Cloning And Functional Analysis Of Walnut JrAMT Gene

Nitrogen is a component of a variety of substances such as proteins and amino acids in living organisms.It is also one of the most important mineral elements in plant growth.It is called a living element.The main nitrogen sources that plants can absorb and utilize are ammonium nitrogen and nitrate nitrogen.At present,the nitrogen utilization rate is generally low during the growth of crops,and the large application of nitrogen fertilizer is still the main means to increase crop yield.Studies have shown that excessive application of nitrogen fertilizer can lead to decreased crop resilience,soil structure damage and water pollution.Therefore,the development of high-efficiency nitrogen crop germplasm innovation has become an important way to improve nitrogen utilization.Juglans regia L.is one of the most important “four nuts” in the world and is a strategic tree for the development of national woody oils.In this thesis,walnuts were used as research objects,and the cloning and overexpression vectors of AMT(ammonium transporter)gene and homologous transformation of walnut somatic embryos were carried out in order to obtain high-efficiency utilization of well-grown walnut ammonium.The main findings of this study are as follows:(1)Successfully cloned the JrAMT gene of walnut,constructed an overexpression vector and carried out subcellular localization analysisThe full-length cDNA of walnut JrAMT gene was successfully cloned by homologous cloning.The results of bioinformatics analysis indicated that the gene was 1464 bp in length.The NCBI online sequence alignment showed that the amino acid sequence encoded by the sequence belonged to Ammonium Transporter Family.The gene encodes a protein with a molecular weight of approximately121.55 kd and a theoretical isoelectric point of 4.75,which is an acidic protein.The overexpression vector 35S-JrAMT-GFP initiated by CaMV35s(strong promoter)was successfully constructed using GFP as a fusion reporter gene.The results of subcellular localization of tobacco epidermis showed that the fusion protein was localized to the cell membrane.(2)Successfully carried out genetic transformation of walnut somatic embryos and obtained positive regenerated plantsThe genetic transformation of 35S-JrAMT-GFP vector was carried out by using the wild-type somatic embryo of walnut as a receptor.The results showed that carbenicillin could effectively inhibit the somatic embryo contamination caused by Agrobacterium;subculture could effectively reduce the browning of transformed embryos and increase the induction rate of transformed embryos;the screening culture with hygromycin could significantly improve The positive rate of transformed embryos;the regenerated plants with the target fragment of about 2000 bp were detected by RT-PCR,and the relative expression of JrAMT gene was significantly higher than that of the control regenerated plants as walnut 35S-JrAMT-GFP positive plants.(3)Functional verification of walnut JrAMT geneThe chlorophyll content,nitrogen content,plant height and dry matter quality of 35S-JrAMT-GFP positive plants were significantly higher by observing the plant height,chlorophyll content,nitrogen content and dry matter quality of 35S-JrAMT-GFP positive plants.In the control plants,it was indicated that the walnut JrAMT gene can promote plant chlorophyll synthesis,nitrogen uptake and vegetative organ establishment,and play an important role in functional establishment.