CN103145817A - Sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and coding gene and application thereof - Google Patents

Abstract

The invention discloses a sickle-alfalfa chloroplast cold-response protein (MfcpCOR14) and a coding gene and an application thereof and belongs to the field of plant genetic engineering. An MfcpCOR14 gene is obtained, the obtained MfcpCOR14 gene is used for constructing a plant expression vector, the constructed plant expression vector is used for converting a plant tissue, and the plant tissue is cultured into a transgenic plant. The expression of the MfcpCOR14 gene can be induced at low temperature. The invention provides a method for culturing a cold and strong light resisting plant by using the MfcpCOR14 gene. After the MfcpCOR14 gene is used for over-expressing the protein of a plant, the growth of the transgenic plant can be superior to that of a wild plant, and the cold and strong light resisting capacity of the transgenic plant can be improved significantly.

Description

The cold response protein MfcpCOR14 of bur clover chloroplast(id) and encoding gene and application

Technical field

The invention belongs to plant genetic engineering field, relate to the cold response protein MfcpCOR14 of a kind of bur clover chloroplast(id) and encoding gene and application.

Background technology

Plant-growth and crop yield meeting are had a strong impact on by various hostile environment conditions, and tropical plants especially easily are subject to the injury of low temperature.Cultivating anti-contrary plant variety is one of major objective of plant husbandry.Yet plant stress tolerance is a quantitative character, relates to the effect of a hundreds of gene at least, and it is very necessary therefore separating tolerance gene from the strong plant of resistance of reverse.

Bur clover (Medicago sativa subsp.falcata) is a kind of very cold-resistant leguminous forage germplasm, also there is distribution in siberian in cold, therefrom clone cold-resistant gene, can be for transgenic breeding provide more good goal gene, and particularly important and urgent for the degeneration-resistant molecular breeding of agriculture and forestry plant.Bur clover mRNA and albumen in cold domestication change (Chinnusamy et al.2007), except a few cold domestication specific gene is cloned with expression analysis (Mohapatra et al.1989, Wolfraim et al.1993), utilize research that bur clover separates cold-resistant gene seldom.

Summary of the invention

For overcoming the shortcoming and defect of prior art, primary and foremost purpose of the present invention is to provide the cold response protein MfcpCOR14 of a kind of bur clover chloroplast(id).

Another object of the present invention is to provide the gene of the cold response protein MfcpCOR14 of the described bur clover chloroplast(id) of coding.

A further object of the present invention is to provide the application of the cold response protein MfcpCOR14 of described bur clover chloroplast(id).

Purpose of the present invention is achieved through the following technical solutions: the cold response protein MfcpCOR14 of a kind of bur clover chloroplast(id), and its aminoacid sequence is as follows:

MATMLTSNTLFQTSNSFPNVPSLTLPKPSRVFLASNWRNASEGTKNTSLSWAYTSSSTKTRRYADGTAGKAGETVNAGIDDIKQFGQDADEKTKDAASSIADKAKENTDKTVEAVGSAGDKAKDYAFDANDKAKEAIGSATDKAKEGFEAATKNTQEAAGSATEALKNAGDQAKEAVEGALDAAKDAVAGKE；

The nucleotide sequence of the cold response protein MfcpCOR14 of described bur clover chloroplast(id) of encoding is as follows:

ATGGCAACAATGTTGACAAGTAACACACTCTTCCAAACCTCAAACTCATTCCCTAATGTCCCTTCACTCACCCTTCCTAAACCCTCCAGGGTCTTCTTAGCTTCCAACTGGAGAAATGCATCAGAAGGAACAAAAAACACTTCACTCAGTTGGGCTTACACTTCTTCTTCTACAAAGACAAGAAGATATGCAGATGGAACAGCCGGCAAAGCCGGAGAAACGGTGAACGCAGGCATAGATGACATCAAACAATTCGGACAAGATGCAGATGAAAAGACAAAGGACGCTGCGAGTTCAATTGCGGATAAAGCAAAAGAAAACACAGACAAGACTGTAGAGGCAGTAGGAAGTGCTGGAGACAAGGCAAAAGATTATGCTTTTGATGCAAATGACAAAGCCAAGGAAGCAATAGGTTCTGCTACTGATAAGGCAAAAGAAGGATTTGAGGCAGCAACGAAGAACACACAGGAGGCTGCTGGGTCAGCGACAGAGGCTCTGAAGAATGCAGGAGATCAGGCAAAGGAGGCGGTGGAAGGAGCGTTGGACGCGGCCAAGGATGCCGTGGCTGGGAAGGAGTAA；

The application of the cold response protein MfcpCOR14 of above-mentioned bur clover chloroplast(id) in Promoting plant growth and raising plant cold resistance and anti-high light ability: this application comprises the gene constructed plant expression vector with MfcpCOR14 of the present invention; With the expression vector conversion of plant tissue that builds; The plant tissue that transforms is cultivated into transfer-gen plant.

The gene constructed plant expression vector of described use MfcpCOR14 of the present invention, available existing plant expression vector construction contains the recombinant expression vector of described gene;

Described plant expression vector includes but not limited to double base agrobacterium vector and the carrier that is used for the unifacial leaf via Particle Bombardment Transformation; Described double base agrobacterium vector comprises pBI121, the pCAMBIA serial carrier; Described plant expression vector also can comprise 3 ' of foreign gene and hold untranslated regional, namely comprises the DNA fragmentation of polyadenylic acid signal and any other effect mRNA processing or genetic expression;

When using described gene constructed recombinant plant expression vector, can use any strong promoter or inducible promoter before its transcription initiation Nucleotide; These promotors include but not limited to cauliflower mosaic virus (CaMV) 35S promoter and ubiquitin (Ubiqutin) promotor, and it can use separately or be combined with other plant promoter; When using gene constructed plant expression vector of the present invention in addition, also can use enhanser, these enhansers zones includes but not limited to ATG initiator codon and neighboring region.Initiator codon must be identical with the reading frame of encoding sequence, to guarantee the translation of whole sequence.Translation control signal and initiator codon can make multiple different source, can be natural, also can synthesize.Translation initiation region can be from the transcription initiation zone or from structure gene.

For the ease of transfer-gen plant being identified and being screened, can process the plant expression vector that uses, comprise adding or replacing the alternative mark of plant.Spendable selected marker comprises the gene of enzyme of polynucleotides encoding herbicide resistant or the antibiotic marker thing with resistance; Described weedicide comprises careless fourth phosphine, glyphosate etc., and described microbiotic comprises kantlex, Totomycin, gentamicin etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

The expression cassette, transgenic cell line and the recombinant bacterium that contain the MfcpCOR14 gene all belong to protection scope of the present invention.

Expression vector of the present invention can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, the various routines such as microinjection, electroporation or special genetic transforming method import vegetable cell or tissue, and the plant tissue that transforms is cultivated into plant.The plant host that is converted can be dicotyledons or monocotyledons.

