Vegetable leaf vein specific promoter and use thereof

A promoter and specific technology, applied in the field of plant genetic engineering, can solve problems such as silencing or co-suppression, and achieve the effect of wide application value

Active Publication Date: 2012-08-08
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, repeated use of the same promoter to drive two or more foreign genes may cause gene silencing or co-suppression (Kumpatla et al., 1998)

Method used

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  • Vegetable leaf vein specific promoter and use thereof
  • Vegetable leaf vein specific promoter and use thereof
  • Vegetable leaf vein specific promoter and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Vein-specific VS promoter cloning

[0035] First, the 1107bp VS promoter was amplified by PCR method using Arabidopsis genomic DNA as a template, and the amplified product was recovered and TA cloned.

[0036] (1) PCR amplification of the target fragment

[0037] Specific primers were designed according to the known sequence of the Arabidopsis VS promoter region, a BamHI restriction site was introduced into the upstream primer, and an Nco I restriction site was introduced into the downstream primer.

[0038] Upstream primer: 5'-AC GGATCC GAACTAGGTCTAGTCACGTGGT-3' (BamHI cutting point introduced)

[0039] Downstream primer: 5'-AC CCATGG GCTGTGTCAATTCTCACTTCTT-3' (introduce Nco I cutting point)

[0040] Genomic DNA of Arabidopsis thaliana was extracted according to a conventional method, and the genomic DNA was used as a template to perform PCR amplification using the above primers to prepare a VS promoter fragment.

[0041] PCR reaction system:

[0042]...

Embodiment 2

[0058] Example 2: Utilize pCAMBIA1301 vector (containing GUS reporter gene) to construct "VS promoter-GUS" fusion gene

[0059] (1) Extract vector pCAMBIA 1301 plasmid from Escherichia coli (the strain can be purchased from biological company or CAMBIA), and recover the large vector fragment (which contains the GUS reporter gene sequence) after double digestion with BamH I / Nco I.

[0060] (2) A plasmid was extracted from the TA clone prepared in Example 1, double digested with BamH I / Nco I, and the VS promoter fragment (same as in Example 1) was recovered by agarose gel electrophoresis.

[0061] (3) The above two fragments were ligated overnight at 16° C. under the catalysis of ligase to complete the construction of the “VS promoter-GUS” fusion gene on the pCAMBIA 1301 vector.

[0062] Connection system:

[0063]

[0064] (4) Transform Escherichia coli DH5α competent cells with the ligation mixture, the method is the same as in Example 1.

[0065] (5) Select the white col...

Embodiment 3

[0071] Embodiment 3: Preparation of transgenic Arabidopsis

[0072] (1) Transform Arabidopsis thaliana with the "VS promoter-GUS" fusion gene constructed in Example 2. The specific transformation method adopts the Floral infiltration (Tague, 2001) method mediated by Agrobacterium, and the obtained seeds are subjected to 50mg / L hygromycetes Plants with normal growth were screened for plant resistance and transferred to soil for culture.

[0073] (2) PCR detection of transgenic plants: cut the leaves of transgenic plants and wild-type plants respectively, and extract genomic DNA from leaves with reference to the method of "Molecular Cloning Experiment Guide (Third Edition)" (Huang Peitang et al., 2002), and carry out PCR with the following primers Reaction, reaction system is as embodiment 1:

[0074] Upstream primer: 5'-ACGGATCCGAACTAGGTCTAGTCACGTGGT-3'

[0075] Downstream primer: 5'-TCGCGATCCAGACTGAATGCC-3'

[0076] The PCR product was subjected to agarose gel electrophores...

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Abstract

The invention discloses a vegetable leaf vein specific promoter and use thereof. With a nucleotide sequence shown by the sequence table 1, the promoter can promote the expression of a target gene in vegetable leaf veins. In the invention, a VS promoter sequence is cloned, and a VS promoter is proved to have unique vein specific activity in arabidopsis thaliana by detecting the expression levels of a 'VS-promoter-GUS' fusion gene in organs of the transgenic arabidopsis thaliana; and meanwhile, the transformation of micro tomato Micro-Tom by the 'VS-promoter-GUS' fusion gene further proves thatthe 'VS-promoter-GUS' fusion gene is specifically expressed only in the veins of transgenic tomatoes. The promoter of the invention is used to construct a 'promoter-target gene to be expressed' fusion gene, and through phytotransformation transgenic plants in which the target genes are specifically expressed in veins can be obtained. The promoter can be used for studying vegetable vascular tissuedifferentiation, solute translocation mechanism and the like and has an important value in study and use of plant genetic engineering.

Description

【Technical field】: [0001] The invention belongs to the field of plant genetic engineering, in particular to obtaining a new plant vein-specific promoter by using molecular biology technology and its application in transgenic plants. 【Background technique】: [0002] Promoter is a specific DNA sequence that directly combines with RNA polymerase and transcription factors to determine whether gene transcription starts or not. In general, a structural gene consists of three parts: a promoter, a coding region, and a terminator. The coding region determines the structure of the protein, and the terminator determines the length of the mRNA. The promoter includes a core promoter region and a regulatory region. The core promoter region produces a basic level of transcription. The regulatory region can respond to different developmental and environmental signals and make corresponding adjustments to the expression level of genes. [0003] The expression of eukaryotic genes, as well a...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/10A01H5/00
Inventor 王宁宁金凤李姝
Owner NANKAI UNIV
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