Gossypium aridum WRKY transcription factor GarWRKY9 for regulating blossoming of plant and application

A transcription factor, plant technology, applied in the field of cotton transcription factor GarWRKY9

Inactive Publication Date: 2015-06-24
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Gossypium aridum WRKY transcription factor GarWRKY9 for regulating blossoming of plant and application
  • Gossypium aridum WRKY transcription factor GarWRKY9 for regulating blossoming of plant and application
  • Gossypium aridum WRKY transcription factor GarWRKY9 for regulating blossoming of plant and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1, the acquisition of GarWRKY9 gene

[0021] 1.1 Extraction of RNA

[0022] extract RNA

[0023] (1) Take 0.5g of fresh cotton tissue, add 0.1g of cross-linked polyvinylpyrrolidone (PVPP), fully grind to powder in liquid nitrogen, quickly transfer the frozen powder into a 10ml centrifuge tube, add 5ml of CTAB extract With 500 μL of 0.1M Tris-HCl of pH 8.0, bathe in water at 65°C for 20 minutes, and mix well by inverting halfway;

[0024] (2) Add an equal volume of chloroform to mix well, and let stand in an ice bath for 10 minutes;

[0025] (3) Centrifuge at 10,000 rpm for 20 minutes at 4°C. Divide into four 1.5ml centrifuge tubes;

[0026] (4) Aspirate the supernatant, add 1 / 3 volume of 8M LiCl and mix, -70°C for 30min or -20°C overnight;

[0027] (5) Centrifuge at 10,000 rpm for 20 minutes at 4°C. Discard the supernatant, wash twice with 70% ethanol, dry the precipitate and dissolve it in 30 μL DEPC water;

[0028] (6) Add 10U DNase without RNase act...

Embodiment 2

[0046] Embodiment 2, the construction of plant expression vector

[0047] 2.1 Construction of pCAMBIA2301-CaMV35S-GarWRKY22 plant expression vector

[0048] Plant expression vector pCAMBIA2301-CaMV35S plasmid (Feng Juan et al., 2013; Cloning and functional analysis of the protein kinase gene GarCIPK8 of the wild species of Gossypium upland). XbaI and KnpI were used to digest pCAMBIA2301-CaMV35S and the target gene fragment GarWRKY9 respectively, recover the large vector fragment and the target gene fragment, connect them with T4 ligase, and transform Escherichia coli Trans1-T1 competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd. Technology Co., Ltd.), and the plant expression vector with the gene of interest was obtained after the recombinant was identified.

[0049] Enzyme digestion of pCAMBIA2301-CaMV35S plasmid and target gene fragment

[0050] The plasmid double enzyme digestion system is as follows:

[0051]

[0052] Enzyme digestion at 30°C, rea...

Embodiment 3

[0070] Embodiment 3, preparation and transformation of Agrobacterium competent

[0071] 3.1 Preparation of Competent Agrobacterium EHA105

[0072] (1) Pick a single colony of EHA105, inoculate it in 5ml LB liquid medium, and culture overnight at 28°C with shaking at 200rpm until the OD600 value is 0.4;

[0073] (2) Inoculate in 400-500ml LB medium (in a 1L Erlenmeyer flask) at a ratio of 1:100, shake the bacteria until the OD600 is 0.6-0.8, and bathe in ice for 10 minutes;

[0074] (3) Collect the bacterial solution in a pre-cooled 50ml centrifuge tube, centrifuge at 5000rpm at 4°C for 5min;

[0075] (4) Discard the supernatant, fully suspend the precipitate with sterile water, centrifuge at 5000 rpm at 4°C for 5 min; repeat this process 3 times.

[0076] (5) Add 1 ml (depending on the number of bacteria) to the washed cells to resuspend the cells containing 10% sterile glycerol.

[0077] (6) Aliquot into 50 μL tubes, quick freeze in liquid nitrogen, and store at -80°C for ...

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Abstract

The invention discloses a transcription factor relevant to blossoming of gossypium aridum, namely gossypium wild species gossypium aridum WRKY gene GarWRKY9, which is characterized in that the transcription factor has a sequence as SEQ ID NO. 2 (Sequence Identifier Number 2) shown in a sequence table. The protein GarWRKY9 relevant to the blossoming of gossypium aridum is separated by using electronic cloning and RT-PCR (Reverse Transcription-Polymerase Chain Reaction) technologies, functional verification is conducted by transgenic arabidopsis, and the transcription factor is proven to be able to advance the blossoming of a plant.

Description

technical field [0001] The invention belongs to the technical field of biological gene engineering, and relates to a cotton transcription factor GarWRKY9 for regulating flowering of plants. Background technique [0002] Flowering is an important process for plants to transform from vegetative growth to reproductive growth. It is an important agronomic trait, which determines whether the crop is suitable for a specific cultivation area and growing season. Plant flowering is the key to the transition of plants from vegetative growth to reproductive growth, and it has strong plasticity. Under the influence of various external environments and internal factors, plants will choose to bloom at an appropriate time to achieve reproductive success. By adjusting the flowering period, the plants can delay or advance flowering, which can control the vegetative growth or reproductive growth of plants, avoid chilling damage or Harm to crops caused by other adversities, and at the same ti...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00
CPCC07K14/415C12N15/827
Inventor 郭琪沈新莲范昕琦徐鹏张香桂
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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