CN107936100A

CN107936100A - Common calanthe herb mannose-binding protein

Info

Publication number: CN107936100A
Application number: CN201711144535.6A
Authority: CN
Inventors: 鲍锦库; 吴传芳; 梁丹凤; 龙鑫; 袁媛
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2017-11-17
Filing date: 2017-11-17
Publication date: 2018-04-20

Abstract

The present invention mainly uses homologous clone and RACE（Rapid Amplification of cDNA Ends）Technology, obtains the cDNA full length sequences and amino acid sequence of common calanthe herb mannose-binding protein, its sequence is respectively such as sequence table SEQ ID NO:1, SEQ ID NO:Shown in 2.Using the present invention, it can fast and effeciently expand to obtain common calanthe herb Mannose-binding protein complete sequence, contribute to the molecular biology research of the plant and its mannose-binding protein with family, be also more widely applied for it and provide strong support.

Description

Common calanthe herb mannose-binding protein

Technical field

The present invention relates to molecular biology gene engineering technology field, and in particular to one kind coding common calanthe herb mannose Protein-bonded DNA sequence dna and amino acid sequence.

Background technology

Common calanthe herb, Calanthe discolor Lindl., belongs to orchid, is a kind of traditional Chinese herbal medicine, Warm-natured, acrid-sweet flavor is nontoxic.Dissipating bind is can be used to, is detoxified, promoting blood circulation, relaxing muscles and tendons.Common calanthe herb is contained as a kind of parts of generic medicinal plants Mannose-binding protein abundance is very low, but its blood coagulation activity is very strong.Unifacial leaf mannose-binding protein family, is in vegetable protein A highly important branch of family, its forming member is very huge.The albumen of this family is combined with monospecific The ability of mannose or manna oligosacchride, and be distributed widely in monocotyledon, while also have a variety of very valuable Biological activity, such as antifungal activity and anti-insect activity.Van Damme, Balzarini J, Smeets et al. are successively from orchid Two kinds are isolated in section's plant helleborine and the stem tuber of two aspidistras with antimycotic mannose-binding protein existing for monomeric form (EHMBP and LOMBP).At present, also not on encode orchid common calanthe herb mannose-binding protein gene total order Row report.

The content of the invention

In view of above-mentioned shortcoming, it is an object of the invention to provide one kind to encode common calanthe herb mannose-binding protein DNA sequence dna and amino acid sequence.

SEQ ID NO in the nucleotide sequence of common calanthe herb mannose-binding protein of the present invention such as sequence table:1 institute Show.

SEQ ID NO in the amino acid sequence of common calanthe herb mannose-binding protein of the present invention such as sequence table:2 institutes Show.

The present invention utilizes homologous clone and RACE by the RNA extracted from the fresh young leaf tissue of common calanthe herb (rapid amplification of cDNA ends) technology, quick clone coding common calanthe herb mannose-binding protein Gene.

The invention has the advantages that：

A kind of orchid common calanthe herb can be rapidly and accurately obtained using technical method of the present invention and primer The full length gene sequence and amino acid sequence of mannose-binding protein.Enrich plant mannose-binding protein family member letter Breath, technical support is provided for subsequent applications and research.

Brief description of the drawings

Fig. 1 is the total serum IgE electrophoresis pattern of the common calanthe herb young leaf tissue of extraction,

Fig. 2 be to common calanthe herb Mannose-binding protein carry out 3 ' RACE cloned sequences electrophoresis pattern, wherein A Figure is the PCR product of first round amplification；B figures are the PCR products of the second wheel amplification.

Fig. 3 is the electrophoresis pattern that 5 ' RACE cloned sequences are carried out to common calanthe herb Mannose-binding protein.

Fig. 4 is the electrophoresis pattern of the nucleotide sequence (cDNA total lengths) of common calanthe herb mannose-binding protein.

Embodiment

1. material and method

1.1 material

Vegetable material：The fresh young leaf tissue of common calanthe herb：Direct clip.

Bacterial strain：Clone strain used is Escherichia coli (Escherichia coli (Migula) Castellani et Chalmers)Top 10。

Plasmid：PEASY-T1 Cloning Vector, size 3928bp, have ampicillin (Amp) and block that is mould Plain (Kal) two kinds of selection markers, easy to the screening of recon, have 5 '-cohesive terminus,cohesive termini of the free T of band, are the special of PCR product Cloning vector (is bought in Beijing Quanshijin Biotechnology Co., Ltd).

