Overexpression Of GhGS1 Gene Improves Cotton Tolerance To Low Nitrogen Stress

Nitrogen,as one of the indispensable chemical elements in plants,not only has a significant impact on many physiological,but also plays a role in a lot of biochemical reactions.In addition,it is not only the main component of nucleic acid,protein,phospholipid and enzyme,coenzyme and prosthetic group such as NAD+,NADP+,FAD and so on,but also the molecular structure of some vitamins,plant hormones and secondary metabolites.What's more,as well known that nitrogen is a significant constituent element of plant chlorophyll,to a great extent,affects the activity of many enzymes in the plant light and dark reaction of photosynthesis.Therefore,reasonable regulation of nitrogen metabolism related genes will be benefit to crop growth and development.Cotton is a nitrogen-sensitive plant.In agricultural production,the extensive use of nitrogen fertilizer is needed to ensure cotton production and quality.However,China as a large cotton production country,using a large number of nitrogen fertilizer brings a lot of negative impact on our country.The demand for nitrogen fertilizer in cotton whereas reducing environmental pollution is a pair of prominent contradictions.How to solve this prominent contradiction? Improving the nitrogen use efficiency in cotton may be the key to solving this contradiction.Glutamine Synthetase(GS)is a key enzyme for the "GS-GOGAT" cycle in the plant nitrogen metabolism pathway.It is the first enzyme that converts the inorganic nitrogen in the external environment into nitrogenous organic matter in the plant.In order to investigate the role of GS1 gene in the process of cotton growth and development,the cotton glutamine synthetase gene(GhGS1)was cloned and the expression vector was constructed to carry out cotton genetic transformation experiment.The main results are as follows:1.Cloning and phylogenetic analysis of GhGS1 geneThe GhGS1 gene(Gh_D07G1773)was cloned from upland cotton(Gossypium hirsutum).Bioinformatics analysis showed that the gene was located on chromosome 7 and contained a 1071 bp ORF encoding 356 amino acid residues with a molecular weight of 39 kDa and the isoelectric point was 5.92.The GhGS1 sequence was highly homologous to rice(Oryza sativa),soybean(Glycine max),Arabidopsis thaliana and maize(Zea mays).The GS gene family in Arabidopsis thaliana was divided into two groups(GS1,GS2).14 GS genes homologous to Arabidopsis were retrieved from the genome of the upland cotton.The phylogenetic tree showed that the GhGS1 gene was cloned in the same subfamily as AtGS1 gene and was closely related to AtGLN1;1 gene.2.Characteristics of GhGS1 gene expressionIn order to explore the expression characteristics of GhGS1 gene in cotton,Real-Time PCR was used to investigate the expression level of target gene in different tissues and organs of cotton.The total RNA of root,stem,leaves,hypocotyl,sepal,petal,pistil,stamen and 0 DPA ovules of cotton were extracted and cDNA was synthesized by reverse transcription.The results indicated that the expression level of GhGS1 was the highest in the root,the higher in the stamen,and the lowest in the leaves.The cotton seedlings were treated with nutrient solution at different levels of nitrogen supply.The results showed that when the cotton seedling was at the level of 5 mM nitrogen,the GhGS1 gene had the highest expression in the lateral roots followed by the main roots.When the cotton seedling was at the low nitrogen level of 0.5 mM and 1mM,regardless of the upper part(cotyledon,hypocotyl)or the underground part(lateral roots,roots),the expression of GhGS1 gene was significantly higher than that of 5 mM nitrogen.This implyed that low concentrations of nitrogen could induce GhGS1 gene expression and this induction was more pronounced in cotyledon.3.Cloning and expression analysis of GhGS1 gene promoter sequenceThe 1965 bp upstream sequence of GhGS1 gene(PGhGS1)was cloned by designated specific primers,using cotton genomic as template.The PGh GS1 sequence was analyzed by the Plantcare cis-acting component database.The results showed that there were some elements about plant hormone,light reaction,stress response element,besides the transcription start site(TATA-box),transcriptional enhancer(CAAT-box).It suggested that the GhGS1 expression was affected by many environmental factors.The GhGS1 promoter fused with GUS gene were introduced into Arabidopsis thaliana and Gossypium hirsutum by Agrobacterium mediated transformation method.Histochemical staining showed that GhGS1 promoter was expressed in various parts in Arabidopsis except mature leaves.And it had strong signals in root,stem,stamen and pistil.Histochemical staining showed that GhGS1 promoter were detected in various parts of cotton,and the stronger signals were found in root,stamens and stem.The results of histochemical staining were consistented with the expression level of GhGS1.4.Under low nitrogen condition,overexpression of GhGS1 gene could improve the tolerance of cotton to a certain extent.Furthermore,in order to study the utilization of nitrogen in GhGS1 gene under low nitrogen condition,the GhGS1 overexpression vector driven by Ca MV35 S was constructed and carried out genetic transformation experiments in cotton.The overexpression GhGS1 transgenic cotton plants were obtained by GUS screening.When planted in the pots with normal fertilization level,overexpression GhGS1 transgenic cotton T1 generation and wild-type cotton grew well and the chlorophyll content was similar.The wild-type cotton and overexpression GhGS1 transgenic cotton T2 generation were disposed under low nitrogen stress.Of course,when the concentration of nitrogen nutrient solution(nitrogen concentration is 5 mM)was appropriate,the overexpression GhGS1 transgenic cotton T2 generation and wild-type cotton were growing well.The chlorophyll content was no significant difference.On the contrary,the leaves of wild-type cotton appeared yellowish,especially the senescence leaves under the low nitrogen level(nitrogen concentration is 0.1 mM).The leaves of overexpression GhGS1 transgenic cotton T2 generation grew well.The chlorophyll content and the total nitrogen content in leaves of transgenic plants were significantly higher than that of wild-type.The above data showed that overexpression of GhGS1 gene not only could increase the chlorophyll content in cotton leaves under low nitrogen stress,which was beneficial to maintain the normal function of leaves,but also the nitrogen content of plants,providing sufficient nitrogen for plant growth.Generally,to some extent,the overexpression of GhGS1 gene could improve the tolerance of cotton under low nitrogen stress.