CN106755074B

CN106755074B - Application of the golden mandarin orange Fm MLP2 albumen in enhancing plant is freeze proof

Info

Publication number: CN106755074B
Application number: CN201611214707.8A
Authority: CN
Inventors: 杨华; 滕进婧; 郭纯; 李梦芸
Original assignee: Hunan Agricultural University
Current assignee: Hunan Agricultural University
Priority date: 2016-12-26
Filing date: 2016-12-26
Publication date: 2019-09-20
Anticipated expiration: 2036-12-26
Also published as: CN106755074A

Abstract

It is a kind of gold mandarin orange Fm MLP2 albumen enhancing plant it is freeze proof in application, belong to field of biotechnology.The amino acid sequence of the albumen can be used for enhancing the frost resistance of target plant as shown in SEQ ID No:6.It is demonstrated experimentally that Fm MLP2 gene is related to plant freezing resistance, after Fm MLP2 gene is overexpressed in plant, the freezing tolerance of plant can be significantly improved.Gold mandarin orange Fm MLP2 albumen and its encoding gene disclosed by the invention are remarkably improved the frost resistance of plant, and important theoretical basis and genetic resources are provided for plant cold resistance breeding work.

Description

Application of the golden mandarin orange Fm MLP2 albumen in enhancing plant is freeze proof

Technical field

The invention belongs to field of biotechnology, it is related to a kind of application of golden mandarin orange Fm MLP2 albumen in enhancing plant is freeze proof.

Background technique

Damage lower than plant caused by zero degrees celsius low temperature is known as freeze injury.Most of whole world area, which can all encounter, to be lower than Zero degree low temperature causes many crops to meet with different degrees of freeze injury, and is often possible to be destructive injury.Therefore, plant Distribution and agricultural production seriously limited by 0 DEG C or less low temperature.People understand plant by studying the frost resistance of plant Freeze proof mechanism reduces freeze injury and causes adverse effect to production, serves agricultural production practice.Thus, plant freezing resistance is always It is the most important thing that people study plant cold resistance.

By the hereditary capacity of plant modification, the ability for resisting freeze injury is made it have, thus utmostly from freeze injury It influences.In recent years, with the development of biotechnology, cultivating transgenic plant using technique for gene engineering becomes cultivation new varieties Effective way.But the research in relation to anti-freeze gene Engineering Breeding is also than relatively limited, main reason is that lacking efficient base Lack enough understandings because of resource, or to its corresponding gene structure, functional mechanism.It excavates new anti-freeze gene and gos deep into recognizing it Functional mechanism has become inevitable trend.

On Miraculin is a kind of plant specificity glycoprotein separated from the plant miracle fruit for originating in West Africa.Nineteen ninety-five Masuda etc. is cloned into On Miraculin gene from miracle fruit, and it is similar that subsequent Gahloth etc. is cloned into On Miraculin from moon tangerine Albumen (miraculin-like protein, MLP) gene, Kuwabara etc. clone Rlem MLP2 from rough lemon (miraculin-like protein 2) and Rlem MLP3 gene, gold build great waves etc. and are cloned into Fc MLP2 from golden bullet.This hair Bright people then has found MLP2 albumen from golden mandarin orange, and is cloned into Fm MLP2 gene.Tsukuda etc. thinks that MLP2 can be secreted into cell The gap of wall and cuticula is the first line of defence for defending following pathogen challenge.Goodwin etc. thinks, MLP2 gene and pathogen Infringement coordinate expression, generate different protease inhibitors to resist the pathogen invasion as caused by microorganism.Isshiki etc. It can inhibit the effect of citrus plant black rot germ spore germination with discovery MLP2 such as Akimitsu.Kuwabara etc. recognizes For Rlem MLP2 plays certain defense reaction when rough lemon is injured by pest or pathogen.Jin Jiantao etc. thinks, MLP2 albumen may play a role in plant anti-insect, anti-microbial pathogen, anti-hunger and in terms of delaying plant senescence.Thus may be used To find out, the function of studying MLP2 albumen and its encoding gene is more, but does not have the report of enhancing freezing tolerance of plant also.

