUS directed by the promoter of MdMLP423 was mainly existed in leaves,roots and fast growing tissues.Deep staining was observed after stress treatments,indicating that MdMLP423 was responded to stresses.The method of qRT-PCR was also used to check the expression of MdMLP423 under different stresses and the results were consistent to GUS staining,indicating that the expression of MdMLP423 was induced by abiotic stresses and exogenous ABA.?5?Overexpression of MdMLP423 increased the resistance of Arabidopsis.Homozygous lines of overexpression of MdMLP423 in Arabidopsis thaliana were obtained by trangenic procedure.Compared with wild-type?WT?,overexpressing MdMLP423 had higer germination rate and longer root length,which increased significantly resistance to different stresses including NaCl,mannitol and exogenous ABA,.No significant differences of anti-stress between MdMLP423 overexpressing lines and the WT lines of 4-week plants were observed.Seed germination and root length experiments showed that MdMLP423 could enhance the stress tolerance of seedlings.?6?MdMLP423 enhanced the stress tolerance by ABA-mediated signaling pathway.In order to explore the mechanism of MdMLP423 stress resistance,the expression level of key genes in ABA signal transduction pathway by qRT-PCR method.Under non stress conditions,the expression of these genes in WT and overexpressed strains was basically the same.However,the expression of the key genes,KINI,AnnAt1 and ROS scavenging genes SOD,CAT,was obviously higher in the MdMLP423 overexpression line than that of WT under NaCl treatment,while the ROS contents was much lower than that of WT.The results suggested that MdMLP423 may enhance stress tolerance and reactive oxygen species scavenging capacity in plants related to ABA signaling pathway.