CN109182359A

CN109182359A - Protein and the application of a kind of pears Cold resistant genes PbrBAM3 and its expression vector, application and coding

Info

Publication number: CN109182359A
Application number: CN201811174612.7A
Authority: CN
Inventors: 黄小三; 赵粱怡; 邢才华; 高浚芝; 张绍铃; 王春孟; 金玉妍; 陶书田; 吴俊�; 吴巨友; 齐开杰
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-10-09
Filing date: 2018-10-09
Publication date: 2019-01-11
Anticipated expiration: 2038-10-09
Also published as: CN109182359B

Abstract

A kind of pears Cold resistant genes PbrBAM3 of the present invention belongs to gene engineering technology field, and the pears Cold resistant genes PbrBAM3 has nucleotide sequence shown in SEQ ID No.1, and the present invention provides a kind of pears Cold resistant genes PbrBAM3, can be improved the cold performance of plant.

Description

A kind of protein of pears Cold resistant genes PbrBAM3 and its expression vector, application and coding And application

Technical field

The present invention relates to field of plant genetic project technology, and in particular to a kind of pears Cold resistant genes PbrBAM3 and its expression Carrier, the protein of application and coding and application.

Background technique

These abiotic stress of arid, with high salt, low temperature have seriously affected growth, development, distribution, yield and the product of plant Matter.In recent years, the frequency of extreme weather is existing, and several abiotic stress act on simultaneously, loses more heavy.Therefore, abiotic stress It is the emphasis that contemporary breeding scholar studies at present.Especially low temperature leads to plant cell osmotic stress and turgescence is lacked of proper care, and destroys and plants The integrality of object cell membrane, make protein active decline or be degraded, peroxide (ROS) excess accumulation leads to oxidative stress. Final photosynthesis of plant is suppressed, cell matrix structure by destroy, metabolic function and matter transportation disorder, thus nutrition Growth and reproductive growth are all suppressed, seriously can lead to plant it is completely dead (Knight et al., 1996；Fowler et al.,2002；Champ,2004；Vogel et al.,2005；Krasensky et al.,2012).In long-term evolutionary process In, plant has formd the efficient and complicated acknowledgement mechanism for resisting injury from low temperature, i.e., on molecule, cell and physiological level A series of adjustment is made, so that reduce low temperature injures (Nakashima et al., 2009) caused by plant.And with this phase The various physiological activities closed are the changed result of gene expression dose in essence.Some small molecules under the conditions of damaging to plants caused by sudden drop in temperature The increase of solable matter (such as soluble sugar content) and inorganic ions can enhance the winter resistance of plant, be the plant responding low temperature side of body Compel the important channel of itself cold tolerance of raising, and soluble maltose just belongs to this kind.

The intracorporal carbohydrate existence form multiplicity of plant, it is many kinds of, and starch is the main of storage in plant Nutrient and the photosynthetic primary product of chloroplaset.Starch is a kind of high polymer of glucose, using fructose-1, 6-diphosphate the bottom of as Object, successively in glucosephosphate isomerase (PGI), phosphoglucomutase (PGM), ADP-Glc pyrophosphorylase (AGPase) and under the catalytic action of starch synthase (SS) it synthesizes, is hydrolyzed into maltose under beta amylase (BAM) effect (Stitt,et al.,2012).In arabidopsis, it is known that beta amylase have 9 (BAM1-9), wherein BAM1, BAM2, BAM3 It is located in chloroplaset with BAM4, prokaryotic expression BAM1, BAM2, BAM3 and BAM4 fusion protein, BAM1, BAM2 and BAM3 have β- Amylase activity and BAM4 does not have, proved using a series of mutant, it is living to influence beta amylase by BAM1 and BAM3 in arabidopsis body Property, and BAM4 adjusting active to beta amylase then depends on BAM1 and BAM3, illustrates beta amylase in Arabidopsis leaf starch Play an important role in degradation process, some beta amylase genes are to play a major role, it is some be help out (Fulton, et al.,2008)。

Starch is hydrolyzed into maltose by beta amylase in chloroplaset, and maltose can be exported by maltose transferase Chloroplaset into cytoplasm, and can in cytoplasm synthesis of glucose etc..Beta amylase is most important in plant leaf blade The exclusive product maltose of amylolytic enzyme, degradable starch plays an important role in terms of plant protection, and maltose is as small point Sub- substance can provide substrate for plant vivo oxidation reduction reaction, and can protect photosynthetic electron transport chain, in the winter resistance of plant In play certain effect (Peng Ting, 2013).Forefathers are the study found that temperature stress can make beta amylase transcriptional level and activity change (Kreps,et al.,2002；Jung, et al., 2003), generation beta amylase, Seki can be especially induced under cryogenic conditions A beta amylase correlation EST is had found when screening low-temperature induction of genes Deng (2001), it was demonstrated that be At BAM3.And At BAM3 The specific expressed accumulation with maltose be closely related, it is thus possible to leaf starch degradation in serve it is conclusive.And Kaplan and Guy (2004) find AtBAM3 significantly by low temperature induction, and arabidopsis during 5 DEG C of low-temperature treatment 192h Maltose content changes parabolically state, meanwhile, they have found maltose, glucose, fruit in AtBAM3 RNA interference system Sugar and the soluble sugar contents such as sucrose and PSII photosynthetic efficiency are all decreased obviously, and AtBAM3 expressed after 4 DEG C of processing 6h it is bright Aobvious to rise (Kaplan, et al., 2004), these all illustrate the expression of low temperature induction AtBAM3 gene and the raising of cold tolerance There is relationship.Wang Yifan the study found that the expression pattern of rice BAM gene by the regulation of a variety of abiotic stress, such as arid, high (Wang Yifan, 2013.) such as temperature, low temperature and salt stress.But the research of also unprecedented BAM gene is reported in pears.

Summary of the invention

The purpose of the present invention is to provide a kind of pears Cold resistant genes PbrBAM3, the cold performance for improving plant can be improved.

The present invention provides a kind of pears Cold resistant genes PbrBAM3, the pears Cold resistant genes PbrBAM3 to have SEQ ID Nucleotide sequence shown in No.1.

The present invention also provides the protein of the pears Cold resistant genes PbrBAM3 coding described in above-mentioned technical proposal to have SEQ Amino acid sequence shown in ID No.2.