Summary of the invention

The object of the present invention is in view of the above shortcomings of the prior art, the new application of golden mandarin orange Fm MLP2 albumen is provided, it should For the amino acid sequence of albumen as shown in sequence table SEQ ID No:6, the new application is that albumen shown in SEQ ID No:6 is enhancing Application in freezing tolerance of plant.

In the above-mentioned methods, the encoding gene of albumen shown in the SEQ ID No:6 is following gene 1) or 2) or 3):

1) its nucleotide sequence is DNA molecular shown in SEQ ID No:5；

2) at least have 70% with the DNA sequence dna 1) limited, at least have 75%, at least having with 80%, at least 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least have 98% or at least have There is 99% identity and encodes the DNA molecular of albumen shown in SEQ ID No:6；

1) or 2) 3) hybridize under strict conditions with the DNA sequence dna limited and encode the DNA of albumen shown in SEQ ID No:6 Molecule.

The stringent condition are as follows: 50 DEG C, in 7%SDS, 0.5M Na₃PO₄Hybridize in the mixed liquor of 1mM ETDA, 65 DEG C, it is rinsed in 0.1 × SSC, 0.1%SDS.

Present invention simultaneously provides a kind of method for cultivating freeze resistant transgenic plant, this method is will to encode above-mentioned shown albumen Channel genes purpose plant, obtain genetically modified plants after cultivating, the freezing tolerances of the genetically modified plants is higher than purpose plant Object.

The above-mentioned purpose plant referred to can be dicotyledon, or monocotyledon.

It is demonstrated experimentally that Fm MLP2 gene is related to plant freezing resistance, after Fm MLP2 gene is overexpressed in plant, Compared to Wild type control plants, Fm MLP2 gene overexpression plant frost resistance is significantly improved.Gold mandarin orange Fm disclosed by the invention MLP2 albumen and its encoding gene are remarkably improved the frost resistance of plant, and important theory is provided for plant cold resistance breeding work Basis and genetic resources.

Detailed description of the invention:

Fig. 1 is the RNA figure under 4 DEG C of different disposal times of golden mandarin orange Miao Yu of the invention.

Fig. 2 is the relative expression's spirogram of golden mandarin orange Fc MLP2 gene of the invention under 4 DEG C of different disposal time.

Fig. 3 is bacterium solution PCR identification p1300M-MLP2-1 conversion Agrobacterium GV3101 gel figure.

Wherein: the 1st swimming lane is Marker, and the 2nd swimming lane is that p1300M empty plasmid expression vector is transformed into Agrobacterium GV3101, 3rd, 4,5,6,7 swimming lanes be that p1300M-MLP2-1 expression vector is transformed into Agrobacterium GV3101.

Fig. 4 is to turn Fc MLP2 gene Arabidopsis plant PCR identification gel figure.

Wherein: the 1st swimming lane is 2K Marker, the 2nd, 3 swimming lanes be wild-type Arabidopsis plants, the 4th, 5,6,7 swimming lanes be to turn Fc MLP2 gene Arabidopsis plant.

Fig. 5 is to turn Fc MLP2 gene Arabidopsis plant and wild-type Arabidopsis plants Phenotypic Observation comparison diagram.

Fig. 6 is to turn Fc MLP2 gene Arabidopsis plant relative conductivity figure.

Fig. 7 is wild-type Arabidopsis plants and turn figure compared with Fc MLP2 gene Arabidopsis plant proteinase activity.

Fig. 8 is to turn Fc MLP2 gene Arabidopsis plant protein relative amount figure.

Specific embodiment

The present invention will be further described with following embodiment, but be not limited to these examples any or similar real Example.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Method therefor is conventional method unless otherwise specified in following embodiments.

Golden mandarin orange (Fortunella margarita (Lour.) Swingle): it is provided by Agricultural University Of Hunan；Bibliography: (Cao,Q,W F Kong,P F Wen,Plant freezening tolerance and genes express in cold acclimation[J].Acta Ecologica sinica,2004,24(4):806-811)。

Arabidopsis wild type (Col-0 is environmental): arabidopsis wild type seeds (Arabidopsis thaliana Ecotype Columbia-0), it is provided by arabidopsis biological study center.

