CN103710366B - Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene - Google Patents

Gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene Download PDF

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CN103710366B
CN103710366B CN201410003074.0A CN201410003074A CN103710366B CN 103710366 B CN103710366 B CN 103710366B CN 201410003074 A CN201410003074 A CN 201410003074A CN 103710366 B CN103710366 B CN 103710366B
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CN103710366A (en
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刘涛
池姗
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Ocean University of China
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract

The invention relates to the field of genetic engineering technology, and particularly relates to a gracilaria chouae uridine diphosphate (UDP)-glucose pyrophosphorylase (UGPase) gene. The nucleotide sequence of the gene and the amino acid sequence of the coding protein are SEQ ID No.1 and SEQ ID No.2 respectively. According to the invention, the gene sequence is cloned through the gene cloning technology, and a prokaryotic expression vector is established; and enzyme activity detection on recombinant protein proves that the gene has the function of catalyzing UDP-glucose and pyrophosphoric acid to form glucose-1-phosphoric acid and UTP and belongs to a key enzyme coding gene of the synthesis path of agar-agar, starch, cellulose, trehalose, sucrose and the like. The gene has an important application value in increasing the content of economic components including algae agar-agar, starch, cellulose, trehalose, sucrose and the like.

Description

A kind of crisp fragrant plant mentioned in ancient texts UDPglucose pyrophosphorylase gene
Technical field
The present invention relates to a kind of crisp fragrant plant mentioned in ancient texts UDPglucose pyrophosphorylase gene.In particular to the nucleotide sequence of the crisp fragrant plant mentioned in ancient texts of one (Gracilaria chouae) UDPglucose pyrophosphorylase gene, and proteins encoded and synthetic starch, Mierocrystalline cellulose, trehalose, sucrose etc. ability and improvement Important Economic composition and degeneration-resistant proterties in application.
Background technology
Clone products synthesis related gene also verifies its function, discloses the relation between gene and product, auxiliaryly carries out breed improvement and has become International Agriculture breeding field and improve one of effective way of economic sector content; Meanwhile, genetic engineering technique production active substance and economic product is utilized to become the core content of modern biotechnology industry development.
UDPglucose pyrophosphorylase (UGPase, UDP-glucose pyrophosphorylase) be a key enzyme in biological carbohydrate metabolism process, major catalytic reaction Glc-1-P+UTP ← → UDPG+PPi in plant and algae, product uridine diphosphoglucose (the UDPG of UDPglucose pyrophosphorylase catalyzed reaction, uridine diphosphateglucose) as the form activating sugar main in body, can be used as glucosyl group donor and participate in agar-agar, sucrose, Mierocrystalline cellulose, hemicellulose, trehalose, pectin substance, callose and glycolipid, the anabolism of glycoprotein.
Marine algae is one of main raw material of the Important Economic resource products such as occurring in nature agar-agar, starch, Mierocrystalline cellulose.Especially be that the agar-agar of main raw material can be widely used in various food and pharmaceutical field with red algae.
The biosynthetic pathway of agar-agar, sucrose, Mierocrystalline cellulose, hemicellulose, trehalose, pectin substance, callose and the different substances such as glycolipid, glycoprotein is different, but is all using the product uridine diphosphoglucose (UDPG) of UDPglucose pyrophosphorylase (UGPase) catalyzed reaction as total initial reaction substrate.UDPglucose pyrophosphorylase (UGPase) catalysis activity directly has influence on the contents level of uridine diphosphoglucose (UDPG), and therefore, UDPglucose pyrophosphorylase (UGPase) is considered to the important key enzyme of a class.
UDPglucose pyrophosphorylase (UGPase) is extensively present in various organism, as green plants (GreenPlants), higher animal (Animals), diatom (Diatom), green alga (Green algae), brown alga (Brown algae), red algae (Red algae), fungi (Fungi), bacterium (Bacteria) etc.At present, in a large amount of green plants and microorganism, clone the gene of this enzyme and demonstrated its function, but what in algae, the clone of this gene carried out is also few, Griffithsia japonica (Griffithsia japonica), Chlamydomonas reinhardtii (Chlamydomonas reinhardtii), Thallus Gracilariae (Gracilariopsis lemaneiformis), fragrant plant mentioned in ancient texts (Gracilaria gracilis) is only had to obtain clone, but do not verify the function of this gene encoding enzyme.
