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Root development-regulating apple polypeptide hormone gene MdCEP7 and application thereof

A polypeptide hormone, apple technology, applied in the field of molecular biology, to inhibit development, reduce photosynthetic energy consumption, and optimize root structure

Active Publication Date: 2017-10-13
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In perennial woody plants, the study of peptide synthesis gene CEP has not been reported

Method used

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  • Root development-regulating apple polypeptide hormone gene MdCEP7 and application thereof
  • Root development-regulating apple polypeptide hormone gene MdCEP7 and application thereof
  • Root development-regulating apple polypeptide hormone gene MdCEP7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Cloning of MdCEP7 gene of embodiment 1 Gala apple

[0031] 1. Gala apple tissue culture leaf RNA extraction

[0032] Extract the total RNA of Gala apple tissue culture leaf by CTAB method, comprise the following steps:

[0033] 1) Take 1.5 g of Gala apple tissue culture seedlings treated with 200mM NaCl salt for 24 hours, put them into a pre-cooled mortar, add liquid nitrogen to grind, and transfer to a pre-cooled 50mL centrifuge tube;

[0034] 2) Quickly add 10 mL of extraction buffer (CTAB 20% w / v, Tris-HCl 0.1 mol / l, EDTA 25 mol / l, NaCl 2 mol / l, mercaptoethanol 2% w / v, PVP 2) preheated to 65°C %w / v, RNase-free double-distilled water to volume, where PVP and mercaptoethanol are used and added), mixed gently, and placed in a water bath at 65°C for 0.5 hours;

[0035] 3) Add a water-saturated phenol / chloroform / isoamyl alcohol (25:24:1) mixture equal to the volume of the centrifuge tube liquid in the previous step, shake in an ice bath for 0.5 hours, centrifuge at 4°C ...

Embodiment 2

[0053] The construction of embodiment 2MdCEP7 gene carrier

[0054] In order to further study the function of MdCEP7 gene, a total of 1863bp fragment including the coding region of MdCEP7 gene was correctly inserted into the expression vector pRI.

[0055] 1. Using DNAMAN software, analyze the restriction site of the MdCEP7 gene, carry out the digestion reaction of the plasmid DNA of pMD18-T-MdCEP7 saved in Example 1, and connect it to the pRI vector to construct the correct recombinant pRI-MdCEP7.

[0056] 2. Transform Agrobacterium LB4404 competent cells with the constructed recombinant pRI-MdCEP7. Perform PCR identification and pick positive colonies for sequencing. The correctly sequenced recombinant pRI-MdCEP7 monoclonal was used for the subsequent transformation of Arabidopsis.

Embodiment 3

[0057] Embodiment 3 obtains transgenic Arabidopsis

[0058] The obtained Arabidopsis seeds were sterilized with 70% alcohol for 3 minutes, 4% sodium hypochlorite for 8-10 minutes (shaking several times during the period), rinsed with sterilized water for 5 times, and blotted dry. Sow on the seed germination medium (spread directly on the surface), light culture (25-28°C, 16h long-day / 8h short-day, 10d), until the seedlings grow. Transplant to substrate and cultivate until flowering.

[0059] Pick Agrobacterium monoclonal colonies and inoculate them in 10mL YEP liquid medium (containing 50mg / L hygromycin), shake at 28°C and 200rpm until the OD600 is 0.6-0.8 (about 48h); take 1mL of the bacterial liquid and add it to 20mL In YEP liquid medium, culture at 28°C and 200 rpm with shaking until the OD600 is 0.6-0.8 (about 5h). The bacteria were collected by centrifugation, suspended and diluted 20 times with the infection solution (containing 0.05 g / mL sucrose, 0.03-0.05% Silweet),...

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Abstract

The invention discloses a root development-regulating apple polypeptide hormone gene MdCEP7 and the application thereof, and belongs to the technical field of molecular biology. MdCEP7 gene is ectopically expressed in Arabidopsis thaliana by a transgenic technology using a 35S strong promoter driving principle to obtain transgenic Arabidopsis thaliana; overexpression of MdCEP7 in the Arabidopsis thaliana affects synthesis and transportation process of plant root auxin; further phenotypic observation shows that the overexpression of MdCEP7 in the Arabidopsis thaliana significantly inhibits elongation of main roots and the number of lateral roots; the results show that the apple MdCEP7 gene plays an important role in affecting the synthesis and transportation of auxin and further regulating plant root development.

Description

technical field [0001] The invention relates to an apple polypeptide hormone gene MdCEP7 for regulating root system development and application thereof, belonging to the technical field of molecular biology. Background technique [0002] Auxin plays an important role in the development of plant roots. The polar transport of auxin exists in the stem, root and leaf of plants, and its polar transport is responsible for the auxin transport carrier located on the plasma membrane. Auxin transporters, including auxin importer (AUX1) and auxin exporter (PINs), affect various aspects of plant growth and development by regulating auxin transport and distribution. The synthesis process of auxin involves the participation of various enzymes, among which TAA1 and YUC genes play a key role, and the synthesis and transport of auxin affect the process of plant root development. [0003] Research results in recent years have shown that polypeptide molecules in plants also play an important ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00
CPCC07K14/415C12N15/8227C12N15/8261
Inventor 王小非郝玉金李睿由春香
Owner SHANDONG AGRICULTURAL UNIVERSITY
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