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A kind of promoter of alfalfa hppd gene and its preparation method and application

An alfalfa, promoter technology, applied in the directions of botanical equipment and methods, biochemical equipment and methods, applications, etc., can solve problems such as backwardness

Active Publication Date: 2018-09-18
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the perennial and polyploid characteristics of alfalfa, the current research on alfalfa genes is still far behind other crops
There are still relatively few reports on the study of the promoter of alfalfa

Method used

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  • A kind of promoter of alfalfa hppd gene and its preparation method and application
  • A kind of promoter of alfalfa hppd gene and its preparation method and application
  • A kind of promoter of alfalfa hppd gene and its preparation method and application

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Embodiment Construction

[0055] The promoter of the HPPD gene of Medicago sativa, the gene sequence of which is shown in SEQ ID NO: 1 in the sequence listing. The sequence was obtained by chromosome walking technology, the specific method is:

[0056] (1) Acquisition of genomic DNA

[0057] Take 1.0 g of fresh alfalfa leaves, and use QIAGEN's DNeasy Plant Maxi Kit to extract total DNA, and the amount of DNA obtained is ≥30 μg.

[0058] (2) Design of specific primers

[0059] According to the known DNA sequence of HPPD gene coding region of alfalfa, three specific primers were designed: SP1: 5'-GTGTTTGTCTTGGTGTTGTTGTTA-3', SP2: 5'-AGAGTAATGGAGCGACGG-CGTAGTTG-3', SP3: 5'-GTTCGGGCTCGATCAAACGACTG-3' ;

[0060] The design direction is the 5' end; the design principle is: SP2 is located inside SP1, and SP3 is located inside SP2; the distance between each two primers is about 100bp, the primer length is 22-26bp, the GC content is 45-55%, and the Tm value is 60- 70°C;

[0061] Primers SP1, SP2 and SP3 ar...

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Abstract

The invention discloses a promoter of alfalfa HPPD gene as well as a preparation method and an application of the promoter. The gene sequence of the promoter is shown in SEQ NO: 1 in the sequence. The preparation method of the promoter comprises steps as follows: (1) acquiring genomic DNA; (2) designing specific primer; (3) performing a first PCR reaction; (4) performing a second PCR reaction; (5) performing a third PCR reaction; (6) performing electrophoresis on 5 mu l of each of first, second and third PRC reaction liquids with 1% agarose gel; (7) recovering clear electrophoretic bands through gel cutting, performing DNA sequencing on PCR products with SP3Primer as a primer; splicing sequences obtained through sequencing, and removing overlapped sequences to finally obtain a promoter sequence of the alfalfa HPPD gene. The promoter of the HPPD gene plays important roles in regulating light / dark reactions as well as multiple stress reactions of the gene and can be used as a promoter of stress response, particularly light control response.

Description

technical field [0001] The invention relates to the field of alfalfa, in particular to an alfalfa promoter and its preparation method and application. Background technique [0002] The promoter is a DNA sequence located upstream of the 5' end of the structural gene, which can be combined with RNA polymerase, so that it can accurately combine with the template DNA and initiate transcription. A typical promoter includes core cis-acting elements such as CAAT-box and TATA-box, which can be roughly divided into constitutive expression promoters, space-time specific expression promoters and inducible expression promoters according to their functions and modes of action. kind. At present, a large number of plant promoters have been isolated and studied, the cauliflower mosaic virus (CaMV) 35s promoter and the cassava vein mosaic virus (CsVMV) promoter are constitutive promoters. The Pyk10 promoter is a tissue-specific expression promoter in Arabidopsis. Constitutively expressed ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82A01H5/00A01H6/54
CPCC12N9/0069C12N15/8223C12N15/8271C12Y113/11027
Inventor 王学敏武语迪姜霁珊王赞高洪文常鑫
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI