| Most terrestrial plants use nitrate as the main nitrogen source,which is not only the nutrient element of plants,but also an important signal to regulate gene expression,plant growth and development.Rice is the crop that consumes the most nitrogen fertilizer in agricultural production.Improving the nitrogen utilization efficiency of rice and reducing the nitrogen fertilizer application on the premise of ensuring the yield and quality of rice can not only reduce the cost of rice planting,but also reduce the environmental pollution caused by eutrophication of water bodies.The main purpose of this study was to clone nitrate transporter from rapidly-growing aquatic plant Hygrophila stricta and transfer it into rice,so as to study the effect of the target gene on rice and its nitrogen use efficiency.In order to achieve this goal,the following studies were carried out:1.Identification and screening of fast-growing aquatic plantsTwenty-one fast-growing aquatic plants were collected and transplanted into glass bottles with 1 g of strong stems and branches from each plant.Net weight was measured every seven days for 28 consecutive days.In 28 days,the wet weight of Ludwigia repens,Hygrophila stricta,Lilaeopsis mauritiana,Rotala rotundifoliavar and Echinodorus amazonicus were increased 44%,46%,64%,52%and 46% respectively.Therefore,we decided to take these plants as the target for subsequent studies.2.Cloning of nitrate transporter gene from Hygrophila strictaTwo nitrate transporter coding genes,HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3,were cloned from Hygrophila stricta by Touch-down PCR and 5’Race and 3’Race.HsNRT1.1-Family 8.1gene encodes 569 amino acids with a molecular weight of 63.43 KD and a PI of 7.86.Sequence alignment showed that the gene had the highest similarities with NRT genes in pepper,tobacco,lotus root,soybean,chickpea,walnut and cotton,with 81.5%、81.1%、80.0%、78.8%、78.6%、77.9% and77.4% respectively.HsNRT1.1-Family 8.3 encodes 584 amino acids with molecular weight of 64.50 KD and isoelectric point of 5.77.It has the highest similarity with pepper,tobacco,walnut,cotton,lotus,soybean and eggplant,85.8%、83.9%、83.4%、83.2%、82.3%、81.2% and 81.0%,respectively.Both HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 are new genes,and they will be used to apply for patents and Gen Bank login numbers in the future.3.Response of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 to nitrate treatment and expression in different tissuesThe expression of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 in roots,stems and leaves of Hygrophila stricta treated with different concentrations of nitrate showed that the expression of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 in high concentration of KNO3 was significantly higher than that in low concentration KNO3 treatment.Tissue specificity analysis showed that HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 were mainly expressed in roots,with the characteristics of root > stem > leaf.4.The functions of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 were preliminarily validated by Hansenula polymorpha.The genome of Hansenula polymorpha has only one nitrate transporter coding gene(YNT1),and the defective Hansenula polymorpha(△ynt)can not grow normally in the presence of nitrogen.Therefore,it can be used to identify whether the coding protein of the cloned gene has the function of nitrate transporter belongs to low affinity or high affinity nitrate in classification.The function of nitrate transporters and coding proteins is strong or weak.In this study,we constructed shuttle expression vectors of yeast using target genes and transformed defective yeast.It was found that both HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 could restore the growth of defective yeast and possess nitrate transporter function.The Km value of HsNRT1.1-Family 8.3 was 1.28 mmol/L and that of HsNRT1.1-Family 8.1 was 1.27 mmol/L,indicating that both of them belong to low affinity nitrate translocation.Using this system,we compared the functions of 10 nitrate transporter proteins cloned in our laboratory:Arabidopsis thaliana At NRT1.1,Cylindrotheca fusiformis Cf NRT,Chlorophytum comosum Cc NRT8.3.1,Cc NRT8.3.2,Cc NRT5.2、Cc NRT8.1,Glossostigma elatinoides Ge NRT2.1,Ge