Tea tree NRT1 gene, protein and gene expression method

A technology of gene expression and tea tree, applied in the field of molecular biology, can solve the problem of lack of research on the characteristics of nitrogen absorption transporter

Inactive Publication Date: 2016-03-09
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The physiological characteristics of tea tree absorption and utilization of nitrogen have been studied, but the research on the characteristics of nitrogen absorption transporters is still relatively lacking. The research on the NRT2 family genes of tea trees has been reported (Feng Suhua et al., 2014; Wang Jin et al., 2014), while the research on tea tree There are very few reports on NRT1 family molecules

Method used

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  • Tea tree NRT1 gene, protein and gene expression method
  • Tea tree NRT1 gene, protein and gene expression method
  • Tea tree NRT1 gene, protein and gene expression method

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Experimental program
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Embodiment 1

[0021] The tea tree NRT1 gene expression method, its steps include:

[0022] (1) Synthesize primer sequences NRT1F and NRT1R, wherein the sequence of NRT1F is shown in SEQ ID No. 3, and the sequence of NRT1R is shown in SEQ ID No. 4;

[0023] (2) Real-time quantitative PCR amplifies the full-length sequence of tea tree NRT1 gene, the PCR system reaction system is 25μL, of which 10×Taqplusbuffer2.5μL, 25mMMgCl 2 1.5μL, 10mM dNTP1μL, 10μM forward primer 1μL, 10μM reverse primer 1μL, template 1μL, sterilized H2O16.75μL, 5U / μL TaqplusDNApolymerase 0.25μL; PCR reaction conditions are: 94℃5min, 94℃30s, 54℃30s , 72℃60s, 30 cycles; 72℃10min; the reaction product was recovered by 1.0% agarose gel electrophoresis, the PCR amplification electrophoresis chart is shown figure 1 ;

[0024] (3) The recovered product is connected to the pMD18-T vector and transformed into E. coli DH5a;

Embodiment 2

[0026] Extract RNA from tea plant roots and leaves for real-time fluorescence quantitative RT-PCR analysis, the results are as follows figure 2 As shown, tea tree is affected by NO 3 - NRT1 expression in roots and leaves decreased significantly after treatment for 5 minutes. The expression level of roots was always lower than that of the control, increasing after 48h and reaching the highest value at 96h. After half an hour, the expression level of NRT1 in the leaves was significantly higher than that of the control, and thereafter the rule of "decrease-rise-decrease" was maintained.

[0027]

[0028]

[0029]

[0030]

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Abstract

The invention discloses a tea tree NRT1 gene, a sequence of which is shown as SEQ ID No.1. The invention also discloses a tea tree NRT1 protein, a sequence of which is shown as SEQ ID No.2. The invention also discloses a gene expression method of the tea tree NRT1. A total length of the tea tree NRT1 gene sequence is 1880bp, 594 amino acids are encoded, the protein molecular weight is 65.92kD, and a theoretic isoelectric point is 9.07043. 12 transmembrane helical regions are contained, wherein a large hydrophilic ring exists between a sixth transmembrane helical region and a seventh transmembrane helical region. The protein is a hydrophilic protein, and the tea tree NRT1 is a constitutive gene. The mechanism for absorbing and assimilating NO3<-> in different tissue parts of a tea tree is shown to be possibly different, and the NRT1 is not only used as a nitrate transfer protein, but also is a signal acceptor sensing the nitrate.

Description

Technical field [0001] The invention relates to a tea tree NRT1 gene, protein and gene expression method, belonging to the field of molecular biology. Background technique [0002] Tea tree [Camelliasinensis (L.)] is a perennial woody plant native to China. It uses leaves as the harvest target and has a large demand for nitrogen. The amount of nitrogen fertilizer applied is closely related to the content of tea quality components, amino acids and tea polyphenols. Therefore, nitrogen fertilizer is the main fertilizer in tea gardens, usually accounting for more than half of the total fertilizer application (Kamauetal., 2008). Tea plants have partial absorption characteristics for different nitrogen sources. The amount of ammonium nitrogen absorbed is more than ten times higher than that of nitrate nitrogen. Studies have shown that the main reason for this difference lies in the difference in absorption and delivery systems (Du Xuhua and Peng Fangren, 2010; Yangetal., 2013). So st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/70
Inventor 杨亦扬王枫胡雲飞万青李荣林阮建云
Owner JIANGSU ACAD OF AGRI SCI
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