Kit and enzyme：Trizol reagents are bought from Invitrogen companies；A small amount of plastic recovery kits are purchased from Axygen Company.

T4 DNA Ligase, M-MLV reverse transcriptase, TdT (nucleotide terminal enzyme (DNA)), Ribonuclease Inhibitor, EasyTaqDNA Polymerase, EasyPfuDNA Polymerase and Fermentas restriction enzymes BamH I, Hind III are purchased from Beijing Quanshijin Biotechnology Co., Ltd.

Remaining chemicals is analytical reagents.

1.2 method

1.2.1 the extraction of common calanthe herb total serum IgE

The explanation of the extraction process reference InvitrogenTrizol kits of common calanthe herb total serum IgE is as follows：

(1) take 0.1mg or so fresh or the common calanthe herb young leaf tissue of -80 DEG C of Cord bloods is immediately placed in full of liquid In the mortar of nitrogen, tissue is fully ground into powdered；

(2) powder is dispensed into the 1.5mL centrifuge tubes that two Liquid nitrogen precoolers are crossed with spoon, and adds 1mL's thereto Trizol reagents, fully shaking mix, and are placed in about 5min on ice；

(3) 200 μ l chloroforms are added into pipe, 5min is placed after fully mixing, then at 4 DEG C, 12,000g centrifuge 5min；

(4) upper strata aqueous phase is transferred in new precooled centrifuge tube, 500 μ l isoamyl alcohol is added, after fully mixing 5min is placed, then at 4 DEG C, 12,000g centrifuge 5min；

(5) supernatant is outwelled, 75% ethanol is added and will precipitate and hang, 4 DEG C, 7,500g centrifugation 3min；

(6) repeat step 5；

(7) centrifuge tube is placed in ventilation and air-dries about 10min by supernatant evacuation as far as possible, make residual ethanol volatilization clean；

(8) the processed water of DEPC (DEPC water) of 20-25 μ lRNAasefree is added into pipe, passes through 1% agarose Gel electrophoresis verifies that the results are shown in Figure 1.

1.2.2 the design of aspecific degenerate primers

Orchid family unifacial leaf known to analysis plants mannose-binding protein sequence, and (sequence used is all from NCBI GenBankhttp://www.ncbi.nlm.nih.gov/), according to conservative region combination PCR primer design principle, design degeneracy Primer is used for the gene cloning of common calanthe herb mannose-binding protein.

Forward primer (forward primer) (FP)：5’-GAC TGC/T AAC/T CTC GTC/G CTC/G-3’；

Reverse transcription primer (TP1)：5’-GCT GTC AAC GAT ACG CTA CGT AAC GGC ATG ACA GTG T (18)-3’；

Reverse primer (reverse primer) (TP2)：5’-GTC AAC GAT ACG CTA CGT AAC G-3’；

Reverse primer (reverse primer) (TP3)：5’-TAC GTA ACG GCA TGA CAG TG -3’.

1.2.3mRNA reverse transcription is into cDNA

The reverse transcription operation of mRNA is following (with reference to the specification of TakaRa M-MLV)：

(1) take and following component is added in 0.5mL centrifuge tubes：

(2) mixture above is placed in 70 DEG C of 10min, is denatured RNA；Then it is immediately placed in 2min on ice；Of short duration centrifugation Mixing liquid is collected to tube bottom, and continues to add following component thereto, the cumulative volume of step (1) (2) is 20 μ l：

(3) flicking tube bottom makes liquid blending, and then of short duration centrifugation collects liquid to tube bottom, is placed in 42 DEG C of insulation 1min；

(4) 96 DEG C of high temperature 5min make reverse transcriptase inactivation so as to stopped reaction；

(5) it is placed on ice through the of short duration reactant that is collected by centrifugation, -20 DEG C can be stored in.

1.2.4 first round pcr amplification reaction

PCR reaction solution is added by following reaction system：

PCR reactions are carried out by the following conditions：By PCR reaction mixtures first through 94 DEG C, thermal denaturation 5min, then expand (94 DEG C, 30s；53 DEG C, 30s；72 DEG C, 45s；Carry out 35 circulations), the 10min at 72 DEG C.Reaction exists GeneAmpPCRSystem2400 thermal cyclers carry out on (Promega companies, Beijing).

