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Application of a Medicago p-Hydroxyphenylpyruvate Dioxygenase Gene

A technology of pyruvate dioxygenase and p-hydroxybenzene, which is applied in the field of molecular biology, can solve the problems of long growth cycle, lagging gene function, and difficulty in genetic transformation, and achieve the effect of increasing the content

Active Publication Date: 2017-09-08
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, alfalfa is a cross-pollinated tetraploid leguminous herbage. Its genetic transformation is difficult, the growth cycle is long, and the research on gene function is lagging behind. So far, the research on the genes related to vitamin E synthesis in alfalfa is still blank.

Method used

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  • Application of a Medicago p-Hydroxyphenylpyruvate Dioxygenase Gene
  • Application of a Medicago p-Hydroxyphenylpyruvate Dioxygenase Gene
  • Application of a Medicago p-Hydroxyphenylpyruvate Dioxygenase Gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Preparation of alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD

[0023] The alfalfa MsHPPD gene provided by the invention is screened by the following method:

[0024] Through BLAST comparative analysis, primers were designed according to the conserved domain of the cloned p-hydroxyphenylpyruvate dioxygenase gene (Genbank accession number: XM_003617343) in alfalfa (RT-PCR primer: F: 5'-CCACTTTCTCCGCATCCACTTGTTTC-3 ', its nucleotide sequence is shown in SEQ ID NO:2 in the sequence listing; R: 5'-CCTCCAATGTCCTAAATATATCCTCAG-3', its nucleotide sequence is shown in SEQ ID NO:3 in the sequence listing). Amplify from alfalfa (Medicago sativa L.cv.Zhongmu No.1, Zhongmu No. 1, Beijing Zhonghu Dongfang Grass Technology Co., Ltd.), and recover a fragment with an expected size of 600bp (recover this length after running electrophoresis Fragments), the recovered fragments were ligated into PMD18-T Vector (Takara), the sequencing results were analyzed by BLASTx in...

Embodiment 2

[0065] Example 2 Agrobacterium-mediated MsHPPD gene transformation of Arabidopsis plants

[0066] 1.1. Materials and reagents

[0067] 1.1.1. Plant material

[0068] The tested alfalfa variety was Medicago sativa L.cv.Zhongmu No.1, Zhongmu No.1.

[0069] 1.1.2. Agrobacterium strains and plasmid vectors

[0070] The Agrobacterium strain used is Agrobacterium tumefaciens: LBA4404 (Beijing Tianenze Gene Technology Co., Ltd.)

[0071] Agrobacterium culture medium:

[0072] Reagent Content (1L) MgSO 4 . 7H 2 o

1g / L Peptone 10g / L Yeast extract 1g / L sucrose 5g / L Agar (solid medium) 15g / L pH 7.0

[0073] 121°C high pressure steam sterilization for 20min; carrier: PBI 121

[0074] 1.2. Experimental method

[0075] 1.2.1 Insert the MsHPPD gene into the pBI121 vector plasmid DNA, the specific steps are: design the upstream primer containing the XbaI restriction site at the 5' end and the downstream primer of the BamHI...

Embodiment 3

[0095] Example 3 Evaluation of vitamin E content in Arabidopsis thaliana in pBI121 vector of MsHPPD gene

[0096] Agrobacterium was transformed with plasmid pBI121 to obtain recombinant Agrobacterium, and Arabidopsis was transformed with recombinant Agrobacterium to obtain a control plant that was transformed into an empty vector. The method was as in Example 1, and the control plant (CK) that was transformed into an empty vector was obtained as Comparative Example 1.

[0097] Evaluate the vitamin E content of the Arabidopsis thaliana of the transfer empty vector obtained in comparative example 1 and the pBI121 carrier Arabidopsis thaliana that the transformation that obtains in embodiment 1 is inserted into MsHPPD gene, specific method is as follows:

[0098] Mix 3g of alfalfa leaves, grind them evenly, then sample 0.03-0.05g, add 1mL of methanol:chloroform (2:1 / V:V) containing 0.01% of 2,6-di-tert-butyl-p-cresol (BHT), avoid Light standing for 20min. Add 0.33mL chloroform a...

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Abstract

The invention discloses a medicago sativa hydroxyphenylpyruvic acid dioxygenase gene MsHPPD. The nucleotide sequence of the dioxygenase gene is as shown in SEQID NO:1 in a sequence table. The invention also discloses a primer pair for cloning the medicago sativa hydroxyphenylpyruvic acid dioxygenase gene MsHPPD, and nucleotide sequences are as shown in the SEQ IDNO:4 and SEQID NO:5 in the sequence table respectively. The invention further discloses a preparation method of the medicago sativa hydroxyphenylpyruvic acid dioxygenase gene MsHPPD, and the application of the medicago sativa hydroxyphenylpyruvic acid dioxygenase gene MsHPPD in the increase of the content of vitamin E in arabidopsis thaliana and improving the stress resistance. According to the medicago sativa hydroxyphenylpyruvic acid dioxygenase gene MsHPPD disclosed by the invention, a base is established for further research on functions of the medicago sativa MsHPPD gene, and the application of the MsHPPD gene in the improvement on the quality of pasture, particularly the application of the MsHPPD gene in the increase of the content of the vitamin E in molecular improved pasture.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to an alfalfa p-hydroxyphenylpyruvate dioxygenase gene MsHPPD and its preparation method and application. Background technique [0002] Vitamin E is a fat-soluble vitamin. Natural products are divided into eight types according to the degree of saturation of the hydrophobic tail and the number and position of methyl groups on the aromatic ring, namely a, β, γ, σ-tocopherol and a , β, γ, σ-tocotrienols, among which a-tocopherol has the highest biological activity. [0003] Vitamin E is an essential micronutrient for the human body, but the human body cannot synthesize it by itself and can only be ingested through diet. Studies have shown that vitamin E can improve the immunity of animals. Relevant studies have shown that the meat quality of livestock supplemented with vitamin E is better than that of normal livestock. Moreover, it can improve the quality of meat and milk and prolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12N15/11C12N15/10C12N15/82A01H5/00
CPCC12N9/0069C12N15/8243C12Y113/11027
Inventor 王学敏姜霁珊贾慧丽冯光燕王赞高洪文
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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