Cloning Of MeNRT2 Genes And Interaction Analysis Between MeNRT2 And MeCIPK Proteins In Cassava

Cassava(Manihot esculenta Crantz)is the sixth largest food crop,with several agronomic traits like a high yield,drought tolerance,combined with Iow-nutrient soils tolerance,widely cultivated in tropical areas around the world.Nitrogen plays an important role in the substance and energy metabolism activities of organisms and is the primary nitrogen for most plants.However,the concentration of NO3-is very easy to change in space and time due to the influence of microorganism,soil type and the amount of fertilizer application.In agricultural cultivation,farmers often exert large amounts of nitrogen fertilizer for high yield,and the average nitrogen utilization rate is only 30%～40%in China.Excessive nitrogen fertilizer has caused leakage of farmland with surface runoff,which results in water and agricultural area source pollution.Therefore,understanding the mechanism of absorption and transport of NO3-has theoretical and practical significance for improving the nitrogen utilization rate of cassava,improving the ecological environment and ensuring the food supply for the population,and provides a theoretical reference for further establishing a low nitrogen efficient farming methods.In this study,four high affinity nitrate transporters genes,namely MeNRT2.1,MeNRT2.2,MeNRT2.5,MeNRT2.6had been isolated from Cassava by PCR cloning technique.Through the exploration of proteins related to nitrate transport,the main results are performed as follows:1.Bioinformatics analysis showed that the MeNRT2 genes encoded about 450～530 amino acids,with two highly conserved regions MFS(G-x-x-x-D-x-G-G-x-R)and NNP(G-W/L-G-N-M/A-G),and with 10～12 transmembrane domains.Wolf pSORT predicted that the MeNRT2 belonged to transmembrane proteins.MeNRT2.1,MeNRT2.3 and MeNRT2.4 were unstable proteins,while MeNRT2.2,MeNRT2.5 and MeNRT2.6 were stable proteins.The cisacting elements in the promoter region of MeNRT2 proteins included core promoters CAAT-box and TATA-box,light response related,hormone response related,and non-biological stress related elements.Phylogenetic tree analysis revealed that the amino acid sequences of MeNRT2 proteins shared higher similarity with the NRT2 of Ricinus communi,Ricinus communis Thobroma cacao and they were the closest in evolutionary relatives.2.Real-time quantitative PCR(qPCR)results showed that when induced by low concentration of NO3-(0.3 mmol·L-),the expression level of MeNRT2 genes in the root was higher than that in the stem and leaf.The expression of genes was up-regulated firstly and then down-regulated,and the trend of change could be called Bell-shaped curve.When induced by high concentration NO3-(3 mmol·L-),the transcription level in root of MeNRT2.5 gene was inhibited by high concentration of NO3-.And the expression of MeNRT2.6 gene was not affected significantly by NO3-concentration.3.We used nating-based split ubiquitin yeast systen(mbSUS)to screen interacting proteins ot MeNRT2,and finally finded that MeNRT2.5 and MeCIPK 1,4,5,6,8,10,11,12,14,15,16,18,19,20,22,23,24,25,26 yeast hybrids could grow on triple deficiency medium(SD/-Leu-Trp-His)only,indicating that MeNRT2.5 protein might interact with MeCIPK proteins,while MeNRT2.1,MeNRT2.2 and MeNRT2.6 proteins might not.