Function Analysis Of The Nitrate Transporter Gene SiNRT1.1Bs In Foxtail Millet

Nitrogen is an essential element in the process of crop growth and development,and it is also the main factor limiting crop yield and quality.In agricultural,farmers use a lot of fertilizer to increase crop yield.However,the large application of N fertilizers not only increase the cost of farmers but also causes severe environmental and ecological pollution such as the eutrophication of marine and fresh water ecosystems and the gaseous loss of N to the atmosphere.Therefore,increasing plant N use efficiency（NUE）is essential for the development of sustainable agricultural.NRT1.1B gene is involved in the absorption,transport and perception of nitrate,and is a key gene for plant nitrogen absorption and utilization.We found that there are two copies of NRT1.1B gene in foxtail millet,namely SiNRT1.1B1（Si9g32650）and SiNRT1.1B2（Si9g32660）,whereas there is only one copy of NRT1.1B in Arabidopsis,rice and sorghum.Based on this speculation,the high-efficiency nitrogen absorption and utilization characteristics of foxtail millet may be related to the copy number of the NRT1.1B gene.The function of SiNRT1.1Bs gene was studied,which laid the foundation for revealing the molecular mechanism of foxtail millet tolerance to low nitrogen and nitrogen-efficient breeding.The main results of this study are as follows:1、NRT1.1B1 and NRT1.1B2 of foxtail millet were successfully transferred to NRT1.1 gene loss-offunction mutant atnrt1.1 by inflorescence infusion.Thirty T₁ ransgenic Arabidopsis plants were obtained,after two generations of screening,and finally two NRT1.1B1 transgenic Arabidopsis homozygous lines were obtained;Thirty-three NRT1.1B2 transgenic Arabidopsis plants of the T₁ generation were obtained,and finally three NRT1.1B2 transgenic Arabidopsis homozygous lines were obtainedwhich provided experimental materials for the later study of SiNRT1.1B gene function.2、NRT1.1 can also bind and transport nitrate homologue chlorate,but chlorate has a toxic effect on plant growth and development.The Arabidopsis atnrt1.1 mutant is less sensitive to chlorate poisoning.We studied the susceptibility of transgenic Arabidopsis to chlorate toxicity and the changes in root growth induced by different concentrations of nitrate.The results showed that both the foxtail millet SiNRT1.1B1 and SiNRT1.1B2 genes have the ability to transport nitrates,which can restore the Arabidopsis sensitivity of mustard atnrt1.1 mutants to chlorate.3、In order to define the subcellular location of the two SiNRT1.1Bs genes in foxtail millet,163 GFPSiNRT1.1B1 and 163GFP-SiNRT1.1B2 fusion vectors were constructed and transferred to Arabidopsis protoplasts.The subcellular localization results indicated that both nitrate transporters SiNRT1.1B1 and SiNRT1.1B2 were localized on the cell membrane.4 、 Hansenula polymorpha nitrate transporter gene YNT1 loss-of-function mutant Δynt can not transport and absorb nitrate when the nitrate concentration is lower than 500 μM,which resulting in failure to grow normally.In order to verify whether the foxtail millet SiNRT1.1B1 and SiNRT1.1B2 genes can transport nitrate,they were transferred to the Hansenula polymorpha mutant Δynt-leu,and genetic complementation experiments were performed.In YGNL medium with 0.5 m M potassium nitrate as the only nitrogen source,the growth of the two-gene transgenic yeast was basically the same as that of the wild type.Further chlorate sensitivity tests showed that SiNRT1.1B1 and SiNRT1.1B2 genes can restore the sensitivity of yeast to chlorate.These results indicate that both SiNRT1.1B1 and SiNRT1.1B2 genes in foxtail millet have nitrate transport functions.5、We transferred the SiNRT1.1B1 and SiNRT1.1B2 genes of foxtail millet into rice.We used RT-PCR to detect the expression level and obtained two SiNRT1.1B1 transgenic lines and five SiNRT1.1B2 transgenic rice lines,which are for follow-up functional research provides test materials.In order to further reveal the influence of the copy number of the SiNRT1.1Bs gene on the nitrogen absorption and transport efficiency of foxtail millet,we constructed two CRISPR/Cas9 gene editing vectors targeting the SiNRT1.1B1 gene,p HUE411-NRT1.1B1-1 and p HUE411-NRT1.1B1-2,two CRISPR/Cas9 gene editing vectors p HUE411-NRT1.1B2-1 and p HUE411-NRT1.1B2-2 targeting SiNRT1.1B2 gene,two CRISPR/Cas9 targeting SiNRT1.1B1 and SiNRT1.1B2 genes simultaneously gene editing vector p HUE411-NRT1.1Bs-1 and p HUE411-NRT1.1Bs-2.Then,they were transformed into foxtail millet protoplasts and tested for gene editing.The results showed that the six gene editing vectors all had shearing activity,which will be used for foxtail millet transformation research.This study proved that both SiNRT.1.1B1 and SiNRT1.1B2 in foxtail millet have nitrate transport functions through Arabidopsis and yeast genetic complementation experiments.The subcellular localization results showed that both SiNRT1.1B1 and SiNRT1.1B2 were localized in the cell membrane,which further implied that both SiNRT.1.1B1 and SiNRT1.1B2 in foxtail millet had the function of nitrate transport.In addition,the expression levels of SiNRT1.1B1 and SiNRT1.1B2 in transgenic rice were tested.And six gene editing vectors were successfully constructed,which will be used for subsequent foxtail millet genetic transformation.In summary,the genes SiNRT.1.1B1 and SiNRT1.1B2 in foxtail millet have the function of nitrate transport,which lays the foundation for further gene function research.