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Promoter of cotton brassinosteroids synthetase GhDWF4 gene and application thereof

A technology of brassinosteroids and promoters, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems affecting plant growth and development

Inactive Publication Date: 2010-01-13
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using strong promoters such as CaMV35S to increase or decrease the expression level of the above genes will often affect the normal growth and development of plants and produce relatively unfavorable traits

Method used

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  • Promoter of cotton brassinosteroids synthetase GhDWF4 gene and application thereof
  • Promoter of cotton brassinosteroids synthetase GhDWF4 gene and application thereof
  • Promoter of cotton brassinosteroids synthetase GhDWF4 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1, Expression Analysis of GhDWF4 Gene in Cotton Plants

[0025] 1. Extraction of Cotton RNA

[0026] Select about 3g of fresh cotton material, quickly grind it into a fine powder in liquid nitrogen, put it into a 50mL centrifuge tube, add 15mL of 65°C preheated RNA extraction solution (2% (W / V) CTAB, 2% (W / V) PVP, 100mmol / L Tris-HCl (pH8.0), 0.5g / L spermidine (Spermidine), 2,0mol / L NaCl, 2% mercaptoethanol (V / V, add before use)), invert and mix . Water bath at 65°C for 3-10 minutes, during which time mix 2-3 times. Chloroform:isoamyl alcohol (24:1) extracted twice (10,000r / min, room temperature, 5min). Take the supernatant, add 1 / 4 volume of 10mol / L LiCl solution, place at 4°C for 6h, and extract 1 times (10,000r / min, room temperature, 5min). Add 2 times the volume of absolute ethanol, and precipitate at -70°C for more than 30 minutes. Centrifuge at 12,000r / min at 4°C for 20min, discard the supernatant. The precipitate was dissolved with 200 μL of DEPC-tr...

Embodiment 2

[0032] The cloning of embodiment 2, GhDWF4 gene promoter sequence

[0033] 1. Extraction of cotton genomic DNA

[0034] Cotton genomic DNA was extracted by the modified CTAB method (Xiao Yuehua and Luo Ming, Cotton Genome PCR Walk Using YADE Method. Acta Genetica Sinica 2002.29: 62-66). Select about 0.5g of young cotton leaves, quickly grind them into fine powder in liquid nitrogen, put them into a 10mL centrifuge tube, add 3mL of 65°C preheated CTAB extract (2% (W / V) CTAB, 2% (W / V) V) PVP, 100mmol / L Tris-HCl (pH8.0), 2.0mol / L NaCl, 2% mercaptoethanol (V / V, add before use)), invert and mix, 65°C water bath for 30min, during which time mix for 2 -3 times. Chloroform:isoamyl alcohol (24:1) extracted twice (10,000r / min, room temperature, 5min). Take the supernatant, add 2 / 3 volume of isopropanol pre-cooled at -20°C, mix well and let stand for 30 minutes. Pick out the flocculent precipitate, rinse with 75% ethanol three times repeatedly, and then rinse once with absolute ethan...

Embodiment 3

[0042] Embodiment 3, the acquisition of GhDWF4 gene promoter analysis transgenic cotton material

[0043] 1. Obtaining the sequence-deleted promoter fragment

[0044] Such as Figure 4 As shown, specific primers GhD4P (SEQ ID NO: 10), GhD4P1 (SEQ ID NO: 11), GhD4P2 (SEQ ID NO: 12), GhD4P3 (SEQ ID NO: 13) were designed according to the cloned GhDWF4 gene promoter sequence , GhD4P4 (SEQ ID NO: 14) and GhD4P5 (SEQ ID NO: 15), using the pBS-GhDWF4 promoter vector as a template, amplification conditions: 94°C pre-denaturation for 5min, 94°C, 30sec, 56°C, 30sec, 72°C , 2min30sec, 30 cycles; 72°C, 10min, PCR amplification to obtain 5 promoter sequence fragments of different lengths. These five promoter sequence fragments were marked as D4P1 (from 2135 to 2600), D4P2 (from 2025 to 2600), D4P3 (from 1488 to 2600), D4P4 (from 930 to 2600) and D4P5 (from 4 to 2600).

[0045] 2. Construction of a vector for fusion of GUS gene with a sequence-deleted promoter

[0046] The five amplifie...

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Abstract

The invention discloses a sequence or a segment of a promoter of a cotton brassinosteroids synthetase GhDWF4 gene. The promoter contains 2600 bases and can be used for adjusting and controlling a target gene to express in a special tissue organ in a plant body so as to further adjust and control the growth of the tissue organ and obtain good agronomic traits.

Description

technical field [0001] The present invention belongs to the field of plant genetic engineering, in particular, the present invention relates to the promoter of brassinosteroid synthase GhDWF4 gene and its fragment, the plant expression vector containing the promoter and its fragment, and the plant expression vector transformed A transformant and a method for preparing a transgenic plant containing the above-mentioned promoter and its fragment. Background technique [0002] Plant genetic engineering needs to obtain a variety of genetic factors that can regulate the expression of functional genes in specific tissues or parts. One major regulatory element is the promoter, which controls the initiation of transcription of a gene. Various promoters need to be used in plant genetic engineering. Among them, the promoter that controls the specific expression of the target gene during the formation of plant vegetative branches is beneficial to control the growth traits of the plant...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/84C12N1/21C12R1/01
CPCC12N15/8222
Inventor 罗明胡明瑜裴炎侯磊李先碧肖月华宋水清李德谋
Owner SOUTHWEST UNIV
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