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CDNA of switchgrass tonoplast Na<+>/H<+> antiporter protein gene PvNHX1 and cloning method thereof

A technology of antiporter and tonoplast membrane, applied in DNA preparation, recombinant DNA technology, DNA/RNA fragments, etc., can solve unfavorable switchgrass vector construction, expression regulation and genetic transformation, and achieve the effect of great application value

Inactive Publication Date: 2015-07-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, so far the tonoplast Na + / H + The study of antiporter genes has not been reported in switchgrass, which is not conducive to the study of vector construction, expression regulation and genetic transformation of switchgrass

Method used

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  • CDNA of switchgrass tonoplast Na&lt;+&gt;/H&lt;+&gt; antiporter protein gene PvNHX1 and cloning method thereof
  • CDNA of switchgrass tonoplast Na&lt;+&gt;/H&lt;+&gt; antiporter protein gene PvNHX1 and cloning method thereof
  • CDNA of switchgrass tonoplast Na&lt;+&gt;/H&lt;+&gt; antiporter protein gene PvNHX1 and cloning method thereof

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Embodiment 1

[0057] (1) Homologous cloning

[0058] According to the tonoplast Na of switchgrass close relatives corn, rice, lemma, zoysia, barley and other plants registered in GenBank + / H + For the full-length sequence of the antiporter gene, DNAMAN software was used for homologous comparison to find the conserved region, and then the software Primer Premier5.0 was used to design primers, and the primers were synthesized by Shanghai Sangong. A total of 12 pairs of primers were designed, two of which had specific bands, and the pair with the best specificity was finally selected for the amplification of the conserved fragments.

[0059] The method for cloning the core fragment of switchgrass AtNHX1 gene comprises the following steps:

[0060] (1) Extract switchgrass total RNA and reverse transcribe it into cDNA;

[0061] (2) Use cDNA as a template to screen primers and optimize the PCR system;

[0062] (3) Recovery and purification of PCR products;

[0063] (4) The PCR product is co...

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Abstract

The invention provides cDNA of a switchgrass tonoplast Na<+> / H<+> antiporter protein gene PvNHX1, and is characterized in that the cDNA has a nucleotide sequence shown in SEQ ID No.1. The invention also provides a cloning method of the cDNA of the switchgrass tonoplast Na<+> / H<+> antiporter protein gene PvNHX1 according to the claim 1, wherein the method comprises the specific steps: (1) extracting total RNA of switchgrass, and carrying out reverse transcription into cDNA; (2) taking the cDNA as a template, and carrying out a high-fidelity full-length PCR reaction by a PvNHX1 gene full-length specific primer; and (3) recovering and purifying the PCR product of the step (2), carrying out an A addition reaction, and thus obtaining the cDNA of the switchgrass tonoplast Na<+> / H<+> antiporter protein gene PvNHX1. The method for obtaining the switchgrass PvNHX1 gene full-length cDNA sequence lays a foundation for vector construction, genetic transformation and the like by utilizing the sequence later on.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to switchgrass tonoplast membrane Na + / H + cDNA of antiporter gene PvNHX1 and its cloning method. Background technique [0002] Originating in North America, switchgrass is a perennial C4 herb. It has strong adaptability, high yield and resistance to barrenness, and is one of the energy crops with the most development potential. According to my country's current national conditions, the development of energy plants should be based on marginal land. The limited salt tolerance of switchgrass limits its planting range. Therefore, it is of great significance to screen out varieties with high stress resistance. Switchgrass is polyploid, and there are large genetic differences between the two ecotypes, so it is extremely complicated to screen varieties from a macro perspective. The proposed solution is to isolate and clone genes related to salt tolerance from switchgrass and perfor...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C12N15/11C12Q1/68
Inventor 张蕴薇黄艳华杨富裕刘斯佳杜娟路海博孙元元
Owner CHINA AGRI UNIV
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