1.2.5 the second wheel PCR reactions

Second wheel PCR reactions are using the first round PCR reaction product described in the present embodiment 1.2.4 as template, by the first round PCR product dilutes 10 times with distilled water, primer TP2 is replaced with the primer TP3 in the present embodiment 1.2.2, again by this reality Apply reaction system in a 1.2.4 and prepare PCR reaction solution progress PCR amplification.

1.2.61% agarose gel electrophoresis detection PCR product

Concrete operations are：The agarose of 1g is weighed in the triangular flask of a 250mL, in addition measures 100mLTE buffer solutions (10mM Tris-HCl, pH=8.0,1mMEDTA) in triangular flask, being heated in micro-wave oven makes it fully be dissolved to uniformly thoroughly Bright shape, is placed in after somewhat cooling down at room temperature, takes the ethidium bromide (EB) of 50 μ l to be added thereto, can fall glue after shaking up.Wait to be gelled After Gu, sample (PCR product, RNA etc.) is added into loading wells, the power supply of electrophoresis tank is opened, makes sample suitable under the proper Current direction swimming, is observed after electrophoresis in ultraviolet imager, as a result as Fig. 2 shows.

1.2.7PCR the recycling of amplified production (with reference to a small amount of plastic recovery kit specifications of Axygen pillars)

(1) Ago-Gel containing target DNA is cut in the UV lamp, is placed in having weighed up the 1.5mL centrifugations of weight Guan Zhong, weighs calculate the weight of blob of viscose again, using the weight as a gel volume (such as 100mg=100 μ l volumes)；

(2) BufferDE-A of 3 gel volumes is added, mixing is placed on about 8min in 75 DEG C of water-baths until blob of viscose is complete Melt, the interruption mixing per 2-3min；

(3) BufferDE-B for adding 0.5 BufferDE-A volume is mixed；When separated DNA fragmentation is less than 400bp When, the isopropanol of 1 gel volume of addition；

Mixed liquor in upper step is transferred to DNA to prepare in pipe (being placed in 2ml centrifuge tubes), 12,000 × g centrifugation 1min, are abandoned Filtrate；

(4) pipe will be prepared and puts back to centrifuge tube, added 500 μ lBufferW1,12,000 × g centrifugation 30s, abandon filtrate；

(5) pipe will be prepared and puts back to centrifuge tube, added 700 μ lBufferW2,12,000 × g centrifugation 30s, abandon filtrate；

(6) 12,000 × g centrifugations 1min washed once with 700 μ lBufferW2 with step 5 same method again；Managed preparing Centrifuge tube is put back to, >=13,000 × g idle running 2min, then pipe will be prepared and be placed under room temperature about 10min, make unnecessary alcohol volatilization dry Only；

(7) pipe will be prepared to be placed in the 1.5ml centrifuge tubes of cleaning, preparing film centre in DNA adds 25-30 μ lElute Or deionized water, it is stored at room temperature 1min, 12,000 × g centrifugation 1min eluted dnas.

1.2.8PCR the connection of amplified production and conversion (reference pEASY-T1CloningVector Mini Kit explanations Book)

Following component is sequentially added in 0.2ml centrifuge tubes：

It is gently mixed, 22-37 DEG C of reaction 5min can complete to connect, and after reaction, centrifuge tube is placed on ice；Connection Good recombinant plasmid is preferably immediately available for converting.

The coupled reaction product of 2-5 μ l is applied directly in the 1.5mlEP pipes equipped with 50ul competent cells, ice bath 30min, 42 DEG C of heat shock 90s, are again placed at 2min on ice, add 600ul nonreactive LB culture mediums, 37 DEG C, 200rpm sways 1h.After 1h, EP pipes are taken out, 2500rpm centrifugation 5min, take down 600ml supernatants, gently blow and beat outstanding bacterium.The solid LB with Amp resistances is coated in train Support on plate, 37 DEG C are incubated overnight.

1.2.9 the identification and sequencing of transformant

The full single bacterium colony of selected shape form rule, and picking expands culture into fluid nutrient medium.After cultivating 5h, take A small amount of bacterium solution carries out double digestion to identify transformant with bacterium colony PCR methods or extraction recombinant plasmid.

After confirming purpose fragment insertion carrier, remaining bacterium solution continues the overnight incubation at 37 DEG C.Overnight culture is served Sea base day biology engineering services Co., Ltd is sequenced, while adds the glycerine of final volume 10% to be stored in -20 DEG C of ice strain Case (short-term preservation).

