The Functional Analysis Of Iron And Copper Transpoter In Pear Pollen

With the rapid development of the economy and urbanization,heavy metals concentration is increased by vehicle exhaust and industrial emissions 'three wastes' in the soil and air,which affects the normal growth and development of the plants.The pollen germination and pollen tube length can be used as a parameter measuring plant reproductive capacity.Now we use pear pollen as experimental materials to study the effect of heavy metals iron(Fe)and copper(Cu)on pollen germination and pollen tube growth.The results showed that the pollen germination and pollen tube elongation were inhibited with the increase of Fe and Cu concentration in growth medium.However,low concentrations of Fe promote the pollen germination and pollen tube growth.The effect of Cu on pollen germination and pollen tube growth of was greater than the influence of Fe.Some heavy metal transporter proteins are the important transporters regulated the metal in cell.In order to study the effect of Fe and Cu on plant pollen,we focused on two heavy metal transporters,yellow stripe-like(transport Fe)and copper transporter(transport Cu)..Yellow stripe-like(YSL)family transporters,belonging to the oligopeptide transporter family,are significant iron transport proteins.In this study,we used the amino acid sequence of Arabidopsis YSL gene AtYSL1 as a query probe to search the YSL gene family in Pyrus bretschneideri.We found 8 YSL genes and mainly analyzed the evolution of 8 YSL genes in pear.Segmental duplications have played a key role in the expansion of the pear YSL family.All of PbrYSLs except PbrYSL1 were expressed in pollen,and PbrYSL4 was significantly responsive to change in the external iron ion supply in pollen.When PbrYSL4 transformed into the yeast mutants Acccl on SD-Ura medium wtih high concentration iron,the yeast mutants PbrYSL4 could restore growth.Morever,PbrYSL4 was localized to the plasma membrane and chloroplasts,thus these results suggest that PbrYSL4 probably transported the iron ions into the cytoplasm of pollen.Copper transporter(COPT),belonging to CTR/COPT,are significant copper transport proteins.In this study,we used the amino acid sequence of Arabidopsis COPT gene AtCOPTl as a query probe to search the COPT gene family in Pyrus bretschneideri.We found 8 COPT genes and mainly analyzed the evolution of COPT genes among different species.The expression of PbrCOPTl and PbrCOPT2 were both responsive to change in the external copper ion supply in pollen,and PbrCOPT1 was more significant than PbrCOPT2.The recombinant plasmids,pCAMBIA1302-PbrCOPTl and pCAMBIA1302-PbrCOPT2,were transported into onion epidermal cells by Agrobacterium infection method.The results showed PbrCOPT1 and PbrCOPT2 were both located in the plasma membrane.But the yeast mutant ?cup2 was not able to restore growth when PbrCOPTl and PbrCOPT2 were transformed into the yeast mutant on SD-Ura medium wtih high concentrations copper.These results indicated that PbrCOPTl and PbrCOPT2 are not able to complement the function of CUP2 in yeast mutant and the function of PbrCOPT1 and PbrCOPT2 should be studied in future.