Genome-wide Identification Of NRAMP Family Genes In Camellia Sinensis And Preliminary Functional Analysis Of CsNRAMP2 And CsNRAMP5 Under Pb Stress

The tea plant [Camellia sinensis(L.)O.Kuntze] is an important crop.It is well known that soil is the main place for tea plants to absorb nutrients.In recent years,with the increasing degree of soil acidification and the rapid development of industry and mining,the content of lead in soil bioavailability has greatly increased.The increase of bioavailable lead content in soil will further promote the absorption and accumulation of lead in tea plants,which is toxic to the growth of tea plants.Therefore,it is significant to understand the molecular mechanism of lead absorption and transport in tea plants,however,the studies about lead absorption and transport are limited.In this study,the one-year-old seedlings of tea plants “zhongcha108” was used as the material to be treated with different concentrations of lead.The effects of lead on the growth of tea plants were studied by phenotypic observation and physiological and biochemical index detection.A kind of membrane integrative protein involved in multiple divalent cations,NRAMP,was identified,and the response patterns of members of this family to lead treatment were analyzed.Finally,the biological functions of Cs NRAMP2 and Cs NRAMP5 were preliminarily analyzed.The main results were as follows.1.To study the effect of lead on the growth and development of tea plants,tea seedlings were treated with Pb(50,100,300,500 mg/L)The results showed that lead treatment significantly inhibited the growth of roots at 56 d.Lead treatment significantly increased the lead content in tea seedlings,which showed a positive correlation with the lead content and treat time.The distribution of lead in different tissues was: roots > old stems > old leaves > young leaves > young stems,while most of lead in tea seedlings was restricted in root.Under 500 mg/L Pb treatment,the chlorophyll content at 7 d was decreased.The contents of catechins and caffeine were lower than those in the control and decreased with the increase of Pb treat time.The activities of SOD and CAT were raised under 50 mg/L Pb treatment and showed no significant change at 500 mg/L.The POD was activated at 500 mg/L Pb treatment.In addition,treatment with a high concentration of Pb(300 mg/L,500 mg/L)significantly increased the contents of amino acids,malondialdehyde,and proline.2.11 Cs NRAMP genes were identified in the Camellia sinensis var.sinensis genome.The number of amino acids in coding regions of 11 Cs NRAMP genes ranged from 279 to 1,373,and the proteins contained a conserved amino acid residues sequence: GQSSTx TGTYA GQFIMx GFLx Lxx KKW.According to phylogenetic tree analysis,this gene family is divided into two groups,Cs NRAMP3,Cs NRAMP4,Cs NRAMP5,and Cs NRAMP8 belong to group 1,and other genes form another group.Transmembrane domain prediction results showed that Cs NRAMP proteins contained 3 to 12 transmembrane regions.3.Quantitative RT-PCR analysis indicated that different Cs NRAMP genes exhibit various expression patterns in tea plants.Three genes,Cs NRAMP3,Cs NRAMP4,and Cs NRAMP5,showed extremely high specificity in roots,while four genes,Cs NRAMP1,Cs NRAMP2,Cs NRAMP10,and Cs NRAMP11 were highly expressed in leaves,moreover,Cs NRAMP6 and Cs NRAMP9 were highly expressed in the stem.Also,two genes,Cs NRAMP7 and Cs NRAMP8 exhibited high expression in both shoots and leaves.Cs NRAMP genes showed different expression characteristics under lead treatment.After 14 d of lead treatment,the expression of Cs NRMAP2,Cs NRMAP3,Cs NRMAP5,and Cs NRMAP8 were significantly up-regulated in leaves,and the expression of Cs NRMAP3,Cs NRMAP5,and Cs NRMAP9 was significantly increased in roots.4.Subcellular localization assay demonstrated that both Cs NRAMP2 and Cs NRAMP5 proteins were located in the plasma membrane.5.The prokaryotic expression results showed that the cells overexpressing genes grew weaker than the control at 5 m M Pb treatment.Cs NRAMP2 and Cs NRAMP5 overexpressing made E.coli cells more sensitive to lead.6.The Cs NRAMP2 and Cs NRAMP5 genes were transferred into Arabidopsis thaliana,and T2 generation plants were obtained after antibiotics screening.