Agrobacterium tumefaciems GV3101 (Agrobacterium tumefaciens GV3101): it is mentioned by Agricultural University Of Hunan For.

Escherichia coli (Escherichia coli) Trans-1 Phage Resistant competence: it is purchased from full Shi Jinsheng Object Technology Co., Ltd., catalog number CD501.

Embodiment 1, the table with fluorescent quantitative PCR technique to Fc MLP2 gene in golden mandarin orange blade under low temperature induction It is analyzed up to amount

Select the growth of temperature control (about 25 DEG C) greenhouse, consistent 2 Nian Shengjin mandarin orange (the Fortunella margarita of growing way thickness (Lour.) Swingle) several plants of seedling be used as experimental material, carry out low-temperature treatment, temperature is set as 4 ± 0.5 DEG C.

It is total that golden mandarin orange Miao is extracted using the mentioning of Beijing Quanshijin Biotechnology Co., Ltd RNA kit (catalog number (Cat.No.): ET121) RNA (see Fig. 1) using the reverse transcription reagent box of Beijing Quanshijin Biotechnology Co., Ltd, remove and using total serum IgE as template The genomic DNA in RNA is removed, reverse transcription obtains cDNA.

According to the homologous protein MLP2-1 gene order of GenBank warehouse publication, design PCR amplification primer, primer by Nanjing Genscript Biotechnology Co., Ltd.'s synthesis, design primer are as follows: Fm MLP2F:5'-ATATTGTCCATTGTCCGAGT-3' (SEQ ID No:1), Fm MLP2R:5'-AAACAGAACATTCAAAGCCG-3'(SEQ ID No:2).With reverse transcription synthesis First chain cDNA is template, and Fm MLP2F and Fm MLP2R are primer, carry out RT-PCR reaction, and RT-PCR response procedures use two Footwork obtains pcr amplification product.PCR reaction system (25.0 μ L): 1.0 μ L of cDNA, forward primer (10 μM) 0.2 μ L, reversed Primer (10 μM) 0.2 μ L, 2 × SYBR Green I Master5.0 μ L, nuclease-free water (Nuclease-free H₂O)18.6 μL.PCR response procedures: 95 DEG C of initial denaturations 5min, 95 DEG C of 10s, 60 DEG C of 30s, 40 circulations.

Expression analysis result of the Fc MLP2 gene in freeze proof process: right at low temperature in order to study Fc MLP2 gene The response of different time processing passes through fluorescence to biennial golden mandarin orange seedling 4 DEG C of low-temperature treatments 0h, 6h, 12h, 18h of progress and for 24 hours The relative expression quantity (being control, expression quantity 1 with (25 DEG C i.e. in figure) of 0h) of quantitative PCR detection gene.Testing result is shown in figure 2, display: Fc MLP2-1 gene in 4 DEG C in 0-6h expression quantity increase, expression quantity slightly declines when then arriving 12h, In the 16h-24h of low-temperature treatment, expression quantity gradually increases steadily.

Embodiment 2, the clone of golden mandarin orange Fc MLP2 gene and analysis

It is total that golden mandarin orange Miao is extracted using the mentioning of Beijing Quanshijin Biotechnology Co., Ltd RNA kit (catalog number (Cat.No.): ET121) RNA, and using total serum IgE as template, reverse transcription obtains cDNA (with embodiment 1).According to the homologous egg of GenBank warehouse publication White MLP2-1 gene order and p1300M carrier sequence, design PCR amplification special primer (have BamH I and Kpn I digestion position Point and protection base), primer is synthesized by Nanjing Genscript Biotechnology Co., Ltd..Forward primer CDS-PF:5'-GGGTACC ATGAAGACTTCATTGATAACAACAC-3'(SEQ ID No:3, leukorrhagia dashed part sequence are BamH I and Kpn I limitation Property inscribe restriction enzyme site and protection base)；Reverse primer CDS-PR:5'-CGGGATCCTTACGCGGTCATTGATATTG-3' (SEQ ID No:4, leukorrhagia dashed part sequence are BamH I and Kpn I restriction enzyme restriction enzyme site and protection base).Again Using reverse transcription at cDNA as template, under the guidance of primer CDS-PF and primer CDS-PR, expand golden mandarin orange Fm with Standard PCR method The cDNA sequence of MLP2 gene carries out 1% agarose gel electrophoresis detection to pcr amplification product, recycles and pure after reaction Change the DNA fragmentation of about 700bp.PCR reaction system (10.0 μ L): 1.0 μ L of cDNA, forward primer (10 μM) 0.2 μ L, reversely draw Object (10 μM) 0.2 μ L, dNTPs (2.5mM each) 0.8 μ L, 10 × Pfu Buffer, 1.0 μ L, Pfu archaeal dna polymerase (5U/ μ L)0.8μL、ddH₂O 6.0μL.PCR response procedures are as follows: 94 DEG C of 3min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min, 30 are followed Ring；72℃5min.

