CN107936100A - Common calanthe herb mannose-binding protein - Google Patents

Abstract

The present invention mainly adopts homologous cloning and RACE (Rapid Amplification of cDNA Ends) technology to obtain the full-length cDNA sequence and amino acid sequence of the nine-son-catenin mannose-binding protein, the sequences of which are shown in the sequence table as SEQ ID NO: 1, SEQ ID NO:2 shown. The present invention can quickly and effectively amplify the complete sequence of the mannose-binding protein gene, which is helpful for the molecular biology research of the plant and the mannose-binding protein of the same family, and also for its wider application Provided strong support.

Description

mannose-binding protein

technical field

The invention relates to the technical field of genetic engineering of molecular biology, in particular to a DNA sequence and an amino acid sequence encoding a mannose-binding protein.

Background technique

Calanthe discolor Lindl., belonging to the Orchidaceae plant, is a traditional Chinese herbal medicine, warm in nature, sweet and pungent in taste, and non-toxic. It can be used for dispelling stagnation, detoxification, promoting blood circulation and relaxing tendons. As a commonly used Chinese herbal medicine, the mannose-binding protein contained in the cinnamon is very low, but its blood coagulation activity is very strong. The monocot mannose-binding protein family is a very important branch of the plant protein family, and its members are very large. This family of proteins has the ability to specifically bind mannose or manno-oligosaccharides, and is widely distributed in monocotyledonous plants, and also has a variety of valuable biological activities, such as antifungal activity and insect resistance activity Wait. Van Damme, Balzarini J, Smeets et al. successively isolated two antifungal mannose-binding proteins (EHMBP and LOMBP) in the form of monomers from the tubers of the orchid plants Huoshaolan and Eryelan. At present, there is no report on the complete sequence of the gene encoding the mannose-binding protein of orchids.

Contents of the invention

In view of the above deficiencies, the object of the present invention is to provide a DNA sequence and amino acid sequence encoding a nonadenin glysmannose-binding protein.

The nucleotide sequence of the noncatenin mannose-binding protein of the present invention is shown in SEQ ID NO: 1 in the sequence listing.

The amino acid sequence of the nine-catenin mannose-binding protein of the present invention is shown in SEQ ID NO: 2 in the sequence listing.

The present invention rapidly clones the gene encoding the mannose-binding protein of C. chinensis by using the RNA extracted from the fresh tender leaf tissue of C. chinensis by homologous cloning and RACE (rapid amplification of cDNA ends).

The present invention has the following beneficial effects:

The full-length sequence and amino acid sequence of a gene of the orchidaceae Nine-catenin mannose-binding protein can be obtained rapidly and accurately by using the technical method and the primers described in the present invention. It enriches the information of plant mannose-binding protein family members and provides technical support for subsequent applications and research.

Description of drawings

Fig. 1 is the total RNA electrophoresis profile of the extracted Cynoptera chinensis tender leaf tissue,

Fig. 2 is the electrophoresis profile of the 3' RACE cloned fragment of the nine sub-catenin mannose-binding protein gene, wherein A is the PCR product of the first round of amplification; B is the PCR product of the second round of amplification.

Fig. 3 is the electrophoresis pattern of the 5' RACE cloned fragment of the nine-catenin glyphosate mannose-binding protein gene.

Fig. 4 is an electrophoresis pattern of the nucleotide sequence (full length of cDNA) of the noncatenin mannose-binding protein.

Detailed ways

1. Materials and Methods

1.1 Materials

Plant material: fresh young leaf tissue of Cynthia chinensis: cut directly.

Bacterial strain: The cloned strain used was Escherichia coli (Migula) Castellani et Chalmers Top 10.

Plasmid: pEASY-T1 Cloning Vector, with a size of 3928bp, has two selection markers of ampicillin (Amp) and kanamycin (Kal), which is convenient for the screening of recombinants, and has a 5'-sticky end with a free T, which is A dedicated cloning vector for PCR products (purchased from Beijing Quanshijin Biotechnology Co., Ltd.).

Kits and enzymes: Trizol reagent was purchased from Invitrogen; small volume gel recovery kit was purchased from Axygen.

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

The rest of the chemicals are analytical reagents.

