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Herba epimedii EsSVP protein, gene coding same and application thereof

A kind of epimedium and gene technology, applied in the field of genetic engineering, can solve the problem that the petals and sepals of Epimedium are not found.

Inactive Publication Date: 2015-04-22
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no reports have been found on the traits of Epimedium petals and sepals and their regulatory mechanisms.

Method used

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  • Herba epimedii EsSVP protein, gene coding same and application thereof
  • Herba epimedii EsSVP protein, gene coding same and application thereof
  • Herba epimedii EsSVP protein, gene coding same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Cloning of Example 1 Epimedium EsSVP Gene

[0026] 1 gene clone

[0027] 1.1 Extraction of total RNA

[0028] (1) Take Epimedium leaves, grind them fully with liquid nitrogen in a mortar baked at 180-200°C, and quickly pour them into a DEPC-treated centrifuge tube. After the liquid nitrogen evaporates, press 1ml reagent / Add an appropriate amount of Trizol (plant RNA extraction reagent) to 100 mg of the material, vortex and mix, and place at room temperature for 30 minutes.

[0029] (2) Centrifuge at 12000rpm for 10min, and suck the supernatant to another centrifuge tube.

[0030] (3) Add 0.2 times the volume of Trizol in chloroform to the supernatant, mix well, and place at room temperature for 5 minutes.

[0031] (4) Centrifuge at 12000rpm for 10min, keep the water phase, and suck it into another centrifuge tube.

[0032] (5) Add an equal volume of isopropanol to the water phase, mix thoroughly, and place at -20°C for 2 hours.

[0033] (6) Centrifuge at 12000rpm,...

Embodiment 2

[0107] Example 2 Agrobacterium-mediated Epimedium EsSVP Gene Transformation (Arabidopsis thaliana)

[0108] 1 Construction of expression vector

[0109] According to the experimental requirements, in this example, the characteristics of multiple cloning restriction sites on the vectors pMV and pBI121 were utilized to digest the cDNA fragments on the cloning vectors and directly connect them to the expression vectors.

[0110] 2 Preparation of Agrobacterium GV3101 and EHA105 Electrically Competent Cells and Transformation of Expression Vectors

[0111] (1) Pick fresh single colonies of Agrobacterium GV3101 and EHA105 in 10mL LB liquid medium, and cultivate them with shaking at 28°C until the late logarithmic period;

[0112] (2) Take 1mL of bacterial liquid and add it to 100mL of fresh LB liquid medium, shake and cultivate to OD at 28°C 600 About 0.5-1.0;

[0113] (3) Transfer the bacterial solution to a 50mL centrifuge tube and place on ice for 30 minutes;

[0114] (4) Cen...

Embodiment 3

[0134] Example 3 Agrobacterium-mediated transformation of Epimedium EsSVP gene (Petunia)

[0135] 1 Agrobacterium-mediated petunia transformation (leaf disk transformation method)

[0136] The explants were cultivated in a greenhouse, and the vigorously growing leaves of wild-type petunia W115 were taken. For specific transformation methods, see Horsch et al.1985.

[0137] 2 tissue paraffin sections

[0138] The pistils at different developmental stages were fixed with FAA fixative, and the paraffin sections were prepared according to Horsch, R.B. et al. (1985). The sections were 8-mm thick and stained with eosin-toluidine blue (Igersheim and Cichocki, 1996).

[0139] Overexpression of 35S::EsSVP transformed petunias did not affect cymose branches and had little or no effect on flowering time, but obviously changed flower development. Calyx enlarged, sometimes leaf-sized ( Figure 7 A, C, D, H). The corolla tube turns green, and the petals cannot fully open (partially lose...

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Abstract

The invention provides herba epimedii EsSVP protein, a gene coding the protein and an application thereof. The herba epimedii EsSVP protein has an amino acid sequence as shown in SEQ ID No. 1, and the gene coding the protein has a sequence as shown in SEQ ID No. 2. According to the invention, herba epimedii EsSVP gene is obtained by cloning for the first time, and the function of the gene is verified in plants (arabidopsis thaliana, petunia) by transgenic technology. The invention provides reference and basis for research of herba epimedii flowering phase, petal and sepal characteristics, and regulation mechanism, and provides possibility for further improvement of ornamental characteristics of ornamental plants such as herba epimedii.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to epimedium EsSVP protein, a gene encoding the protein and application thereof. Background technique [0002] Epimedium belongs to Ranunculaceae and Berberidaceae Berbridaceae. The plants of this genus have peculiar flower shapes, rich flower colors and leaf colors, and most of them are evergreen herbs. , which is resistant to shade and has strong adaptability (Stearn, 2002), has been widely used in potted plants, special gardens, rock gardens, flower borders and ground covers in Europe, America, and parts of China (Ren Lin et al., 2008). China is one of the more important origin centers of epimedium germplasm resources. Among the more than 50 known species, as many as 40 are native to China, accounting for about 80% of the total. Rich and excellent wild resources It provides the possibility to develop Epimedium plants as new ornamental plants. [0003] Epimedium is an ancient ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82C12N1/15C12N1/19C12N1/21A01H5/00
CPCC07K14/415C12N15/827
Inventor 李志能
Owner SOUTHWEST UNIV
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