DNA fragmentation after purification is connected into carrier T, connection product converts Escherichia coli Trans-1 Phage Competent cell is equably coated on the LB solid medium containing Amp antibiotic by Resistant competent cell, to flat Plate surface is dry, is inverted plate in 37 DEG C of overnight incubations.The positive single colonie grown on picking antibiotic plate shakes bacterium, with shaking Bacterium solution carry out PCR identification as template, PCR is accredited as positive bacterium solution, and to take 1mL to send limited to long Satraplatin still biotechnology Company is sequenced.

Sequencing result shows that the Fm MLP2 gene DNA fragment of above-mentioned about 700bp contains shown in SEQ ID No:5 868bp nucleotide sequence, the 868bp nucleotides sequence are classified as the coded sequence of Fc MLP2 gene, and coding has SEQ ID No:6 Shown in the albumen that is made of 236 amino acid, which has the protein structure domain of Kuniz type.The albumen is on NCBI Positioning number is AF283533_1.

Embodiment 3, building Fm MLP2 expression vector and p1300M-MLP2 expression vector convert Agrobacterium GV3101

The building of Fm MLP2 expression vector: reagent is mentioned with the plasmid of TIANGEN Biotech (Beijing) Co., Ltd. is small Box extracts the plasmid of the positive bacterium solution of embodiment 2.Using restriction enzyme BamH I and Kpn I double digestion pGM-Fc MLP2 Plasmid, pCAMBIA1300M carrier (p1300M is provided by Agricultural University Of Hunan), by after digestion p1300M carrier segments with MLP2 is recycled respectively.P1300M carrier segments after digestion need to carry out dephosphorylation.Double enzyme digestion reaction system (50.0 μ L): 25.0 μ L of Plasmid DNA, 10 × K Buffer, 2.5 μ L, 0.5 μ L of BamH I, 0.5 μ L of Kpn I, ddH₂O 21.5μL.Dephosphorization Acidification reaction system (60 μ L): 50.0 μ L of digestion products, 10 × Buffer, 5.7 μ L, 1.0 Phophatase μ L, ddH₂O 3.3 μL。

The p1300M carrier segments after double digestion are attached with MLP2-1 with T4 ligase, coupled reaction system (10 μ L): 2.0 μ L of p1300M carrier segments, 4.0 MLP2-1 μ L, 5 × T₄Buffer 2.0μL、T₄1.0 μ L of ligase, ddH₂O 1.0μ L converts connection product with Escherichia coli Trans-1 Phage Resistant competence, and picking single colonie is shaken Bacterium uses the bacterium solution shaken to carry out PCR identification as template, is accredited as positive bacterium colony to bacterium solution PCR and carries out plasmid extraction, then Digestion identification is carried out again.Choosing digestion result is that positive bacterium solution 1mL is sent to the still Bioisystech Co., Ltd's sequencing of long Satraplatin, is surveyed Fm MLP2 expression vector p1300M-MLP2-1 is constructed successfully sequence as the result is shown.Glycerol is added by isometric in rest part, It is stored in -80 DEG C of ultra low temperature freezers.

