CN108034000A - Chinese cymbidium mannose-binding protein - Google Patents

Abstract

The present invention obtains the conserved sequence of the mannose-binding protein gene by comparing the sequences of the mannose-binding protein genes of the Orchidaceae family plants, designs degenerate primers, and clones the mannose-binding protein gene by 3'RACE and 5'RACE methods. The nucleotide sequence of the mannose-binding protein gene of Molan chinensis is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the mannose-binding protein in Molan chinensis obtained by translation is shown in SEQ ID NO: 2 in the sequence listing. The invention not only enriches the mannose-binding protein members in the Orchidaceae family, but also provides a material basis for the subsequent application and research of the gene and its protein in resistant transgenic agriculture. Other unreported mannose-binding proteins are provided for reference.

Description

Molan mannose binding protein

technical field

The invention belongs to the field of mannose-binding protein, and in particular relates to the gene and protein sequence of the mannose-binding protein from molantium.

Background technique

Molan (Cymbidium sinense (Jackson ex Andr.) Willd.), also known as Yearling Orchid, belongs to Angiospermae, Monocotyledoneae, Microspermae, Orchidoideae , Jianlan subgenus (Jensoa) ground plants. Growing in moist but well-drained shady places beside forests, shrubs or valleys.

Existing reports have shown that monocot mannose-binding mannose-binding protein (mannose-binding protein) exists in monocotyledonous orchids, such as Orchid chinensis, Cynthia chinensis, etc., and the monocotyledon mannose-binding protein is mannose The sugar-binding protein specifically combined with its derivatives has a specific and strong inhibitory effect on tumor cells and retroviruses, and has good resistance to diseases and insect pests of agricultural crops. There have been no published reports on the gene encoding the mannose-binding protein of Moranthia spp.

Contents of the invention

In view of the above deficiencies, the object of the present invention is to provide a molan mannose-binding protein gene and its protein sequence.

The present invention obtains the conserved sequence of the mannose-binding protein gene by comparing the sequences of the mannose-binding protein genes of the Orchidaceae family plants, designs degenerate primers, and clones the mannose-binding protein gene by 3'RACE and 5'RACE methods. The nucleotide sequence of the mannose-binding protein gene of Molan chinensis is shown in SEQ ID NO: 1 in the sequence listing, and the amino acid sequence of the mannose-binding protein in Molan chinensis obtained by translation is shown in SEQ ID NO: 2 in the sequence listing.

The present invention has the following beneficial effects:

Using the method of the present invention, the gene and protein sequence of the mannose-binding protein in the Orchid plant can be quickly and accurately cloned, which not only enriches the members of the mannose-binding protein in the Orchidaceae family, but also contributes to the subsequent development of the gene and its protein The application and research in resistant transgenic agriculture provides a material basis, in addition, it can also provide a reference for the discovery of other unreported mannose-binding proteins in orchids.

Detailed ways

Example 1: Molan total RNA extraction, quality detection and reverse transcription

1. Materials and methods

1.1 Materials

1.1.1 Plant material: The whole plant of Molan was collected from Mount Emei, Sichuan, China, and was used directly or quickly frozen and stored at -80°C.

1.1.2 Enzymes and kits: RNA extraction kits were purchased from TaKaRabao Bioengineering Co., Ltd. (Dalian, China), MLV reverse transcriptase, TdT (nucleotide terminal transferase), RNase Inhibitor

1.2 Method

1.2.1 Molan total RNA extraction

Smash the whole plant of Molanca officinalis, use the RNA extraction kit (TaKaRabao Bioengineering Co., Ltd.) to extract the total RNA, and then prepare the following reaction solution in a microcentrifuge tube to remove the DNA in the total RNA:

React the reaction solution in the above microcentrifuge tube with 37°C for 20-30min, add 50μl DEPC-H ₂ O, add 100μl phenol, chloroform, isoamyl alcohol (25:24:1), centrifuge, take the upper layer and transfer to another In a microcentrifuge tube, add 10 μl of 3M sodium acetate solution (pH=5.2), then add 250 μl of absolute ethanol, place at -20°C for 30-60 minutes, centrifuge to recover the precipitate, wash the precipitate with 70% ethanol, and dry it in vacuum to obtain sample RNA .