One of product of UDPglucose pyrophosphorylase (UGPase) catalyzed reaction uridine diphosphoglucose (UDPG), as the initial substrate of the economic sectors such as synthesis of sucrose, Mierocrystalline cellulose, hemicellulose, pectin substance, makes green plants become the main raw material extracting above-mentioned substance.Simultaneously, in the biosynthesizing of the important seaweed gel-agar-agar of synthesis, UDPglucose pyrophosphorylase (UGPase) is considered to the rate-limiting enzyme in floridosides building-up process, control the synthesis of important product UDP-D-glucose, for the agar content improved in red algae, there is important value.At present, in plant, the functional verification of UDPglucose pyrophosphorylase (UGPase) gene is mainly through detecting the activity of its catalysis reversed reaction UDPG and tetra-sodium formation Cori ester and UTP, but in red algae, brown alga, the clone of UDPglucose pyrophosphorylase (UGPase) gene extremely lacks, and its function is confirmed not yet.
Summary of the invention
For the technical problem existed in currently available technology, the invention provides a kind of UDPglucose pyrophosphorylase gene, a kind of UDPglucose pyrophosphorylase albumen of its codified, described albumen has the activity of catalysis UDPG and tetra-sodium formation Cori ester and UTP, and the application of this gene can be used for improving agar-agar, sucrose, Mierocrystalline cellulose, hemicellulose, trehalose, pectin substance, callose and glycolipid, glycoprotein equal size.
An object of the present invention is to provide a kind of UDPglucose pyrophosphorylase gene, and described gene is the UDPglucose pyrophosphorylase gene separated from crisp fragrant plant mentioned in ancient texts (Gracilaria chouae), called after GcUGP; The nucleotide sequence of described gene is as shown in SEQ ID NO:1.
Two of object of the present invention is the albumen providing a kind of described genes encoding, nucleotide sequence coded by shown in SEQ ID NO:1 of described albumen, and its aminoacid sequence is as shown in SEQ ID NO:2; It has the activity of reversible catalysis UDPG and tetra-sodium formation Cori ester and UTP.
Three of the object of the invention is described gene and the application of proteins encoded in synthesis agar-agar, starch, Mierocrystalline cellulose, trehalose, sucrose thereof.
Four of the object of the invention is that described gene and proteins encoded thereof are being improved economic sector proterties and carrying out the application in fermentation engineering.
The present invention is by the method for gene clone, a UDPglucose pyrophosphorylase gene has been cloned into from crisp fragrant plant mentioned in ancient texts, and the albumen demonstrating its coding by experiment has the activity of catalysis UDPG and tetra-sodium formation Cori ester and UTP, it is the key gene of synthesis agar-agar, starch, Mierocrystalline cellulose, trehalose, sucrose.Clone and analyze crisp fragrant plant mentioned in ancient texts UDPglucose pyrophosphorylase gene and contribute to the red algae agar-agars such as the crisp fragrant plant mentioned in ancient texts of deep understanding, starch, Mierocrystalline cellulose, trehalose, Sucrose synthesis mechanism, also can provide genetic resources for associated products genetically engineered and molecular breeding simultaneously.The present invention is separated to UDPglucose pyrophosphorylase gene first from crisp fragrant plant mentioned in ancient texts (Gracilaria chouae), and by prokaryotic expression carrier, Enzyme assay is carried out to recombinant protein, confirm that it has the active function of catalysis UDPG and tetra-sodium formation Cori ester and UTP, there is the using value of agar-agar, starch, Mierocrystalline cellulose, trehalose, sucrose genetically engineered and molecular breeding.
Accompanying drawing explanation
Fig. 1 is the pcr amplification figure of GcUGP gene cDNA total length of the present invention.
Fig. 2 is GcUGP gene of the present invention and encoding amino acid sequence (being respectively initiator codon and terminator codon in square frame) thereof.
Fig. 3 is that after GcUGP gene transformation pET32a carrier of the present invention, PCR detects positive colony pcr amplification figure (1 swimming lane is pET32a BamHI and NotI double digestion product; 2 swimming lanes are crisp fragrant plant mentioned in ancient texts UGPase gene transformation pET32a carrier BamHI and NotI double digestion product; M is DNA standard molecular weight).
