A kind of geranyl pyrophosphate synthase gene and its application from Fujian Aureus aureus

A technology of geranyl pyrophosphate and base pyrophosphate, which is applied in the field of genetic engineering to achieve the effects of easy amplification and simple operation

Active Publication Date: 2021-08-06
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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  • A kind of geranyl pyrophosphate synthase gene and its application from Fujian Aureus aureus
  • A kind of geranyl pyrophosphate synthase gene and its application from Fujian Aureus aureus
  • A kind of geranyl pyrophosphate synthase gene and its application from Fujian Aureus aureus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The present embodiment provides the extraction of total leaf RNA of Clematis clematis, using the Trizol extraction kit of Dalian Bao Biological Cable Co., Ltd., comprising the following steps:

[0027] (1) 100 mg of fresh Clematis clematis leaves were ground into powder and put into (1) a 1.5 mL centrifuge tube, then immediately added 1 mL of RNAiso Plus, mixed 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 12,000 g at 4° C. for 5 minutes.

[0029] (3) Transfer 800ml of the supernatant into a new centrifuge tube. Do not absorb the precipitate, then add 200ml of chloroform into the supernatant, shake and mix vigorously until the homogenate is emulsified into milky white, and then stand at room temperature for 5 minutes to obtain a mixture. liquid.

[0030] (4) The mixture was centrifuged at 12000 g at 4° C. for 15 min, and the homogenate was divided into three la...

Embodiment 2

[0036] This embodiment provides a method for sequencing the transcriptome of A. roxburghii, which includes the following steps:

[0037] Extract the total RNA from the leaves of Aureus clematis, test the quality of RNA extraction, and meet the requirements for library construction (RNA concentration>250ng / μL, total amount>20μg, OD260 / OD280 between 1.8 and 2.2, good integrity, RIN>6.5) . Then, use magnetic beads to enrich poly(A) mRNA and break it into fragments, and use this as a template to sequentially synthesize the first cDNA strand and the second cDNA strand, after purification, elution, end repair, and addition of poly(A) ) followed by ligation of sequencing adapters. Fragments with a size of 200bp to 700bp were selected for PCR amplification, a cDNA sequencing library was established, and Ilumina HiSeq 2000 was used for sequencing. This part of the experiment was entrusted to Main Technology Service Cable Company to complete.

[0038] Use the short reads assembly sof...

Embodiment 3

[0040] This embodiment provides a method for synthesizing the first strand of cDNA of A. roxburghii, comprising the following steps:

[0041] With the purified total RNA of Clematis clematis leaves obtained in Example 1 as a template, oligo(dT)18 was used as a reverse transcription primer, and PrimeScript™ Reverse Transcriptase (TakaRa China) was used according to SMART TM PCR cDNASynthesis Kit (Clontech USA) was used to synthesize the first strand of cDNA. The total volume of the reaction system was 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) Incubate the reverse transcription mixture 1 in step (1) at 65°C for 5 minutes, then quickly cool it on ice for 2 minutes, and centrifuge for a few seconds to gather the mixture of template RNA and primers at the bottom of the PE tube;

[0...

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Abstract

The invention provides a geranyl pyrophosphate synthase gene derived from Fujian Aureus aureus and its application, and relates to the technical field of genetic engineering. The geranyl pyrophosphate synthase gene has the nucleotide sequence shown in SEQ ID NO.1 and the amino acid sequence shown in SEQ ID NO.2. The invention provides an important basis for the selection and breeding of clematis varieties, and also provides a basis for the research on transgenic plant strains enriched in terpenoids.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, and in particular to a geranyl pyrophosphate synthase gene derived from A. auranthus fujian and application thereof. Background technique [0002] Fujian clematis (Anoectochilus roxburghii) is a perennial herbaceous plant that grows in Fujian and belongs to the species of Anoectochilus roxburghii in the family Orchidaceae. The important pharmacologically active substances in Aureus aureus Fujian are mainly: steroidal compounds, triterpenoids, flavonoids, sugar components, alkaloids, cardiac glycosides, esters, taurine, various amino acids, Trace elements and inorganic elements, etc. Known among the people as "King of Medicine", "Golden Grass", "God Grass", "Bird Ginseng" and other laudatory titles. Among them, steroids, triterpenes, polysaccharides, flavonoids, etc. in A. roxburghii are considered to be important active substances, and the synthesis of these substances directly affe...

Claims

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Application Information

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