Family Analysis Of Magnesium Transpoter In Pear And Function Verification Of PbrMGT7

The magnesium ion(Mg2+)is the most abundant divalent cation in living cells and it plays essential roles in various cellular functions.Mg2+ homeostasis in plant cells is crucial for plant growth.The MRS2/MGT family proteins localize to various membranes and they function during Mg2+ transport in model plants Arabidopsis thaliana and Oryza sativa,but their functions are largely unknown in fruit trees.In this study,we performed a genome-wide analysis to identify the MGT gene family members in the pear Pyrus bretschneideri.Meanwhile,the function of a member PbrMGT7 in pear pollen was verified.The primary results were as follows:1.Sixteen MGT genes were indentified in Pyrus bretschneideri employing BLASTP and HMMsearch.In order to classify the MGT genes identified,we searched MGT genes in other ten species,including six dicots(Arabidopsis thaliana,Populus trichocarpa and four other Rosaceae species:Malus domestica,Fragaria vesca,Prunus mume and Prunus persica),three monocots(Oryza sativa,Zea mays and Brachypodium distachyon),one bryophyte(Physcomitrella patens)and one lycophyte(Selaginella moellendorffi),and a total of another 81 MGT genes were identified in the same way.A phylogenetic tree based on 97 protein sequences of the 11 species was then constructed employing the software RAxML.According to the phylogenetic tree,all the MGT genes were separated into five groups,named Groups Ⅰ,Ⅱ,Ⅲ,IV and V.Group Ⅱ contains 7 PbrMGT genes that is the most and Group V follows with a number of 5 and Group IV contain 4 PbrMGT genes,while neither of GroupⅠ nor Group Ⅲ contain any PbrMGT gene.Then the gene duplication events and expression patterns of the PbrMGT family was analysised.Six tissues including roots,stems,leaves,pollen tubes,stigmas and fruit were used in Semi-quantitative reverse-transcription PCR analysis and results showed that no PbrMGT gene was proved expressed in any unique tissue including pollen,and expression of the variety of the PbrMGT genes was detected in more than four tissues tested,while PbrMGT5 was observed only in leaf and pollen,and PbrMGT4 found just in leaf and fruit.Most of the PbrMGT genes observed expressed in pollen,except PbrMGT4,PbrMGT10,PbrMGT11,PbrMGT15 and PbrMGT16.Expression of PbrMGT7 and PbrMGT9 was detected in all six tissues tested.2.The effect of Mg2+ on pear pollen and expression of PbrMGTs was studied.Results showed that low Mg2+ concentrations(＜5mM)could promote both the pollen germination and length of pollen tubes compared with no added Mg2+,and 1 mM was the most suitable concentration for pear pollen growth.When the Mg2+ concentration arrived higher than 10 mM,growth of the pollen would be significantly inhibited and no germinated pollen could be observed when it came to 100 mM.the expression of several genes altered at a level of more than 1.5 times with additional Mg2+ including,including PbrMGT3,PbrMGT7,PbrMGT12 and PbrMGT13 and the expression of PbrMGT7 responsed the most to the additional Mg2+ that was significantly altered by more than two times both in a promoting concentration and an inhibiting and concentration,proving that PbrMGT7 may be functional gene in pear pollen to transport magnesium.Besides,the expression level of some genes such as PbrMGT 6,PbrMGT8,PbrMGT9 and PbrMGT 14 did not change obviously and oher PbrMGTs including PbrMGTl,PbrMGT2,PbrMGT4,PbrMGT5,PbrMGT1O,PbrMGT11,PbrMGT15 and PbrMGT16 were hardly expressed in pollen under magnesium treatment.To select genes for futher study,we searched the RPKM value of the 16 PbrMGT genes in pear pollen,finding that PbrMGT7 owned the highest score thus was finally picked out.3.The function of PbrMGT7 in pear pollen was verified.PbrMGT7 could functionally complement a bacterial strain deficient in Mg2+,indicating a role in magnesium transport.Co-localization experiment confirmed that PbrMGT7 was localized in the mitochondrial,suggesting that PbrMGT7 could mediate Mg2+-trafficking between the mitochondria and cytosol.All these results together suggest that PbrMGT7 was related to Mg2+ homeostasis in pear pollen tube growth.