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Prunus pseudocerasus L. DELLA proteins as well as coding genes and application thereof

A technology that encodes genes and proteins, applied in applications, genetic engineering, DNA preparation, etc., can solve problems such as poor salt and alkali resistance of Colt rootstocks, susceptibility to root cancer, and high soil fertility requirements

Inactive Publication Date: 2012-10-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional dense planting method mostly uses dwarf rootstocks to control the size and height of the tree. However, there are only a handful of sweet cherry dwarf rootstock varieties that have been successfully bred in China, and the application range is small. Most of the production uses rootstock resources imported from abroad. , such as Gisela series, Colt
However, due to the difference in geographical environment, these rootstocks still have certain limitations in the actual production and application in my country. For example, Gisela series rootstocks have high requirements for soil fertility, and there are generally rootstocks with "small feet" after grafting scion varieties, while Colt rootstocks Poor salt and alkali tolerance, susceptible to root cancer
Chinese cherry 'Prunus pseudocerasus L.' is a semi-wild small tree distributed in the Beijing area. It has strong resistance to stress and pests, and has a good grafting affinity with sweet cherry varieties, but it is used as a sweet cherry variety. The rootstock variety, the grafted tree grows vigorously, the crown is tall and large, and the dwarfing effect is not obvious

Method used

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  • Prunus pseudocerasus L. DELLA proteins as well as coding genes and application thereof
  • Prunus pseudocerasus L. DELLA proteins as well as coding genes and application thereof
  • Prunus pseudocerasus L. DELLA proteins as well as coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Embodiment 1, the acquisition of cherry DELLA protein PpGAI and its coding gene

[0047] 1. Obtaining the middle fragment of PpGAI gene

[0048] Chinese cherry 'Prunus pseudocerasus L.' (Prunus pseudocerasus L.) was used as the test material (collected from the Plant Tissue Culture Technology Laboratory of Haidian District, Beijing), and the total RNA was extracted by CTAB method. The extracted RNA pellet was dissolved with 30 μl of ultrafiltered water (RNA free). Take 1-2 μl of dissolved RNA, add 1 μl of 6× electrophoresis loading buffer, mix well, and detect RNA by 1.5% agarose gel electrophoresis. Observed under the gel imaging system, the electrophoresis results are as follows figure 1 As shown, two bands of 28S rRNA and 18S rRNA can be clearly seen from the figure, and the content ratio of 28S rRNA and 18S rRNA is about 1:1-2:1, without tailing and smearing. UV Spectrophotometry OD 260 / OD 280 = 1.95, OD 260 / OD 230 =2.00, the concentration is 1131ng / μl, ind...

Embodiment 2

[0098] The prokaryotic expression analysis of embodiment 2, PpGAI gene

[0099] 1. Construction of prokaryotic expression vector pGEX-4T-1 / PpGAI

[0100] Primers PF1 (SEQ ID NO: 15) and PR1 (SEQ ID NO: 16) were designed according to the full-length ORF sequence of the PpGAI gene, and introduced into the restriction sites BamH I and salI of the pGEX-4T-1 prokaryotic expression vector.

[0101]

[0102] The cDNA obtained by reverse transcription in Example 1 was used as a template, and the full-length PpGAI gene was amplified by PCR reaction using PF1 and PR1 as primers. Add the following components (50 μl system) to a 200 μl centrifuge tube:

[0103]

[0104] The PCR program is: 94°C 5min; 94°C 30sec, 62°C 45sec, 72°C 2min, 35Cycles; 72°C 10min. After detecting the PCR product by 1% agarose gel electrophoresis, recover and purify the target fragment, connect it to the pGEX-4T-1 prokaryotic expression vector to construct a recombinant plasmid, and identify positive clone...

Embodiment 3

[0107] Example 3, Functional Verification of PpGAI Gene Transformation in Arabidopsis

[0108] 1. Construction of pCAMBIA1301 / PacGAI plant expression vector

[0109] Primers PF2 (SEQ ID NO: 17) and PR2 (SEQ ID NO: 18) were designed according to the full-length ORF sequence of the PpGAI gene, and introduced into the restriction sites Nco I and Bgl II of the pGEX-4T-1 prokaryotic expression vector.

[0110]

[0111] The cDNA obtained by reverse transcription in Example 1 was used as a template, and the full-length PpGAI gene was amplified by PCR reaction using PF2 and PR2 as primers. Add the following components (50 μl system) to a 200 μl centrifuge tube:

[0112]

[0113] The PCR program is: 94°C 5min; 94°C 30sec, 62°C 45sec, 72°C 2min, 35Cycles; 72°C 10min. After detecting the PCR product by 1% agarose gel electrophoresis, recover and purify the target fragment, connect it to the pCAMBIA1301 plant expression vector to construct a recombinant plasmid, and identify posit...

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Abstract

The invention discloses Prunus pseudocerasus L. DELLA proteins as well as coding genes and an application thereof in the technical field of conservation and utilization of genetic resources of agricultural wild plants. In the invention, key negative regulator DELLA proteins and the coding genes thereof in a gibberellin signal transduction path can be obtained by clone from cherries at the first time, which is called as PpGAI. The PpGAI is transformed into Escherichia coli BL21 so as to prove that the PpGAI has bioactivity and can code proteins with expected molecular weight sizes; and the PpGAI genes are transformed into wild arabidopsis thaliana so as to prove that the DELLA proteins of the coding genes can result in the obvious dwarf character of the arabidopsis thaliana. According to the invention, excellent dwarf genes are provided for fruit tree genetic engineering, and a new path is created for the dwarf and thick planting of the cherries.

Description

technical field [0001] The invention belongs to the technical field of protection and utilization of genetic resources of agricultural wild plants, and specifically relates to PpGAI of cherry DELLA protein and its coding gene and application. Background technique [0002] Gibberellins (Gibberellins, GAs), as a large class of tetracyclic diterpenoid phytohormones, play an extremely important role in the growth and development of plants, and act on the entire life cycle of plants, such as seed germination, stem Elongation, leaf extension, flower and fruit development (Fleet C M, SUN T P.A DELLAcate balance: the role of gibberellin in plant morphogenesis. Current Opinion Plant Biology, 2005, 8: 77-85). At present, the synthesis and metabolic pathways of GAs have been studied clearly, and the key metabolic enzymes and corresponding genes have been cloned one after another. Therefore, the current research focuses on the molecular mechanism of the gibberellin signal transduction p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/63C12N1/21C07K14/415C12N15/10C12N15/84A01H5/00C12R1/19
Inventor 李天红钟翡沈欣杰赵凯
Owner CHINA AGRI UNIV
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