1.2 Method

1.2.1 Extraction of total RNA from Cynoptera chinensis

The extraction process of the total RNA of Cynoptera chinensis refers to the instructions of the InvitrogenTrizol kit, as follows:

(1) Take about 0.1 mg of fresh or -80°C cryopreserved young Leaf tissue of Dictaria chinensis and quickly place it in a mortar filled with liquid nitrogen, and grind the tissue into powder;

(2) Divide the powder into two liquid nitrogen pre-cooled 1.5mL centrifuge tubes with a medicine spoon, add 1mL of Trizol reagent to them, shake and mix well, and place on ice for about 5 minutes;

(3) Add 200 μl of chloroform to the tube, mix well, let it stand for 5 minutes, and then centrifuge at 12,000 g for 5 minutes at 4°C;

(4) Transfer the upper aqueous phase to a new pre-cooled centrifuge tube, add 500 μl of isoamyl alcohol, mix well and let stand for 5 minutes, then centrifuge at 12,000 g for 5 minutes at 4°C;

(5) Pour off the supernatant, add 75% ethanol to suspend the precipitate, and centrifuge at 7,500g for 3min at 4°C;

(6) Repeat step 5;

(7) Pour out the supernatant as much as possible, and place the centrifuge tube in a ventilated place to air dry for about 10 minutes, so that the residual ethanol is evaporated;

(8) Add 20-25 μl of RNAasefree DEPC-treated water (DEPC water) to the tube, and verify by 1% agarose gel electrophoresis. The results are shown in FIG. 1 .

1.2.2 Design of non-specific degenerate primers

Analyze the known mannose-binding protein sequences of Orchidaceae monocotyledonous plants (the sequences used are all from NCBIGenBank http://www.ncbi.nlm.nih.gov/ ), and design degenerate primers based on the principle of PCR primer design combined with conserved regions Cloning of the gene for the mannose-binding protein of the genus-catenin.

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 (TP2): 5'-GTC AAC GAT ACG CTA CGT AAC G-3';

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

1.2.3 Reverse transcription of mRNA into cDNA

The reverse transcription operation of mRNA is as follows (refer to the instructions of TakaRa M-MLV):

(1) Take a 0.5mL centrifuge tube and add the following ingredients:

(2) Place the above mixture at 70°C for 10 minutes to denature the RNA; then quickly place it on ice for 2 minutes; briefly centrifuge to collect the mixed liquid to the bottom of the tube, and continue to add the following components to it, the total of steps (1) and (2) The volume is 20 μl:

(3) Gently flick the bottom of the tube to mix the liquid, then centrifuge briefly to collect the liquid to the bottom of the tube, and incubate at 42°C for 1 min;

(4) High temperature at 96°C for 5 minutes to inactivate reverse transcriptase and stop the reaction;

(5) The reaction was collected by brief centrifugation and placed on ice, which could be stored at -20°C.

1.2.4 The first round of PCR amplification reaction

Add the PCR reaction solution according to the following reaction system:

The PCR reaction was carried out according to the following conditions: the PCR reaction mixture was subjected to heat denaturation at 94°C for 5 minutes, and then amplified (94°C, 30s; 53°C, 30s; 72°C, 45s; for 35 cycles), and then at 72°C Next 10min. The reaction was performed on a GeneAmpPCRSystem2400 thermal cycler (Promega, Beijing).

1.2.5 The second round of PCR reaction

The second round of PCR reaction takes the first round of PCR reaction product described in this embodiment 1.2.4 as a template, and the first round of PCR product is diluted 10 times with distilled water, with the primer TP3 described in this embodiment 1.2.2 Instead of primer TP2, PCR reaction solution was prepared again according to the reaction system in Example 1.2.4 for PCR amplification.

1.2.61% agarose gel electrophoresis to detect PCR products

The specific operation is: weigh 1g of agarose in a 250mL conical flask, and additionally measure 100mLTE buffer solution (10mM Tris-HCl, pH=8.0, 1mM EDTA) in the conical flask, heat it in a microwave oven to fully dissolve it to Uniform and transparent, place at room temperature and cool down slightly, add 50 μl of ethidium bromide (EB) into it, shake well and then pour the glue. After the gel is solidified, add the sample (PCR product, RNA, etc.) to the spotting hole, turn on the power of the electrophoresis tank, and make the sample swim in the direction of the current at an appropriate voltage. After the electrophoresis is completed, observe it in the ultraviolet imager. 2 shows.