1.2.2 UV spectrophotometric detection of total RNA quality

Use distilled water to dilute the RNA sample to be tested by a certain number of times to the dilution volume, which can be used to measure the OD value. Take distilled water as a blank control, and measure the OD value of the RNA sample at wavelengths of 260nm, 280nm, and 310nm respectively. According to the formula

ssRNA＝40×(OD ₂₆₀ -OD ₃₁₀ )×dilution factor

Calculate the RNA concentration, and judge the purity of the extracted RNA according to OD ₂₆₀ /OD _280. The quality of OD ₂₆₀ /OD ₂₈₀ is about 2.0, which is better, and then determine whether it is necessary to re-extract RNA.

1.2.3 Reverse transcription of total RNA to synthesize cDNA

Step (1): Add the following ingredients to a 0.5ml centrifuge tube:

Universal Primer 1: 5'-GCTGTCAACGATACGCTACGTAACGGCATGACAGTGT ₁₈ -3'

Step (2): Place the mixture described in step (1) at 70°C for 10 minutes to denature the RNA; place it on ice for 2 minutes; collect the mixture to the bottom of the tube after centrifuging for 5 seconds, and add the following components:

After adding the above ingredients, the total volume of steps (1) (2) is 24 μl.

Step (3): Shake gently, mix well, centrifuge for 5 seconds, collect the compound to the bottom of the tube, and incubate at 42°C for 1 min;

Step (4): Add 1 μl MLV (reverse transcriptase), and incubate in a water bath at 42°C for 1-2 hours;

Step (5): place at 70°C for 15 minutes to terminate the reaction;

Step (6): Centrifuge for 5 seconds, collect the cDNA of the reaction and place it on ice, and store it at -20°C for future use.

Example 2: 3' RACE PCR reaction

1. Materials and methods

1.1 Materials

1.1.1 Source of mannose-binding protein gene

In NCBI's gene database (https://www.ncbi.nlm.nih.gov/gene/), search for the mannose-binding protein genes of plants in the Aramiaceae family, perform BLAST comparison, and obtain the conservation of the mannose-binding protein genes The sequence was used to design a degenerate primer, and the degenerate primer was: 5'-GACTGCAACCTCGTCCTC-3'.

1.1.2 Medium

SOC liquid medium: tryptone 10g/L, yeast extract 5g/L, NaCl 0.5g/L, 250mmol/L KCl solution 10ml, adjust pH to 7.0 with 5mmol/L NaOH, 2mol/L sterilized 5ml of MgCl ₂ solution, 20ml of 1mol/L glucose solution sterilized by suction filtration.

LB solid medium: tryptone 10g/L, yeast extract 5g/L, NaCl 5g/L, adjust pH to 7.0 with 5mol/L NaOH, after autoclaving, add 10g/L agar powder.

1.1.3 Enzymes and kits

Taq DNA polymerase and 3'RACE kit were purchased from TaKaRa Bao Bioengineering Co., Ltd. (Dalian, China), and the column gel recovery kit was purchased from Omiga Company

1.1.4 Strains

Escherichia coli (Escherichia coli) Top10 (purchased from Invitrogen)

1.1.5 Plasmid vector

PUC18-T vector (purchased from TaKaRa Bao Biological Engineering Co., Ltd.)

1.2 Method

1.2.1 The first round of PCR amplification reaction

Prepare the PCR amplification reaction solution according to the following scheme:

Carry out the PCR reaction according to the following conditions: place the cDNA product at 90°C for 30 minutes to heat denature, then amplify (94°C for 30 seconds; 55°C for 30 seconds; 72°C for 1 min; repeat 30 times), and then place at 72°C Set for 10min. The reaction was performed on a GeneAmp PCRSystem2400 thermal cycler (Promega, Beijing).

1.2.2 The second round of PCR amplification reaction

The second PCR reaction uses the first-round PCR reaction product described in this embodiment 1.2.1 as a template, and the first-round PCR reaction product is diluted 10 times with distilled water, and the general primer 2 is used to replace the present embodiment 1.2.1 For the universal primer 1, the PCR amplification reaction solution was prepared again according to the conditions in 1.2.1 of this example, and the second round of PCR amplification was performed. The universal primer 2 is: 5'-GTCAACGATACGCTACGTAACG-3'.

1.2.3 Recovery and connection of PCR amplified fragments

After 1% agarose gel electrophoresis, the PCR product obtained by the second round of PCR reaction was purified with a column gel recovery kit (Omiga company, the experimental steps were carried out according to the company's column gel recovery kit instructions), and then The purified fragments were cloned into the PUC18-T vector vector (the cloning step was performed according to the instructions of the PUC18-Tvector kit of Taka Rabao Bioengineering Co., Ltd.).

