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Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof

A technology of promoter and purpose, applied in the field of molecular biology, can solve the problem of few researches on the flowering regulation of Dendrobium officinale, and achieve the effect of reducing the consumption of resources and energy and accelerating the breeding process.

Pending Publication Date: 2021-12-10
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, so far, there are few studies on the regulation of flowering in Dendrobium candidum, and the study on the DoWOX4 gene promoter of Dendrobium candidum has not been reported.

Method used

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  • Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof
  • Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof
  • Dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining and structural analysis of the Dendrobium officinale promoter ProDoWOX4 sequence

[0035] Tissue culture seedlings of Dendrobium officinale Kimura et Migo were grown on the medium of 1 / 2MS+0.1% activated carbon+2% sucrose+0.6% agar powder (pH5.4), and placed in the tissue culture room for culture (conditions were 24±2°C, 12 hours light).

[0036] Extraction of Genomic DNA from Dendrobium officinale. In this experiment, the hand-held method was used to extract the genomic DNA of Dendrobium officinale. The experimental steps are as follows:

[0037](1) Grind the protocorm of Dendrobium officinale into powder in liquid nitrogen, quickly transfer it to a 2 mL centrifuge tube, add 700 μL of 2×CTAB extract (preheated at 65°C in advance), and mix thoroughly by inversion several times. The mixture was then placed in a water bath at 65°C for 15 minutes, during which time it was shaken every 5 minutes.

[0038] (2) Take out the mixed solution, cool it at ro...

Embodiment 2

[0055] Embodiment 2 constructs the plant expression vector containing promoter ProDoWOX4 sequence and vector transforms to Agrobacterium

[0056] The target expression vector used in this example is the binary expression vector pCAMBIA1301 (the vector contains 35S promoter element and GUS site, Kana resistance).

[0057] First, design primers to amplify the promoter ProDoWOX4 with pCAMBIA1301 vector adapter, the template is the extracted DNA of Dendrobium officinale, the high-fidelity enzyme used for amplification is Toyobo High-fidelity DNA Polymerase Kit (KOD FX), and the reaction system is 50 μL , see the instructions for the specific reaction system and operation steps. ProDoWOX4 promoter cloning primers with vector 1301 linker are:

[0058] ProDoWOX4-1301-FP1: 5'- CGGTACCCGGGGATCC TTTCTTATGATCTATGCTCC-3',

[0059] ProDoWOX4-1301-RP1: 5'- CCTCCAGATCTACCATGG ATGGAACTTGAGGCATCTTCC-3', the underline indicates the linker used to construct the vector. The PCR product was...

Embodiment 3

[0061] Example 3 pCAMBIA1301:ProDoWOX4:GUS for Arabidopsis genetic transformation and molecular identification

[0062] The Agrobacterium containing the plant expression vector obtained in Example 2 is introduced into Arabidopsis thaliana by the method of inflorescence infection, so as to realize the genetic transformation of Arabidopsis thaliana. The steps of Arabidopsis inflorescence infection method are as follows:

[0063] (1) Scribe and activate the target Agrobacterium liquid stored at -80°C, pick a single colony in LB liquid medium (Kana resistance), and place it on a shaker (28°C, 200rpm) for shaking overnight (approx. 16h), until the OD600 of the bacterial solution is about 0.8.

[0064] (2) Centrifuge the cultured Agrobacterium liquid (5000 rpm, 10 min) to collect the target bacteria.

[0065] (3) Prepare 100 mL of osmotic culture solution (1 / 2 MS+5% sucrose), and add 20 μL of surfactant Silwet L-77 to resuspend Agrobacterium.

[0066] (4) Take Arabidopsis thalian...

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Abstract

The invention discloses a dendrobium officinale flower tissue preference and stress inducible promoter ProDoWOX4 and application thereof, and belongs to the technical field of molecular biology. The promoter ProDoWOX4 provided by the invention has the length of 2003bp and can induce preference expression of a corresponding target gene in sepals and petals of floral organs, and due to the dominant expression, consumption of other resources and energy of plants can be reduced, and an appropriate regulation and control element is provided for construction of a plant genetic engineering expression vector; and the promoter has important significance on improvement and improvement of excellent characters such as salt tolerance or cold tolerance of plants, so that the breeding process of excellent varieties can be accelerated. The invention also provides a nucleic acid construct containing the promoter ProDoWOX4, a recombinant expression vector, a host cell, a genetic engineering cell and an amplification primer, and application of the substances.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a Dendrobium candidum flower tissue-preferring and stress-inducible promoter ProDoWOX4 and an application thereof. Background technique [0002] In eukaryotes, the transcription of gene expression is regulated by a promoter (Wray et al., 2003). The promoter contains a specific DNA sequence, which contains the binding site of the RNA polymerase protein complex, and plays a key regulatory role in the correct expression of the natural gene or the transgene. With the deepening of promoter research, it has been rapidly developed in the application of genetic engineering. Genetic transformation is an important method in plant breeding. From basic research to crop improvement, the use of appropriate promoters is a key factor to ensure the success of genetic transformation (Potenza et al., 2004, Porto et al., 2014). Therefore, the development and utilization of an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/84C12N15/11C12N1/21C12N5/10A01H5/02A01H6/20C12R1/01C12R1/19
CPCC07K14/415C12N15/8223C12N15/8273
Inventor 段俊曾丹琦何春梅
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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