Fig. 4 is SDS-PAGE detection figure after GcUGP gene transformation e. coli bl21 expression product purifying of the present invention.
Fig. 5 is the Western-Blot detection figure of GcUGP gene transformation e. coli bl21 expression product of the present invention.
Fig. 6 is that the differing temps of GcUGP gene transformation e. coli bl21 expression product of the present invention is to the detection figure of enzymic activity.
Fig. 7 is that the pH of GcUGP gene transformation e. coli bl21 expression product of the present invention is to the detection figure of enzymic activity.
Fig. 8 is the detection figure of the different metal ions enzyme activity of GcUGP gene transformation e. coli bl21 expression product of the present invention.
Fig. 9 is the different concentration of substrate Enzyme activities figure of GcUGP gene transformation e. coli bl21 expression product of the present invention.
Figure 10 is the different concentration of substrate double reciprocal curve figure of GcUGP gene transformation e. coli bl21 expression product of the present invention.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these examples are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted specific experiment condition in the following example, usual conveniently condition, Molecular Cloning: A Laboratory guide (Sambrook J, et al.2008.Molecular Cloning:A Laboratory Manual, condition 3rdEd.), or according to the condition that manufacturer advises.
Embodiment 1: the cloning and analysis of full length gene coding region
Crisp fragrant plant mentioned in ancient texts gathers from Shantou City, Guangdong Province, and acquisition time is in August, 2011.Adopt Trizol method to extract crisp fragrant plant mentioned in ancient texts sporophyte total serum IgE, use TAKARA company PrimeScript II1 ststrand cDNA Synthesis test kit with the first chain cDNA of crisp fragrant plant mentioned in ancient texts sporophyte total serum IgE reverse transcription for template, adopt Touchdown round pcr to carry out the amplification of the CDS full length sequence of crisp fragrant plant mentioned in ancient texts GcUGP gene, amplimer comprises 2 groups of (5 '-TAGAATTCATGAATCGCGACTCCAGCTC-3 ' and 5 '-TGCGGCCGCTTAATGCGGAATCAC-3 '; 5 '-TAGAATTCATGAATCGCGACTCCAGCTCG-3 ' and 5 '-AGCGGCCGCATGCGGAATCACATG-3 ').Pcr amplification program is: 94 DEG C of 3min; 94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min, 15 circulations, each cycle annealing temperature reduces by 1 DEG C; 94 DEG C of 30s, 45 DEG C of 30s, 72 DEG C of 2min, 20 circulations; 72 DEG C of 10min.PCR primer is after 1% agarose gel electrophoresis detects, and the blob of viscose of cutting containing object band under ultraviolet lamp, uses sepharose to reclaim test kit and reclaim object fragment, in-20 DEG C of preservations.The object fragment reclaimed, spend the night in 16 DEG C of metal baths and be connected to cloning vector pMD19-T, and be transformed in competent escherichia coli cell E.coli Top10, coat on the LB solid medium containing 100mg/mL Amp, 37 DEG C of incubated overnight, after the blue hickie screening of IPTG/X-gal, a picking 4-10 positive colony checks order.By sequencing result by sequence alignment, be separated to a crisp fragrant plant mentioned in ancient texts UGP gene, called after GcUGP.GcUGP CDS sequence is 1488bp, and its nucleotide sequence is as shown in SEQ ID NO:1, and 496 amino acid of encoding, take ATG as initiator codon, TAA is terminator codon.
The preparation of embodiment 2:GcUGP proteins encoded and analysis
Crisp fragrant plant mentioned in ancient texts GcUGP PCR primer is after the agarose gel electrophoresis detection of 1%, object band is cut under ultraviolet lamp, sepharose reclaims, recovery product GcUGP and pET32a plasmid carry out BamHI and NotI double digestion, after 37 DEG C of metal bath 3-4h, detect with 1% agarose gel electrophoresis, use sepharose to reclaim test kit and reclaim.Be connected with plasmid pET32a by object fragment GcUGP, 16 DEG C are spent the night, the recombinant plasmid called after pET32a-GcUGP built.
By recombinant plasmid transformed E. coli expression strains BL21, picking BL21 positive colony, shake bacterium and preserve bacterial strain.PCR detects recon.PCR primer detects in 1% agarose gel electrophoresis, the imaging of automatic gel image analysis instrument.Picking electrophoresis detection inserts the correct cloning and sequencing of band, and detect with or without sudden change, whether frameshit frame changes.