1.2.7 Recovery of PCR amplification products (refer to the instructions of Axygen Column Mini Gel Recovery Kit)

(1) Cut out the agarose gel containing the target DNA under the ultraviolet light, and place it in a 1.5mL centrifuge tube that has been weighed, weigh again to calculate the weight of the gel block, and use this weight as a gel volume (such as 100 mg = 100 μl volume);

(2) Add 3 gel volumes of BufferDE-A, mix well and place in a 75°C water bath for about 8 minutes until the gel blocks are completely melted, and mix intermittently every 2-3 minutes;

(3) Add 0.5 BufferDE-A volume of BufferDE-B and mix well; when the separated DNA fragment is less than 400bp, add 1 gel volume of isopropanol;

Transfer the mixture in the previous step to a DNA preparation tube (placed in a 2ml centrifuge tube), centrifuge at 12,000×g for 1 min, and discard the filtrate;

(4) Put the preparation tube back into the centrifuge tube, add 500μl BufferW1, centrifuge at 12,000×g for 30s, and discard the filtrate;

(5) Put the preparation tube back into the centrifuge tube, add 700μl BufferW2, centrifuge at 12,000×g for 30s, and discard the filtrate;

(6) Wash again with 700μl BufferW2 in the same way as step 5 and centrifuge at 12,000×g for 1min; put the preparation tube back into the centrifuge tube, spin it at ≥13,000×g for 2 minutes, and then place the preparation tube at room temperature for about 10 minutes to evaporate the excess ethanol ;

(7) Place the preparation tube in a clean 1.5ml centrifuge tube, add 25-30 μl of Elute or deionized water to the center of the DNA preparation membrane, let it stand at room temperature for 1 min, and centrifuge at 12,000×g for 1 min to elute the DNA.

1.2.8 Ligation and transformation of PCR amplified products (refer to pEASY-T1CloningVector small kit manual)

Add the following ingredients in sequence in a 0.2ml centrifuge tube:

Mix gently and react at 22-37°C for 5 minutes to complete the connection. After the reaction, place the centrifuge tube on ice; the connected recombinant plasmid is best used for transformation immediately.

Add 2-5 μl of the ligation reaction product directly to a 1.5ml EP tube containing 50ul competent cells, bathe in ice for 30min, heat shock at 42°C for 90s, place on ice again for 2min, add 600ul of anti-LB medium, and heat at 37°C, Shake at 200rpm for 1h. After 1h, take out the EP tube, centrifuge at 2500rpm for 5min, remove 600ml of supernatant, and blow the suspended bacteria gently. Spread on solid LB plates with Amp resistance and culture overnight at 37°C.

1.2.9 Identification and sequencing of transformants

Select a single colony with regular shape and shape, and pick it into a liquid medium for expansion. After culturing for 5 hours, take a small amount of bacterial liquid and use the colony PCR method or extract the recombinant plasmid for double enzyme digestion to identify transformants.

After confirming that the target fragment was inserted into the vector, the remaining bacterial solution was cultured overnight at 37°C. The overnight culture was sent to Shanghai Jitian Bioengineering Technology Service Co., Ltd. for sequencing, and the strain plus 10% glycerol in the final volume was stored in a -20°C refrigerator (short-term storage).

The resulting sequence was searched for Blast similarity on NCBI (National Center for Biotechnology Information, USA), and DNAMAN5.2.2.0 and other software were used for sequence analysis. According to the actual sequence, it was further confirmed that the inserted fragment was a mannose-binding protein 3' end.

1.2.10 5'RACEPCR reaction

1.2.10.1 Primer design

The specific primers required for the 5'-RACE reaction were designed according to the 3'-terminal sequence of the nine sub-catenin mannose-binding protein obtained by 3'-RACE sequencing, and were used for nested PCR, as follows:

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

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

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

1.2.10.2 mRNA reverse transcription into single-stranded cDNA and purification

Using NP1 described in Example 1.2.10.1 as a reverse transcription primer, reverse transcription was performed according to the method described in Example 1.2.3. After the reverse transcription is completed, add 2-3 μL of RNaseA to the obtained cDNA, remove the RNA in a 37°C water bath for 30 minutes, and then directly recover and purify it with a small gel recovery kit. The final elution volume depends on the amount of cDNA.

1.2.10.3 Homopolymer tailing of cDNA (TdTTailingofcDNA)

The amount of cDNA used in the TdTTailing reaction can be determined according to the amount of total RNA extracted in the first step.

(1) Add the following ingredients to a 0.2mL centrifuge tube:

(2) Incubate at 94°C for 2-3 minutes, quickly place on ice to cool for 2 minutes, collect samples after brief centrifugation, and place on ice;

(3) Add 1 μl TdT and mix gently (final volume 20 μl), incubate at 37°C for 30 minutes;

(4) TdT was heat-inactivated at 60°C for 10 minutes, and the tailed product was collected by brief centrifugation and placed on ice.

1.2.10.4 PCR reaction of cDNA tailed product

Using the tailed cDNA in Example 1.2.10.3 as the reaction substrate, using TP1 in Example 1.2.2 and NP2 in Example 1.2.10.1 as primers, according to the PCR in Example 1.2.4 Conditions for PCR amplification. The reaction product was stored in a -20°C refrigerator for future use.

