Identification And Functional Analysis Of Nitrate Transporter Gene Family In Cassava

Nitrogen is one of the important nutrient elements for plant growth and development,the level of nitrogen supply directly affects the yield and quality of crops.Increasing the application of nitrogen fertilizer is the main means to increase crop yield.However,with the increasing amount of nitrogen fertilizer application year by year,crop yield did not increase significantly,but brought about some problems,such as soil acidification,greenhouse gas emission,water eutrophication and production cost increase.Therefore,to greatly improve crop nitrogen use efficiency and cultivate nitrogen efficient varieties is an effective economic means to increase crop yield and reduce excessive use of nitrogen fertilizer.Cassava(Manihot esculenta)is tolerant to drought and low nitrogen,and has strong tolerance to a variety of abiotic stresses.Mining the key genes of cassava-related nitrogen efficient utilization is of great significance for the improvement and cultivation of nitrogen-efficient germplasm resources.In this study,the whole genome of cassava nitrate transport protein MeNPF gene family was identified by bioinformatics method.We systematically analyzed the phylogenetic relationship,gene structure,protein conserved motif,chromosome location,gene replication events and promoter cis-acting elements of the family members.MeNPF6.2 and MeNPF6.3genes were cloned from cassava.The gene function of MeNPF6.2 was preliminarily analyzed by using transcriptome sequencing,real-time quantitative PCR,subcellular localization,cassava and rice overexpression.The main results are as follows:(1)A total of 72 members of MeNPF family were identified from the whole genome level of cassava,all of which contained PTR2 domain.These genes are unevenly distributed on 18 chromosomes of cassava.We found a total of 21 pairs of segmental duplication genes,but no tandem duplication genes were found.According to the classification and phylogenetic analysis of At NPF genes in Arabidopsis thaliana,MeNPF genes were divided into 8 subfamilies(Ⅰ-Ⅷ),and the number of MeNPF genes in subfamily Ⅴ was the largest.The same subfamily has similar gene structure and protein conserved motif.The prediction results of cis-acting elements in promoters suggest that MeNPF gene may be related to the response of abiotic stress and hormonal signal transduction.(2)The transmembrane domains and subcellular localization of MeNPF6.2 and MeNPF6.3proteins were analyzed by bioinformatics methods.We found that both MeNPF6.2 and MeNPF6.3 contained 12 transmembrane domains,which may be plasma membrane proteins.Through the transient expression of gene-e GFP fusion vector in tobacco protoplasts and stable expression in Arabidopsis,we verified that MeNPF6.2 and MeNPF6.3 were located on the cell membrane.(3)Real-time quantitative PCR detection showed that MeNPF6.3 gene was mainly expressed in the roots of cassava seedlings,and the expression was the highest under 0.2 mmol/L nitrate treatment,and the expression in stems and leaves was not induced by external nitrate concentration,while MeNPF6.2 gene was mainly induced in leaves.The results showed that the expression of these two genes was induced by nitrate.(4)The phenotypic analysis of MeNPF6.2 overexpression transgenic cassava showed that under normal growth condition,the growth of overexpressed lines was significantly better than that of non-transgenic control,and the leaf area,root length and root number were significantly increased.Under low nitrogen treatment,the transgenic lines and control were subjected to a certain degree of nitrogen stress,but the transgenic lines showed strong tolerance to low nitrogen,and their root length,root number and leaf area were significantly higher than those of control.(5)Through the phenotypic observation of transgenic rice lines with overexpression of MeNPF6.2,we found that there was no significant difference in grain length between transgenic rice and non-transgenic control,but grain width was significantly higher than that of control,and100-grain weight was also significantly increased.(6)In order to find the interacting proteins of MeNPF6.2,three interacting proteins,including ribosomal protein L7Ae/L30e/S12e/Gadd45 family proteins(T24D18＿3),myosin 1(MYA1)and arabinogalactoprotein 15(AGP15),were preliminarily screened by membrane system yeast two-hybrid library technique.The above results suggest that MeNPF6.2 gene plays an important role in response to low nitrate stress.Overexpression of MeNPF6.2 enhances low nitrate tolerance of cassava.Heterologous expression in rice increases 100-grain weight,which can be used to improve crop tolerance to low nitrate.