1.2.4 Preparation of Competent Cells

Competent cells were prepared according to the CaCl ₂ method (Wang Qian, Modern Medical Experimental Methods, People's Health Publishing House, 2008, 496-497).

1.2.5 Conversion

Mix 10 μl of the junction fragment obtained in Example 1.2.3 with 50 μl of competent cells prepared in Example 1.2.4, place on ice for 30 minutes, then place in a water bath at 42°C for 90 seconds, and place on ice for 1- After 2 minutes, add 800 μl of SOC liquid medium and incubate for 1 hour; take 100 μl of the above culture solution and spread it on an LB plate (containing Amp); place it upside down at 37°C for 1 hour to absorb excess liquid, and then culture it upside down for 12 hours to obtain transformants.

1.2.6 Positive clone identification and sequencing analysis

A single colony with regular shape was taken to identify transformants by colony PCR and enzyme digestion. After confirming the desired insert by colony PCR and enzyme digestion, refer to Sambrook et al. :895–909.) for stab culture, and cultured at 37°C for 12 hours.

The puncture culture was sent to Shanghai Jitian Bioengineering Technology Service Co., Ltd. for sequencing. Sequences were analyzed using BLAST of NCBI. At the same time, DNAMAN 5.2.2.0 software and Vector NTI 9.0 were used for sequence analysis.

Example 3: 5' RACE PCR reaction

1. Materials and methods

1.1 Materials

1.1.1 The PCR amplification template is the PCR product in 1.2.2 of this embodiment.

1.1.2 Enzymes and kits

RNase A (nucleotidase), TdT (nucleotide terminal transferase), and 5'RACE kits were purchased from TaKaRabao Bioengineering Co., Ltd. (Dalian, China)

1.2 Method

1.2.1 Primer design

According to the above 3'RACE PCR amplification reaction product sequence, design the specific primers required for the 5'RACE reaction, as follows:

GSP: 5'-GACCTCGCCTATTCATCAGT-3'

GSP1: 5'-TTCACTCACCTTGTTCTGCTC-3'

GSP2: 5'-AATGGGGTGGCTGTATATAAC-3'

1.2.2 Homopolymer tailing of cDNA

TdT (nucleotide terminal transferase) was used to tail the total cDNA obtained in Example 1, and the amount of cDNA used in the TdT tailing reaction was determined according to the abundance of the target mRNA. Add the following ingredients and mix gently:

Incubate at 94°C for 2-3min, cool on ice for 1min, collect samples after centrifugation for 5 seconds, place on ice; add 1 μl TdT to mix gently, incubate at 37°C for 30min; finally heat inactivate TdT at 70°C for 10min , samples were collected by centrifugation for 5 seconds and placed on ice.

1.2.3PCR amplification reaction

Use the GSP2 primers described in Example 1.2.1 and Universal Primer 1 described in Example 1 1.2.3 to carry out the first round of PCR according to the conditions described in Example 2 1.2.1, and distilled water after the reaction is completed The PCR product was diluted 10 times; use the GSP primer described in Example 1.2.1 and the universal primer 2 described in Example 2 1.2.2 to perform the second round of PCR according to the conditions described in Example 2 1.2.1.

Recover and purify the PCR product according to the method described in Example 2 1.2.3, clone it into the PUC18-T vector, and transform it into competent Top10 cells according to the method described in Example 2 1.2.5, according to Example 2 1.2.6 The method carries out positive clone identification and sequencing analysis.

Embodiment 4: Obtaining of full-length cDNA sequence

1. Materials and methods

1.1 Materials

1.1.1 PCR amplification template

The 3' RACE amplification product in Example 2 is spliced with the 5' RACE amplification product in Example 3

1.2 Method

1.2.1 Primer design

According to the sequence obtained by 3'RACE amplification in Example 2 and the 5'RACE amplification sequence in Example 3, the full length was spliced, and the full-length primers were designed according to the full-length sequence:

Forward primer: 5'-GAGACAAACCAACCCCCTAAT-3'

Reverse primer: 5'-CGCATACGCACACGACCTC-3'

1.2.2 Full-length cDNA sequence acquisition

Carry out PCR amplification according to the conditions described in 1.2.1 in Example 2 again, recover and purify the PCR product according to the method described in 1.2.3 in Example 2, and clone it into the PUC18-T vector carrier, according to 1.2.5 in Example 2 Said method was transformed into competent Top10 cells, positive clone identification and sequencing analysis were carried out according to the method described in 1.2.6 in Example 2, and the full-length nucleotide sequence (cDNA sequence) of the mannose-binding protein gene of Molan chinensis was obtained, as shown in the sequence Listed as shown in SEQ ID NO:1.

