Cloning And Function Analysis Of Nitrate Transporter Gene MeNRT2.1 In Cassava(Manihot Esculenta Crantz)

Cassava(Manihot esculenta Crants)is tropical crop,with a typical high photosynthetic capacity,high starch yield,drought tolerant and well growth on low-nutrient soils.Cassava has great potential applications in response to the food crisis,energy crisis,and feed supply,industrial starch and ethanol.Nitrogen is one of the most critical elements and the largest elements plants demand.And it is the main limiting factor for growth and yield.The research about molecular mechanism of nitrogen absorption and transport mainly focused on the nitrate transport protein family.It has been concentrated on the studies of NRT1 and NRT2 families in Arabidopsis plants.Rice and other crops' NRTS genes have also been being studied.Cassava has the genetic characteristics of NUE that makes it become the African first food crops and pioneer crops.However,the research on molecular mechanism of NRTs family genes in cassava is less.To insight into the molecular basis for tolerance of poor soils and improve the utilization rate of nitrate in cassava,the "hua nan 5" cassava cultivar grown in medium containing different nitrate concentrations was used as experimental material in this study.The full-length cDNA encoding a high-affinity nitrate transporter gene,designated MeNRT2.1,was isolated from cassava by using bioinformatics and RT-PCR techniques.Through the quantitative real-time PCR analysis,subcellular localization of fusion protein,GUS staining technique and transgenic technology to explore the nitrogen transfer function of the gene so that providing the reference for further screening NRT2 genes and increasing production of genetic modification of cassava.Of course,it has an important significance for high resistance varieties of cassava breeding.The main research results are as follows:1)The nitrate nitrogen has an important effect on the growth and development of cassava tissue.The relative growth of cassava cultivar is proportional to the concentration of NO3-range between 0-10 mmolL-1.The cassava cultivar root numbers will be inhibited by much too high or low NO3-.2)A NRT2 gene is isolated from cassava by using RT-PCR techniques.MeNRT2.1 was 1899 bp in length and contained an open reading frame of 1593 bp.Sequence analysis revealed that the ORF of MeNRT2.1 encode 530 amino acid residues.Phylogeneticanalyses showed that MeNRT2.1 was highly homologous to MtNRT2,AtNRT2,TcNRT2 and PtNRT2.MeNRT2.1's closest homolog is Theobroma cacao NRT2.1,with 90%amino acid identity.3)Quantitative real-time PCR analysis showed that the MeNRT2.1 transcript of root under 0.2 mmolL-1 NO3-1 treatment is the highest among three nitrate concentrations,and has a low level on 10 mmolL-1 in root.Little MeNRT2.1 mRNA was found in other examinated tissues.Therefore,the expression of MeNRT2.1 is induced and specific pattern in root.Subcellular localization of the fusion protein of MeNRT2.1 with GFP only appears in the cytomembrane.Maybe the gene has a function of NO3-transport.4)The CRISPR/Cas9 editing system was built,to provide the basis for the research of gene function.5)The promoter of MeNRT2.1 was isolated from cassava by using RT-PCR technique,and it was 1876 bp.The expression vector including GUS was constructed,and the gene was transformed into Arabidopsis,Staining analysis showed that the promoter has the foundation to express in root;it was up-regulated expression in roots?stems and leaves under 0.2 mmolL-1 and 10 mmolL-1 NO3-1 treatment for 12 hours.However,it was expresssed in old leaves of transgentic Arabidopsis seedings after 12 hours without NO3-.6)It's a longer period for the transformation of cassava,and are still in tissue differentiation stage.Through the construction of expression vector,agrobacterium-mediated transformation,to obtain transgenic Arabidopsis thaliana.The phenotypic analysis shows that the transgenic Arabidopsis on the MS medium grew significantly better than that of wild type.The results of nitrogen starvation showed that the transgenic Arabidopsis can survive,but the wild type were dead.And transgenic Arabidopsis thaliana had accelerated the absorption of chlorate to damage the chlorophyll in the experiment.Quantitative PCR showed that the MeNRT2.1 was high-efficiency expression in the transgenic Arabidopsis plants.We preliminarily obtained that the MeNRT2.1 can promote the NO3-absorption in the transgenic Arabidopsis plants.