Preferably, the protein is improving the application in plant cold resistance ability.

The present invention also provides the acquisition methods of the pears Cold resistant genes PbrBAM3 described in above-mentioned technical proposal, including it is following Step:

Using pears cDNA as template, PCR amplification is carried out with pears Cold resistant genes primer pair, obtains pears Cold resistant genes PbrBAM3；

The pears Cold resistant genes primer pair includes pears Cold resistant genes upstream primer and pears Cold resistant genes downstream primer, the pears Cold resistant genes upstream primer has nucleotide sequence shown in SEQ ID No.3, and the pears Cold resistant genes downstream primer has SEQ Nucleotide sequence shown in ID No.4.

The present invention also provides a kind of expression vector containing pears Cold resistant genes PbrBAM3 described in above-mentioned technical proposal, The expression vector includes pears Cold resistant genes PbrBAM3 described in above-mentioned technical proposal.

Preferably, the expression vector is that pears Cold resistant genes described in above-mentioned technical proposal are inserted into plasmid pBI121 PbrBAM3 is built-up.

The present invention also provides the pears Cold resistant genes PbrBAM3 described in above-mentioned technical proposal in improving plant cold resistance ability Application.

Preferably, it is described improve plant cold tolerance method the following steps are included:

The pears Cold resistant genes PbrBAM3 and pBI121 carrier is attached, expression vector is obtained；

The expression vector is transferred in Agrobacterium tumefaciems, recombination Agrobacterium tumefaciems is obtained；

The recombinational agrobacterium is infected into plant.

Preferably, the plant includes tobacco or pears.

The present invention also provides the expression vectors described in above-mentioned technical proposal to improve the application in plant cold resistance ability.

The present invention provides a kind of pears Cold resistant genes PbrBAM3, can be improved the cold performance of plant.

Detailed description of the invention

Fig. 1 is flow chart of the invention；

Fig. 2 is the expression pattern that PbrBAM3 of the present invention responds Different stress processing, wherein Fig. 2 a is at low temperature (4 DEG C) Reason；Fig. 2 b is dehydration；Fig. 2 c is ABA processing；Fig. 2 d is maltose processing；

Fig. 3 is PbrBAM3 gene subcellular localization of the present invention, wherein Fig. 3 A, GFP gene (control) is at ultraviolet light (UV) Light (figure left), light field (in) under imaging, right figure both is superimposed imaging；Fig. 3 B, PbrBAM3 gene is at ultraviolet light (UV) (left side), light field (in) under imaging, right figure both is superimposed imaging；

Fig. 4 is β-amylase activity detection that the present invention turns PbrBAM3 genetic tobacco, wherein Fig. 4 a is that growth cabinet is normal The starch of warm dark culturing 12h tobacco leaf dyes；Fig. 4 b is the starch dyeing of tobacco leaf after constant temperature illumination cultivation 48h；Figure 4c is tobacco fresh young leaflet tablet transmission electron microscope observing (CW: cell wall of the normal-temperature light according to 12 hours；SG: amylum body).；Fig. 4 d It is tobacco beta amylase determination of activity under normal condition；Fig. 4 e is the measurement of tobacco maltose content under normal condition；Fig. 4 f is The measurement of tobacco soluble sugar content under normal condition；

Fig. 5 is β-amylase activity detection that the present invention turns PbrBAM3 gene Ussurian pear, wherein Fig. 5 a is growth cabinet The starch of room temperature dark culturing 12h Ussurian pear blade dyes；Fig. 5 b is the starch dye of Ussurian pear blade after constant temperature illumination cultivation 12h Color；Fig. 5 c is Ussurian pear fresh young leaflet tablet transmission electron microscope observing (CW: cell wall of the normal-temperature light according to 12 hours；SG: starch Grain)；Fig. 5 d is Ussurian pear beta amylase determination of activity under normal condition；Fig. 5 e is Ussurian pear maltose content under normal condition Measurement；Fig. 5 f is the measurement of Ussurian pear soluble sugar content under normal condition；

Fig. 6 be that the present invention turns PbrBAM3 genetic tobacco low-temperature treatment phenotype and physiological index determining, wherein Fig. 6 A-C It is the phenotype of the forward and backward transgenosis of Potted tobacco low-temperature treatment；Fig. 6 D is tobacco processing after stain；Fig. 6 E is surviving for low-temperature treatment Rate；Fig. 6 F is the forward and backward phenotype of single plant tobacco low-temperature treatment；Fig. 6 G is relative conductivity measurement after tobacco low-temperature treatment；Fig. 6 H It is the measurement of mda content after tobacco low-temperature treatment；Fig. 6 I is the measurement of proline content after tobacco tobacco low-temperature treatment；

Fig. 7 is that the present invention turns PbrBAM3 gene Ussurian pear low-temperature treatment phenotype and physiological index determining, wherein Fig. 7 A is Phenotype before transgenosis Ussurian pear low-temperature treatment；Fig. 7 B is the phenotype after transgenosis Ussurian pear low-temperature treatment；Fig. 7 C is transgenosis Ussurian pear low-temperature treatment after stain；Fig. 7 D is relative conductivity measurement after transgenosis Ussurian pear low-temperature treatment；Fig. 7 E is the transgenosis autumn The measurement of mda content after sub- pears low-temperature treatment；Fig. 6 F is the measurement of proline content after transgenosis Ussurian pear low-temperature treatment；

Fig. 8 is that the present invention turns in PbrBAM3 gene plant histochemical stain and measurement analysis H after low-temperature treatment₂O₂With O^2-Accumulation, Fig. 8 a are whole strain NBT dyeing after transgene tobacco low-temperature treatment；Fig. 8 b is whole strain after transgene tobacco low-temperature treatment DAB dyeing；Fig. 8 C is transgene tobacco low-temperature treatment rear blade NBT and DAB dyeing；Fig. 8 d is transgenosis Ussurian pear low-temperature treatment Rear blade NBT and DAB dyeing；Fig. 8 e is content of hydrogen peroxide measurement after transgene tobacco low-temperature treatment；Fig. 8 e is transgenosis cigarette Content of hydrogen peroxide measures after careless low-temperature treatment；Fig. 8 f is the measurement of transgene tobacco low-temperature treatment rear blade superoxide anion resisitance； Fig. 8 g is the measurement of transgenosis Ussurian pear low-temperature treatment rear blade content of hydrogen peroxide；

Fig. 9 is the measurement that the present invention turns CAT, POD, SOD enzyme activity after low-temperature treatment in PbrBAM3 gene plant, wherein Fig. 9 A is the measurement of CAT enzyme activity after transgene tobacco low-temperature treatment；Fig. 9 B is the survey of POD enzyme activity after transgene tobacco low-temperature treatment It is fixed；Fig. 9 C is the measurement of SOD enzyme activity after transgene tobacco low-temperature treatment；Fig. 9 D is CAT enzyme after transgenosis Ussurian pear low-temperature treatment Measurement living；Fig. 9 E is the measurement of POD enzyme activity after transgenosis Ussurian pear low-temperature treatment；Fig. 9 F is transgenosis Ussurian pear low-temperature treatment The measurement of SOD enzyme activity afterwards.