As shown in the sequence listing SEQ ID NO:1, the full length of the gene of Molan mannose-binding protein is 586bp, including an open reading frame of 531bp, and the coding is composed of 176 amino acid sequences as shown in the sequence listing SEQ ID NO:2 of mannose-binding protein. The start codon of the gene sequence is located at 35-37bp, and the stop codon is located at 563-565bp. There is a 34bp non-coding region upstream of the start codon; a 21bp non-coding region downstream of the stop codon.

<110> Sichuan University

<120> Molan mannose-binding protein

<160>2

<170> PatentIn version 3.5

<210> 1

<211> 586

<212>DNA

<213> Orchidaceae, Cymbidium sinense (Jackson ex Andr.) Willd.

<400> 1

gagacaaacc aaccccctaa ttaattaacc agccatgggt atcttctcca ttatcaggat 60

ccttctcctc tgcgccgcat ccctcacaat ccttcttgcc aacccatcgt ccggccagtg 120

caacaaccac ttgctctccg gtgagcgcct tagcccaggc caatccctca caagtggcaa 180

catggagttc atcatgcaat acgactgcaa tctcgtcctc tatgataacg ggaaaccgat 240

ctgggcttcg ggcacttatc atagcggctc tggctgctac gtcgccatgc agaccgacgg 300

caacctcgtc atctacgaca atagaaataa tcccttatgg gcgagcaaca ctaatgggga 360

aaatggaaac tatatcctta tactgcaaaa agatcgcaat cttgttatat acagccaccc 420

catttgggct acggggacta attacgctgg ttcggtcgct gtcgtcgtcg ccgctgcgcg 480

taatgggacg gtggggattt cgggggcgga gcagaataag gtgagggaaa taaggaagat 540

tctgaagatt atgactgatg aataggcgag gtcgtgtgcg tatgcg 586

<210>2

<211> 176

<212> PRT

<213> Orchidaceae, Cymbidium sinense (Jackson ex Andr.) Willd.

<400>2

Met Gly Ile Phe Ser Ile Ile Arg Ile Leu Leu Leu Cys Ala Ala Ser

1 5 10 15

Leu Thr Ile Leu Leu Ala Asn Pro Ser Ser Gly Gln Cys Asn Asn His

20 25 30

Leu Leu Ser Gly Glu Arg Leu Ser Pro Gly Gln Ser Leu Thr Ser Gly

35 40 45

Asn Met Glu Phe Ile Met Gln Tyr Asp Cys Asn Leu Val Leu Tyr Asp

50 55 60

Asn Gly Lys Pro Ile Trp Ala Ser Gly Thr Tyr His Ser Gly Ser Gly

65 70 75 80

Cys Tyr Val Ala Met Gln Thr Asp Gly Asn Leu Val Ile Tyr Asp Asn

85 90 95

Arg Asn Asn Pro Leu Trp Ala Ser Asn Thr Asn Gly Glu Asn Gly Asn

100 105 110

Tyr Ile Leu Ile Leu Gln Lys Asp Arg Asn Leu Val Ile Tyr Ser His

115 120 125

Pro Ile Trp Ala Thr Gly Thr Asn Tyr Ala Gly Ser Val Ala Val Val

130 135 140

Val Ala Ala Ala Arg Asn Gly Thr Val Gly Ile Ser Gly Ala Glu Gln

145 150 155 160

Asn Lys Val Arg Glu Ile Arg Lys Ile Leu Lys Ile Met Thr Asp Glu

165 170 175

Claims (3)

1. A molan mannose-binding protein, characterized in that it has the nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing. 2. A molan mannose-binding protein, characterized in that it has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing. 3. A molan mannose-binding protein gene is obtained by comparing the sequences of the mannose-binding protein genes of Orchidaceae family plants to obtain the conserved sequence of the mannose-binding protein gene, and using this to design degenerate primers. 3'RACE and 5'RACE cloned.