The acquisition of described MfcpCOR14 gene fragment and the preparation method of plant expression vector comprise the following steps:

(1) design of primers

1. MfcpCOR14 amplification upstream primer RT81:CACAAACTCGAAAAACTTAAGAACCA;

2. MfcpCOR14 amplification downstream primer RT82:CTTACTCCTTCCCAGCCACG;

3. introduce the upstream primer pMD1:GATC of BamH I restriction enzyme site GGATCCGAGGATCTACTAGTCATATGG;

4. original BamH I restriction enzyme site that suddenlys change is introduced the downstream primer pMD2:GTGA of Xba I restriction enzyme site TCTAGAGCTCGGTACCCGG AAATCCGA;

(2) acquisition of MfcpCOR14 gene fragment:

Take the cDNA of bur clover (Medicago falcata) as template, use primer 1. with the primer MfcpCOR14 gene fragment that 2. increases, and connect and enter in the pMD20-T carrier, build and obtain the pMD-MfcpCOR14 carrier;

(3) structure of plant expression vector pBI-MfcpCOR14

Take the pMD20-MfcpCOR14 carrier as template, use primer 3. 4. to make the MfcpCOR14 gene fragment introduce Xba I and two restriction enzyme sites of BamH I with primer, and then build acquisition plant expression vector pBI-MfcpCOR14;

Described transgenic plant are transgene tobacco;

The acquisition of described transgene tobacco comprises the following steps:

(1) method that turns of electricity consumption imports pBI-MfcpCOR14 in agrobacterium tumefaciens EHA105, obtains to contain the positive bacterium colony of expression vector pBI-MfcpCOR14 Agrobacterium;

The Agrobacterium EHA105 that contains expression vector pBI-MfcpCOR14 that (2) will activate contaminates tobacco aseptic seedling leaf dish, through cultivating altogether and antibiotic-screening, obtains transgene tobacco.

The contriver has cloned the cDNA sequence of a new gene from bur clover, can not find out the protein sequence high with its homology at ncbi database; This genetic expression is subjected to low temperature induction, and the albumen preliminary assay of its translation is positioned at chloroplast stroma, the maturation protein molecular size is 14kDa, therefore with the cold response protein 14(chloroplastic of its called after chloroplast(id) cold responsive14kDa protein), gene name MfcpCOR14.

The present invention has following advantage and effect with respect to prior art:

The contriver has cloned the cDNA sequence MfcpCOR14 of the cold response protein MfcpCOR14 of chloroplast(id) from bur clover.The expression of MfcpCOR14 gene is subjected to low temperature induction, built the expression vector that is suitable for dicotyledons, transform dicotyledonous model plant tobacco, the growth of transfer-gen plant is better than the wild-type plant, and transfer-gen plant has obviously improved winter resistance and anti-high light ability simultaneously.

Description of drawings

Fig. 1 is the schematic diagram of plant expression vector pBI-MfcpCOR14.

Fig. 2 is the pcr amplification agarose gel electrophoresis figure of MfcpCOR14 gene open reading frame sequence of the present invention, and wherein, M is the standard DNA molecule; The amplified fragments of the 1st, MfcpCOR14 gene.

Fig. 3 is that the PCR of the plant expression vector of MfcpCOR14 gene identifies agarose gel electrophoresis figure, and wherein, M is the standard DNA molecule; 1-8 is positive recombinant; The 9th, pMD-MfcpCOR14 carrier, positive contrast; The 10th, negative control.

Fig. 4 is the feature histogram of low temperature induction MfcpCOR14 genetic expression; Alphabetical a, the b of post top and c represent the significant difference (P≤0.05) between different treatment, and namely data post top is alphabetical represents there is no significant difference when identical, and there is significant difference in data post top letter expression simultaneously.

Fig. 5 is the transgene tobacco PCR qualification result agarose gel electrophoresis figure that imports the MfcpCOR14 gene, and wherein, M is the standard DNA molecule; W represents wild-type tobacco; 6-3,11-2,13-2 represent the different MfcpCOR14 genetic tobaccos that turn.

Fig. 6 is that the Southern hybridization and the Northern hybridization that import the transgene tobacco of MfcpCOR14 gene are identified, wherein, W represents wild-type; 6-3,11-2,13-2 represent the different MfcpCOR14 genetic tobaccos that turn; Figure (a) is Southern hybridization, and figure (b) is Northern hybridization.

Fig. 7 is that the Western hybridization that imports the transgene tobacco of MfcpCOR14 gene is identified, wherein, W represents wild-type; 6-3,11-2,13-2 represent the different MfcpCOR14 genetic tobaccos that turn.

Fig. 8 is the transgene tobacco growth measurement that imports the MfcpCOR14 gene, and wherein, W represents wild-type tobacco; 6-3,11-2,13-2 represent the different MfcpCOR14 genetic tobaccos that turn; Figure (a) be 4,5,6 the week age seedling overground part fresh weight, figure (b) be 4,5,6 the week age shoot root section fresh weight, figure (c) is 4,5, the 6 whole strain fresh weights of week seedling in age, figure (d) be 10,14 the week age seedling overground part fresh weight, figure (e) be 10,14 the week age shoot root section fresh weight, figure (f) be 10,14 week seedling in age whole strain fresh weights, figure (g) 4,5,6,10,14 age in week the seedling upgrowth situation; Alphabetical a, b, c, d and the e of post top represents the significant difference (P≤0.05) between different treatment, and namely data post top is alphabetical represents there is no significant difference when identical, and there is significant difference in data post top letter expression simultaneously.

Fig. 9 is transgene tobacco winter resistance and the anti-high light analysis that imports the MfcpCOR14 gene, and wherein, W represents wild-type tobacco; 6-3,11-2,13-2 represent the different MfcpCOR14 genetic tobaccos that turn; Figure (a) is the Net Photosynthetic Rate of deepfreeze (3 ℃), and figure (b) is relative conductivity and each strain photo that freezes processings (4 ℃ 3 hours), and figure (c) is the relative conductivity that freezes processing (serial sub-zero temperature), and scheming (d) is high light (1200 μ molm ^-2s ^-1) relative conductivity processed, figure (e) is high light (1200 μ molm ^-2s ^-1) the maximal photochemistry efficiency Fv/Fm that processes; Alphabetical a, b, c, d, e, f, g and the h of post top represents the significant difference (P≤0.05) between different treatment, and namely data post top is alphabetical represents there is no significant difference when identical, and there is significant difference in data post top letter expression simultaneously.

Embodiment

The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.

Bur clover or bur clover seed: kind is the Hulun Buir, animal and veterinary institute forage germplasm resources storehouse, the Chinese Academy of Agricultural Sciences Beijing.

Intestinal bacteria (Escherichia coli) DH10B: available from the super bio tech ltd that grinds in Shanghai.

PBI121 expression vector: available from the Beijing DingGuo ChangSheng Biology Technology Co., Ltd.

Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105: available from sky, Beijing bounties Gene Tech. Company Limited.

Tobacco or tobacco (Nicotiana tabacum L.) seed: kind is middle cigarette 90, academy of agricultural sciences, Guangdong Province crop investigations institute.

