Biological information of lettuce NRT2/3 and preliminary functional validation
[Objectives]Nitrate nitrogen is the primary nitrogen source for dryland plants,NRT2/3 plays a crucial role in plant nitrate absorption and the regulation of nitrogen uptake.We analyzed the members of lettuce NRT2/3 family using bioinformatics,studied their physicochemical properties,and gene structure evaluation,and assessed the functions of select NRT2/3 genes utilizing VIGS technology.[Methods]The NRT2/3 proteins from Arabidopsis and rice,as well as the annotated NRT proteins from lettuce were downloaded from the NCBI website.Bioinformatics methods were utilized to identify the members of the lettuce NRT2/3 family,and a multi-sequence alignment of the NRT2/3 proteins from lettuce and Arabidopsis was conducted using DNAMAN software.Furthermore,a multiple sequence alignment of the NRT2/3 proteins from lettuce,rice,and Arabidopsis was conducted using MEGA11 software,followed by the construction of a phylogenetic tree.Then,their coincidences of the common NRT2/3 proteins were proved in physicochemical properties,functional domains,conserved motifs,hydrophilicity,cis-acting elements,and gene structure,using online software.The chromosome localization,the secondary structure,phosphorylation sites,and transmembrane regions of lettuce NRT2/3 family proteins were predicted.Drawing from our team's prior transcriptome sequencing findings,the NRT2/3 genes,which had comparatively high expression levels,were targeted for expression analysis:1)The seedlings of the two lettuce varieties grown to 20 days were planted on substrates and irrigated by NO3-0,0.35,0.7,and 11.5 mmol/L solutions for the invetigated of expression levels.2)The expression levels of these genes in the two lettuce varieties were reduced using TRV-VIGS transient silencing method,subsequently,the lettuce seedlings were grown in normal nitrogen nutrient solutions for 21 days,and then invetigated the expression levels,N uptakes and growth indexes.[Results]This study identified a total of 8 lettuce NRT2/3 family members,consisting of 7 LsaNRT2 proteins and 1 LsaNRT3.1 protein.These members were unevenly distributed across 4 chromosomes.Their physicochemical properties,functional domains,transmembrane regions,phosphorylation sites,cis-acting elements,and gene structure,et al.basically conformed to the common characteristics of the NRT2/3 family.The transcriptome sequencing results showed that among the 8 identified genes,LsaNRT2.4 and LsaNRT3.1 had notably high expression levels in leaves of the two lettuce varieties.LsaNRT2.4 and LsaNRT3.1 genes responded to low NO3-concentrations to a certain extent.Following VIGS silencing,the expression levels of LsaNRT2.4 and LsaNRT3.1 in lettuce leaves were significantly reduced,as were the plant height,leaf length,leaf width,chlorophyll-a content,and leaf nitrate content.Notably,the nitrate content in leaves of lettuce(TRV::LsaNRT3.1)was significantly lower than that of lettuce(TRV::LsaNRT2.4).Observations of the phenotype revealed that lettuce silenced using VIGS displayed symptoms like smaller leaves and withered leaf tips,and the lettuce(TRV::LsaNRT3.1)exhibited a more pronounced effect.[Conclusions]Most characteristics of lettuce NRT2/3 family members align with the common traits of plant NRT2/3 family,with LsaNRT2.4 and LsaNRT3.1 genes showing significantly higher expression levels in the leaves compared to the other 6 genes;LsaNRT2.4 and LsaNRT3.1 play pivotal roles in lettuce nitrate absorption,and responsed to deficient and low NO3-stress very quickly,especially in their expressions in roots.The increased expressions of LsaNRT2.4 and LsaNRT3.1 genes,improved the N uptake and growth of lettuce.
万方数据
维普