Gained sequence carries out Blast similarity searchings on NCBI (American National Biotechnology Information center), adopts at the same time Sequence analysis is carried out with softwares such as DNAMAN5.2.2.0, further confirms that the fragment of insertion is that nine sons are a chain of according to actual sequence 3 ' ends of careless mannose-binding protein.

1.2.105 ' RACEPCR reactions

1.2.10.1 design of primers

3 ' the terminal sequence of common calanthe herb mannose-binding protein being sequenced according to 3 '-RACE designs 5 '-RACE reactions Required specific primer, it is as follows for nest-type PRC：

Reverse transcription nested primer (NP1)：5’-CACGCCACCATCTTCTTC-3’；

Nested primer (NP2)：5’-CTCGGCAAGGCGTAAATG-3’；

Nested primer (NP3)：5’-TAGCTTCCGCTGGCCCTGT-3’.

1.2.10.2mRNA reverse transcription goes out single-stranded cDNA and purifying

By the use of the NP1 described in the present embodiment 1.2.10.1 as reverse transcription primer, according to side described in the present embodiment 1.2.3 Method carries out reverse transcription.The RNaseA of 2-3 μ L is added in the cDNA that reverse transcription is completed to obtain backward, 37 DEG C of water-bath 30min remove RNA Afterwards, with the direct recovery purifying of a small amount of plastic recovery kits, final elution volume is depending on the amount of cDNA.

1.2.10.3cDNA homopolymeric tailing (TdTTailingofcDNA)

The cDNA amounts used in TdTTailing reactions can be depending on the amount of the total serum IgE extracted in the first step.

(1) following component is added into 0.2mL centrifuge tubes：

(2) 2-3min is incubated at 94 DEG C, is immediately placed in cooled on ice 2min, through of short duration collected after centrifugation sample, is positioned over On ice；

(3) plus 1 μ lTdT are gently mixed (20 μ l of final volume), 37 DEG C of insulation 30min；

(4) 60 DEG C of 10min heat inactivations TdT, it is of short duration that tailing product is collected by centrifugation and is put on ice.

1.2.10.4cDNA the PCR reactions of tailing product

Reaction substrate is made with the tailing cDNA in the present embodiment 1.2.10.3, with the TP1 in the present embodiment 1.2.2 and this reality The NP2 applied in a 1.2.10.1 makees primer, according to the PCR conditions in the present embodiment 1.2.4, carries out PCR amplification.Reaction product is put Saved backup in -20 DEG C of refrigerators.

1.2.10.5 nest-type PRC reacts

Take the 1 μ l of PCR product in the present embodiment 1.2.10.4 to be diluted in 9 μ lTE buffer solutions, dilute 10 times.Implemented with this The NP2 in TP2 and the present embodiment 1.210.1 in example 1.2.2 makees primer, according to the PCR conditions in the present embodiment 1.2.4, into The PCR amplification of the row first round.Distilled water after the completion of reaction by reaction product sterilizing dilutes 10 times, with the present embodiment 1.2.2 In TP3 and 1.2.10.1 in NP3 make primer, again with the PCR conditions in the present embodiment 1.2.4, carry out PCR amplification.Instead Answer product to be placed in -20 DEG C of refrigerators to save backup.

1.2.11PCR the electrophoresis detection of amplified fragments, recycling and connection

According to progress described in the present embodiment 1.2.6,1.2.7 and 1.2.8.Electrophoresis detection result such as Fig. 3 shows.

1.2.12PCR conversion, identification and the sequencing of amplified fragments

Carried out according to requirement in the present embodiment 1.2.8 and 1.2.9.

1.2.13 the acquisition of common calanthe herb Mannose-binding protein full length sequence

According to the sequencing result of 3 ' and 5 ' RACE in the present embodiment, design this section of splicing sequence and include including code area just To primers F LFP：5 '-ATGAAGATGAACATACTCCTCTGC-3 ' and reverse primer FLRP：5’- TTACTCATCACCCATAACCTTCACAA-3’.PCR amplification is carried out again according to the PCR conditions in the present embodiment 1.2.4, with The common calanthe herb cDNA of reverse transcription is template, uses EasyPfu DNA Polymerase (2.5U/l), annealing temperature setting instead 50 DEG C of progress PCR amplifications, obtain the common calanthe herb mannose-binding protein full-length gene of complete and accurate.