To successfully be connected to the BL21 bacterial strain of object fragment, and cultivate to shake bacterium according to 1:1000 ratio (10ml LB liquid nutrient medium+10ul AP+10ul bacterium liquid), surveyed OD value, until OD600nm value reaches 0.6(3-4h every one hour).With the IPTG concentration induction bacterium liquid of 0.1mM, inductive condition is 25 DEG C, 160rpm, induction 6-8h.Get 2ml bacterium liquid 4 DEG C of 12000rpm5min after induction terminates centrifugal, abandon supernatant, pipe is inverted on thieving paper; Precipitation adds 1/2 volume and shifts to an earlier date 1 good × PBS(PH=7.5 of precooling) in (namely 2ml centrifugal after precipitation in add 1ml1 × PBS), fully mix.The broken bacterium liquid of ultrasonic method, collect the supernatant liquor after fragmentation, with the membrane filtration of 0.45 μm, after Ni column purification, the protein sample collected is put into dialysis tubing and is dialysed, and to remove unwanted ion, obtains restructuring GcUGP albumen, its aminoacid sequence, as shown in SEQ ID NO:2, adopts SDS-PAGE, Western-Blot to detect the expression of recombinant protein.
The functional verification of embodiment 3:GcUGP proteins encoded
UGP enzyme activity determination: reaction system is as follows: 100mM1 × PBS, 0.85mM UDPG, 0.5mM PPi, 5mM Mgcl2,0.3mM NADP, 5unit PGM, restructuring GcUGP albumen prepared by 5unit GDH and appropriate embodiment 2, total reaction system is 1mL, adds substrate M-6-P initial action.Initial action after hatching 2min after the system of above-mentioned removing substrate being mixed under relevant temperature condition, with corresponding damping fluid for blank, in the change of 340nm place difference assaying reaction 0min, 6min and 12min light absorption value, each reaction arranges 4 Duplicate Samples.After testing, enzyme is lived as 487.16U/g, and be 1.67umol to the Km of UDPG, optimal reactive temperature is 40 DEG C, and optimal pH is 8.0.This enzyme is high temperature enzyme, basic protein; Mg 2+, Ca 2+, Mn 2+and Zn 2+can promote that enzyme is lived, Pb 2+, Cu 2+suppress it active.
The gene that current UGP enzyme is relevant and albumen obtain detection in some plants and bacterium; such as bacterium Streptococcusequi subsp.Zooepidemicus(is see non-patent literature: Ma Z; Fan HJ, Lu CP.Molecular cloningand analysis of the UDP-Glucose Pyrophosphorylase in Streptococcus equi subsp.zooepidemicus.Mol Biol Rep.2011Apr; 38 (4): 2751-60.); bacterium Sphingomonas paucimobilis(is see non-patent literature: Marques AR; Ferreira PB; S á-Correia I, Fialho AM.Characterizationof the ugpG gene encoding a UDP-glucose pyrophosphorylase from the gellan gum producerSphingomonas paucimobilis ATCC31461.Mol Genet Genomics.2003Mar; 268 (6): 816-24.); bacterium Mycobacterium tuberculosis(is see non-patent literature: Lai X; Wu J; Chen S; Zhang X; WangH.Expression, purification, and characterization of a functionally activeMycobacterium tuberculosis UDP-glucose pyrophosphorylase.Protein Expr Purif.2008Sep; 61 (1): 50-6.); plant barley (barley) is (see non-patent literature: Decker D; Meng M; Gornicka A; Hofer A; Wilczynska M, Kleczkowski LA.Substrate kinetics and substrate effects onthe quaternary structure of barley UDP-glucose pyrophosphorylase.Phytochemistry.2012Jul; 79:39-45.); But its respective enzymic activity is all far smaller than the sea-tangle UGP gene originally researched and analysed, and the Km value of UGP enzyme prepared by above-mentioned several UGP enzyme and the present invention contrasts as follows:
Source Km value (μm ol)
The embodiment of the present invention 3 1.67
Bacterium Streptococcus equi subsp.Zooepidemicus 8.5
Bacterium Sphingomonas paucimobilis 7.5
Bacterium Mycobacterium tuberculosis 11.87
Barley (barley) 250
Although the invention describes concrete example, having a bit is obvious to those skilled in the art, namely can make various changes the present invention and change under the premise without departing from the spirit and scope of the present invention.Therefore, claims cover all these variations within the scope of the present invention.

Claims (3)

1. encode the gene of UDPglucose pyrophosphorylase in crisp fragrant plant mentioned in ancient texts, it is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:1.
2. by an albumen for genes encoding described in claim 1, it is characterized in that, the aminoacid sequence of described albumen is as shown in SEQ ID NO:2, and it has the function of reversible catalysis UDPG and tetra-sodium formation Cori ester and UTP.
3. the application of albumen in synthesis agar-agar, starch, Mierocrystalline cellulose, trehalose, sucrose of UDPglucose pyrophosphorylase gene according to claim 1 and coding thereof.
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