NRT1.1,Hygrophila stricta HsNRT1.1-Family-8.1 and HsNRT1.1-Family-8.3,were transported into defective Hansenula polymorpha yeast.The growth curves of trapped yeast,wild yeast and recombinant yeast showed that the recombinant yeast constructed by nitrate transporter Cc NRT8.1 from Chlorophytum comosum grew fastest and entered the plateau stage in the shortest time.It was speculated that the coding protein of this gene had stronger nitrate transporting function.5.Transient expression of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 in Rice Callus.To study the molecular biological effects of target genes on nitrate uptake mechanism in rice,monocotyledon plant expression vectors p CAMBIA 3301-HsNRT1.1-Family 8.1,p CAMBIA3301-HsNRT1.1-Family 8.3 driven by ubiquitin promoter were constructed respectively.Agrobacterium-mediated transformation of rice callus was carried out for 7 days.The transient expression of GUS gene in rice callus was detected and HsNRT1.1-Family 8.1 and HsNRT1.1-Family8.3 were expressed.The expression of GUS gene was detected in rice with RT8.3 gene,but not in non-GM control.6.Real-time fluorescence quantitative RT-PCR to detect the effects of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 on endogenous genes in Rice.According to the existing literature,TGA1/4,NRT2,CIPK23,CPSF30,NRG2,NIA1 and Ni R play an important role in regulating the expression of low affinity nitrate transporters in plant nitrogen response.In order to study the effects of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 on rice,HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 genes were transfected into rice callus by Agrobacterium-mediated method,respectively,on 1 d,2 d,3 d,5 d and 7 d after infection.Real-time fluorescence quantitative RT-PCR was used to study the seven genes.The results showed that the expression of endogenous genes TGA1/4,NRT2.1,CPSF30,CIPK23,NRG2,NIA1 and Ni R in rice callus was significantly increased at 3 d compared with that in the blank vector p CAMBIA3301(Table3-4,Table 3-5,Figure 3-18).Compared with the control,the expression level of Os CPSF30,Os NRG2,Os TGA1/4,Os CIPK23,Os NIA1,Os NRT2.1 and Os Ni R increased 12.78,12.15,5.86,3.91,3.74,1.61 and 0.51 fold respectively at the 3rd day of rice callus transformation of HsNRT1.1-Family 8.1.The expression levels of rice endogenous genes NRG2,NIA1,CIPK23,CPSF30,TGA1/4,NRT2.1 and Ni R increased 9.77,8.03,7.83,7.64,6.41,1.41and1.19 fold respectively(Table 3-6)after 3 days of rice callus transformation.In addition,HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 had greater effects on CPSF30,NRG2,TGA1/4 and CIPK23 than NIA1 and NRT2.1,while HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 had less effects on NIA1 and NRT2.1.Since the expression of all genes increased significantly 3 days after the infection of Agrobacterium tumefaciens,this time node can be used as an important time node in subsequent experiments to study the effects of other genes on endogenous genes in rice.These results indicated that HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 genes from Hygrophila stricta could induce the up-regulation of rice endogenous genes after being transferred into rice callus.It was speculated that HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 genes could interact with these transcription factors and protein kinases in rice after being transferred into rice callus,and positively regulate the expression of CPSF30,NIA1,NRG2,TGA1/4 genes,thus participating in the expression of CPSF30,NIA1,NRG2 and TGA1/4 genes.Nitrogen use and nitrate signal transduction in rice affect the expression of other endogenous genes.Up to now,we have not obtained HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 transgenic rice,and the effects of HsNRT1.1-Family 8.1 and HsNRT1.1-Family 8.3 on nitrogen use efficiency of transgenic rice still need to be studied.Because nitrate transporters are widely involved in NO3-uptake,transport and intracellular redistribution,cloning nitrate transporter genes from a variety of biological resources,especially from plants that can grow rapidly under low nitrogen environment,is of great significance for improving nitrogen use efficiency of crops by genetic engineering. |
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