Specific embodiment

The present invention provides a kind of pears Cold resistant genes PbrBAM3, the pears Cold resistant genes PbrBAM3 to have SEQ ID Nucleotide sequence shown in No.1, particular sequence are as follows:

ATGACGTTAACTCTCCGTTCTTCAACTTCGTTCGTCAATCTTAAAGACCATGAGGGCCTTAAAACCTC CGGCGACTCCCCCGGCACGATATGCTTTGCACAAATCAAGCCATCATGCTGTGTCCGGGCAAAGAGTTCAATGCAA GAAACACGGCTCTTGCACGAGAACTTAATGACCATCTCTGATGGAAGGAGAGAGTTGCTTCATGCACTCCCCGGTA TTTCTCACACCAGCTCCGACAACAAGGTGCCTGTGTACGTGATGCTTCCGCTCGACACGGTGAGTCACGGAGGGCA TTTGAATAAGCCGAGAGCAATGAATGCGAGTTTGATGGCCCTGAAGCAAGCAGGGGTTGAAGGAGTTATGGTGGAT GTGTGGTGGGGATTGGTGGAGGAGGACGGACCTTCCAAGTACAACTGGGAAGGGTATGCCGAGCTTGTGCAGATGG TGCAAAAGCACGGCATGAAGATCCAAGTCGTCATGTCTTTTCATCAGTGCGGAGGAAATGTTGGAGACTCATGCAG CATTCCTTTACCTCCATGGGTGCTTGAAGAAGTCAGCAAGAACCCTGACCTTGTTTACACAGACAAATCAGGCAGG CGGAATCCTGAGTACATATCCTTGGGATGTGATTCATTGCCTGTTCTCGGAGGAAGAACTCCCATTCAGGTCTACA CCGATTACATGAGGAGCTTCCACGACAGATTCATTGACTACTTGGGCGACGTTATTGTGGAAATTCAAGTAGGCAT GGGTCCTTGTGGGGAGCTCAGATATCCAGCTTATCCAGAAAGCAACGGAACTTGGAGGTTTCCTGGAATCGGGGAA TTCCAATGTTATGACAAGTACATGAGAGCTTCCTTGGAAGCATCAGCAGAGGCACTGGGGAAGAGAGATTGGGGAA GAAGTGGACCCCATGATTCCGGCCAGTACAATCAATTTCCTGAAGACACCGGATTTTTCAAAAGAGAGGGAACATG GAACACCGAGTATGGACAGTTCTTCCTGGAATGGTACTCCGGAAAGTTACTAAGACACGGAGATAGAATATTGTCA GCTGCGAAAGGAGTATTCCAAGGATCTGGAGCTAAACTATCCGGAAAGATTGCTGGGATTTACTGGCACTACGGAT CAAGATCACATGCGGCTGAATTAACTGCAGGCTACTACAACACTAGACACAGAGATGGTTACGTACCAACAGCCAT GATGTTCAGCAAAAACATGGTTGTATTAAACTTCACCTGCATGGAAATGAAAGATAGTGAACAGCCTGAACACGCA AATTGCTCACCAGAAGGTCTGGTGAGGCAGGTAAGGATGGCGACCAAGAGTGCTGGAATTGACCTAGCAGGAGAAA ATGCCCTGGAGAGGTATGACACTGGTGCGTTTGCACAAGTTTTAGCAACAAGCAGATCAGATTCCGGCAATTCACT AAGTGCATTTACATATCTCCGAATGAACAAGAGATTGTTCGAGGGGGATAACTGGAGGAACATGGTTGAATTTGTG AGAAGCATGGCAGAAGGTGGCCGGAACAGAAGTCTTTCAGAGTGCGACTCTACCGGAAGCGACCTCTTTGTTCGTT TTATCAAAGAGAAGAAAGTCGAGAAAACCAAGGAAACTGTTCTTGTGTAG。

In the present invention, the pears Cold resistant genes PbrBAM3 can be improved the ability of plant cold resistance.In the present invention, institute The overall length for stating the pears Cold resistant genes PbrBAM3 is 1638bp, encodes 545 amino acid.

In the present invention, the pears are preferably birch-leaf pear (Pyrus betuLaefoli).

The present invention is not particularly limited the pears cDNA method extracted, using the method for producing traditional extraction plant cDNA ?.

In the present invention, the pears Cold resistant genes upstream primer has nucleotide sequence shown in SEQ ID No.3, specifically Sequence is as follows:

5’-ATGACGTTAACTCTCCGTTCTTCAAC-3’。

In the present invention, the pears Cold resistant genes downstream primer has nucleotide sequence shown in SEQ ID No.4, specifically Sequence is as follows:

5’-CACAAGAACAGTTTCCTTGGTTTTC-3’。

In the present invention, the thinking of the pears Cold resistant genes primer pair design is preferred are as follows: using arabidopsis BAM3 as keyword Pears gene database is searched for, it is the most similar to obtain 8 sequences, and highest scoring is named as PbrBAM3.According to PbrBAM3 Gene order designs the primer pair for expanding the sequence using Primer Premier 5.0.

In the present invention, described to carry out the preferably every 50 μ L of system that PCR amplification uses with pears Cold resistant genes primer pair and include: 5 × Q5Reaction Buffer, 10 μ L, 10mM dNTP 1 μ L, 10 μM of 2.5 μ L of pears Cold resistant genes upstream primer, 10 μM of pears are anti- Cold 0.5 μ L, Q5High-Fidelity DNA Polymerase of downstream of gene primer 2 .5 μ L, template cDNA 0.5 μ L, ddH₂O Supply 50 μ L.

