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Dammarenediol synthase gene of panax japonicus var and applications thereof

A gene, dammarane-type technology, applied to the cloning and application of dammarenediol synthase gene in Panax notoginseng

Inactive Publication Date: 2015-01-21
黄璐琦 +10
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] So far, the research on the biosynthesis pathway of the active ingredient of Panax notoginseng is still blank, and there is no related report on the cloning of the dammarenediol synthase (DS) gene in Panax notoginseng

Method used

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  • Dammarenediol synthase gene of panax japonicus var and applications thereof
  • Dammarenediol synthase gene of panax japonicus var and applications thereof
  • Dammarenediol synthase gene of panax japonicus var and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0032] Transcriptome sequencing and data analysis of the rhizome of Panax notoginseng

[0033] 1. Sample collection

[0034] The four-year-old Panax japonicus.C.A.Mey.var.bipinnatifidus (Seem.)C.Y.Wu et K.M.Feng) was collected from Enshi, Hubei. The rhizomes, leaves, flowers, and fruits were taken separately and frozen in liquid nitrogen, and then stored in a -80°C refrigerator for later use.

[0035] 2. Isolation and detection of total RNA of Panax notoginseng

[0036] Fully grind all kinds of samples stored at -80°C in liquid nitrogen, and then use the optimized Trizol method to extract total RNA from the samples. The whole process is guaranteed to be carried out under low temperature conditions, and a certain concentration of PVP solution (polyvinylpyrrolidone) is added. , and appropriately increase the concentration of β-mercaptoethanol. After removing PVP and β-mercaptoethanol by centrifugation, use high-concentration NaAc solution to precipitate RNA, and DNase to remov...

Embodiment 2

[0042] Cloning of Panax notoginseng dammarenediol synthase gene:

[0043] Using the forward primer P1: 5'-ATGTGGAAGCTGAAGGTTGC-3', the reverse primer P2: 5'-TTAAATTT TGAGCTGCTGGTGG-3', using the Panax notoginseng rhizome cDNA library as a template to amplify the full-length sequence of the candidate gene for PCR amplification . The amplification system is as follows: 10×buffer 2.5ul, dNTP 1ul, primers P1 and P2 1ul each, Taq enzyme 0.5ul, template 1ul, and the rest made up with water, the total volume is 25ul. Reaction conditions: 30 reaction cycles, denaturation at 94°C for 1 min, annealing at 42°C for 2 min, extension at 75°C for 3 min. Finally, extend at 75°C for 10 min.

[0044] After cloning the full-length sequence of the candidate gene, link it to the cloning vector pT7-Blue and transform it into E. coli competent cells E.coli DH5α, the steps are as follows:

[0045] a) Take 100 μL of competent cell suspension from a -80°C ultra-low temperature refrigerator, thaw and...

Embodiment 3

[0053] Bioinformatics analysis of DS gene

[0054] The Panax notoginseng dammarenediol synthase (DS) gene obtained in the present invention has a full length of 2310bp, its sequence is shown in SEQ ID NO.1, wherein the open reading frame is located at 1-2310bp, and the encoded protein sequence is Shown in SEQ ID NO.2. Blast the full-length sequence of dammarenediol synthase that has been spliced ​​and analyzed in the NCBI database. The gene has a typical ISOPREN_C2_like superfamily domain, such as figure 2 .

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Abstract

The invention discloses a dammarenediol synthase gene of panax japonicus var and applications thereof. The dammarenediol synthase gene of panax japonicus var is prepared by the following steps: carrying out transcriptome sequencing and Denovo splicing on panax japonicus var rhizome by utilizing the second-generation Sole xaHiSeq2000 for the first time; analyzing and finding out a candidate gene for encoding the dammarenediol synthase (DS) in panax japonicus var and performing in vitro cloning, to finally obtain the dammarenediol synthase gene of panax japonicus var, wherein the sequence of the gene is SEQIDNO.1. The gene is genetically transformed into the panax japonicus var through agrobacterium tumefaciens mediated transformation, to obtain transgenic plant with high content of dammarane saponin, thus providing a technical support for the industrial production of the dammarane saponin.

Description

technical field [0001] The invention belongs to the field of biological technology, and mainly relates to the cloning and application of a dammarenediol synthase (Dammarenediol synthase, DS) gene in Panax notoginseng. Background technique [0002] Panax japonicus.C.A.Mey.var.bipinnatifidus (Seem.) C.Y.Wu et K.M.Feng) belongs to the Araliaceae Panax L. plant, is one of the traditional precious medicinal materials, has a high medicinal value Use value, such as heat-clearing and detoxifying, smoothing Qi and invigorating the stomach, promoting blood circulation and eliminating fatigue, nourishing and strengthening, etc. Its main active ingredients are saponins, of which oleanane-type pentacyclic triterpene saponins are the main ones, and at the same time contain a small amount of da Marane-type tetracyclic triterpenoid saponins. [0003] Panax notoginseng is a species recorded in the Chinese Pharmacopoeia. It has broad application prospects and considerable economic value. At...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/82A01H5/00
CPCC12N9/88C12N15/8243C12Y402/01125
Inventor 陈平黄璐琦张绍鹏赵小龙汪琪朱闻君伍翀王如峰邓琛张西锋
Owner 黄璐琦
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