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Cucumber AG-like gene CsMADS24 overexpression carrier and application thereof

An expression vector and plant expression vector technology, applied in the field of cucumber AG-like gene CsMADS24 overexpression vector, can solve the problem of less research on the regulation of flower development by MADS-box gene

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

Cucumber is an important vegetable crop, and there are relatively few studies on the regulation of flower development by MADS-box genes

Method used

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  • Cucumber AG-like gene CsMADS24 overexpression carrier and application thereof
  • Cucumber AG-like gene CsMADS24 overexpression carrier and application thereof
  • Cucumber AG-like gene CsMADS24 overexpression carrier and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment １

[0018] Example 1: Expression vector PHB-CsMADS24 build

[0019] (1) Primer design: According to the sequence of cucumber CsMADS24 (Csa017355) published on CuGI (http: / / cucumber.genomics.org.cn / page / cucumber / index.jsp), design primers at both ends:

[0020] CsMADS24-F: 5'-aaaaCTGCAGATGAGTTGTTATGAGGAAG-3' (with PstI site)

[0021] CsMADS24-R: 5'-aaaaTCTAGATTACACAAGTTGAAGAGAG-3' (with XbaI site).

[0022] (2) Extraction of total RNA from cucumber flower buds

[0023] The cucumber variety used is Huabei type cucumber. Take 1 mg of flower buds with a length of about 0.7 mm, freeze them in liquid ammonia immediately after collection, and use TRIzol (Invitrogen, USA) reagent method to extract total RNA: add 1.5 ml Trizol, and place at room temperature for 5 min to fully lyse. Centrifuge at 12,000rpm for 5 minutes and discard the pellet. Add 200 ul of chloroform, vortex and mix well, and place at room temperature for 15 min. Centrifuge at 12,000 g for 15 min at 4°C. Aspira...

Embodiment 2

[0036] Example 2: PHB-CsMADS24 transfection Agrobacterium GV3101

[0037] (1) Preparation of Agrobacterium Competent Cells

[0038] 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.

[0039] (2) Transformation and identification of Agrobacterium

[0040] Add 10 μl of plasmid DNA to 200 μl of competent Agrobacterium, mix well, bathe in ice for 30 minutes, freeze in liquid nitrogen for 3-5 minutes, bat...

Embodiment 3

[0041] Embodiment 3: containing PHB-CsMADS24 Transformation of Arabidopsis thaliana with Agrobacterium GV3101

[0042] (1) Planting of Arabidopsis

[0043] ①) The Arabidopsis used is Columbia The wild-type Arabidopsis was preserved by the Key Laboratory of Crop Physiology, Ecology, Genetics and Breeding of Jiangxi Agricultural University. The seeds harvested in the current year were vernalized for 72 h at 4 degrees after planting, and the seeds in the next year were vernalized for 24 h 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.

[0044] ②Put the nutrient soil in a plastic pot, add nutrient solution into the tray, and start planting after the nutrient soil absorbs water and beco...

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Abstract

The invention discloses a cucumber AG-like gene CsMADS24 overexpression carrier and application thereof to flower organ modification, and belongs to the technical field of biology. The carrier is a plant expression carrier containing double 35S promoters and a cucumber gene CsMADS24. A CsMADS24 transgenic plant obtained by overexpressing the CsMADS24 in Arabidopsis thaliana is in a flowering state at the early development stage of flowers; and at the later development stage of the flowers, only few relatively thin and long flowers can be formed, the petals and the stamens of most of the flowers cannot normally develop, and only sepals and carpel develop. The result shows that CsMADS24 play an important control role on the aspect of flower organ development. A novel flower organ variant material obtained by virtue of the gene CsMADS24 can be applied to breeding of crops and ornamental plants and has certain agricultural and ornamental values.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a cucumber AG-like gene CsMADS24 overexpression vector and application thereof. Background technique [0002] The normal development of flowers is the basis for the reproduction of plants, and it is also a critical period for agricultural production to obtain products and increase yields. The genes that control flower development are identified by means of molecular biology and by changing their expression, the flower type can be changed purposefully, and the new materials that produce variation can be used for breeding applications of crops and ornamental plants. With the gradual deepening of the research on the related issues of flower development, it was found that the development of flower organs is controlled by a class of MADS-box homeotic genes. The MADS-box gene family is named after the initials of the first four members identified: yeast MCM1 gene, Arabidopsis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00
Inventor 胡丽芳刘世强贺浩华杨寅桂蒋伦伟王义华
Owner JIANGXI AGRICULTURAL UNIVERSITY
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