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Rape sodium-hydrogen pump transport protein coding sequence and application thereof

A technology of sequence and coding, applied in the field of preparation of the protein and nucleic acid sequence

Inactive Publication Date: 2003-11-12
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Before the present invention was published, there was no disclosure or report of the Brassica napus Na mentioned in this patent application. + / H + Antiporter protein sequence and its nucleic acid sequence

Method used

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  • Rape sodium-hydrogen pump transport protein coding sequence and application thereof
  • Rape sodium-hydrogen pump transport protein coding sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Brassica napus Na + / H + Cloning of antiporter gene

[0043] 1. Tissue separation (isolation)

[0044] Brassica napus was obtained from Sichuan University, and the leaf material of Brassica napus was immediately frozen in liquid nitrogen. .

[0045] 2. RNA isolation (RNA isolation)

[0046] Take part of the tissue, grind it with a mortar, add it to a 50ml tube filled with lysate, shake it fully, and then transfer it into a glass homogenizer. After homogenization, transfer to a new 50ml tube and extract total RNA (TRIzol Reagents, Gibco, NY, USA). The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis.

[0047] 3. Cloning of Full-length cDNA

[0048] According to Arabidopsis Na + / H + Antiporter amino acid conservative sequence, design primers, use the principle of homologous gene cloning, and use the RACE method (GibcoBRL kit)

[0049] Perform full-length cDNA cloning in three stages:

[0050] (1) 3′-RACE

[0051] 2001BN3' (...

Embodiment 2

[0059] Brassica napus Na + / H + Sequence information and homology analysis of antiporter gene

[0060] The new Brassica napus Na of the present invention + / H + The full-length cDNA of antiporter is 1819bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at nucleotides 169-1716. Deduction of Brassica napus Na + / H + The amino acid sequence of antiporter has a total of 515 amino acid residues, a molecular weight of 57148 Daltons, and an isoelectric point (pI) of 6.30. See SEQ ID NO.4 for the detailed sequence.

[0061] Brassica napus Na + / H + The full-length cDNA sequence of antiporter and its encoded protein were searched for nucleotide and protein homology in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases using BLAST program , it was found that it was related to Arabidopsis Na + / H + antiporter has 89% identity at the nucleotide level (S...

Embodiment 3

[0063] Brassica napus Na + / H + Eukaryotic cell expression of antiporter protein or polypeptide in tobacco cells and salt tolerance identification of transgenic plants

[0064] Contains the gene of interest (Brassica napus Na + / H + antiporter gene) expression vector construction

[0065] According to Brassica napus Na + / H + The full-length sequence (SEQ ID NO.3) of antiporter, design the primer that amplifies the complete coding reading frame, and respectively introduce restriction endonuclease sites on the forward and reverse primers (this may depend on the vector selected), In order to construct the expression vector. Using the amplified product obtained in Example 1 as a template, after PCR amplification, Brassica napus Na + / H + The antiporter cDNA is cloned into an intermediate vector (such as pBluescript), and further cloned into a binary expression vector (pBI121, Jefferson R.A., 1987, Plant Molecular Biology Reporter 5: 387-405.), and the expression vecto...

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Abstract

The present invention provides a novel Na+ / H+ antiporter protein expressed in the rape, coding sequence and its application for improving adverse resistance (salt-resistance and alkali-resistance) of plant. Said invention relates to the fusion gene construction body containing the described gene and new recombinant expresion vector with said construction body, also relates to the plant cell converted by said expression vector and the transgenic plant of the described gene produced by converted cell and its progeny, including plant seed and plant tissue. Said invention can make said gene be expressed in plant, and the obtained transgenic plant has enhanced adverse-resistance.

Description

technical field [0001] The invention relates to the fields of molecular biology, enzymology, physiology, genetic engineering and the like. Specifically, the present invention relates to a Na + / H + antiporter protein (Brassica napus Na + / H + antiporter, BnNHX1) and its nucleic acid sequence. The present invention also relates to the preparation method and use of the protein and nucleic acid sequence. Background technique [0002] In recent years Na + / H + The application research of antiporter (sodium hydrogen pump transporter) in biology is developing rapidly, and its biggest feature is that it can convert Na + Efflux is pumped into the vacuole "reservoir" and is associated with the alkalinization of the vacuole, and this protein plays an important role in restoring cellular ion balance (Plant Physiol, 1985, 78: 163-167). [0003] In the present invention, we cloned Arabidopsis Napus from Brassica napus + / H + Antiporter gene homologue BnNHX1. [0004] Befor...

Claims

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Application Information

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IPC IPC(8): A01H1/00C07K14/415C12N15/29C12N15/63
Inventor 唐克轩王劲左开井孙小芬
Owner FUDAN UNIV
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