The clone of embodiment 1MfcpCOR14

One, bur clover cDNA template preparation

With 60 ℃ of hot-water soaks 5 minutes, vernalization under 28 ℃ of dark conditions was when seed shows money or valuables one carries unintentionally with bur clover (Medicago falcata) seed, point is sowed in the plastic tub (diameter 15cm) that fills earth, cultivate natural lighting, temperature 25-28 ℃ in the greenhouse.It is that (intensity of illumination is 200 μ molm for the growth cabinet of 5 ℃ that the bur clover plant in seedling age 8-10 week is put into temperature ^-2s ^-1) in, light/dark cycle is illumination in 12 hours, carries out 5 ℃ of subzero treatment, processes continuously 2 days.Get the mature leaf of yellow clover, extract total RNA with the TRE-Trizol method, obtain reverse transcription the first chain cDNA as template with M-MLV reversed transcriptive enzyme test kit (Promega company) reverse transcription ,-20 ℃ save backup.

Two, design amplification MfcpCOR14 gene primer

The sequence (ES323281) that the cold induced gene cDNA of bur clover that builds according to this laboratory reduces the MfcpCOR14 gene cDNA fragment of finding in the library, carry out the BLASTX comparison on NCBI, and the highest sequence of download corresponding nucleotide sequence identity, puncture vine clover MTR2g014050.According to the sequence of the MfcpCOR14 gene cDNA fragment zone design primer corresponding to puncture vine clover MTR2g014050 open reading frame two ends:

MfcpCOR14 amplification upstream primer RT81:CACAAACTCGAAAAACTTAAGAACCA;

MfcpCOR14 amplification downstream primer RT82:CTTACTCCTTCCCAGCCACG;

Three, amplification obtains MfcpCOR14 gene and carrier construction

Carry out pcr amplification with the template of above-mentioned step 1 preparation and the primer of step 2 design.

PCR reaction system (25 μ L): Ex Taq archaeal dna polymerase (TaKaRa company, 5U μ L ^-1) 0.1 μ L, 10 * Ex Taq DNA polymerase buffer liquid, 2.5 μ L, dNTPs(10mmolL ^-1) 0.25 μ L, upstream primer RT81(10 μ molL ^-1) 0.8 μ L, downstream primer RT82(10 μ molL ^-1) 0.8 μ L, reverse transcription the first chain cDNA1 μ L, ddH ₂O complements to 25 μ L.

The PCR response procedures: 94 ℃ 3 minutes; 94 ℃ 20 seconds, 57 ℃ 30 seconds, 68 ℃ 40 seconds, 35 circulations; 68 ℃ 10 minutes.Amplified production detects (as Fig. 2) with 1% agarose gel electrophoresis.

Obtain the PCR fragment (Fig. 2) consistent with expection fragment length (605bp).After the PCR product that obtains is reclaimed test kit (Dongsheng company) recovery with DNA gel, with T4DNA ligase enzyme (TaKaRa company), PCR being reclaimed fragment is connected with linear pMD20-T carrier (TaKaRa company), reaction system is as follows: 1 μ L10 * T4 ligase enzyme damping fluid, PCR reclaims fragment 150ng, 1 μ L T4DNA ligase enzyme, the linear pMD20-T carrier of 1.5 μ L (10mmolL ^-1), adding sterilized water to cumulative volume is 10 μ L, 16 ℃ of connections are spent the night.

The preparation of competent escherichia coli cell: the intestinal bacteria DH10B bacterium liquid of going bail for transfering loop and being stored in-70 ℃ of Ultralow Temperature Freezers, draw plate, overnight incubation in 37 ℃ of incubators on the SOB solid medium; The single colony inoculation of picking intestinal bacteria DH10B with 200rpm shaking culture 6 hours, is inoculated in 200mL SOB liquid nutrient medium on 37 ℃ of constant-temperature tables in the SOB of 1mL liquid nutrient medium, on 37 ℃ of constant-temperature tables with the 200rpm shaking culture to OD ₅₅₀=0.8.Collect thalline in centrifugal 10 minutes with 2500rpm, add 10% glycerine washing of precooling, centrifugal collection bacterium.Repeat with 10% glycerine washing, centrifugal collection bacterium.With the bacterium packing, save backup in-70 ℃ at last.

Connect the product electric shock and transform intestinal bacteria: get 20 μ L competent escherichia coli cells to electric shock cup (an aperture 2mm), add 1 μ L to connect product, transform with electric shock instrument (MicroPulser, Bio-RAD company) electric shock.Shock parameters is: resistance 200 Ω, 1800V, 25 μ F.Thalline after electric shock changes in 1mL SOC liquid, on 37 ℃ of constant-temperature tables with 200rpm shaking culture 1 hour.Getting 100 μ L bacterium liquid coats and contains 24mgL ^-1IPTG and 40mgL ^-1The LB solid of X-gal (contains 100mgL ^-1Penbritin) on substratum, be inverted overnight incubation in 37 ℃ of incubators.

The screening of recombinant plasmid pMD-MfcpCOR14, purifying and order-checking: the bacterium colony that is locus coeruleus does not contain Insert Fragment, and only picking is the bacterium colony of hickie, is bacterium colony PCR and detects.The positive bacterium colony of picking PCR is inoculated in 1mL and contains 100mgL ^-1In the LB liquid nutrient medium of penbritin, with 200rpm shaking culture 5 hours, be bacterium liquid PCR take RT81/RT82 as primer and detect on 37 ℃ of constant-temperature tables.Draw 5 μ L positive bacteria liquid, be inoculated in 3mL and contain 100mgL ^-1In the LB liquid nutrient medium of penbritin, spend the night with the 200rpm shaking culture on 37 ℃ of constant-temperature tables.With alkaline lysis extracting plasmid, 4 ℃ save backup.The recombinant plasmid called after pMD-MfcpCOR14 of purifying delivers Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and checks order, and sequencing result reverse complemental chain is as follows:

CACAAACTCGAAAAACTTAAGAACC ATGGCAACAATGTTGACAAGTA ACACACTCTTCCAAACCTCAAACTCATTCCCTAATGTCCCTTCACTCACCC TTCCTAAACCCTCCAGGGTCTTCTTAGCTTCCAACTGGAGAAATGCATCAG AAGGAACAAAAAACACTTCACTCAGTTGGGCTTACACTTCTTCTTCTACAA AGACAAGAAGATATGCAGATGGAACAGCCGGCAAAGCCGGAGAAACGGT GAACGCAGGCATAGATGACATCAAACAATTCGGACAAGATGCAGATGAAA AGACAAAGGACGCTGCGAGTTCAATTGCGGATAAAGCAAAAGAAAACAC AGACAAGACTGTAGAGGCAGTAGGAAGTGCTGGAGACAAGGCAAAAGAT TATGCTTTTGATGCAAATGACAAAGCCAAGGAAGCAATAGGTTCTGCTACT GATAAGGCAAAAGAAGGATTTGAGGCAGCAACGAAGAACACACAGGAGG CTGCTGGGTCAGCGACAGAGGCTCTGAAGAATGCAGGAGATCAGGCAAA GGAGGCGGTGGAAGGAGCGTTGGACGCGGCCAAGGATGCCGTGGCTGGG AAGGAGTAAG

Sequencing result and cDNA reduce the sequence (ES323281) of MfcpCOR14 gene cDNA fragment in the library and compare, and prove that the PCR product that amplifies is the MfcpCOR14 gene, and sequencing result shows that the MfcpCOR14 gene is reversely connected on pMD20-T.With the PCR product sequence that the Omiga2.0 software analysis increases, obtained open reading frame (ORF) sequence of MfcpCOR14 gene, as underscore mark part in above-mentioned sequencing sequence.Encode the gene of the cold response protein MfcpCOR14 of described bur clover chloroplast(id) by 605 based compositions, the protein coding region of MfcpCOR14 (Sequence coding for amino acids in protein wherein, CDs) by 579 based compositions of 26～604,192 amino acid of encoding.Have 55 amino acid whose chloroplast(id) signal peptides with the coded albumen of ChloroP analysis software prediction MfcpCOR14,26～190 bit bases of corresponding above-mentioned sequencing result reverse complemental chain, after the processing of chloroplast(id) signal peptide, the maturation protein molecular size of prediction is 14kDa.