According to the present embodiment 1.2.6, the method in 1.2.7 and 1.2.8 carries out the electrophoresis detection of amplified production, cuts glue successively Recycling and connection, and convert into competent cell.According in the present embodiment 1.2.9 method carry out positive colony identification and Sequencing analysis.Obtain the full length nucleotide sequence (cDNA sequence) of common calanthe herb Mannose-binding protein, electrophoresis inspection Result such as Fig. 4 is surveyed to show.

From sequence table SEQ ID NO:It is complete that nucleotide sequence shown in 1 can be seen that obtained common calanthe herb cDNA Long sequence is made of 513 nucleotide, comprising a complete open reading frame, is encoded such as sequence table SEQ ID NO:2 institutes The mannose-binding protein precursor protein being made of 170 amino acid shown.The domain analysis shows carried out to CDA, CDA ammonia There are a complete mannose-binding protein Functional domains in base acid sequence, and with snowdrop mannose-binding protein (Galanthusnivalis agglutinin, GNA) equally, has 3 typical " QXDXNXVXY " mannose-binding activities Center, belongs in evolution than more conservative snowdrop correlation mannose-binding protein.

<110>Sichuan University

<120>Common calanthe herb mannose-binding protein

<160>2

<170>SIPOSequenceListing 1.0

<210> 1

<211> 513

<212> DNA

<213>Orchid family common calanthe herb（Orchidaceae Calanthe discolor Lindl.）

<400> 1

atgaagatgaacatactcctctgcaccgcatatctcacactcctcatcaccccatcatca 60

ggccaactctacaaccacttgctctctggtcagcgcctcaacacaggccaatctcttgta 120

cagagcggctatatcttcatcatccaaaatgactgcaatcttgtcctctacaatttgggt 180

actgcaatttgggcttcgggaacccatggtaggggtaacggatgctatcttatcatgcag 240

cgagacggtaaccttgttgtctatgacagaaataatcgcgccatatgggcaagtaacact 300

aaccgcagaactggaaactatatcctcatactacaaaaagatcgtaatgttgttatatat 360

agtgtacccacttggtctacgaggactaataccgttgactcggctgatgttgtcattgcc 420

cctacacaaaatgggacagtgctgccttcgggtgcggagcagaataaggtgagggaaatg 480

gggaagattgtgaaggttatgggtgatgagtaa 513

<210> 2

<211> 170

<212> PRT

<213>Orchid family common calanthe herb（Calanthe discolor Lindl.）

<400> 2

Met Lys Met Asn Ile Leu Leu Cys Thr Ala Tyr Leu Thr Leu Leu Ile

1 5 10 15

Thr Pro Ser Ser Gly Gln Leu Tyr Asn His Leu Leu Ser Gly Gln Arg

20 25 30

Leu Asn Thr Gly Gln Ser Leu Val Gln Ser Gly Tyr Ile Phe Ile Ile

35 40 45

Gln Asn Asp Cys Asn Leu Val Leu Tyr Asn Leu Gly Thr Ala Thr Trp

50 55 60

Ala Ser Gly Thr His Gly Arg Gly Asn Gly Cys Tyr Leu Ile Met Gln

65 70 75 80

Arg Asp Gly Asn Leu Val Val Tyr Asp Arg Asn Asn Arg Ala Ile Trp

85 90 95

Ala Ser Asn Thr Asn Ala Arg Thr Gly Asn Tyr Ile Leu Ile Leu Gln

100 105 110

Lys Asp Arg Asn Val Val Ile Tyr Ser Val Pro Thr Trp Ser Thr Arg

115 120 125

Thr Asn Thr Val Asp Ser Ala Asp Val Val Ile Ala Pro Thr Gln Asn

130 135 140

Gly Thr Val Leu Pro Ser Gly Ala Glu Gln Asn Lys Val Arg Glu Met

145 150 155 160

Gly Lys Ile Val Lys Val Met Gly Asp Glu

165 170

Claims

1. a kind of common calanthe herb mannose-binding protein, it is characterised in that it has SEQ ID NO in sequence table:Shown in 1 Nucleotide sequence.

2. a kind of common calanthe herb mannose-binding protein, it is characterised in that it has SEQ ID NO in sequence table:Shown in 2 Amino acid sequence.

3. by the RNA extracted from the fresh young leaf tissue of common calanthe herb, using homologous clone and RACE technologies, nine sons are obtained The cDNA full length sequences and amino acid sequence of a chain of grass mannose-binding protein.