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Peony PsRD22 gene and application thereof

A psrd22, peony technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of stunted flower bud development, small flower leaflets, flowers but no leaves, etc.

Active Publication Date: 2016-07-20
SHANGHAI BOTANICAL GARDEN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to lack of understanding of the mechanism of dormancy relief in peony flower buds, it is often impossible to completely break the dormancy in flower buds, resulting in poor bud development, resulting in abnormal flowering, flowers without leaves, small flower leaflets, short flowering period, and even failure to induce flowering (Zhao Haijun, Zhang Wantang) , Zheng Guosheng, etc. Peony deep dormancy characteristics and release methods. Shandong Forestry Science and Technology, 2000, 5: 44-46)

Method used

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  • Peony PsRD22 gene and application thereof
  • Peony PsRD22 gene and application thereof
  • Peony PsRD22 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Preparation and analysis method of peony PsRD22 gene

[0048] Grind peony flower buds with liquid nitrogen in a mortar to powder, and use the plant RNAout kit to perform RNA extraction as required.

[0049] Fully dissolve the total RNA with RNase-free double distilled water.

[0050] Remove possible residual DNA with DNaseI.

[0051] The RNA quality was preliminarily detected by agarose gel electrophoresis, the RNA concentration and the 260 nm and 280 nm light absorption values ​​of the RNA were detected by the NanoDrop instrument, and the RNA purity was predicted according to OD260 / OD280. Confirm that the RNA concentration is not lower than 4ng / μL, the total amount is higher than 20μg, the OD260 / 280 is between 1.8 and 2.2, the integrity is good (28S:18S>1.0), and there is no protein and DNA contamination.

[0052] The obtained RNA was used as a template for reverse transcription, and cDNA was obtained and stored in a -20°C refrigerator for later use.

[00...

Embodiment 2

[0062] Example 2 Real-time fluorescent quantitative PCR detection of the expression pattern of PsRD22

[0063] Take an appropriate amount of plant tissue, grind it into powder with liquid nitrogen in a mortar, and use the plant RNAout kit to extract RNA according to the requirements.

[0064] Removal of residual DNA, gel running and quantification were as described in Example 1.

[0065] First-strand cDNA was synthesized using PrimeScript Reverse Transcriptase Kit (Takara).

[0066] The real-time fluorescence quantitative PCR system is 20 μL, including about 100 ng of cDNA, 0.2 μmol / L forward and reverse primers and 10 μL of 2×SYBRpremixExTaqTM (Takara).

[0067] Amplification program: pre-denaturation at 95°C for 30s, followed by 40 cycles of amplification (95°C for 5s, 60°C for 40s).

[0068] Each amplification was performed in 3 technical replicates and in 2 biological replicates.

[0069] The primer sequence of the expression molecular marker GYRD of PsRD22 is as follow...

Embodiment 3

[0080] Example 3 The subcellular localization method of PsRD22 gene in tree peony

[0081] (1) Transient expression of PsRD22 gene in tobacco

[0082] The coding region of PsRD22 (without the terminator) was cloned into the pENTER vector (Invitrogen Company), and 1 μL of the gel-recovered product (about 20 ng), 1 μL of the pENTER vector and 0.5 μL of salt solution were reacted at 22°C for 2 hours, and then sent to the company for sequencing after transformation. The correctly sequenced plasmid was recombined by LR reaction: 1 μL of the correctly sequenced plasmid, 1 μL of the target vector pK7FWG2 containing 35S promoter and GFP, 0.5 μl of LR recombinase, and reacted at 22°C for 2 hours to obtain the recombinant plasmid pK7FWG2-RD22.

[0083] Select healthy tobacco 4-6 weeks after seedling transplantation, use a syringe needle to pierce 4-6 small holes on the back of each leaf, and inject the Agrobacterium suspension transferred into the pK7FWG2-RD22 plasmid into the leaves wi...

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Abstract

The invention discloses a PsRD22 gene and an encoded protein thereof for sustained high expression in a flower bud low-temperature dormancy releasing process, and further designs an expression molecular marker GYRD of the gene. The PsRD22 gene has the advantages that the low-temperature catalytic peony technique is assisted by the expression molecular marker for the first time; after proofing by experiments, the encoded protein of the gene can obviously improve the resistance of plants to drought threat, and the excellent candidate gene is provided for the stress resistance breeding of peony.

Description

technical field [0001] The invention belongs to the fields of plant molecular biology, plant genetic engineering and biotechnology, and relates to a plant dormancy release response and stress resistance gene, in particular to a tree peony PsRD22 gene and its application. Background technique [0002] Peony (Paeoniasuffruticosa Andr.) is a traditional famous flower unique to China, and also a world-famous flower. It has beautiful leaves, large flowers and bright colors, and has the reputation of "the king of flowers". Tree peony is a perennial deciduous subshrub of Paeoniaceae Paeoniaceae. There are 8 original species in total, which are divided into leathery disk subgroup and fleshy disk subgroup, all of which are native to China. China is not only the origin and distribution center of wild species of peony in the world, but also the cultivation center of horticultural varieties of peony in the world (Wang Lianying, Yuan Tao, "Chinese Peonies and Paeoniae", Beijing Jindun Pu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00C12Q1/68C12N15/11
CPCC07K14/415C12N15/827C12N15/8273C12Q1/6895
Inventor 高燕蒋昌华宋垚叶康秦俊奉树成
Owner SHANGHAI BOTANICAL GARDEN
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