In the present invention, the program for carrying out PCR amplification with pears Cold resistant genes primer pair preferably includes: 98 DEG C of 30s；98 DEG C 10s, 60 DEG C of 30s, 72 DEG C of 60s, totally 35 circulations；72℃2min；4℃5min.

The present invention also provides the pears Cold resistant genes PbrBAM3 of above-mentioned technical proposal to improve answering in plant cold resistance ability With.

In the present invention, the method for the cold tolerance for improving plant preferably includes following steps:

The recombinational agrobacterium is infected into plant.

The condition that the present invention is attached the pears Cold resistant genes PbrBAM3 and pBI121 carrier is not particularly limited, Using conventional method.

The present invention is transferred to the method in Agrobacterium tumefaciems to the expression vector and is not particularly limited, and is using conventional method It can.

The method that the present invention infects plant to the recombinational agrobacterium is not particularly limited, using conventional method.

In the present invention, the plant includes tobacco and pears.

The present invention also provides the protein of the pears Cold resistant genes PbrBAM3 of above-mentioned technical proposal coding to have SEQ ID Amino acid sequence shown in No.2, particular sequence are as follows:

MTLTLRSSTSFVNLKDHEGLKTSGDSPGTICFAQIKPSCCVRAKSSMQETRLLHENLMTISDGRRELL HALPGISHTSSDNKVPVYVMLPLDTVSHGGHLNKPRAMNASLMALKQAGVEGVMVDVWWGLVEEDGPSKYNWEGYA ELVQMVQKHGMKIQVVMSFHQCGGNVGDSCSIPLPPWVLEEVSKNPDLVYTDKSGRRNPEYISLGCDSLPVLGGRT PIQVYTDYMRSFHDRFIDYLGDVIVEIQVGMGPCGELRYPAYPESNGTWRFPGIGEFQCYDKYMRASLEASAEALG KRDWGRSGPHDSGQYNQFPEDTGFFKREGTWNTEYGQFFLEWYSGKLLRHGDRILSAAKGVFQGSGAKLSGKIAGI YWHYGSRSHAAELTAGYYNTRHRDGYVPTAMMFSKNMVVLNFTCMEMKDSEQPEHANCSPEGLVRQVRMATKSAGI DLAGENALERYDTGAFAQVLATSRSDSGNSLSAFTYLRMNKRLFEGDNWRNMVEFVRSMAEGGRNRSLSECDSTGS DLFVRFIKEKKVEKTKETVLV。

In the present invention, the isoelectric point of the protein is 4.99, molecular weight 28.9KDa.

The present invention also provides the protein described in above-mentioned technical proposal to improve the application in plant cold resistance ability.

In the present invention, the expression vector is preferably inserted into pears cold-resistant described in above-mentioned technical proposal in plasmid pBI121 Gene PbrBAM3 is built-up.The present invention does not have the method that pears Cold resistant genes PbrBAM3 is inserted into plasmid pBI121 There is particular determination, using conventional method.

The present invention also provides expression vectors described in above-mentioned technical proposal to improve the application in plant cold resistance ability.

Combined with specific embodiments below to of the present invention kind of pears Cold resistant genes PbrBAM3 and its expression vector, application It is further described in detail with the protein of coding and application, technical solution of the present invention includes but is not limited to following implements Example.

Embodiment 1

The clone of pears PbrBAM3 full length gene cDNA

Using arabidopsis BAM3 as keyword search pears gene database, it is the most similar to obtain 8 sequences, selects score most High nucleotide sequence, is named as PbrBAM3.According to PbrBAM3 gene order, is designed and expanded using Primer Premier 5.0 Increase the primer pair of the sequence.

Specific step is as follows:

It using birch-leaf pear cDNA as template, is expanded using high fidelity enzyme, amplification system is shown in Table 1, and amplification program is shown in Table 2, expands Increase primer sequence are as follows:

(SEQ ID No.3) PbrBAM3F:5 '-ATGACGTTAACTCTCCGTTCTTCAAC-3 '

(SEQ ID No.4) PbrBAM3R:5 '-CACAAGAACAGTTTCCTTGGTTTTC-3 '

UsingDNA gel QIAquick Gel Extraction Kit (Axygene, USA) obtains product to amplification and purifies back Receive, purified product withCarrier (TransGen, China) is attached, and linked system is shown in Table 3,25 DEG C of incubations E. coli competent Trans5 α is converted after 30min.

Escherichia coli method for transformation is as follows:

1) by 5 μ L connection products be added the ice bath melted E. coli competent Trans5 α of 50 μ L (TransGen, China it) in cell, mixes gently, places 30min on ice；

2) it in 42 DEG C of water-baths after heat shock 45s, then places 2 minutes in ice, which not shake centrifuge tube；

3) the not antibiotic LB liquid medium of 500 μ L, 37 DEG C of shaking tables is added, 200rpm cultivates 1h, makes bacteria resuscitation；

4) competent cell after taking 100~200 μ L to recover uniformly is applied to the LB solid medium containing corresponding antibiotic, Culture dish inversion is put in 37 DEG C of constant incubators, overnight incubation；

1 gene magnification system of table

2 gene magnification PCR program of table

The LB liquid training containing corresponding antibiotic is added in 2.0mL centrifuge tube in monoclonal on 12~16h picking plate after conversion Base is supported, 37 DEG C of shaking table shake cultures then carry out positive identification to bacterium solution muddiness.Reagent uses 2 × TSINGKE Master Mix (Tsingke, China), reaction system are shown in Table 4, PCR program and are shown in Table 5.After obtaining positive colony, positive colony is sent to holding up The sequencing of company of section, according to sequencing result, obtains the full length gene and DNA sequence dna of PbrBAM3.

Table 3Carrier linked system

4 positive identification reaction system of table

5 gene magnification PCR program of table

Clone obtains the gene order of the gene in pears, finds after being sequenced, obtains one section of 1638bp sequence, sequence is such as Shown in SEQ IDNo.1, the protein of 545 amino acid is encoded, sequence is as shown in SEQ IDNo.2, the molecule of the protein Amount is 132.51kDa, isoelectric point 4.99.

