Function Analysis Of GhNRT2.5 In Cotton Response To Low Nitrogen Stress

Nitrogen is a constituent of proteins,nucleic acids,phospholipids,and other organic nitrogen compounds necessary for the growth and development of plants.These substances are the structural or functional components on which living cells depend,thus nitrogen is called the element of life.Amino acid synthesis has the most demand for nitrogen because it is not only the basis for protein synthesis,but also a precursor to many other compounds.Nitrogen is also an essential element of nucleotides,coenzymes and co-groups?NAD⁺,NADP,FAD?,and it is also a structural component of chlorophyll molecules.In addition,some plant hormones?such as auxin and cytokinin?,vitamin molecules contain nitrogen,or are derived from nitrogen-containing compounds.Nitrate is the main nitrogen source for much higher plants.Understanding the nitrate absorption mechanism in the soil and the plant's response to nitrogen stress is very important for improving plant nitrogen absorption and utilization efficiency and reducing nitrogen fertilizer application.Cotton as a nitrogen-sensitive plant often needs to increase the application of nitrogen fertilizer to increase yield,but too much or too little nitrogen fertilizer will cause the decline in cotton yield.Cotton farmers often tend to increase nitrogen fertilizer because they cannot determine the appropriate nitrogen application rate.While cause cotton tarnishing late maturity,yield decline and unabsorbed nitrogen with environmental erosion caused by soil erosion.Therefore,"less fertilization,high yield cotton"has become the focus of breeding research at this stage.As a high-affinity transporter,NRT2 uses nitrate as a specific substrate.When available nitrates are limited,the high-affinity transport system is activated and plays a leading role.NRT2.5has been shown to play an important role in plants responding to severe nitrogen deficiency stress in Arabidopsis.The study of GhNRT2.5 gene is helpful to understand the molecular mechanism of cotton response to nitrate signal and it's role in cotton nitrogen uptake.To this end,this paper uses cotton genetic transformation methods to change the expression of NRT2.5 in cotton,and explores the role of NRT2.5 in cotton under low nitrogen stress.The main results are as followed:1.Cloning and analysis of cotton GhNRT2.5 geneFirstly,in order to investigate the transcriptome data of cotton varieties with different nitrogen use efficiency under low nitrogen stress,the nitrate transporters of the cotton NRT2 family were found.Only the nitrate transporters encoded by Gohir.D12G148900 were found in low nitrogen supply.The level of expression was higher than the normal nitrogen supply level.It is speculated that the transporter encoded by Gohir.D12G148900 in cotton plays an important role in responding to low nitrogen stress or improving nitrogen fertilizer utilization efficiency in cotton.Then the evolutionary tree of Arabidopsis NRT2 family and cotton NRT2 family was analyzed,and suggested that the protein encoded by Gohir.D12G148900 gene has the closest relationship with Arabidopsis NRT2.5.Therefore,Gohir.D12G148900 was named GhNRT2.5.Then,using the protein online prediction website TMHMM to analyze the amino acid sequence encoded by the GhNRT2.5 gene,it was found that GhNRT2.5 has a total length of 506 amino acids and is a membrane-bound protein with 10transmembrane domains.2.Expression characteristics and functional analysis of GhNRT2.5 geneThe heat map predicted that GhNRT2.5 was highly expressed in cotton stalks,followed by leaves and roots.The actual analysis of cotton tissues by quantitative RT-PCR showed that the expression of GhNRT2.5 in cotton leaves and roots was the highest,which was slightly different from the heat map results.When the cotton seedlings of GhNRT2.5 gene silenced by VIGS,the expression of GhNRT2.5 gene in the roots and leaves of cotton seedlings injected with pTRV₂-GhNRT2.5 was significantly lower than that of the control,and its expression level was less than half of the control.Moreover,it showed classical nitrogen deficiency phenotypes such as yellowing of leaves,and the chlorophyll content was lower than that of the control,suggesting that silencing the GhNRT2.5 gene would cause cotton seedlings to be more sensitive to nitrogen deficiency.After treating cotton under different nitrogen supply levels,it was found that the expression level of GhNRT2.5 gene was higher than that under normal nitrogen supply level in a short time?within 2 days?and low nitrogen supply level,but in a long time?3 days?treatment the expression level of GhNRT2.5 gene did not continue to increase,and the expression under normal nitrogen supply level was higher than the low nitrogen supply level,indicating that GhNRT2.5 gene was involved in the short-term response process of cotton to low nitrogen.In order to better understand the function of GhNRT2.5 gene,transient expression in tobacco and heterologous expression of GhNRT2.5:GFP fusion gene in Arabidopsis,fluorescence observations showed that cotton GhNRT2.5 is a plasma membrane-localized nitrate transporter protein.3.Cloning and expression analysis of GhNRT2.5 gene promoter sequenceIn order to further clarify the regulatory mechanism of GhNRT2.5 gene during cotton growth,the 2500bp upstream regulatory region of cotton GhNRT2.5 gene was cloned to obtain the promoter of this gene?pGhNRT2.5?,and then a plant expression vector fused with GUS gene was constructed?PGhNRT2.5::GUS?and transfer it to Arabidopsis thaliana.GUS staining of the obtained homozygous offspring revealed that the gene was mainly expressed in tissues and organs of Arabidopsis anthers,pods,axillary buds,rosette leaves,and roots.According to the analysis of the cis-acting elements in the GhNRT2.5 promoter,it was found that there are multiple regulatory elements related to aging.Therefore,exogenous ABA was used to simulate the plant senescence process,and it was found that the Arabidopsis seedlings with exogenous ABA addition had different staining at the growth points of the plants in a short time compared with the control.This indicates that the GhNRT2.5 gene can respond to plant senescence.The leaves of different parts of Arabidopsis plants which had reached the full flowering stage were soaked with GUS dying solution overnight and found that the rosette leaves of Arabidopsis thaliana were most stained,and the stem and inflorescence leaves were stained lightly,implying that the GhNRT2.5 gene was expressed in the senescent relatively live leaves,which is consistent with the result that the GhNRT2.5 gene promoter can respond to the plant senescence process.4.GhNRT2.5 gene may play an important role in response to low nitrogen stress in cottonThe expression of endogenous NRT2.5 in cotton was controlled by molecular means,and an excess,interference,gene editing plant expression vector of GhNRT2.5was constructed and cotton genetic transformation was performed,and corresponding transgenic plants were screened and identified.By observing the phenotypes of existing cotton plants,it was found that the cotton that had undergone gene editing showed significantly yellow leaves than the other two types of cotton.The leaves of the same location of wild-type cotton growing at the same time were selected to measure their chlorophyll content.The content of chlorophyll b and carotenoids in cotton plants decreased compared with wild type.Then,the activity of nitrogen-metabolizing enzymes in the transgenic cotton was determined.It was found that the cotton activity of gene editing in glutamate synthase,nitrate nitrogen,and nitrite reductase was lower than that of wild type,while as increase in excess cotton plants.The above data showed that reducing the expression of GhNRT2.5 will reduce the nitrate absorption and chlorophyll synthesis in leaves.In addition,GhNRT2.5 can participate in the key processes of cotton nitrogen assimilation and translocation,which can improve cotton's ability to resist low nitrogen stress to a certain extent.