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Geranyl pyrophosphate synthase gene from anoectochilus roxburghii and application of geranyl pyrophosphate synthase gene from anoectochilus roxburghii

A technology of geranyl pyrophosphate and geranyl pyrophosphate, applied in the field of genetic engineering, to achieve the effect of easy amplification and simple operation

Active Publication Date: 2020-03-20
SANMING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the isolation and function of GPPS proteins have only been studied in a limited number of plant species

Method used

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  • Geranyl pyrophosphate synthase gene from anoectochilus roxburghii and application of geranyl pyrophosphate synthase gene from anoectochilus roxburghii
  • Geranyl pyrophosphate synthase gene from anoectochilus roxburghii and application of geranyl pyrophosphate synthase gene from anoectochilus roxburghii
  • Geranyl pyrophosphate synthase gene from anoectochilus roxburghii and application of geranyl pyrophosphate synthase gene from anoectochilus roxburghii

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] This embodiment provides the extraction of RNA from the total leaves of Anoectochilus, using the Trizol extraction kit of Dalian Bao Biocable Company, which includes the following steps:

[0027] (1) 100 mg of fresh leaves of Anoectochilus are grinded into powder and put into (1) 1.5 mL centrifuge tube, then immediately add 1 mL of RNAiso Plus, and mix by inversion to obtain a homogenate.

[0028] (2) The well-mixed homogenate was allowed to stand at room temperature for 5 minutes, and then centrifuged at 12000g at 4°C for 5 minutes.

[0029] (3) Transfer 800ml of the supernatant to a new centrifuge tube. Do not aspirate the precipitate. Then add 200ml of chloroform to the supernatant, shake vigorously until the homogenate emulsifies and become milky white, and then let it stand at room temperature for 5 minutes to obtain a mixture liquid.

[0030] (4) Centrifuge the mixed solution at 12000g at 4°C for 15 minutes. At this time, the homogenate is divided into three layers. From...

Embodiment 2

[0036] This embodiment provides a transcriptome sequencing method of Anoectochilus Fujian, which includes the following steps:

[0037] Extract the total RNA from the leaves of Anoectochilus fujianensis, check the quality of RNA extraction, and meet the requirements of building a database (RNA concentration> 250ng / μL, total amount> 20μg, OD260 / OD280 between 1.8~2.2, good integrity, RIN> 6.5). Then, use magnetic beads to enrich poly(A)mRNA to break into fragments. Using this as a template, the first cDNA strand and the second cDNA strand are sequentially synthesized, after purification, elution, end repair, and poly(A) ) Then connect the sequencing adapter. Select 200bp~700bp size fragments for PCR amplification, establish cDNA sequencing library, and use IIIumina HiSeq 2000 for sequencing. This part of the experiment was commissioned by Main Technology Services Cable.

[0038] Use the short reads assembly software Trinity (v2.4.0) for De novo assembly, and after obtaining the Con...

Embodiment 3

[0040] This embodiment provides a method for synthesizing the first strand of cDNA of Anoectochilus fujianensis, which includes the following steps:

[0041] The purified total RNA of Anoectochilus leaves obtained in Example 1 was used as a template, oligo(dT)18 was used as a reverse transcription primer, and PrimeScriptTM Reverse Transcriptase (TakaRa China) was used according to SMART. TM PCR cDNA Synthesis Kit (Clontech USA) instructions for the synthesis of the first strand of cDNA. The total volume of the reaction system is 20 μL.

[0042] (1) Prepare reverse transcription mixture 1 in a 0.2mL PE tube according to Table 1 below;

[0043] (2) Prepare reverse transcription mixture 2 in another 0.2mL PE tube according to the reagents in Table 1 below;

[0044] (3) The reverse transcription mixture 1 in step (1) is incubated at 65°C for 5 minutes and then quickly chilled on ice for 2 minutes, and centrifuged for a few seconds to make the mixture of template RNA, primers, etc., gathe...

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Abstract

The invention provides a geranyl pyrophosphate synthase gene from anoectochilus roxburghii and an application of the geranyl pyrophosphate synthase gene from anoectochilus roxburghii, and relates to the technical field of genetic engineering. The geranyl pyrophosphate synthase gene has a nucleotide sequence as shown in SEQID NO.1, and an amino acid sequence as shown in SEQID NO.2. An important basis is provided for variety breeding of the anoectochilus roxburghii, and a base is provided for researching terpenoid enriched transgenic plant strains.

Description

Technical field [0001] The present invention relates to the technical field of genetic engineering, and in particular to a geranyl pyrophosphate synthase gene derived from Anoectochilus Fujian and its application. Background technique [0002] Anoectochilus roxburghii (Anoectochilus roxburghii) is a perennial herbaceous plant that grows in Fujian and belongs to the species of Anoectochilus roxburghii. The important pharmacologically active substances in Anoectochilus fujianensis are mainly: steroids, triterpenoids, flavonoids, carbohydrates, alkaloids, cardiac glycosides, esters, taurine, various amino acids, Trace elements and inorganic elements, etc. It is known as "the king of medicine", "golden grass", "sacred grass" and "bird ginseng" in folk. Among them, the steroids, triterpenes, polysaccharides, and flavonoids in Anoectochilus in Fujian are considered to be important active substances. The synthesis of these substances directly affects the medicinal value of Anoectochil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12Q1/6851C12R1/19
CPCC12N9/1085C12N15/70C12Q1/6851C12Y205/01001C12Q2563/107C12Q2531/113
Inventor 张君诚杨琳邹涵卓张杭颖邢建宏宋育红
Owner SANMING UNIV
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