Embodiment 2

Different adverse environmental factors handle the qRT-PCR analysis of lower PbrBAM3 gene (SEQ ID No.1)

The expression pattern of PbrBAM3 gene is analyzed using the method for real-time fluorescence quantitative PCR (qRT-PCR), it is fixed Amount reagent is QuantiNovaTM SYBR Green PC (QIGEN, Germany), and method is shown in Table referring to specification, reaction system 6。

6 quantitative PCR reaction system of table

For the Tublin using in pears as reference gene, the cDNA that reverse transcription obtains is template.Each sample in triplicate, reacts Program is shown in Table 7.Using 2 after having reacted^-ΔΔCtAlgorithm calculates gene expression.Reference gene qRT-PCR primer are as follows:

(SEQ ID No.5) Tublin-F:5 '-TGGGCTTTGCTCCTCTTAC-3 '

(SEQ ID No.6) Tublin-R:5 '-CCTTCGTGCTCATCTTACC-3 '

7 quantitative PCR response procedures of table

Its expression pattern at low temperature is studied, and finds to extend with the low-temperature treatment time, PbrBAM3 gene Expression quantity also gradually increase, expression quantity has reached peak value when to 72h, then slowly reduces, this and its table in express spectra Expression patterns are consistent (Fig. 2 a).Meanwhile finding that PbrBAM3 is also induced by dehydration in the research to other Stress treatments Expression, although fold induction is not high, is held in the trend (Fig. 2 b) continued to increase.PbrBAM3 is in ABA processing early stage Respond faint, 12~response for 24 hours is strong, and expression later declines (Fig. 2 c).Maltose is the reaction substrate of beta amylase, theoretical For upper, due to feedback inhibition effect, external source addition substrate may be such that the expression of beta amylase reduces, and in 200mM malt In the processing of sugar, when PbrBAM3 and the when of increased and reduced, but whole is (Fig. 2 d) on a declining curve.

Embodiment 3

PbrBAM3 gene subcellular localization

The region ORF (being free of terminator codon) of PbrBAM3 is expanded, and constructs p35S-PbrBAM3-GFP carrier, In GV3101, PbrBAM3-GFP with compare GFP distinguish instantaneous conversion to Ben's tobacco epidermal leaf cells.

Agrobacterium infects tobacco mesophyll cell and carries out as follows:

(1) Agrobacterium monoclonal is inoculated in 3ml LB/Kan/Rif fluid nutrient medium (containing 50mg/ on picking fresh culture L kanamycins and 50mg/L rifampin), 28 DEG C are cultivated 1~2 day, 220rpm/ minutes, activate bacterium solution；

(2) activation bacterium solution is taken, 50:1 is inoculated into (kanamycins containing 50mg/L in 50ml LB/Kan/Rif fluid nutrient medium With 50mg/L rifampin), 28 DEG C are cultivated 8~12 hours, 220rpm/ minutes, bacterium solution OD600 are detected during culture and is arrived between 0.6 Between 0.8；

(3) bacterium solution is transferred in 50ml centrifuge tube, 5000rpm/ minutes, is centrifuged 5 minutes, removes supernatant；

(4) 10ml cleaning solution (10mM MES+10mM MgCl2) is added, thallus is resuspended, 4000rpm/ minutes, be centrifuged 5 points Clock removes supernatant, then is repeated once with 5ml cleaning solution, is eventually adding 3ml cleaning solution, sufficiently inhales and plays mixing；

(5) resuspended bacterium solution is drawn, 19:1 is added in cleaning solution, measures OD600；

(6) adjusting the final concentration OD600 of two kinds of bacterium solutions is about 0.8,5 μ l acetosyringones of addition (acetosyringone, AS), after mixing, room temperature 4 hours, wait to be injected；

(7) infected liquid is packed into syringe (5ml), 2~3 leaves are injected in a combination, and blade, which randomly selects, to be distributed in In different plants, frame takes the blade being of moderate size, and pressing syringe will be in liquid injection to tobacco leaf lower epidermis；

(8) after injecting, tobacco is placed at 25 DEG C and is cultivated, 48~72 hours；

(9) confocal laser scanning microscope reporter gene positioning scenarios are then used.

The result shows that have fluorescence in entire cell when control vector converts, Fig. 3 A is as a result seen, and recombinant vector conversion In cell as a result fluorescence can only be shown in Fig. 3 B in chloroplaset, illustrate that PbrBAM3 is positioned at chloroplaset.

Embodiment 4

Plant conversion carrier building

Plant binary expression vector pBI121 used in genetic transformation, design primer PbrBAM3-pBI121-F/R will Among two restriction enzyme sites of Xba I and Sma I that the amplification of PbrBAM3 full length gene is inserted on the carrier, design of primers is as follows. Using birch-leaf pear cDNA as template.

(SEQ ID No.7)PbrBAM3-pBI121-F:

5’-GCTCTAGAATGACGTTAACTCTCCGTTCTTCAAC-3’

(SEQ ID No.8)PbrBAM3-pBI121-R:

5’-TCCCCCGGGCACAAGAACAGTTTCCTTGGTTTTC-3’

Amplified fragments recycling,Carrier connection and conversion, positive colony detection and sample presentation sequencing and etc. Method referring to embodiment 1.

After sequencing result is correct, reagent (Axygen, USA) box is extracted in a small amount with AxyPrep Plasmid DNA to positive strain Plasmid is extracted, plasmid is named as pBI121-PbrBAM3.With restriction enzyme Xba I and Sma I respectively to plasmid pEASY- PbrBAM3 and pBI121 carries out double digestion, and digestion rear electrophoresis simultaneously recycles, and digestion system is shown in Table 8.TaKaRa T4 is used later DNA Ligase (TaKaRa, Japan) connection DNA fragmentation and recycling carrier, overnight, linked system is shown in Table 9 for 16 DEG C of reactions.It will be even Object of practicing midwifery converts E. coli competent Trans5 α, and picking monoclonal chooses positive monoclonal in containing after carrying out positive identification Block and shake bacterium in the LB liquid medium of that (Kan) antibiotic, extract plasmid, so far overexpression vector pBI121-PbrBAM3 is constructed It completes.It builds and surveys carrier to be transferred to Agrobacterium competence GV3101 spare.

