Gossypium hirsutum GhTCP2 gene promoter and application thereof

A technology of promoters and genes, applied in the field of genetic engineering, can solve problems such as the ability to resist biotic and abiotic stresses

Active Publication Date: 2014-08-13
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The Bt gene of the transgenic insect-resistant cotton currently on the market is driven by the 35S promoter of the cauliflower virus, which is expressed in various tissues of the plant, so that other tissues or organs that are not eaten by the cotton bollworm overexpress heterologous Bt gene, which may affect the growth and development of cotton, and the ability to resist biotic and abiotic stress

Method used

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  • Gossypium hirsutum GhTCP2 gene promoter and application thereof
  • Gossypium hirsutum GhTCP2 gene promoter and application thereof
  • Gossypium hirsutum GhTCP2 gene promoter and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Acquisition of cotton GhTCP2 gene promoter

[0031] The GhTCP2 gene promoter of the present invention is cloned from the commercially available upland cotton (Gossypium hirsutum) variety Xinluzao 13 by using the Genome Walking method. The central conserved sequence of GhTCP2 gene (its sequence is shown in SEQ ID No. 2), 5'RACE and 3'RACE flanking sequences amplified in our laboratory were spliced ​​with SeqMan software, and primer GhTCP2ProSP1 was designed according to the sequence obtained by splicing , GhTCP2ProSP2 and GhTCP2ProSP3. Using the total DNA of cotton genome as a template, the gene promoter region was amplified by three nested PCR reactions using the same AP primers of the Genome Walking Kit. After diluting the 1stPCR reaction solution by 1000 times, take 1 μL as the template of the 2nd PCR reaction to prepare the 2nd PCR reaction solution. After diluting the 2nd reaction solution by 1000 times, take 1 μL as the template of the 3rd PCR reaction ...

Embodiment 2

[0050] Example 2 Cotton GhTCP2 gene semi-quantitative PCR

[0051] 1. Use the Biotec Universal Plant Total RNA Extraction Kit (spin column type) to extract total RNA from the roots, stems, leaves, terminal buds, lateral buds, and flower buds of Xinluzao No. 13 cotton grown for 85 days. The specific operation steps are as follows:

[0052] (1) Take 100 mg of plant tissue and grind it quickly and fully in liquid nitrogen. During the grinding process, keep liquid nitrogen in the mortar.

[0053] (2) Transfer the homogenized sample into a 1.5mL RNase free centrifuge tube, add 1mL of lysate to mix by inversion, and incubate at 65°C for 5min to completely decompose the ribosomes. Centrifuge at 12,000 rpm for 10 min at 4°C, and transfer the supernatant to an RNase free filter column.

[0054] (3) Centrifuge at 10,000 rpm for 45 seconds at 4°C, and collect the filtrate in a collection tube.

[0055] (4) Add 1 volume of 70% ethanol to the collection tube and mix well, then transfer t...

Embodiment 3

[0082] Embodiment 3 Cotton GhTCP2 gene promoter transforms Arabidopsis thaliana

[0083] The GhTCP2 gene promoter was cloned into the plant expression vector pBI121 (gifted by Academician Li Jiayang, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) to obtain the expression vector pBI121-GhTCP2, which was amplified in Escherichia coli DH5α. Through the Agrobacterium-mediated transformation method, the GhTCP2 gene promoter carried by pBI121 was transformed into Arabidopsis thaliana, and a total of 20 transgenic Arabidopsis plants were obtained.

[0084] The process of cloning the gene into the vector, propagating the vector in E. coli DH5α, and Agrobacterium-mediated transformation is as follows:

[0085] 1. Construction of the carrier

[0086] Design upstream and downstream primers GhTCP2ProEBF and GhTCP2ProEBR according to the GhTCP2 gene promoter sequence, obtain the GhTCP2 promoter fragment by PCR, introduce EcoRV (blunt end) at the 5' end, intr...

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Abstract

The invention relates to a gossypium hirsutum GhTCP2 gene promoter and application thereof. The sequence of the gossypium hirsutum GhTCP2 gene promoter is shown as SEQ ID No. 1. The invention discloses that the GhTCP2 gene promoter can make specific expression in axillary buds and flower buds of plants (arabidopsis, gossypium hirsutum, etc.), can provide a promoter for specific expression of an insect-resistant gene in gossypium hirsutum axillary buds and flower buds, helps to reduce the negative impact on gossypium hirsutum resulted from insect-resistant gene expression in other tissues, and improve the insect-resistant ability of gossypium hirsutum under the premise of without influence on cotton yield.

Description

technical field [0001] The invention belongs to the field of genetic engineering, in particular to the application of cotton GhTCP2 gene promoter and tissue-specific expression pattern thereof. Background technique [0002] Cotton (Gossypium Hirsutum) is one of the most important economic crops in the world, and its fiber is the main raw material for the human textile industry. At the same time, cotton seeds and oilseeds are also indispensable excellent resources in people's daily production and life, and play an important role in animal husbandry and diesel production. However, there are many diseases and insect pests in cotton, especially insect pests, which directly affect cotton yield. Among them, cotton bollworm is an important borer pest in cotton bud and boll stage. It mainly eats buds, flowers and bolls, and also feeds on young leaves. This insect is the dominant species of cotton bollworm in China, and its damage has been rampant in recent years. It is very import...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/63C12N5/10A01H5/00
Inventor 李学勇邢进赵金凤刘伟娜
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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