Cloning And Functional Study Of NRTs Gene

In this paper,aiming at the key problems of excessive application of nitrogen fertilizer in rice,increasing planting costs and serious environmental pollution,the nitrate transporter-encoding gene NRT was cloned from a variety of biological resources,and NRT transgenic rice was created to study the effect of the target gene on the morphology and yield of rice.And the interaction between rice endogenous genes at molecular level.The following studies have been carried out:1.The functional complementation test using defective yeastThere is only one nitrate transporter coding gene（YNT1）in the genome of Hansenula polymorpha.There was only one nitrate transporter gene（YNT1）in the genome of Hansenula polymorpha,which was unable to absorb nitrate in defective Hansenula polymorpha（（35）ynt Leu-）,and needed Leu in culture medium to grow and reproduce normally.In order to identify whether diatom CfNRT2.1 has the function of nitrate transporter,a yeast shuttle expression vector with yeast YNT promoter driving the expression of CfNRT2.1 gene was constructed and transformed into defective yeast（（35）ynt Leu-）to express CfNRT2.1.The genetically defective yeast regained the ability to absorb nitrate,indicating that CfNRT2.1 has the function of a nitrate transporter.2.Subcellular localization of CfNRT2.1A plant expression vector was constructed to express the CfNRT2.1-GFP fusion protein driven by p35S and transformed into Arabidopsis protoplasts.It was found that CfNRT2.1-GFP was specifically expressed on the protoplast membrane,indicating that the protein encoded by the CfNRT2.1 gene was a membrane protein.3.CfNRT2.1 significantly improves the biomass,100-seed weight and grain size of transgenic riceUnder the conditions of strictly controlled nitrogen fertilizer application,the test found that the chlorophyll of CfNRT2.1 transgenic rice and non-transgenic rice Yundao No.1 at the three-leaf and one-heart stage were 49.19±2.97 SPAD and 41.09±2.31 SPAD,The plant height was 24.36±3.49 cm and 22.1±2.33 cm.Compared with non-transgenic rice,the chlorophyll content of transgenic rice increased by 19.71%,the plant height increased by 9.96%.The 100-kernel weights of the mature seeds were 2.90±0.11 and 2.60±0.08,Compared with non-GM rice,transgenic rice rice has darker green and stronger leaves.and the kernel lengths were 7.60±0.13 mm and 7.27±0.22 mm.The 100-kernel weight increased by 11.36%and the kernel length increased by 4.53%.All the above results were three biological replicates,and the difference of analysis of variance was very significant.These results indicated that CfNRT2.1 could promote rice growth and grain development under low nitrogen condition.4.Transcriptome sequencing and molecular identification of differential genesUsing CfNRT2.1 transgenic rice and non-transgenic rice Yundao 1 with different growth stages（three-leaf and one-heart stage and tillering stage）and different treatments（KNO₃ KCl）as materials,transcriptome sequencing was performed,and differentially expressed genes were screened.Real-time fluorescent quantitative RT-PCR detection was carried out on some genes.5.Cloning of NRT genes of Echinodorus grisebachii Small and small Spirodela polyrrhiza SchleidUsing the roots of fast-growing aquatic plant Echinodorus grisebachii Small and small Spirodela polyrrhiza（L.）Schleid as materials,degenerate primers were designed,and 5’-Race,3’-Race and nested PCR were used comprehensively.Two nitrate transporter coding genes Sp NRT2.3 and Eg NRT1-family8.1 were cloned.Gen Bank search found that Sp NRT2.3 has the amino acid sequence of high affinity nitrate transporter with Spirodela intermedia,Colocasia esculenta,Dioscorea cayenensis subsp.Rotundata and Musa acuminata subsp.malaccensis.The similarity is the highest,88.30%-74.56%.Eg NRT1-family8.1 has the highest amino acid sequence similarity to the low-affinity nitrate transporter in Cocos nucifera,Phoenix dactylifera,Elaeis guineensis,Chlorophytum comosum,which is 78.11-79.33%.This is the first time that NRT genes have been cloned from these species,providing new genetic resources for obtaining nitrogen and efficient use of transgenic plants.6.Creation of CfNRT2.1,CcNPF1.1-FAMILY8.1 and GeNRT2.1 transgenic riceIn order to better compare the functions of Chlorophytum CcNPF1.1-Family8.1,Dwarf Pearl GeNRT2.1 and Diatom CfNRT2.1,using the rice japonica model variety Nipponbare as the recipient,these 3 genes were transferred into rice,and 3 were obtained respectively.PCR and Basta test strips of26,15 and 15 transgenic rice strains of T0 generation of two genes found that the target gene was successfully integrated into the transgenic rice genome and expressed.In the future,these genes will be used for nitrogen utilization in rice.More in-depth research is carried out on the impact of this.