The plant expression vector (pBI-MfcpCOR14) of embodiment 2MfcpCOR14 gene builds

Due to MfcpCOR14 gene Opposite direction connection on the pMD20-T carrier, according to pMD20-T carrier insertion point both wings sequence, design the amplimer with BamH I and Xba I restriction enzyme site:

Introduce the upstream primer pMD1:GATC of BamH I restriction enzyme site GGATCCGAGGATCTACTAGTCATATGG;

Original BamH I restriction enzyme site that suddenlys change is introduced the downstream primer pMD2:GTGA of Xba I restriction enzyme site TCTAGAGCTCGGTACCCGG AAATCCGA;

The cloning vector pMD-MfcpCOR14 that builds in the embodiment 1 is as template, take pMD1 and pMD2 as the upstream and downstream primer, carries out the amplification of MfcpCOR14 gene PCR, and purifying reclaims the MfcpCOR14 gene fragment that amplification obtains.

MfcpCOR14 gene fragment and pBI121 expression vector that above-mentioned recovery obtains are used respectively restriction enzyme Xba I (TaKaRa company) and BamH I (TaKaRa company) double digestion, reclaim respectively MfcpCOR14 gene purpose fragment and enzyme and cut rear pBI121 carrier large fragment, after connecting with T4DNA ligase enzyme (TaKaRa company), the MfcpCOR14 gene is inserted between the Xba I and BamH I of expression plasmid pBI121CaMV35S promotor downstream multiple clone site, build and obtain recombinant expression vector, called after pBI-MfcpCOR14.Respectively take RT81/RT82 and pBI121 support C aMV35S sequence upstream primer ZG13(CTCGGATTCCATTGCCCAGCT)/RT82 detects as primer carries out bacterium liquid PCR, proves to have obtained recon (Fig. 3).Further Insert Fragment is checked order, result shows, the sequence of the sequence of Insert Fragment and MfcpCOR14 coding region is in full accord, and the restriction enzyme site at Insert Fragment two ends is also entirely true, thereby proof has been built into pBI-MfcpCOR14 expression vector, the schematic diagram of pBI-MfcpCOR14 expression vector such as Fig. 1.

Embodiment 3MfcpCOR14 is subjected to the expression of low temperature induction

One, the bur clover template under the acquisition cold condition

It is growth cabinet (the 200 μ molm of 5 ℃ that the bur clover plant in seedling age 8-10 week is put into temperature ^-2s ^-1) in, all are made as illumination and interlunation illumination in 12 hours.Processed 4 days continuously, carry out 5 ℃ of subzero treatment.Get respectively the mature leaf of subzero treatment 0h, 12h, 24h, 48h, 96h, add liquid nitrogen to grind sample, extract total RNA of blade with TRE-Trizol reagent, use PrimeScript ^TMRT reagent Kit(TaKaRa company) reverse transcription acquisition reverse transcription the first chain cDNA is the cDNA template, and-20 ℃ save backup.

Two, design specific detection primer

According to the MfcpCOR14 gene cDNA sequence and cut a type clover Actin(MTR3g095530) sequence, go out the quantitative primer of detection with Beacon Designer7.0 software design:

The quantitative primer ZG1861:ACGCAGGCATAGATGACAT in the upstream of MfcpCOR14 gene;

The quantitative primer ZG1862:CAGAACCTATTGCTTCCTTGG in the downstream of MfcpCOR14 gene;

The quantitative primer ZG1613:ATTCACGAGACCACCTAC in the upstream of Actin gene;

The quantitative primer ZG1614:GAGCCACAACCTTAATCTTC in the downstream of Actin gene.

Three, quantitative PCR detection differential expression

The template cDNA of step 1 preparation in embodiment 3 is diluted to 15ng μ L ^-1The template that is used for quantitative PCR.Reaction system be 10 μ L:SYBR Premix Ex Taq(2 *) 5 μ L, upstream and downstream primer (10 μ molL ^-1) each 0.5 μ L, cDNA template 1 μ L, sterilized water 3 μ L.The Mini Option Real-Time PCR System that uses instrument to produce as Bio-Rad company, the PCR reaction conditions is set to: 95 ℃ 10 seconds; 94 ℃ 5 seconds, 57 ℃ 25 seconds, 40 circulations.Not add the cDNA template as negative control, each sample arranges 3 repetitions, with Actin as reference gene.Reaction is carried out the solubility curve analysis after finishing, and pcr amplification efficient is more than 95%.Utilize Bio-Rad CFX Manager (version1.6) software automatically to calculate the relative expression quantity of gene.

The result of real-time quantitative PCR shows, MfcpCOR14 expresses and obviously induced in 12 hours in subzero treatment, and subzero treatment 48 hours and 96 hours, expression amount maintained highest level (Fig. 4) always.Result shows, the expression of low temperature induction MfcpCOR14 gene.

Generation and the Molecular Detection of embodiment 4 transgene tobaccos

One, the generation of transgene tobacco

The preparation of Agrobacterium competent cell: agrobacterium tumefaciens (Agrobacterium tumefaciens) the EHA105 bacterium liquid that is stored in-70 ℃ of Ultralow Temperature Freezers of going bail for transfering loop is rule on the YEB flat board, cultivated 48 hours for 28 ℃, picking list bacterium colony, be inoculated in 50mL liquid SOB, 28 ℃ of overnight incubation are drawn 0.5mL bacterium liquid, are inoculated in 500mL liquid SOB, cultivated 8 hours for 28 ℃, to OD ₆₀₀Be 0.6, cooling 10 minutes of ice bath is poured in the 200mL centrifuge tube of sterilization after balance 4 ℃ into, 4000rpm, centrifugal 10 minutes, collect thalline, remove SOB, be inverted on paper handkerchief, the control solid carbon dioxide divides, and adds 50mL10% glycerine, in shaking on ice, the suspension thalline, 4 ℃, 4000rpm, centrifugal 15 minutes, collect thalline, abandon 10% glycerine, repeated washing once adds 2mL10% glycerine, the suspension thalline, packing, 20 μ L/ pipes add after the liquid nitrogen quick-frozen in-70 ℃ of Refrigerator stores.

