Identification And Expression Analysis Of Zinc Transporter MtHMA2 And MtMTP3 In Medicago Truncatula.

Zinc is an essential micronutrient for plant growth and development.Both Zn deficiency and Zn excess are harmful to plants.AMF(Arbuscular mycorrhiza fungi,AMF)can symbiosis with more than 80% terrestrial plants to form arbuscular mycorrhizal(Arbuscular,mycorrhiza,AM)symbionts.Under zinc deficiency condition,AMF transferred the absorbed zinc to plants,while under zinc excess conditions,AMF reduced the uptake and translocation of zinc.The molecular mechanism of mycorrhizal on plant absorbing Zn was rare researched.The effects of AM on rice and Medicago truncatula Zn transporters were analyzed.The results got as follow:1.A MTP transporter MtMTP3 was identified in model plant Medicago truncatula.Bioinformatics analysis showed that MtMTP3 encoded 385 amino acids.Phylogenetic analysis and sequence alignment showed that MtMTP3 belonged to zinc CDF transporter and had the typical characteristics of zinc family CDF.Expressing of MtMTP3 in yeast increased the resistance of yeast to zinc.Using promoter of MtMTP3 to drive the expression of GUS reporter gene and RT-PCR showed that MtMTP3 was mainly expressed in cortex of root.When growing at high concentration of zinc,manganese or iron deficiency condition,the expression of MtMTP3 were increased in root.when Inoculation with Rhizophagus irregularis,the expression of MtMTP3 were decreased in root,even in high Zn condition.The arbuscular decreased under high zinc condition.Semiquantitative RT-PCR showed that the expression of MtMTP3 were significantly increased under low Pi condition.These results showed that MtMTP3 may function by limiting zinc transporting to shoot when zinc excess in the root,which is similar to AtMTP3.Mycorrhiza may depress the expression of MtMTP3 by enhancing the absorption of phosphate,thus promoting the uptake of Zn and transportation to shoot.2.A P1B-ATPase transporter MtHMA2 was identified in model plant Medicago truncatula.Phylogenetic analysis and sequence alignment showed that MtHMA2 belonged to P1B-ATPase family.Yeast complementation experiments showed that MtHMA2 can transporter Zn and Pb.Using promoter of MtHMA2 to drive GUS reporter gene showed that MtHTP2 was expressed higher in cortex of root.Semiquantitative RT-PCR and real-time PCR showed that MtHMA2 was depressed on high zinc condition in root,Pb can increase the expression of MtHMA2 in root,while Cd increased the expression of MtHMA2 on shoot.Overexpression of MtHMA2 in root will increase the resistance to zinc at low concentration,however,the leaves show symptoms of Zn poisoning at high Zn concentration.Mycorrhizal increased the expression of MtHMA2,while under high zinc condition,mycorrhiza depressed the expression of MtHMA2.Maybe the mycorrhizal fungi reduced the translocation of Zn to the upper part of the soil under high zinc treatment.3.Semiquantitative RT-PCR analysis the effects of AM on plants rice and Medicago truncatula ZIP transporters as well as rice HMAs,finding that AMF increased the expression of OsHMA2.Function analysis of OsHMA2 upstream regular elements,funding that AMF reduced the OsHMA2 transcriptional activity in Medicago truncatula.