Molecular Cloning And Expression Analysis Of Molybdenum Transporter In Lotus Japonicus

Molybdenum(Mo) is an essential trace element for plant growth and development,and its physiological function is mainly realized through some Mo-containing enzymes.Especially, it is a great influence on nitrogen fixation and the formation of root nodules in legumes. Lotus japonicus is a perennial herb belonging to Legume, which is usually used as a model plant with a small genome and known genome sequences, but the molecular mechanism of Mo uptake and transport has not been reported in L. japonicus. In this experiment, L. japonicus was used as a material, which was grown on the MS solid media with the Mo concentration of 0 nmol/L(-Mo), 103 nmol/L(+Mo) and 1030 nmol/L(10×Mo) to observe its growth. In addition, the contents of Mo and the activities of nitrate reductase(NR), aldehyde oxidase(AO) and xanthine dehydrogenase(XDH) in roots and leaves were mensurated. Total RNA was extracted from the leaves of L. japonicus cultured on the +Mo medium for 45 days. The full-length c DNA of Lj MOT1 was obtained by a homologous cloning method and RACE technology, and its bioinformatics was analyzed.At the same time, the expression of Lj MOT1 was determined under the conditions of different Mo concentration, and Lj MOT1 was transformed into tobacco and Arabidopsis mutants for subcellular localization and functional identification, respectively. The main results were as follows:(1) L. japonicus was cultured on MS solid media with different Mo concentrations. The results indicated that plants growing on-Mo for 45 d appeared some deficiency symptoms,such as short roots, few lateral roots and leaf yellowing. On +Mo or 10×Mo, the growth of L. japonicus had no obvious differences and was not damaged, too. These results reveal that the normal growth of L. japonicus for the demand of Mo is essential, and is tolerant of the slightly highe Mo concentration.(2) On the three media(-Mo, +Mo and 10×Mo), the Mo contents were relatively lower in leaves than those in roots of L. japonicus. The Mo contents in leaves on –Mo were respectively 0.5 and 0.2 times higher than those on +Mo and 10×Mo, and the Mo contents in roots on –Mo were respectively 1.3 and 1.45 times higher than those on +Mo and10×Mo. At the same time, the activities of NR, AO and XDH were measured in leaves and roots under three culture conditions. The results showed that the activities of Mo-containing enzymes were positive correlated with the contents of Mo in different tissues, which were regulated by the capacity of effective absorption and transport of Mo in plant.(3) A Mo transporter gene Lj MOT1(Gen Bank accession No. KM379115) was cloned, and the full-length c DNA was 1824 bp, including an 1407 bp open reading frame(ORF), 79 bp 5'untranslatedregion(UTR) and 323 bp 3' UTR and 15 bp poly A. According to whole genome database of L. Japonicus, Lj MOT1 was localized on the third chromosome. The Lj MOT1 gene was predicted to encode 468 amino acids with a relative molecular weight of49657.7Da, and the isoelectric point(PI) of Lj MOT1 was 9.14, and the molecular formula was C2270H3653N583O619S20 with 7145 atoms.(4) The Lj MOT1 protein and MOT1 proteins reported had two conserved motifs:PMPVQPMKAIAAVA and WFGAMPCCHGAGGLAGQYRFG, which were unique conserved domains of Mo transporters, but they did not belong to any known protein families. In addition, the alignment of amino acid sequences showed the Lj MOT1 protein had the highest identities with the Gm MOT1 protein, and reached 81.14%. Phylogenetic analysis indicated that L. japonicus was the closest relationship of soybean, and clustered together with dicotyledonous plants, which conformed to the traditional evolution.(5) The results of q RT-PCR illuminated that the expression level of Lj MOT1 in roots appeared a rising trend with increasing Mo concentrations on the media. The expression level of Lj MOT1 in leaves on +Mo and 10×Mo media had no significant difference, but they were obviously higher than those on-Mo medium.(6) A eukaryotic expression vector p Cambia1304-Lj MOT1 was constructed, which was transiently expressed in tobacco, and the result showed that the Lj MOT1 protein was located on the plasma membrane by a laser scanning confocal microscope. At the same time, The Lj MOT1 was transformed into Arabidopsis mutant mot1 by Agrobacterium-mediated method, and 4 positive plants were successfully screened.