The plasmid electric shock transforms Agrobacterium: with 1 μ L pBI-MfcpCOR14 plasmid (20ng μ L ^-1) add in the Agrobacterium competent cell that 20 μ L thaw, flicking the tube wall mixing, ice bath was transferred in electric shock cup (aperture 2mm) after 90 seconds, was placed between the electrode of electric shock instrument (MicroPulser, Bio-RAD company), and selection procedure Agr shocks by electricity.After electric shock finishes, rapidly 1mL YEB liquid nutrient medium is poured into the electric shock cup, then be transferred to liquid-transfering gun and shake in tube, 28 ℃ of soft shaking culture 2 hours.Draw 0.3mL bacterium liquid, be coated on the YEB flat board and (contain 35mgL ^-1Paraxin and 50mgL ^-1Kantlex) on.Be inverted in 28 ℃ of incubators and cultivated 36 hours.

The evaluation of positive bacterium colony: the single bacterium colony on the picking flat board, respectively take RT81/RT82 and pBI121 support C aMV35S sequence upstream primer ZG13(CTCGGATTCCATTGCCCAGCT)/RT82 detects as primer carries out bacterium liquid PCR.Further draw PCR positive bacteria liquid to 3mL YEB liquid nutrient medium and (contain 35mgL ^-1Paraxin and 50mgL ^-1Kantlex) in, 28 ℃ of shaking culture 36 hours.Get 2mL bacterium liquid with alkaline lysis extracting plasmid, carry out enzyme with restriction enzyme Xba I (TaKaRa company) and BamH I (TaKaRa company) and cut detection, determine to contain in positive colony plant expression vector pBI-MfcpCOR14.Get the 0.8mL agrobacterium liquid, add 0.2mL80% glycerine, be stored in-70 ℃ of Ultralow Temperature Freezers after mixing standby.

The activation of Agrobacterium and suspension: get the Agrobacterium EHA105 stock solution that contains expression vector pBI-MfcpCOR14, (contain 35mgL at the YEB flat board ^-1Paraxin and 50mgL ^-1Kantlex) upper line, 28 ℃ of cultivations.Choose and separate good single bacterium colony, be inoculated in 2mL liquid YEB(and contain 35mgL ^-1Paraxin and 50mgL ^-1Kantlex) in, 28 ℃ of dark cultivations 24 hours of vibration.Draw bacterium liquid 50 μ L, coat the YEB flat board and (contain 35mgL ^-1Paraxin and 50mgL ^-1Kantlex) on, be inverted dark the cultivation 36 hours for 28 ℃.The Agrobacterium that newly grows is scraped off, suspend with the MS liquid nutrient medium and dilute, make OD ₆₀₀Be 0.8, make Agrobacterium bacterium liquid.

The generation of transgene tobacco: tobacco (Nicotiana tabacum L.) seed is inoculated on the MS substratum after 70% alcohol and the sterilization of 2% clorox, 28 ℃ of illumination cultivation approximately 1 month.When launching 5-7 sheet true leaf, namely can be used for Agrobacterium-mediated Transformation.Get the blade of tobacco aseptic seedling, be cut into 1cm with scissors ²The leaf dish of size and stabs aperture with sharp mouth tweezers on the surface, and the manufacturing wound changes in the Agrobacterium bacterium liquid of above-mentioned preparation and soaks, and frequently shakes up therebetween.Soak after 15 minutes, suck the unnecessary bacterium liquid of leaf panel surface with aseptic thieving paper, slightly dry up, (the MS minimum medium adds 0.1mgL to be placed in the common substratum that has padded filter paper ^-1Naphthylacetic acid and 1mgL ^-1Benzyladenine, pH5.8) on, 28 ℃ of dark were cultivated 2 days altogether.Will be through common material transfer of cultivating to selecting substratum (MS minimum medium interpolation 0.1mgL ^-1Naphthylacetic acid, 1mgL ^-1Benzyladenine, 250mgL ^-1Cephamycin and 100mgL ^-1Kantlex, pH5.8), 28 ℃ of illumination cultivation.20 days subcultures once.The seedling that success is broken up goes to root media (MS minimum medium interpolation 250mgL ^-1Cephamycin and 100mgL ^-1Kantlex is taken root on pH5.8), and when growing 5-7 sheet true leaf, the uncork hardening is transplanted in soil.

The growth of transgene tobacco: the transgene tobacco seedling of transplanting is cultivated in the greenhouse, and the initial stage gives certain sheltering from heat or light, and the seedling after survival is transferred to gradually under natural lighting and grows.In process of growth, regularly water, apply fertilizer.Adopt the selfing sowing, namely before petal opens, to the inflorescence bagging; After fruit forms, then remove bagging.Sowing after fruit maturation namely obtains Transgenic Tobacco Seeds.

Two, the PCR of transgene tobacco detects

Micromethod DNA rapid extraction: get the leaf disk of transgene tobacco and wild-type plant thereof with 1.5mL centrifuge tube lid, add 400 μ L to be preheating to the extracting solution (200mmolL of 80 ℃ ^-1Tris-HCl pH8.0,250mmolL ^-1NaCl, 25mmolL ^-1EDTA, massfraction are 0.5% SDS), fully to grind in the 1.5mL centrifuge tube with the little hammer that grinds, 70 ℃ of xeothermic baths moved on to ice bath and placed 2 minutes after 10 minutes.Centrifugal 1 minute of 13000rpm gets 300 μ L supernatant liquors to new centrifuge tube, adds the equivalent Virahol, mixing, standing 5 minutes of room temperature.Centrifugal 2 minutes of 13000rpm abandons clean supernatant liquor, DNA is precipitated be dissolved in 50 μ L TE solution (1molL after air-dry ^-1NaCl, 10mmolL ^-1Tris-HCl pH8.0,1mmolL ^-1EDTA), be stored in-20 ℃, standby.

PCR detects: design upstream primer according to CaMV35S promoter sequence on the pBI121 carrier: pBI121 support C aMV35S sequence upstream primer ZG13:CTCGGATTCCATTGCCCAGCT;

With MfcpCOR14 amplification downstream primer RT82 pairing.Take the DNA of above-mentioned micromethod rapid extraction as template, carry out pcr amplification.Reaction system (20 μ L) is: Taq archaeal dna polymerase 0.1 μ L, 10 * buffer2 μ L, dNTPs(10mmolL ^-1) 0.25 μ L, upstream primer ZG13 and downstream primer RT82(10 μ molL ^-1) each 0.5 μ L, DNA1 μ L, ddH ₂O complements to 20 μ L.The PCR response procedures: 94 ℃ 3 minutes; 94 ℃ 20 seconds, 55 ℃ 30 seconds, 72 ℃ 40 seconds, 35 circulations; 72 ℃ 5 minutes.Detect the PCR product with 1% agarose gel electrophoresis.

