Saffron, saffron endophytic fungi GGPPS (geranylgeranyl pyrophosphate synthase) gene, gene cloning method and application

A technology of endophytic fungi and cloning method, which is applied in the field of genetic engineering and can solve the problem of undiscovered function verification.

Active Publication Date: 2017-01-04
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As to whether there is geranylgeranyl pyrophosphate synthase in saffron and the functional verification of the geranylgeranyl pyrophosphate synthase gene in saffron, there is no discovery in the current patent literature.

Method used

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  • Saffron, saffron endophytic fungi GGPPS (geranylgeranyl pyrophosphate synthase) gene, gene cloning method and application
  • Saffron, saffron endophytic fungi GGPPS (geranylgeranyl pyrophosphate synthase) gene, gene cloning method and application
  • Saffron, saffron endophytic fungi GGPPS (geranylgeranyl pyrophosphate synthase) gene, gene cloning method and application

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Experimental program
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Embodiment 1

[0036] The extraction of embodiment 1 saffron endophytic fungus total RNA

[0037] The biological name of the endophytic fungus of saffron is Cadophora luteo-olivacea., and its designated name is 3P1CQ1. It is currently preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures. The preservation number is: CGMCCNO.11606, and the preservation date is: 2015 November 24; Preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The cultivation and storage conditions of the saffron endophytic fungus are plate storage, and the medium conditions are: 200g of potatoes, 20g of glucose, 5g of potassium dihydrogen phosphate, 1.46g of anhydrous magnesium sulfate, 10mg of VB1, 18g of agar, and single steamed water to 1L.

[0038] Take an appropriate amount of saffron endophytic fungus mycelium and put it into a pre-cooled mortar, pour liquid nitrogen into it, grind it quickly to powder, add 1ml RNAiso Plus, and follo...

Embodiment 2

[0039] Example 2 Cloning of the GGPPS Conserved Fragment of the Endophytic Fungus Saffron

[0040] Genomic DNA of endophytic fungus mycelium of saffron was extracted by CTAB method as a template, and primers Fg1: 5'-AAATCAGTTCTGCGACCTGTTCC-3' and Rg1: 5'-CGGTCTTGTTTCCAACCATTTC-3' were used to design primers in conserved regions for PCR amplification. The reaction system is as follows: Template 1 μL, primers Fg1 (20 μM) and Rg1 (20 μM) each 0.5 μL, TakaraPremix Taq 25 μL, ddH 2 O 22 μL, the PCR amplified band was recovered by gel, ligated and transformed into DH5α competent cells, and then sent for sequencing to obtain the GGPPS conserved fragment sequence.

Embodiment 3

[0041] Example 3 Cloning of the 3' end of the GGPPS gene of the saffron endophyte

[0042] The 3′RACE (rapid-amplification of cDNA ends, RACE) specific primer GSP2 was designed according to the GGPPS conserved fragment sequence: 5′–CGACCTGTTC CAGAGGGCG ATTG–3′. Then, 3'-RACE cDNA and 5'-RACE cDNA were synthesized by reverse transcription using the protocol provided by SMART RACE cDNA Amplification Kit (CLONTECH, USA). 3′-RACE PCR reaction system: Template (3′-RACE cDNA) 4 μL, primer Long UP (4 μM) 1 μL, primer Short UP (20 μM) 1 μL, primer GSP2 (20 μM) 1 μL, 10×Buffer 5 μL, dNTP (2.5 mM each) 4 μL, Taq DNA polymerase 25 μL, ddH 2 O 9 μL, PCR amplification conditions: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, extension at 72°C for 3 min, 5 cycles; denaturation at 94°C for 30 s, annealing at 70°C for 30 s, extension at 72°C for 2 min, 5 cycles; denaturation at 94°C 30s, annealing at 68°C for 30s, extension at 72°C for 2min, 25 cycles; finally extension...

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Abstract

The invention relates to a saffron, a saffron endophytic fungi GGPPS (geranylgeranyl pyrophosphate synthase) gene, a gene cloning method and application, and belongs to the technical field of gene engineering. The invention discloses two genes including the saffron and GGPPS in metabolic pathway of the saffron endophytic fungi; the two genes have nucleotide sequences shown as SEQ ID NO.4 and SEQ ID NO.3, have the identical open reading frames shown as SEQ ID NO.1 and code amino acid sequences shown as code SEQ ID NO.2. The color genetic complementary reaction verifies that the GGPPS gene coding protein has a typical GGPPS enzyme function; FPP (farnesyl pyrophosphate) and IPP (isopentenyl pyrophosphate) can be catalyzed to be synthesized into the GGPPS (C<20>).

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a GGPPS gene of saffron and saffron endophytic fungus, a gene cloning method and application. Background technique [0002] Geranylgeranyl pyrophosphate synthase (GGPPS) is a key enzyme in the synthesis of terpenes, gibberellins, vitamin E and other compounds. Geranylgeranylpyrophosphate synthase can catalyze the condensation of 15-carbon farnesyl pyrophosphate (FPP) and 5-carbon isopentenyl pyrophosphate (IPP) to generate 20-carbon geranylgeranyl Pyrophosphate (GGPP), GGPP is a key precursor for the biosynthesis of terpenoids and their carotenoids, see figure 1 . At present, the existence of this gene has been found in bacteria, fungi, algae and higher plants, and it is involved in the biosynthesis of different compounds. [0003] Chinese patent CN 101475946A discloses a Danshen geranyl geranyl pyrophosphate synthase gene and its encoded protein and it...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/113C12N1/14C12N15/10C12P23/00C12P27/00C12P17/06C12R1/645
CPCC12N9/1085C12P17/06C12P23/00C12P27/00C12Q1/686C12Y205/01029C12Q2531/113
Inventor 赵军曾子金焉兆萍
Owner SHANGHAI NORMAL UNIVERSITY
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