Bioinformatics And Functional Analysis Of Mn-CDF Subfamily Of MTP Protein Family In Pear

Pear tree is an important crop which is widely grown around the world.However,environmental pollution,the lack of mineral elements and heavy metal poisoning,which causes the decline of tree vigor and the decrease in fruit yield and quality,become important factors affecting the quality and yield of pear.Manganese(Mn)is an essential trace element for plant metabolism and development,but excessive Mn is toxic to plant and blocks plant growth.Plant metal tolerance proteins(MTPs)play significant roles in heavy metal homeostasis and tolerance.As a part of the MTP family,the Mn-CDF subfamily plays an important role in improving the plant’s tolerance to Mn toxicity.However,the systematic analysis of the Mn-CDF subfamily in the pear genome(Pyrus bretschneideri Rehd.)has not been reported yet.The pear genome(Dangshan pear)was successfully sequenced in 2012.Based on this study,a comprehensive bioinformatics analysis of the Mn-CDF subfamily was performed in the pear genome.The function of pear Mn-CDF genes were initially investigated through yeast functional complementation assays.In addition,the transcript level of the Mn-CDF genes that have been proven to be resistant to high manganese toxicity was analyzed in different tissues of pear seedlings.The main results are as follows:(1)First,the multiple sequence alignments of the two conserved domains(Cation_efflux;ZT dimer)of Mn-CDF proteins were searched in Pfam 31.0 program online,and then they were used for establishing the hidden Markov model(HMM)profiles.Then,referring to the two HMM profiles,HMER 3.1 tool was used to search the pear protein database for candidate proteins.A total of 11 candidates were found to meet both of the above conditions.Combined with the characteristic sequence D***D(*stands for any amino acid)contained in the Mn-CDF protein,the same protein and proteins lacking in the characteristic sequence in the pear protein database were removed.Finally,7 Mn-CDF proteins were identified in the pear genome.According to the similarity in protein sequences and the nomenclature of Arabidopsis and rice,Mn-CDF genes in the pear genome were named as follows:PbMTP8.1,PbMTP8.2,PbMTP9,PbMTP10,PbMTP11.1,PbMTP11.2,PbMTP11.3,respectively.(2)Sequence analysis of the Mn-CDF protein of pear showed that the length of the protein sequences encoded by the Mn-CDF genes was 394-416 amino acids.All of these proteins contained 6 transmembrane domains(TMDs)and were predicted to be localized in plant cell endomembrane systems such as tonoplast or Golgi membrane.By analyzing these protein sequences,we found that the signature motif D***D(*represents any amino acid)exists in both TMD-Ⅱ and TMD-Ⅴ.Compared with the corresponding protein sequences of Arabidopsis,the Mn-CDF members of pear show 61%-85%identity and 72%-91%similarity in sequences.Multiple sequence alignments of Mn-CDF protein from Arabidopsis,rice and pear were performed,and then the phylogenetic tree was established by the neighborjoining method using MEGA 7.0.The Mn-CDF genes of pear were divided into two groups based on the classification rules of Mn-CDF subfamily genes in plants and the branches of the evolutionary tree.Among them,PbMTP8.1 and PbMTP8.2 were classified as Group8.PbMTP9,PbMTP10,PbMTP11.1,PbMTP11.2,and PbMTP11.3 belong to Group9.Structural analysis of Mn-CDF genes in the pear genome shows that the genes in Group8 contain 7 exons,while the genes in Group9 contain 6 exons.Motif analysis showed that protein sequences of Mn-CDF subgroup in pear contain six conserved motifs,which was consistent with the result in Arabidopsis and rice.The above bioinformatics analysis demonstrated that the Mn-CDF genes in plant were highly conservative in evolution.(3)Gene localization in chromosomes suggested that except for PbMTP11.2 and PbMTP11.3 distributed on unassembled scaffold 467.0,the Mn-CDF genes of pear were evenly distributed on different chromosomes.The construction and the analysis of threedimensional(3D)structure models of pear Mn-CDF proteins indicated that these models were similar in structures.Besides,these models that we built were reliable.(4)The Mn-CDF genes were cloned from the pear genome and constructed with the yeast expression vector pFL61,and then the recombinant vectors were transferred to 7 kind of yeast mutants,separately.The results showed that other than PbMTP11.3,the other genes could promote the tolerance of yeast mutant ⊿pmr1 to high concentration of Mn.Except PbMTP11 homologs(PbMTP11.1,PbMTP11.2,PbMTP11.3),the other pear Mn-CDF genes also improved the tolerance of yeast mutant ⊿ccc1 to high concentration of iron(Fe2+).However,all of these genes failed to enhance the resistance of the other yeast mutants to high concentrations of zinc(Zn),cadmium(Cd),copper(Cu),cobalt(Co)or nickel(Ni)ions.(5)In order to study the expression of these genes in different tissues of pear seedlings,we tested these genes’ expression in the transcriptional level using quantitative RT-PCR method.Quantitative RT-PCR analysis showed that Mn-CDF genes of pear had different expression patterns in root,stem and leaf tissues.The above results reveal the important roles of Mn-CDF subfamily in the maintenance of Mn2+balance and distribution in pear tree,laying the foundation for further research.