The Cloning And Functional Analysis Of NRT Genes From Chlorophytum Comosum

Nitrate transporters play a particularly important role in the absorption of nitrates by plants.It has been reported that the overexpression of nitrate transporter gene can significantly increase the nitrogen use efficiency and yield of crops.The cloning of nitrate transporter genes from specific biological resources is of great importance for the use of bioengineering technology to improve nitrogen use efficiency of crop,enhance plant nutrition resistance,increase production and reduce environmental pollution caused by excessive application of nitrogen and so on.Since the absorption and utilization of nitrogen is directly related to the growth rate of plants,this study first screened the growth rates of 20 species of fast-growing plants such as Anubias nana,Myriophyllum verticillatum L.,and Chlorophytum comosum.It was found that the growth rates of Chlorophytum comosum,Myriophyllum verticillatum L.,Japanese court grasses,and Eucheuma grasses were relatively fast,and the wet weights increased more.The wet weights of 0.5g robust stems increased by 56%,52%,72%and 84%respectively after 4 weeks of growth.In particular,it needs to be pointed out that Chlorophytum comosum has some features,for example:well-developed root system,rapid growth,strong growth capacity,and long-term hydroponic survival.Compared with other plants tested,hydroponics showed that the roots were soft,white,crystal clear,and grew stronger than soil culture.So the four low-affinity nitrate transporter genes have been successfully cloned from Chlorophytum comosum using the degenerate primers amplification techniques and RACE technology?all of them have applied for national invention patents?.The function and expression specificity of these NRT genes were studied.The main results include:1.We triumphantly cloned a new nitrate transporter gene from Chlorophytum comosum,named CcNPF8.3.1.The gene sequence is 2155 bp,which contains 1749bp complete open reading frame and encoding 582 amino acids.By comparison,the protein encoded by this gene has the highest homology with low affinity NRT in Capsicum annuum,Arabidopsis thaliana and Ananas comosus,with a concordance rate of 79.8%,75.0%,and 69.9%.The nucleotide sequence homology was 53.64%,43.79%and 67.74%.By transferring pYNR-CcNPF8.3.1 into Hansenula polymorpha which lacks the YNT1 gene,we found that defective yeast can restore normal growth.It explains that CcNPF8.3.1 has the function of transporting nitrates with the Km value of 1.1 mmol/L.Real-time PCR?q-PCR?analysis of the expression of the target gene in roots and leaves revealed that when the concentration of KNO₃ in the hydroponic solution was 2 mmol/L,the expression of CcNPF8.3.1 in the root was significantly higher than that in the leaves.It is 3.58 times that in the leaves.In addition,the expression level of this gene in roots and leaves was related to the concentration of KNO₃ in water culture solution.Compared with the addition of 0.5mmol/L KNO₃,when 2mmol/L KNO₃ was in the hydroponic medium,the target gene expressed was 4.12 times higher in roots and 3.23 times higher in leaves.2.Successfully cloned a new nitrate transporter gene CcNPF8.3.2 from Chlorophytum comosum using Touch-down PCR technology.The gene sequence is2314 bp and it contains 1629 bp complete open reading frame,encoding 543 amino acids.The highest homology was found with the low-affinity NRT sequences of Nelumbo nucifera,Ananas comosus and Capsicum annuum.The agreement rates were82.4%,82.2%,and 79.8%.The nucleotide sequence homologies were 57.41%,67.46%and 57.42%,respectively.Transfer of this gene into defect type yeast strains,it can restore the growth of the defective yeast,indicating that the gene has a nitrate transport function with a Km value of 1.1 mmol/L.The results of real-time PCR indicated that the expression of CcNPF8.3.2 was higher in the plant's roots,it is 15.51times the amount expressed in the leaves.Compared with the addition of 0.5 mmol/L KNO₃,the addition of 2 mmol/L KNO₃ in the hydroponic medium was 2.32-fold higher in the roots and 13.23-fold higher in the leaves.3.In this experiment we successfully cloned a new nitrate transporter protein encoding gene named CcNPF5.2 from Chlorophytum comosum.The gene is 2147bp in nucleotide sequence,containing the full open reading frame of 1656 bp for a 552amino acids polypeptide.The highest homology with the low-affinity NRT sequences of Gossypium hirsutum,Nicotiana tabacum and Nelumbo nucifera was 70.0%,68.6%,and 67.4%.The nucleotide sequence homology were 49.12%,49.41%and 56.08%,respectively.The gene was transformed into a deficient form of Hansenula polymorpha??35?ynt?,the transgenic yeast was still able to resume normal growth.It demonstated that the gene has a nitrate transport function with a Km value of 1.2mmol/L.Experiment results of gene expression levels indicated that CcNPF5.2 had a higher expression level in the roots and the expression of this gene was upregulated in2mmol/L nitrate.The expression level of the target gene was 1.14 times higher in roots and 1.89 times higher in leaves.4.As described in the experimentation,a full length CcNPF8.1 clone has been constructed,which contains 2076 bp.It contains a 1701bp complete open reading frame and encodes 567 amino acids.The protein encoded by this gene had the highest homology with those of low-affinity nitrate transporters from Ananas comosus,Gossypium hirsutum and Zea mays,which were 81.8%,75.9%and 75.7%,respectively.The nucleotide sequence homology was 75.51%,50.29%and 61.42%,respectively.pYNR-CcNPF8.1 was electroporated into the defective yeast,and the transgenic yeast was able to resume growth in the nitrate-containing medium,it indicated that the gene has a nitrate-transporting function with a Km value of 1.4mmol/L.This shows that the CcNPF8.1 gene is a low-affinity nitrate transporter gene.The results of real-time PCR showed that the expression level of CcNPF8.1 was higher in the roots,it is 5.59 times the amount expressed in the leaves.The expression of the gene was up-regulated in roots and leaves when it was induced by high concentration of nitrate?2mmol/L?.The gene of interest was 1.15 times higher in roots and 3.49 times higher in leaves.5.The plant expression vectors p35S::CcNPF8.3.1/pBI121,p35S::CcNPF8.3.2pBI121,p35S::CcNPF5.2/pBI121 and p35S::CcNPF8.1/pBI121 were respectively constructed,in which these genes were driven by the 35S promoter.These vectors were separately transferred into tobacco cultivar NC89 and Obtained 48,56,42 and 37 strains of 4 transgenic tobacco plants,respectively.Identification of tobacco bacterial resistance using T₁ generations of four transgenic tobacco plants.The disease index of the four transgenic tobaccos was found to be 81.11,84.44,77.78and 80.00,the disease index of wild type tobacco was 92.22.It indicates that the expression of NRT gene increased the nutritional resistance of the plant to some extent.