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Cucumber CsMADSi gene overexpression vector and an application thereof

A technology of expression vector and plant expression vector, applied in the field of cucumber E gene CsMADS1 overexpression vector

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

Although genes related to important economic traits of cucumber are being cloned gradually, there are few reports on genes related to cucumber flowering

Method used

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  • Cucumber CsMADSi gene overexpression vector and an application thereof
  • Cucumber CsMADSi gene overexpression vector and an application thereof
  • Cucumber CsMADSi gene overexpression vector and an application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment １

[0022] Example 1: Expression vector pCAMBIA1301-CsMADS1 build

[0023] (1) Primer design: According to the cucumber CsMADS1 sequence (Csa004117) published on CuGI (http: / / cucumber.genomics.org.cn / page / cucumber / index.jsp), design primers at both ends:

[0024] CsMADS1-F: 5'-aaaaCCATGGATGGGAAGAGGAAGAGTAG-3' (with NcoI site)

[0025] CsMADS1-R: 5'-aaaaAGATCTTCAAAGCATCCAACCAGGGAG-3' (with BglII site).

[0026] (2) Extraction of total RNA from cucumber flower buds

[0027]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 extract total RNA by using TRIzol (Invitrogen, USA) reagent method: 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...

Embodiment 2

[0040] Example 2 : pCAMBIA1301-CsMADS1 transfection Agrobacterium GV3101

[0041] (1) Preparation of Agrobacterium Competent Cells

[0042] 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.

[0043] (2) Transformation and identification of Agrobacterium

[0044] 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 minu...

Embodiment 3

[0045] Embodiment 3: containing pCAMBIA1301-CsMADS1 Transformation of Arabidopsis thaliana with Agrobacterium GV3101

[0046] (1) Planting of Arabidopsis

[0047] ①The Arabidopsis thaliana 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.

[0048] ②Put the nutrient soil in a plastic pot, add nutrient solution into the tray, and start planting after the nutrient soil absorbs ...

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Abstract

The invention discloses a cucumber E family gene CsMADSi overexpression vector and an application thereof in modification of floral organs, belonging to the technical field of biology. The vector is a plant expression vector containing a 35S promoter and a cucumber E family gene CsMADS1. A CsMADS1 transgenic plant obtained by overexpression of CsMADS1 in arabidopsis thaliana cannot shoot and bloom normally, and many clustered acicular leaves or vestigial flowerlet branchlets grow abnormally at top meristems. The blooming time of a plant can be controlled by using the vector disclosed by the invention to cultivate a special transgenic plant, so that the cucumber E family gene CsMADSi overexpression vector and an application thereof in modification of floral organs have certain agricultural value and ornamental value.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, in particular to a cucumber E-type gene CsMADS1 overexpression vector and application thereof. Background technique [0002] Flowering is a very important developmental process of higher plants transitioning from vegetative growth to reproductive growth, and flowering at the right time is extremely important for the survival and successful reproduction of most plants. The regulation of flowering time is a very complicated process, which is affected by both its own genetic factors and external environmental factors. So far, six genetic pathways including photoperiod, vernalization, temperature, gibberellin, autonomy and age have been found to participate in the regulation of flowering time. [0003] MADS-box genes are an important class of transcriptional regulators in eukaryotes, which play an important role in growth and development regulation and signal transduction, and exist in an...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/66A01H5/00
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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