Flower-specific expression vector of cucumber CsLOX1 gene and application of flower-specific expression vector

An expression vector and plant expression vector technology, applied in the field of cucumber CsLOX1 gene flower-specific expression vector, can solve the problem of few functional reports and the like

Inactive Publication Date: 2015-03-25
JIANGXI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research focus of lipoxygenase gene mainly focuses on its participation in the regulation of aging and st...

Method used

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  • Flower-specific expression vector of cucumber CsLOX1 gene and application of flower-specific expression vector
  • Flower-specific expression vector of cucumber CsLOX1 gene and application of flower-specific expression vector
  • Flower-specific expression vector of cucumber CsLOX1 gene and application of flower-specific expression vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1: Construction of flower-specific expression promoter expression vector 1301-AP3

[0021] (1) Primer design: Refer to Hill (1998) and Duan (2008), download the flower-specific promoter AP3 promoter and its 5' end sequence (GI: 223046640) from Genbank Data, take 726 before the transcription start site bp, designed Arabidopsis flower-specific promoter AP3-specific primers:

[0022] AP3-F: 5'-aaaGGATCCAACTTGTGATTCCTTCTTAA-3' (with BamHI site)

[0023] AP3-R: 5'- aaaCTGCAGATTCTTCTCTCTTTGTTTAATC -3' (with PstI site)

[0024] (2) Extraction of Arabidopsis genome

[0025] The Arabidopsis used was Columbia The wild-type Arabidopsis was preserved by the Key Laboratory of Crop Physiology, Ecology, Genetics and Breeding of Jiangxi Agricultural University. Add 1 ml of 75% alcohol to the 1.5 ml centrifuge tube containing Arabidopsis seeds, sterilize for 5 minutes, and invert the centrifuge tube up and down to make the seeds fully contact with the alcohol. A...

Embodiment 2

[0054] Example 2 : 1301-AP3-CsLOX1 transfection Agrobacterium GV3101

[0055] (1) Preparation of Agrobacterium Competent Cells

[0056] Pick a single colony of Agrobacterium GV3101 and inoculate it in 5ml of YEB medium, shake it overnight at 28°C, inoculate it in 50 ml of YEB medium at a ratio of 1:100, and inoculate it at 28°C for about 6-7h until OD600=0.4 -0.6. Place the bacterial solution on ice for 30 minutes; centrifuge at 5,000 rpm at 4°C for 5 minutes, discard the supernatant, and suspend the bacterial cells in 10 ml of 0.15 M NaCl; centrifuge at 5,000 rpm at 4°C for 5 minutes, discard the supernatant, and use 1 ml 20 mM CaCl 2 , 4°C) gently suspend, aliquot 200μl per tube, or add sterile glycerol with a final concentration of 20%, and store at -70°C.

[0057] (2) Transformation and identification of Agrobacterium

[0058] Add 10 μl of plasmid DNA to 200 μl of Agrobacterium competent, mix well, ice-bath for 30 minutes, freeze in liquid nitrogen for 3-5 minutes, b...

Embodiment 3

[0059] Example 3: Transformation of Arabidopsis thaliana with Agrobacterium GV3101 containing 1301-AP3-CsLOX1

[0060] (1) Planting of Arabidopsis

[0061] ①The seeds harvested in the current year were vernalized for 72 hours at 4 degrees after planting, and the seeds in the next year were vernalized for 24 hours after planting. Then they were transferred to an artificial culture room at a relative humidity of 80%, a constant temperature of 20-24°C, a light intensity of 80-200 μmol / M2 / S, and a light cycle of 8 hours in the dark and 16 hours in the light. The soil used was a mixture of 3 parts vermiculite, 1 part perlite and 2 parts black soil.

[0062] ②Put the nutrient soil in a plastic pot, add nutrient solution into the tray, and start planting after the nutrient soil absorbs water and becomes moist.

[0063] ③ Put the Arabidopsis seeds on the flat paper, and use a toothpick to spot the Arabidopsis seeds on the soil. Cover it with a plastic wrap, and cultivate it in th...

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Abstract

The invention discloses a flower-specific expression vector of a cucumber CsLOX1 gene, an application of the flower-specific expression vector in transformation of floral organs and belongs to the technical field of biology. The vector is a plant expression vector containing arabidopsis thaliana flower-specific promoter AP3 and the cucumber CsLOX1. By expressing the CsLOX1 gene in arabidopsis thaliana, the obtained CsLOX1 transgenic plant has closed petals, the development of petals is incomplete or is deleted, stamens are degenerated, the number of pistils is increased, ovules and stigmas which abnormally and ectopically grow are obviously visible, which indicates that CsLOX1 plays an important regulatory role in the development of floral organs of cucumber. The novel material prepared through the CsLOX1 gene and capable of varying the floral organs can be applied to breeding of crops and ornamental plants.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a flower-specific expression vector of cucumber CsLOX1 gene and application thereof. Background technique [0002] Lipoxygenase (LOX) is a kind of non-heme ferritin widely present in animals and plants. It participates in the LOX pathway in the fatty acid oxidation pathway, and specifically catalyzes the enzymes containing cis, cis-1,4-pentadiene structure Oxygenation reactions of polyunsaturated fatty acids. The lipoxygenase gene family exists widely in the plant kingdom. It is a multi-gene family with two types of 9-LOX and 13-LOX, and there are different numbers of isoenzymes. The expression of plant LOX genes runs through the entire process of plant life history, and is present at all stages of plant growth and development, including seed germination, tuber formation, nodule development, flower development, fruit ripening, and plant senescence. Expression of the L...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/82C12N15/66A01H5/00
Inventor 胡丽芳刘世强贺浩华蒋伦伟黄长干肖伟
Owner JIANGXI AGRICULTURAL UNIVERSITY
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