Three, the Southern of transgene tobacco hybridization and Northern hybridization

The extraction of genomic dna: adopt the CTAB method to extract DNA, concrete grammar is as follows: get 1g tobacco (wild-type tobacco and transgene tobacco) blade, with the quick grind into powder of liquid nitrogen, (massfraction is 1.5% CTAB, 75mmolL to add 4mL to be preheating to 1.5 * CTAB extracting solution of 70 ℃ ^-1Tris-HCl pH8.0,1molL ^-1NaCl, 15mmolL ^-1EDTA), be transferred to the 50mL centrifuge tube after the grinding pulping, then use 3mL1.5 * CTAB extracting solution to clean mortar, go to the 50mL centrifuge tube, mixing.65 ℃ of water-baths 1 hour are interrupted mixing.After water-bath finishes, add the 5mL chloroform, screw the pipe lid, the mixing 10 minutes of turning upside down, centrifugal 15 minutes of 5000rpm.The careful supernatant of drawing is to new 50mL centrifuge tube, and recording volume, and the massfraction that adds 1/10 volume is 10% CTAB solution, shakes up.The chloroform that adds 4/5 volume, mixing 10 minutes turns upside down.Centrifugal 15 minutes of 5000rpm.The careful supernatant of drawing is to the 50mL centrifuge tube, and recording volume, and (massfraction is 1%CTAB, 50mmolL to add the precipitated liquid of equivalent ^-1Tris-HCl pH8.0,10mmolL ^-1EDTA), the mixing that softly turns upside down, room temperature was placed 15 minutes, precipitation DNA.Centrifugal 10 minutes of 5000rpm carefully outwells supernatant, exhausts supernatant with liquid-transfering gun.Add the high salt TE(1molL of 2mL to precipitation ^-1NaCl, 10mmolL ^-1Tris-HCl pH8.0,1mmolL ^-1EDTA) and 5 μ L10mgmL ^-1RNAase(10mmolL ^-1Tris-HCl pH7.5 and 15mmolL ^-1The NaCl solution preparation is through 15 minutes deactivation DNA enzymes of 100 ℃ of water-baths), jog is to precipitation dissolving (30-40 minute) fully in 55 ℃ of shaking tables.The dehydrated alcohol that adds 2 times of volumes, mixing gently turns upside down.Go out cotton-shaped DNA with rifle head hook, then rinsing in 70% ethanol moves to the 1.5mL centrifuge tube.Of short duration centrifugal, exhaust supernatant, suitable air-dry precipitation is used 0.1-0.2mL TE(10mmolL ^-1Tris-HCl pH8.0,1mmolL ^-1EDTA) dissolution precipitation.Get the DNA solution of 10 times of 2 μ L dilutions, use 0.8% agarose gel electrophoresis, detect the quality of DNA, survey OD _260nmAnd OD _280nm, determine purity and the concentration of DNA.

The extraction of total RNA: get 0.1g tobacco (wild-type tobacco and transgene tobacco) blade, with the quick grind into powder of liquid nitrogen, extract total RNA of blade with TRE-Trizol reagent, and determine concentration and the purity of total RNA.

The enzyme of genomic dna is cut and transferring film: get 15 μ g genomic dnas, add with aseptic deionized water to cumulative volume 33 μ L, 2 μ L EcoRI(or XbaI), 4 μ L10 * enzyme cutting buffering liquids, 37 ℃ of enzymes are cut and are spent the night.Add the point sample damping fluid, point sample is to 0.8% sepharose, with 4Vcm ^-1Electrophoresis 5 hours is until the indicating dye bromjophenol blue is swum to gel length 2/3 distance.With capillary tube technique, DNA is transferred on Hybond XL nylon membrane (Amersham).Concrete grammar is as follows: glue is lain on the filter paper that soaks into NaOH the 0.4molL of filter paper two ends immersion transfer cell ^-1In NaOH solution.Cover the surrounding of having sealed nylon membrane with preservative film after nylon membrane, in case the transfer system short circuit.Cover filter paper, and catch up with bubble, spread the approximately thick thieving paper of 7cm, and press the weight of 200g, continue transferring film 16 hours.After shifting end, with 2 * SSC(300mmolL ^-1Sodium-chlor, 30mmolL ^-1Trisodium Citrate pH7.0) after simple rinsing, then contains massfraction 0.5%SDS with the 0.5 * SSC(that boils) the solution soaking several minutes.Natural air drying 5 minutes is wrapped with preservative film, carries out mark, be stored in 4 ℃ standby.

The sex change electrophoresis and transferring film of total RNA: with conventional denaturing formaldehyde electrophoretic method, Bu Tong big or small RNA molecular separation is opened, forwarded to Hybond N with capillary tube technique ⁺On nylon membrane (Amersham).Specific as follows: take the 1.2g agarose, heating is dissolved in 85mL DEPC treated water, adds 10mL10 * MOPS solution after cooling, and 5mL37% formaldehyde after the 4 abundant mixings of μ L GoldView, is prepared into 1.2% denaturing formaldehyde gel.Get 30 μ g RNA samples, add successively 2 μ L10 * MOPS solution, 2.5 μ L37% formaldehyde, 7.5 μ L methane amides add DEPC and process water, make cumulative volume to 50 μ L, in 65 ℃ of insulations 10 minutes, make the RNA sex change after mixing.Then be placed in cooled on ice 5 minutes, and added electrophoresis load sample damping fluid.With the RNA sample loading of each sex change to denaturant gel, with 4Vcm ^-1Electrophoresis 3h is until the indicating dye bromjophenol blue is swum to gel length 2/3 distance.Electrophoresis is washed gel twice with aseptic deionized water after finishing, and each 10 minutes, then use 10 * SSC solution to wash 10 minutes.Gel is lain on the filter paper that shifts bridge, drive bubble away.With a large little Hybond N close with gel ⁺(Amersham) nylon membrane is layered on glue, catches up with bubble.On the nylon membrane upper berth 2 the same with the nylon membrane size and with the abundant wetting filter paper of 10 * SSC, the surrounding of having sealed nylon membrane with preservative film is in case the transfer system short circuit.Spread the approximately thick thieving paper of 7cm on filter paper, and press the weight of 200g, continue transferring film 16 hours.After completing RNA and shifting, nylon membrane is placed on the moistening filter paper of 2 * SSC, be placed in that will to have facing up of RNA to carry out in UV-crosslinked instrument UV-crosslinked, the UV-light total energy of employing is 800kJ.Nylon membrane is placed on DEPC again and processes rinsing in water, natural air drying 5 minutes is wrapped with preservative film, carries out mark, be stored in 4 ℃ standby.

Probe mark: the MfcpCOR14 fragment that obtains with clone in embodiment 1 step (the PCR product that namely obtains with primer pair RT81 and RT82 and template bur clover cDNA) 50ng(25 μ L reaction system) as the template of probe mark, adopt Random Primer DNA Labeling Kit Ver.2.0(TaKaRa) come label probe with reference to the method for specification sheets.Every 25 μ L reaction systems contain 5 μ L[α- ³²P] dCTP isotropic substance (Beijing Fu Rui company) carries out mark, and reaction adds isopyknic 0.8molL after finishing ^-1NaOH solution makes the probe molecule sex change, is used for hybridization analysis.

Hybridization: nylon membrane is put into hybrid pipe, add appropriate prehybridization solution (every 1cm ²Membrane area adds the 0.1mL prehybridization solution), 42 ℃ of prehybridizations 4 hours add the good probe of mark in hybrid pipe, 42 ℃ of hybridization 16 hours.Hybridize completely, pour out hybridization solution, with the 2 * SSPE(300mmolL that contains massfraction 0.5%SDS ^-1NaCl, 20mmolL ^-1Na ₂HPO ₄, 2mmolL ^-1EDTA, pH7.4) respectively wash once at 42 ℃ and 65 ℃, each 15 minutes, washed 15 minutes at 65 ℃ with the 0.2 * SSPE that contains massfraction 0.2%SDS for the third time.After washing the film end, with counter detection of radioactive intensity, use the preservative film coating, after press mold developed 1 day, with FX automatic imaging system (BIO-RAD company) scanning analysis results of hybridization.