8 double digestion system of table

9 T4 ligase linked system of table

Embodiment 5

The genetic transformation of tobacco

1) bacterial strain prepares: taking out the pBI121-PbrBAM3 carrier kept from -80 DEG C, is stained with a small amount of Agrobacterium with oese Liquid is crossed on LB solid medium (kanamycins containing 50mg/L, 50mg/L rifampin, 25mg/L gentamicin), 28 DEG C of trainings Support 2~3d；Picking monoclonal, in new LB solid medium (kanamycins containing 50mg/L, 50mg/L rifampin, 25mg/L celebrating Big mycin) on cross and cross again, cultivate 2~3d, thallus scraped with the knife blade of sterilizing, is placed in not antibiotic In MS fluid nutrient medium, 28 DEG C, 200r/min cultivates 1~2h, sufficiently shakes scattered thallus, and adjust OD600 with MS fluid nutrient medium It is worth 0.6~0.8 in case infecting use；

2) explant prepares: choosing the sterile tobacco to grow fine, takes maximum 2~3 blades, remove master pulse and limb Edge is cut into 0.5cm²The square of left and right size, be put into it is sterile and added in the triangular flask of a small amount of MS fluid nutrient medium, for infecting With；

3) it infects and co-cultures: bacterium solution cultured in the first step being poured into the triangular flask equipped with explant, is infected 10min, continuous jog in infection processs.After infecting, blot the bacterium solution that explant has with the filter paper of sterilizing, blade back downwards, It is put on the total training culture medium (MS+2.0mg/L 6-BA+0.3mg/L NAA) for being covered with aseptic filter paper, dark culture 2 in culturing room~ 3d；

4) screening and culturing: whole explants collection after co-culturing 3d is put into sterile triangular flask, with sterile water wash 3 ~5 times, the water on explant surface is finally blotted with aseptic filter paper, is placed in screening and culturing medium (MS+400mg/L Cef+100mg/L Km+2.0mg/L 6-BA+0.3mg/LNAA) on cultivate；

5) culture of rootage: the long resistant buds to 1~2cm long are scaled off, MS+200mg/LCef+0.05mg/L is placed in Culture of rootage in NAA culture medium.

Contain 3.0% sucrose and 0.8% agar in above-mentioned culture medium, and pH value is adjusted to 5.9~6.0.Culture medium high temperature After high pressure sterilization, when it is cooled to 60 DEG C or less, the antibiotic of filtration sterilization is added, dispenses spare.

After resistant buds are taken root, and when growing 2~3 blades, a small amount of blade is taken to carry out DNA extraction, DNA extraction step is such as Under:

1) a small amount of tobacco leaf is taken to be put into 1.5mL centrifuge tube, liquid nitrogen grinding to powdered, addition 600 μ L CATB extraction Liquid, CTAB extract liquid making method and are shown in Table 10；

2) 65 DEG C of water-bath water-bath 90min are put into after mixing well, during which every 30min is mixed by inversion once；

3) after the completion of water-bath, 700 μ L24:1 (chloroform: isoamyl alcohol) is added and mixes extract, is acutely mixed by inversion, under room temperature 12000r/min is centrifuged 15min, draws supernatant liquor (about 500 μ L) and is transferred in new 1.5mL centrifuge tube；

4) the pre- cold isopropanol isometric with supernatant is added, after mixing of turning upside down, is put in -20 DEG C of refrigerator precipitating (precipitatings Time can extend)；

5) it is taken out after the completion of precipitating, 12000r/min is centrifuged 10min.Supernatant is outwelled, 75% ethyl alcohol of 1mL pre-cooling is added, Cleaning 2~3 twice, abandons alcohol, in air-dried in draught cupboard；

6) 20~30 μ L ddH are added in every pipe₂O dissolving DNA, the DNA dissolved save -20 DEG C of refrigerators.

Concentration Testing, each sample take 2 μ L, pass through detected through gel electrophoresis.

The identification of transgene tobacco positive plant, using the DNA of said extracted as template, with two pairs of primer detections, 35S starting Sub- forward primer gene mentation reverse primer and PbrBAM3 special primer.Primer sequence is as follows:

(SEQ ID No.9)35S-F:5’-TCCTCGGATTCCATTGCCCAGC-3’

(SEQ ID No.3) forward primer:

5'-ATGACGTTAACTCTCCGTTCTTCAAC-3'；

(SEQ ID No.4) reverse primer:

5’-CACAAGAACAGTTTCCTTGGTTTTC-3’。

10 CTAB extract recipe of table

More plants of positive plants are obtained by PCR identification, #3, #/7 is chosen and #11 system is further analyzed sxemiquantitative point Analysis finds that the gene overexpression in tobacco really, harvest overexpression plant T2 are used for subsequent analysis for seed.

Embodiment 6

The genetic transformation of Ussurian pear

(1) it blade preculture: chooses Ussurian pear young leaflet tablet (after subculture 2~3 weeks), in crosscutting three knife of vein, with distal shaft Face (leaf back) contacts culture medium, is laid in precultivation medium dark culture 5~7 days.

(2) activation of bacterium solution: the Agrobacterium kind for depositing in -80 DEG C is subjected to primary scribing line and is placed on 28 DEG C of incubator cultures 36~48 hours, in 3ml LB/Kan/Rif, 28 DEG C were cultivated 1-2 days picking monoclonal, 220rpm/ minutes, activated bacterium solution；It takes Bacterium solution is activated, 50:1 is inoculated into 50ml LB/Kan/Rif fluid nutrient medium (kanamycins containing 50mg/L and 50mg/L benefit good fortune It is flat), 28 DEG C are cultivated 8~12 hours, and 220rpm/ minutes, bacterium solution OD600 is detected during culture between 0.6 to 0.8.

(3) it disseminates: bacterium solution being transferred in 50ml centrifuge tube, 5000rpm/ minutes, be centrifuged 5 minutes, remove supernatant, use NN69 fluid nutrient medium is resuspended, and bacterium solution is made to be diluted to OD600=0.8-1.0.The blade of preculture is placed in the bacterium solution after dilution In infect 15-30min (moderately adjusting according to bacteria solution active), take out blade and drain that (not draining the later period has agriculture bar on filter paper Bacterium is overflowed), dark culture 2~3 days on symbiotic culture medium.

(4) blade in symbiotic culture medium later period subculture: is transferred to screening and culturing medium dark culture 14~21 days after dark culture (according to the actual situation, if callusization serious 14 days, normal 21 days).It is transferred to cultivate 2~3 weeks under light and sprout.Cut bud identification Positive seedling is transferred in positive seedling subculture medium afterwards and expands numerous, is transferred in root media when stem length is to 2~3cm, about 14 days After take root, transplanted in soil (pay attention to keep humidity) after one month.

