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A light-inducible promoter with a specific expression of alfalfa plastocyanin and a pair of primers for rapid cloning of the promoter

A plastocyanin and promoter sequence technology, applied in the field of medicine, can solve problems affecting the normal growth and development of plants, plant variation, etc.

Inactive Publication Date: 2016-04-27
SHENYANG PHARMA UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the continuous and efficient expression of exogenous genes in recipient plants often causes plant variation and affects the normal growth and development of plants.

Method used

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  • A light-inducible promoter with a specific expression of alfalfa plastocyanin and a pair of primers for rapid cloning of the promoter
  • A light-inducible promoter with a specific expression of alfalfa plastocyanin and a pair of primers for rapid cloning of the promoter

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Experimental program
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Embodiment 1

[0018] (1) PCR method to clone the promoter sequence;

[0019] a) reaction system;

[0020] Sterilized deionized water 33.75ul, alfalfa genomic DNA 5ul, 10×PCR buffer 5ul, dNTPmix 4ul, upstream and downstream primers 1ul each, rTaq 0.25ul;

[0021] b) Reaction conditions: pre-denaturation at 95°C for 2min, denaturation at 95°C for 30s, annealing at 53°C for 30s, extension at 72°C for 1min, cycle 30 times, extension at 72°C for 10min;

[0022] c) Two-way primer upstream primer 5'-CCCAAGCTTAAAATAATAATGTGTGTTATGAGA-3', downstream primer 5'-cgggatccgctctgacacaaagcctt-3'.

[0023] (2) PCR reaction products were detected by agarose gel electrophoresis and sent to Gene Company for sequencing and sequence analysis;

[0024] (3) Construction of recombinant plasmids for transformation; including a) extraction of plasmids; b) enzyme digestion; c) enzyme linkage; the characteristics are: a) SDS alkali denaturation method to extract pBI121 plasmid in Escherichia coli; b) HindⅢ-BamHI doub...

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Abstract

The invention discloses a promoter sequence with photoinduction specificity and leaf tissue specificity for promoting expression of alfalfa plastocyanin and a pair of primers for quickly cloning the promoter. The document reports that the plastocyanin is a plastid protein related to plant photosynthesis, and has been proved to be a photoinduction expression protein. The alfalfa plastocyanin promoter has 9 specific elements, including TATAbox, CAATbox and other promoter necessary elements, a specific element G-box(ctttatca) related to photoresponse, and GT-1 basic sequence (aatccaca) and the like related to tissue specificity expression; and the experiment proves that the promoter has certain photoinduction specificity and leaf tissue specificity, and can drive the specific expression of the reported gene GUS in transgenic alfalfa leaf. The primers are specific primers designed in reference to related sequences of Medicago truncatula genome, and are suitable for quickly cloning the alfalfa plastocyanin promoter by a PCR (polymerase chain reaction) method.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a light-induced promoter sequence for promoting the specific expression of alfalfa plastocyanin and a pair of primers for rapidly cloning the promoter. Background technique [0002] Using transgenic plants as bioreactors, introducing exogenous genes into plants to express exogenous recombinant proteins, and producing medicinal proteins or vaccines has become a hotspot in the research of transgenic plants. However, in the process of expressing foreign genes in recipient plants, adverse phenomena such as low expression efficiency, unstable expression products, and even gene inactivation or silencing often occur, resulting in the inability of transgenic plants to be put into practical use. Therefore, it is often necessary to construct an expression vector capable of expressing a heterologous protein at a high level. In order to better increase the expression activity of foreign gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/11
Inventor 苏昕游松薛柏忠王彩云刘鑫禚瑞芳
Owner SHENYANG PHARMA UNIVERSITY
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