P1300M-MLP2 expression vector converts Agrobacterium GV3101: the p1300M-MLP2-1 expression vector that will be built, Agrobacterium tumefaciems GV3101 is transformed into using electric shocking method.It is MLP2-1 gene pairs plant growth hair to more accurately navigate to The influence educated, while p1300M empty plasmid expression vector is transformed into Agrobacterium tumefaciems GV3101.By bacterium solution PCR gel electrophoresis Find that p1300M-MLP2-1 and p1300M empty plasmid has the purpose band for meeting size, sees Fig. 3, as a result table after imaging Bright p1300M-MLP2-1 expression vector and p1300M empty plasmid have all been transferred to Agrobacterium GV3101, can carry out follow-up test.

The acquisition and identification of embodiment 4, Fm MLP2 transgenic arabidopsis

One, the acquisition of transgenic arabidopsis

The inflorescence infusion method mediated using Agrobacterium GV3101 is by p1300M-MLP2-1 expression vector and p1300M empty plasmid It is integrated into Colombia's wildtype Arabidopsis thaliana genome.Harvest and its dry seed after plant is mature, in superclean bench Its uniform sowing is screened on the MS solid medium containing hygromycin (concentration 50mg/L) after carrying out Aseptic sterilisation, Observe the state of its growth.After 2 weeks or so that growth conditions are good, the dark green sprigging of blade allows it suitable in the soil It is grown in condition.Same mode step sizing, when screening for 3 generation, discovery turns base on resistant MS solid medium Wild type is then all died due to plant can survive, and shows that transgenic plant is homozygous plants.

Two, transgenic plant PCR is identified

The genomic DNA through antibiotic-screening for positive transgenic plant is extracted, while being with wild type (Wild Type) Control carries out PCR identification using CDS-PF/DS-PR as primer.As a result 4 plants of p1300M-MLP2-1 have been identified in T1 generation and has turned open country Raw type Arabidopsis plant is positive, these plant are still positive in PCR identification, and by sequencing result analysis still in sun Property (see Fig. 4).

Embodiment 5, Fm MLP2 transgenic arabidopsis functional analysis

It will turn MLP2 gene plant in -6 DEG C of processing 3h, and transfer it under conditions of normally cultivating and restored, It is compareed after restoring 1 day and 5 days with wildtype Arabidopsis thaliana (Wild Type).

1. the freeze proof phenotypic analysis of transgenic plant

Fig. 5 is seen, by phenotypic analysis as can be seen that transgenic arabidopsis has apparent frost resistance than wild type.

2. transgenic plant physiological and biochemical index is analyzed

2.1, conductivity is surveyed

Conductivity size has reacted the extent of the destruction of film, by data analysis shows, the conductance of wild type (Wild Type) Rate is greater than transgenic plant, illustrates that WT lines extent of the destruction of film in refrigerating process is bigger (see Fig. 6).

2.2, proteinase activity

Turn MLP2-1 plant proteinase activity to be suppressed, and WT lines (WT) proteinase activity it is relatively high (see Fig. 7).

2.3, protein relative amount

In frost free period, it is unobvious to turn the reduction of MLP2-1 plant protein content；In convalescence, protein content gradually go up to Horizontal before enduring cold, plant is radiated vigour again.And WT lines (Wild Type) are fast in the decline of frost free period protein content Speed；In convalescence, protein content further declines, and last cell is thoroughly dead (see Fig. 8).

Conclude that based on the above results it is lower due to turning MLP2-1 plant proteinase activity, functional protein content compared with Height, transgenic plant are more tolerant of arctic weather than wild type, illustrate that MLP2 gene has antifreezing function.

SEQUENCE LISTING

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<120>application of the golden mandarin orange Fm MLP2 albumen in enhancing plant is freeze proof

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<170> PatentIn version 3.3

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Claims

1. encoding application of the gene of golden mandarin orange Fm MLP2 albumen in enhancing plant is freeze proof, the gene order such as SEQ ID No:5 It is shown.

2. a kind of method for cultivating freeze resistant transgenic plant, it is characterised in that: this method is by coding shown in SEQ ID No:5 The channel genes purpose plant of golden mandarin orange Fm MLP2 albumen, through cultivation after obtain genetically modified plants, the genetically modified plants it is freeze proof Ability is higher than purpose plant.

3. according to the method described in claim 2, it is characterized by: the purpose plant is that dicotyledon or unifacial leaf are planted Object.