Culture medium prescription:

(1) common pears seedling subculture medium: MS+2.0mg/L 6BA+0.2mg/L IBA+30g/L sucrose+7.0g/L fine jade Rouge；

(2) precultivation medium: NN69+3.0mg/L TDZ+0.3mg/L IBA+30g/L sucrose+7.0g/L agar；

(3) NN69 fluid nutrient medium: NN69+3.0mg/L TDZ+0.3mg/L IBA+100mM/L AS+30g/L sucrose；

(4) symbiotic culture medium: NN69+3.0mg/L TDZ+0.3mg/L IBA+100mM/L AS+30g/L sucrose+7.0g/ L agar；

(5) screening and culturing medium: NN69+3.0mg/L TDZ+0.3mg/L IBA+30g/L sucrose+7.0g/L agar+10mg/ L kanamycins+100-150mg/L Cef；

(6) positive seedling subculture medium: MS+2.0mg/L 6BA+0.2mg/L IBA+30g/L sucrose+7.0g/L agar+ 5mg/L kanamycins+100mg/L Cef；

(7) root media: 1/2MS+0.5mg/L IBA+30g/L sucrose+7.0g/L agar+5mg/L kanamycins+ 100mg/L Cef；

The extraction and identification of transgenosis Ussurian pear plant DNA, with above-described embodiment 5.

Embodiment 7

PbrBAM3 beta amylase Activity determination

To detect whether the albumen of PbrBAM3 gene coding of the present invention has the function of Starch Hydrolysis, respectively to transgenosis cigarette Grass and Ussurian pear are from starch dyeing, transmission electron microscope microscopic observation and physiological measurement (beta amylase activity, maltose content and solvable Property sugar) three aspects are verified.

The hydrolysing activity of PbrBAM3 can be reflected from side by studying transgene tobacco starch accumulation situation.Work as dark processing After 12h, wild-type tobacco and tri- PbrBAM3 tobacco overexpression strain starch of #3, #7, #11 are completely hydrolyzed, are not dyed to Blue (Fig. 4 a).But it cannot effectively degrade in the starch of continuous light 48h poststaining, wild-type tobacco, whole strain is all contaminated At navy blue, and transgenosis system tobacco is not dyed to blue, illustrates that starch has obtained effective degradation (Fig. 4 b).Ussurian pear dyeing As a result therefore (Fig. 5 a, b) consistent with tobacco, no matter in tobacco or Ussurian pear, can accelerate plant after overexpressing PbrBAM3 The degradation of starch.For the accumulation of the starch from ultra microstructure level, our PbrBAM3 by normal-temperature light according to 12h surpass Expression tobacco and Ussurian pear have carried out transmission electron microscope observing.White in the overexpression strain Chloroplast of tobacco and Ussurian pear is bright It selects (amylum body) to be considerably less than wild type (Fig. 4 c and 5c), which is consistent completely with starch dyeing.Then we are from just facing Ussurian pear beta amylase activity is overexpressed under normal condition to be determined.Beta amylase activity in transgenosis system Ussurian pear is aobvious It writes and is higher than wild type (Fig. 5 d), while its hydrolysate maltose content also significantly rises (Fig. 5 e), and three transgenic lines The soluble sugar content in blade is also above wild type (Fig. 5 f) under normal operation.In addition, to β-shallow lake in transgene tobacco In powder enzymatic activity, maltose content and soluble sugar content measurement result and Ussurian pear consistent (Fig. 4 d-f), illustrate that PbrBAM3 has There is the activity of beta amylase, meanwhile, the overexpression of PbrBAM3 may will affect the entire glycometabolism approach of transgenic plant.

Embodiment 8

Transgene tobacco cold hardness evaluation

The potting transgene tobacco and wild-type tobacco (WT) of 40d seedling age are used for low-temperature resistance identification.In low-temperature treatment Before, the tobacco and wild-type tobacco for overexpressing PbrBAM3 gene do not have apparent phenotypic difference, but after 0 DEG C of processing 36h, wild Raw type comes to harm more serious than transgenosis system, and most of blade is all in water stainization state, and transgenosis system only has part Water stainization is presented in tobacco, and room temperature is restored two days later, and wild type is all dead, and transgenosis system has part to restore normal (Fig. 6 A-C), single The result of strain low-temperature treatment is also the same (Fig. 6 F).Typan blue is dyed after processing, and the color of wild type is very deep, and turns base Because being to illustrate wild type after low-temperature treatment almost without color, there are more dead cells, and transgenosis system dead cell is seldom, Show the injury very little (Fig. 6 D) being subject to.Survival rate is counted after recovery, transgenic plant has higher survival rate, wherein #3, # 7 systems are that 40% or more, #11 reaches 60% or more (Fig. 6 E).Conductance measurement finds phase of the wild-type tobacco after low-temperature treatment It is higher to conductivity, illustrate that more serious Cell Membrane Injury occurs in wild-type tobacco, so as to cause more serious electricity Solve matter leakage (Fig. 6 G).Relative to wild-type tobacco, the MDA content of transgene tobacco accumulation is lower, illustrates wild-type tobacco film Lipid peroxidation is high, and the membranous system extent of damage is high (Fig. 6 H).Since proline hydrophily is extremely strong, plasm colloid and tissue can be stablized Interior metabolic process can reduce freezing point, thus proline increases under cryogenic, in plant tissue, and plant can be improved Winter resistance, after low-temperature treatment, the proline content of transgenosis system is all higher than wild type, illustrates that the cold tolerance of transgenosis system is stronger (Fig. 6 I).

In transgenic line, conductivity is lower and high survival rate show they may have than wild type have it is stronger anti- The ability of ROS.Plant leaf is dyed with DAB and NBT histochemical staining method, is respectively intended to detection hydrogen peroxide (H2O2) and the content of superoxide anion (O2-).Such as Fig. 8 a-c, with DAB and NBT histochemical stain, transgenosis system tobacco contaminates Color is obviously lighter than wild type, and the measurement of content of hydrogen peroxide is also below wild type (Fig. 8 e), and superoxide anion resisitance is higher than wild Type (Fig. 8 f).Meanwhile the activity of superoxide dismutase (SOD), peroxidase (POD), hydrogen peroxide (CAT) is divided Analysis.Measurement result shows, superoxide dismutase (SOD), peroxidase (POD), hydrogen peroxide in transgene tobacco (CAT) activity is intended to (Fig. 9 A-C) higher than wild type, and plant activity in vivo oxygen residual is lower, and cellular damage is smaller.These knots Fruit shows that the PbrBAM3 gene of overexpression can effectively enhance transgenosis cold tolerance.

