PcFPS gene primer pair and application of gene amplified by primer pair to increase of content of patchouli alcohol in pogostemon cablin

A technology of patchouli alcohol and primer pair, which is applied in the field of genetic engineering and can solve the problems of insufficient patchouli alcohol yield and the like

Active Publication Date: 2019-02-22
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention provides a pair of primers for the PcFPS gene in order to solve the problem of lower yields of pothurian alcohol. The primer pair can be specifically amplified from Patchouli to obtain the PcFPS gene. The synthesis of colechinol is related. Through the primer pair provided by the present invention to amplify the PcFPS gene and construct its transgenic plant, the patchouli transgenic plant with high content of pocchinol can be obtained, and the problem of insufficient yield of pocchinol can be solved

Method used

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  • PcFPS gene primer pair and application of gene amplified by primer pair to increase of content of patchouli alcohol in pogostemon cablin
  • PcFPS gene primer pair and application of gene amplified by primer pair to increase of content of patchouli alcohol in pogostemon cablin
  • PcFPS gene primer pair and application of gene amplified by primer pair to increase of content of patchouli alcohol in pogostemon cablin

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Embodiment 1

[0046] 1.1 Experimental method and process

[0047] 1.1.1 Plant material and treatment

[0048] The material used in the present invention is Hainan Patchouli. Use robust patchouli branches, cut them in the sand, transplant them in the field after the roots of the plants grow, and extract RNA from the leaves of the plants after the plants mature.

[0049] 1.1.2 Test reagents

[0050] TRIZOL was purchased from Tiangen Biochemical Technology Co., Ltd.

[0051] EXTaq DNA polymerase and DH-5α competent pMD18-T were purchased from Dalian Biotech.

[0052] The reverse transcription kit was purchased from Thermo Fisher Scientific Co., Ltd.

[0053] Gel recovery kit was purchased from Omega Corporation.

[0054] 1.1.3 RNA extraction and cDNA synthesis

[0055] Total RNA was extracted by the modified TRIZOL method. Based on the TRIZOL method, two chloroform extractions were performed, and further extraction was performed using phenol:chloroform:isoamyl alcohol (25:24:1).

[005...

Embodiment 2

[0075] Except that the primer pair adopts the primers of the control group, other steps are the same as in Example 1. The forward primer of the primer pair of the control group is shown in SEQ ID NO.5, and the reverse primer is shown in SEQ ID NO.6:

[0076] Forward primer SEQ ID NO.5: 5'-CAGTTGGCGACTACTACAA-3';

[0077] Reverse primer SEQ ID NO.6: 5'-TGAACAACACTTGGCTAATG-3'.

[0078] The PCR amplification primer of embodiment 1 and embodiment 2 carries out gel electrophoresis detection, and the result is as follows Figure 4 Shown, wherein 1, 2, 3 swimming lanes are the PCR amplification result of the primer pair that embodiment 1 adopts; 4, 5, 6 swimming lanes are the PCR amplification result of the control primer pair that embodiment 2 adopts. It can be seen that the primer pair provided by the present invention has strong specificity and clear bands; while the primer pair of the control group has poor specificity, in which multiple bands appear, and the size of the fragm...

Embodiment 3

[0080] The expression vector of Patchouli FPS gene was constructed using the in-fusion kit of Baoriyi Biotechnology Co., Ltd.:

[0081] (1) Recover the pYES2 plasmid with a single restriction endonuclease to linearize the pYES2 plasmid;

[0082] (2) Design specific primers with carrier sequences according to the carrier construction technology, and use the bacterial solution as a template to amplify the FPS sequence;

[0083] (3) Link the FPS sequence amplified in step (2) to the linearized pYES2 plasmid obtained in step (1) to obtain the expression vector pYES2-PcFPS;

[0084] (4) Transform the expression vector pYES2-PcFPS into Escherichia coli DH5α by the heat shock method, grow overnight on LB solid medium containing kanamycin, and then pick and multiply the white spot, and verify the correct positive by bacterial liquid PCR and sequencing clone.

[0085] The plasmid pYES2-PcFPS was transformed into the competent Agrobacterium tumefaciens GV3101 by electric shock method,...

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Abstract

The invention provides a PcFPS gene primer pair and an application of a gene amplified by the primer pair to increase of the content of patchouli alcohol in pogostemon cablin, and relates to the technical field of gene engineering. The nucleotide sequences of an upstream primer and a downstream primer of the PcFPS gene primer pair are as shown in SEQ ID No. 3 and SEQ ID No. 4. The primer pair canspecifically amplify the FPS gene related to patchouli alcohol synthesis, and is high in specificity and does not easily amplify non-specific strips as compared with other primers. The invention further provides an application of the PcFPS gene and the primer pair thereof to increase of the content of the patchouli alcohol in the pogostemon cablin. The PcFPS gene primer pair can perform specific amplification to obtain the PcFPS gene, the PcFPS gene is transferred into the pogostemon cablin to construct transgenic pogostemon cablin, patchouli alcohol synthesis can be effectively improved by over-expression of the PcFPS gene, and the content of the patchouli alcohol in the transgenic pogostemon cablin is increased.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a primer pair of PcFPS gene and the application of the gene amplified by the primer pair to increase the content of pokichol in patchouli. Background technique [0002] Terpenoids are relatively important compounds in Chinese herbal medicines. It has been found that a variety of terpenoids are active ingredients in Chinese herbal medicines. At the same time, they are also an important class of natural spices and indispensable raw materials for cosmetics and food industries. [0003] Patchouli alcohol, Patchouli alcohol, also known as patchouli alcohol, is a tricyclic sesquiterpene compound present in natural plants, and its molecular formula is C 15 h 26 O, molecular weight 222.37, structural formula name 1,6-Methanonaphthalen-1(2H)-ol,octahydro-4,8a,9,9-tetramethyl-,(1R,4S,4aS,6R,8aS)-, colorless Crystals with a light patchouli aroma. Insoluble in water, soluble i...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/54C12N15/10C12N15/82A01H5/00A01H6/50
CPCC12N9/1085C12N15/10C12N15/8243C12Y205/01C12Q2531/113
Inventor 吴友根杨豫章于靖张军锋杨东梅
Owner HAINAN UNIVERSITY
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