1.2.10.5 Nested PCR reaction

Take 1 μl of the PCR product in Example 1.2.10.4 and dilute it into 9 μl of TE buffer, and dilute it 10 times. Using TP2 in Example 1.2.2 and NP2 in Example 1.210.1 as primers, the first round of PCR amplification was performed according to the PCR conditions in Example 1.2.4. After the reaction is completed, the reaction product is diluted 10 times with sterilized distilled water, and the TP3 in the present embodiment 1.2.2 and the NP3 in the 1.2.10.1 are used as primers, and the PCR conditions in the present embodiment 1.2.4 are carried out again. Amplify. The reaction product was stored in a -20°C refrigerator for future use.

1.2.11 Electrophoretic detection, recovery and connection of PCR amplified fragments

Proceed as described in this example 1.2.6, 1.2.7 and 1.2.8. The results of electrophoresis detection are shown in Figure 3.

1.2.12 Transformation, identification and sequencing of PCR amplified fragments

Carry out according to the requirements in 1.2.8 and 1.2.9 of this embodiment.

1.2.13 Acquisition of the full-length sequence of the mannose-binding protein gene

According to the sequencing results of 3' and 5' RACE in this example, design the forward primer FLFP: 5'-ATGAAGATGAACATACTCCTCTGC-3' and the reverse primer FLRP: 5'-TTACTCATCACCCATAACCTTCACAA-3 including the coding region of this spliced sequence '. Carry out PCR amplification again according to the PCR conditions in this example 1.2.4, use the reverse transcribed Diycinella cDNA as a template, use EasyPfu DNA Polymerase (2.5U/l) instead, and set the annealing temperature to 50°C for PCR Amplified to obtain a complete and accurate full-length gene of the nine-catenin mannose-binding protein.

According to the methods in 1.2.6, 1.2.7 and 1.2.8 of this example, electrophoresis detection, gel cutting recovery and ligation of the amplified products were performed sequentially, and transformed into competent cells. The identification and sequencing analysis of positive clones were carried out according to the method in 1.2.9 of this example. The full-length nucleotide sequence (cDNA sequence) of the gene of the 9-catenin mannose-binding protein was obtained, and the electrophoresis detection result is shown in FIG. 4 .

It can be seen from the nucleotide sequence shown in SEQ ID NO: 1 in the sequence table that the obtained full-length sequence of Cynopteris cDNA consists of 513 nucleotides, contains a complete open reading frame, and encodes the sequence The mannose-binding protein precursor protein consisting of 170 amino acids shown in the list SEQ ID NO:2. The domain analysis of CDA shows that there is a complete functional domain of mannose-binding protein in the amino acid sequence of CDA, and it has three typical "QXDXNXVXY" like Galanthusnivalis agglutinin (GNA) The mannose-binding active center belongs to the evolutionarily conserved snowdrop-related mannose-binding protein.

<110>Sichuan University

<120> mannose-binding protein

<160>2

<170>SIPOSequenceListing 1.0

<210> 1

<211> 513

<212>DNA

<213>Orchidaceae Calanthe discolor Lindl.

<400> 1

atgaagatgaacatactcctctgcaccgcatatctcacactcctcatcaccccatcatca 60

ggccaactctacaaccacttgctctctggtcagcgcctcaacacaggccaatctcttgta 120

cagagcggctatatcttcatcatccaaaatgactgcaatcttgtcctctacaatttgggt 180

actgcaatttgggcttcgggaacccatggtaggggtaacggatgctatcttatcatgcag 240

cgagacggtaaccttgttgtctatgacagaaataatcgcgccatatgggcaagtaacact 300

aaccgcagaactggaaactatatcctcatactacaaaaagatcgtaatgttgttatatat 360

agtgtacccacttggtctacgaggactaataccgttgactcggctgatgttgtcattgcc 420

cttacacaaaatgggacagtgctgccttcgggtgcggagcagaataaggtgagggaaatg 480

gggaagattgtgaaggttatgggtgatgagtaa 513

<210> 2

<211> 170

<212> PRT

<213>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 (3)

1. A noncatenin mannose-binding protein, characterized in that it has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. 2. A noncatenin mannose-binding protein, characterized in that it has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 3. Obtained the full-length cDNA sequence and amino acid sequence of the mannose-binding protein of Cynopteryx japonicus by using the homologous cloning and RACE technology from the RNA extracted from the fresh young leaf tissue of Cynopteryx japonicus.