Adopt the PCR method to detect (using primer pair ZG13 and RT82) transgene tobacco, positive plant can amplify the special band of 900bp left and right, and the wild-type control plant can not expand and this special band (Fig. 5), proves that the MfcpCOR14 gene imports in transgene tobacco.

The Southern results of hybridization shows (as Fig. 6 a), the specific hybridization signal that there is no the MfcpCOR14 gene in wild-type tobacco, the specific hybridization signal that the MfcpCOR14 gene is arranged in transgenic tobacco plant shows that the MfcpCOR14 gene has been incorporated in the genome of transgene tobacco.

The Northern results of hybridization shows (as Fig. 6 b), there is no the specific hybridization signal of MfCPCOR14 gene in wild-type tobacco, and the specific hybridization signal of MfCPCOR14 gene is arranged in transgene tobacco, shows that the MfCPCOR14 gene can express in transgene tobacco.

Four, the Western of transgene tobacco hybridization

The preparation of polyclonal antibody: take by 100 amino acid whose peptide sections of 57～156, MfcpCOR14 albumen as antigen, 194～493 bit base recombinant peptide section purifying and immune rabbits with sequencing result reverse complemental chain in corresponding embodiment 1, through getting the whole blood purifying after 4 immunity, obtain MfcpCOR14 polyclonal antibody ((Shanghai) Co., Ltd. prepares by the Ai Bimate biological medicine).

The extraction of total protein: get 0.1g tobacco (wild-type tobacco and transgene tobacco) blade, with the quick grind into powder of liquid nitrogen, with 0.1mL RIPA albumen extract (150mmolL ^-1NaCl, 50mmolL ^-1Tris, 5mmolL ^-1EDTA, 1%NP-40, massfraction are 0.5% Sodium desoxycholate, massfraction is 0.1%SDS, pH7.5) extract the total protein of blade, and (massfraction is 0.01% Coomassie brilliant blue G250 with the Xylene Brilliant Cyanine G method, 5% ethanol, 10% phosphoric acid) measure protein content under the 595nm wavelength.

SDS-polyacrylamide gel electrophoresis and the electrotransfer of total protein: with conventional SDS-polyacrylamide gel electrophoresis, Bu Tong big or small albumen sepn is opened, used the water-bath type electrotransfer to BioTrace ^TMOn NT nylon membrane (Pall).Specific as follows: as to use Mini-

The concentrated glue of system Casting stand gel mould preparation 15% acrylamide separation gel and 5% acrylamide.Get 100 μ g protein samples, add 10 μ L sample buffer (0.5mmolL ^-1Tris-HCl pH6.8,25% glycerine, massfraction are 4%SDS, massfraction is 0.01% tetrabromophenol sulfonphthalein, 5% mercaptoethanol), add distilled water and make cumulative volume to 20 μ L, in 95 ℃ of insulations 5 minutes, make protein denaturation after mixing.Then be cooled to room temperature, 12000rpm may disturb the insolubles of electrophoresis in centrifugal 10 minutes with removal.To the SDS-polyacrylamide gel, first electrophoresis to tetrabromophenol sulfonphthalein dyestuff enters separation gel from concentrated glue under the 70V constant voltage with the protein sample loading of each sex change, then continues electrophoresis to tetrabromophenol sulfonphthalein with the 100V constant voltage and migrate to the separation gel bottom.Take out gel in transfering buffering liquid (25mmolL ^-1Tris, 192mmolL ^-1Glycine, 20% methyl alcohol) balance is 20 minutes, cuts 1 large little BioTrace close with gel ^TMNT nylon membrane (Pall) soaked 15 minutes at transfering buffering liquid.After moistening sponge pad and the big or small filter paper close with gel, press from both sides lay sponge pad, filter paper, gel, nylon membrane, filter paper, sponge pad successively in transfer, shut and shift folder, put into transfer groove 200mA Constant Electric Current under cooling conditions and turn 2 hours.

Immunodetection: take out nylon membrane after electrotransfer, nylon membrane is placed in confining liquid, and (massfraction is 5% skim-milk, 0.05%Tween20,20mmolL ^-1Tris, 500mmolL ^-1NaCl, pH7.5) room temperature sway the sealing 1 hour after, go to TBST(0.05%Tween20 20mmolL ^-1Tris, 500mmolL ^-1NaCl, pH7.5) in the MfcpCOR14 polyclonal antibody of 1:500 dilution, room temperature jolting 2 hours.Wash 5 times with TBST, each jolting went to TBST(0.05%Tween20 20mmolL after 5 minutes ^-1Tris, 500mmolL ^-1NaCl, pH7.5) in the goat anti-rabbit igg two anti-(Kirkegaard and Perry Laboratories) of horseradish peroxidase conjugation of 1:10000 dilution, room temperature jolting 1.5 hours.Wash 5 times with TBST, each jolting was used chemical luminous substrate after 5 minutes

West Pico Chemiluminescent Substrate(Thermo) reaction presses the X-exposure to develop after 5 minutes.

The Western results of hybridization is seen Fig. 7, and result shows transgene tobacco 6-3,11-2, and MfcpCOR14 protein expression in 13-2, corresponding wild-type plant W do not have MfcpCOR14 to express.

The growth of embodiment 5 transgene tobaccos, winter resistance and anti-high light are identified

One, growth measurement

Transgene tobacco and wild type seeds thereof are sowed in filling the plastic tub of earth (diameter 15cm), and overlay film is cultivated in the greenhouse, natural lighting, temperature 25-30 ℃.After 1 week, tobacco seedling is transplanted in the seedling-growing container (every lattice length of side 6cm) that fills earth, every lattice 1 strain is cultivated in the greenhouse, natural lighting, temperature 25-30 ℃.Respectively at 4,5, during 6 all seedling ages, carefully plant is taken out, with the earth wash clean, dry the rear every strain overground part of weighing, underground part and whole strain weight respectively.Simultaneously, after 1 week of sowing, tobacco seedling is transplanted in the plastic tub (diameter 15cm) that fills earth, every basin 1 strain is cultivated in the greenhouse, natural lighting, temperature 25-30 ℃.Respectively at 10, during 14 all seedling ages, carefully plant is taken out, with the earth wash clean, dry the rear every strain overground part of weighing, underground part and whole strain fresh weight respectively.

Upgrowth situation to transgene tobacco under home is observed, and finds that its growing way obviously is better than wild-type (Fig. 8 g).To 4 weeks, 5 weeks, 6 weeks, 10 weeks and tobacco plant fresh weight weighing result demonstration in 14 ages in week, 4 all seedling age phase transgene tobacco fresh weights do not have difference with wild-type, and 5 weeks, 6 weeks, 10 weeks and 14 all seedling age phase transgene tobacco fresh weights are significantly higher than wild-type (Fig. 8 a-f).Result shows that the growth of transgene tobacco is better than wild-type.

