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Transcription factor GhbHLH18 for expression of elongation period of cotton fibers and application thereof

A transcription factor, cotton technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., to achieve the effect of great application prospects

Active Publication Date: 2018-09-25
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether these transcription factors regulate cotton fiber growth and development remains to be further studied

Method used

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  • Transcription factor GhbHLH18 for expression of elongation period of cotton fibers and application thereof
  • Transcription factor GhbHLH18 for expression of elongation period of cotton fibers and application thereof
  • Transcription factor GhbHLH18 for expression of elongation period of cotton fibers and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Cloning of Example 1 Transcription Factor GhbHLH18

[0038] (1) RNA extraction and inversion of upland cotton (Gossypium hirsutum)

[0039] (1) RNA extraction of upland cotton

[0040] The laboratory uses the CTAB-LiCl method to extract cotton RNA to reduce the impact of secondary metabolites on RNA extraction, and then uses the column method to purify the RNA for subsequent experiments. The following are the specific experimental steps:

[0041] 1) Preparation: pipette tips, tubes, EP tubes, and glass bottles for containing reagents were soaked in 0.1% DEPC water for 8 hours and then autoclaved twice. The mortar, pestle, tweezers, and medicine spoon used in the experiment were baked in a blast drying oven at 150°C for 4 hours.

[0042] 2) Pipette 20mL RNA extraction working solution into a 50mL centrifuge tube, and put it in a 65°C metal water bath to preheat.

[0043] 3) Immediately put the collected experimental sample into a mortar filled with liquid nitrogen,...

Embodiment 2

[0068] Example 2: Construction of the pHELLSGATE12-GhbHLH18 vector by the Getaway method

[0069] (1) Acquisition of gene fragments containing attB linkers

[0070] First, gene primers added with an attB linker are designed, and the nucleotide sequences of the primer pair are shown in SEQ ID NO.4 and SEQ ID NO.5. The fragment recovered in Example 1 was used for PCR amplification with primers added with an attB linker to obtain a gene fragment containing an attB linker. The PCR product is subjected to gel electrophoresis, and the gel is cut and recovered to obtain the fragment obtained by PCR.

[0071] (2) BP reaction construction entry vector

[0072] The entry vector was constructed using BP Clonase from Invitrogrn. The reaction system is as follows:

[0073]

[0074] React at 16°C for 2 hours to construct the entry vector.

[0075] (3) LR reaction constructs interference carrier

[0076] The carrier is pHELLSGATE12vector. The enzyme digestion system is as follows...

Embodiment 3

[0079] Embodiment 3: Obtaining of GhbHLH18 transcription factor RNAi plants

[0080] 1) Transform Agrobacterium with pHELLSGATE12-GhbHLH18 vector

[0081] The interference vector sequenced correctly in Example 2 was selected, and the interference vector was extracted for subsequent Agrobacterium transformation. Similar to transforming E. coli. Take 10 μL of the interfering carrier and add it to 100 μL of Agrobacterium GV3101 competent cells, mix gently and then let stand on ice for 30 minutes. Immediately after liquid nitrogen treatment for 3 minutes, heat shock at 37°C for 10 minutes, followed by ice bathing for 1 to 3 minutes. Add 800 μL LB non-resistance liquid medium, and incubate on a shaker at 180 rpm at 28°C for 3 to 5 hours. After centrifuging at 4000rpm for 3 minutes, discard most of the supernatant, suspend the bacteria and spread evenly on a solid LB resistance plate, incubate at 28°C for 2-3 days.

[0082] The monoclonal bacteria were cultured in the resist...

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Abstract

The invention provides a transcription factor GhbHLH18 for expression of the elongation period of cotton fibers and application thereof, belongs to the field of plant molecular biology and gene engineering and discloses a transcription factor GhbHLH18 related to the development of the cotton fibers and playing an important role in the fiber-elongation process and application thereof. The nucleotide sequence of the transcription factor GhbHLH18 is shown in SEQ ID NO.1. The transcription factor GhbHLH18 has the following characteristics that (A) the expression condition in cotton tissues and fibrocytes in the fiber-elongation process is very clear, and the transcription factor is not reported on plants such as cotton; (B) the transcription factor provided by the invention plays an importantrole in adjustment in the cotton fibers-elongation process and has an influence on the quality of the cotton fibers.

Description

technical field [0001] The present invention relates to the fields of plant molecular biology and genetic engineering, in particular to a transcription factor GhbHLH18 expressed during cotton fiber elongation and its application, more specifically to a transcription factor that can be expressed in cotton fibers and affect the quality of cotton fibers GhbHLH18 and the application of the transcription factor in plant genetic improvement. Background technique [0002] Cotton fiber is the most important natural textile raw material. Studying the characteristics of fiber differentiation and development will help to explore the characteristics of early fiber differentiation and development, and provide a basis for genetic improvement and breeding. Cotton fiber is essentially a single-cell structure developed and differentiated from the epidermal cells of the ovule located in the ovary. Cotton fiber is mainly composed of polysaccharides (cellulose, hemicellulose), lignin, cell wal...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/10C12N15/82C12N15/66A01H5/00A01H6/82
CPCC07K14/415C12N15/1096C12N15/66C12N15/8261C12Q2521/107
Inventor 左开井高正银孙文杰宋晓云
Owner SHANGHAI JIAO TONG UNIV
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