Two, cold resistance is measured

Transgene tobacco and wild-type plant seedling thereof were grown in the greenhouse after 12 weeks, move on to temperature and be growth cabinet (the light intensity 500 μ mol photonm of 3 ℃ ^-2s ^-1) in, growth cabinet illumination every day and interlunation are respectively 12 hours.Continuous low temperature was processed 4 days, measured Net Photosynthetic Rate, estimated cold resistance.Specific as follows, use the portable photosynthesizer of Li-Cor6400P (production of U.S. Li-Cor company) to measure the photosynthetic rate of following the 3rd leaf in plant top, measuring light intensity is 700 μ molm ^-2s ^-1, adapting to after 30 minutes and measure, each strain plant is repeated 3 strains, calculating mean value.

After deepfreeze, the Net Photosynthetic Rate of transgene tobacco is apparently higher than wild-type tobacco.Process wild-type tobacco net photosynthetic rate after 4 days for 3 ℃ and be down to 28% before processing, and the transgene tobacco net photosynthetic rate be down to respectively process front 47.5%, 45.9% and 46.6%(Fig. 9 a).Result shows that transgene tobacco has higher photosynthetic rate than wild-type tobacco at low temperatures, has improved cold resistance.

Three, frost resistance is measured

Be that 2% chlorine bleach liquor sterilized 20 minutes with transgene tobacco and wild type seeds thereof through massfraction, then use sterile water wash 4 times, be seeded on the MS substratum, 28 ℃ of illumination cultivation, intensity of illumination are 200 μ molm ^-2s ^-1, the photoperiod is 12 hours.After plant to be planted grows true leaf, forward in new MS substratum every ware 12 strains, every numbering 3 wares to.The seedling of cultivating for 5 weeks is put into growth cabinet (200 μ molm ^-2s ^-1) the middle low temperature that adapts to, cultivated 12 hours for 15 ℃, cultivated 8 hours for 8 ℃, cultivate after 4 hours for 4 ℃, be placed in-4 ℃ of processing 3 hours.Clip plant overground part is put in the triangular flask that the 25mL deionized water is housed, and establishes 3 repetitions, surveys electricity with DDS-11A type conductivity meter in the shaking table jog after 2 hours and leads R1.Boiling water bath was cooled to room temperature after 20 minutes, surveyed electricity and led R2.The calculation formula of relative conductivity is (R1/R2) * 100%.

3 of the leaf disks that following the 3rd leaf diameter of transgene tobacco and wild-type plant top thereof of getting 12 weeks of growth in the greenhouse is 1cm are put into test tube, blade can not be overlapping, be placed in the mixture of ice and water balance and add a small ice toward test tube after 1 hour, allow leaf disk touch ice cube as far as possible, move in the freeze cycle instrument and process.Measure respectively the relative conductivity of 0 ℃ ,-2 ℃ ,-4 ℃ ,-6 ℃ ,-8 ℃ ,-10 ℃ processing.Concrete process as follows: 0 ℃ of balance 1 hour, take out 0 ℃ of sample; Per hour to fall the speed cooling of 2 ℃, sampling after relevant temperature is processed 1 hour.Move in triangular flask after managing interior ice cube dissolving after sampling, add deionized water to 25mL, survey electricity with DDS-11A type conductivity meter in the shaking table jog after 2 hours and lead R1; Boiling water bath was cooled to room temperature after 20 minutes, surveyed electricity and led R2.The calculation formula of relative conductivity is (R1/R2) * 100%.Each each strain plant 3 strain of test, test repeats 3 times.

After freezing processing, tobacco leaf film system comes to harm, and relative conductivity obviously raises.To 5 age in week seedling carry out-4 ℃ process 3 hours after, the wild-type relative conductivity is 27.8%, the transgene tobacco relative conductivity is respectively 15.5%, 17.1%, 16%, is starkly lower than wild-type (Fig. 9 b).Leaf disk is carried out a series of sub-zero temperatures process, the wild-type relative conductivity is significantly higher than transgene tobacco (Fig. 9 c).Result shows that transgene tobacco is subjected to the freeze injury degree lighter than wild-type, has improved frost resistance.

Four, anti-high light is identified

Get transgene tobacco and the wild-type plant in 12 weeks of growth in the greenhouse, ripe the 3rd leaf of leaf that launch, be the leaf disk of 1cm with punch tool cut-off footpath, the blade face faces up and is placed in the culture dish that fills distilled water, halogen tungsten lamp irradiation with two 1000W, between light source and blade face, alternating floor flows water layer to keep leaf table temperature in room temperature, and the leaf surface light intensity is 1200 μ molm ^-2s ^-1Respectively at irradiation 0, sampling in 1.5,3 hours, measure relative conductivity and Fv/Fm.After sampling, 5 leaf disks are put in the triangular flask that the 25mL deionized water is housed, established 3 repetitions, survey electricity with DDS-11A type conductivity meter in the shaking table jog after 2 hours and lead R1; Boiling water bath was cooled to room temperature after 20 minutes, surveyed electricity and led R2; The calculation formula of relative conductivity is (R1/R2) * 100%.Simultaneously, use the leaf folder with leaf disk dark adatpation 20 minutes after sampling, the portable Modulate fluorometer of FMS-2 (Britain Hansatech company) irradiating and detecting light (＜0.05 μ molm is used in 5 repetitions ^-2s ^-1) record initial fluorescence (initial fluorescence yield, Fo), then shine saturation pulse light (3000 μ molm ^-2s ^-1) record maximum fluorescence (maximal fluorescence yield, Fm), the difference of maximum fluorescence and initial fluorescence is variable fluorescence (variable fluorescence, Fv), thereby calculate maximal photochemistry efficiency (maximal photochemical efficiency, Fv/Fm), all measured values are calculated by instrument.

After high light is processed, the wild-type tobacco relative conductivity increases rapidly, processes to rise to 16%, 3 hour in 1.5 hours and rise to 30.7%; And the increase of transgene tobacco relative conductivity is gentler, process be respectively after 3 hours 17.5%, 15.2% and 19.2%(Fig. 9 d).Correspondingly, descend rapidly after wild-type tobacco Fv/Fm high light is processed, and the decline of transgene tobacco Fv/Fm is gentler, after high light is processed, transgene tobacco Fv/Fm is significantly higher than wild-type (Fig. 9 e).Result shows that transgene tobacco is subjected to the high light injury light than wild-type, and anti-high light ability improves.

Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (4)

1. the cold response protein MfcpCOR14 of bur clover chloroplast(id), is characterized in that its aminoacid sequence is as shown in SEQ ID NO.1.

2. encode the nucleotide sequence of the cold response protein MfcpCOR14 of bur clover chloroplast(id) claimed in claim 1 as shown in SEQ ID NO.2.

3. the application of the cold response protein MfcpCOR14 of bur clover chloroplast(id) claimed in claim 1 in Promoting plant growth and raising plant cold resistance and anti-high light ability.

4. the application of the cold response protein MfcpCOR14 of bur clover chloroplast(id) according to claim 3 is characterized in that: this application comprises the gene constructed plant expression vector of MfcpCOR14 that obtains with the present invention; With the expression vector conversion of plant tissue that builds; The plant tissue that transforms is cultivated into transgenic plant.

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