Embodiment 9

The identification of transgenosis Ussurian pear low-temperature resistance

Further to probe into whether PbrBAM3 can also improve the cold tolerance of pears, by the gene stable conversion to Ussurian pear In overexpressed, and the overexpression Ussurian pear of potting earth culture (OE2, OE9 and OE19 system) and wild type Ussurian pear WT are carried out Freeze proof analysis.Before low-temperature treatment, the Ussurian pear and wild type Ussurian pear for overexpressing PbrBAM3 gene do not have phenotypic difference, but After low-temperature treatment, wild type comes to harm more serious than transgenosis system, and most of blade is all in water stainization state, is had There is shrinkage wilting in blade, and the Ussurian pear of transgenosis system shows good (Fig. 7 A, B).Typan blue is carried out after low-temperature treatment Dyeing, the color of wild type is very deep, and transgenosis system very slight color, illustrates wild type after low-temperature treatment, has more dead thin Born of the same parents, and transgenosis system dead cell is seldom, shows the injury very little (Fig. 7 C) being subject to.Proline determination as a result, it has been found that, low-temperature treatment Afterwards, the proline content of transgenosis system is all higher than wild type, and it is small point related to illustrate that transgenosis system accumulates more osmotic adjustments Sub- substance reduces freezing point, improves cold tolerance (Fig. 7 D).Conductance measurement finds wild type Ussurian pear after low-temperature treatment Relative conductivity it is higher, illustrate that more serious Cell Membrane Injury occurs in wild type Ussurian pear, the knot with tobacco measurement Fruit is consistent (Fig. 7 E).The mda content of wild type Ussurian pear is apparently higher than transgenosis Ussurian pear, illustrates wild type Ussurian pear film System damage degree is high, and low temperature is smaller to the injury of wild type Ussurian pear, and transgenosis Ussurian pear cold tolerance is stronger (Fig. 7 F).With DAB and NBT histochemical staining method dyes plant leaf, be respectively intended to detection hydrogen peroxide (H2O2) and super oxygen yin from The content of sub (O2-), either DAB or NBT dyeing, the dyeing of transgenosis system Ussurian pear are all obviously lighter than wild type, peroxidating The measurement of hydrogen content is also below wild type (Fig. 8 d, g), and superoxide anion resisitance is higher than wild type (Fig. 8 h).To superoxides discrimination Change enzyme (SOD), peroxidase (POD), hydrogen peroxide (CAT) activity analyzed.Measurement result shows that the transgenosis autumn is sub Superoxide dismutase (SOD), peroxidase (POD), hydrogen peroxide (CAT) activity in pears are intended to higher than wild type (Fig. 9 D-F) illustrates that plant activity in vivo oxygen residual is lower, and cellular damage is smaller.These results indicate that the PbrBAM3 being overexpressed Gene can effectively enhance transgenosis cold tolerance.

By above embodiments, it can be concluded that, a kind of pears Cold resistant genes PbrBAM3 provided by the invention can be improved plant Cold performance.

The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Sequence table

<110>Agricultural University Of Nanjing

<120>protein and the application of a kind of pears Cold resistant genes PbrBAM3 and its expression vector, application and coding

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1638

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atgacgttaa ctctccgttc ttcaacttcg ttcgtcaatc ttaaagacca tgagggcctt 60

aaaacctccg gcgactcccc cggcacgata tgctttgcac aaatcaagcc atcatgctgt 120

gtccgggcaa agagttcaat gcaagaaaca cggctcttgc acgagaactt aatgaccatc 180

tctgatggaa ggagagagtt gcttcatgca ctccccggta tttctcacac cagctccgac 240

aacaaggtgc ctgtgtacgt gatgcttccg ctcgacacgg tgagtcacgg agggcatttg 300

aataagccga gagcaatgaa tgcgagtttg atggccctga agcaagcagg ggttgaagga 360

gttatggtgg atgtgtggtg gggattggtg gaggaggacg gaccttccaa gtacaactgg 420

gaagggtatg ccgagcttgt gcagatggtg caaaagcacg gcatgaagat ccaagtcgtc 480

atgtcttttc atcagtgcgg aggaaatgtt ggagactcat gcagcattcc tttacctcca 540

tgggtgcttg aagaagtcag caagaaccct gaccttgttt acacagacaa atcaggcagg 600

cggaatcctg agtacatatc cttgggatgt gattcattgc ctgttctcgg aggaagaact 660

cccattcagg tctacaccga ttacatgagg agcttccacg acagattcat tgactacttg 720

ggcgacgtta ttgtggaaat tcaagtaggc atgggtcctt gtggggagct cagatatcca 780

gcttatccag aaagcaacgg aacttggagg tttcctggaa tcggggaatt ccaatgttat 840

gacaagtaca tgagagcttc cttggaagca tcagcagagg cactggggaa gagagattgg 900

ggaagaagtg gaccccatga ttccggccag tacaatcaat ttcctgaaga caccggattt 960

ttcaaaagag agggaacatg gaacaccgag tatggacagt tcttcctgga atggtactcc 1020

ggaaagttac taagacacgg agatagaata ttgtcagctg cgaaaggagt attccaagga 1080

tctggagcta aactatccgg aaagattgct gggatttact ggcactacgg atcaagatca 1140

catgcggctg aattaactgc aggctactac aacactagac acagagatgg ttacgtacca 1200

acagccatga tgttcagcaa aaacatggtt gtattaaact tcacctgcat ggaaatgaaa 1260

gatagtgaac agcctgaaca cgcaaattgc tcaccagaag gtctggtgag gcaggtaagg 1320

atggcgacca agagtgctgg aattgaccta gcaggagaaa atgccctgga gaggtatgac 1380

actggtgcgt ttgcacaagt tttagcaaca agcagatcag attccggcaa ttcactaagt 1440

gcatttacat atctccgaat gaacaagaga ttgttcgagg gggataactg gaggaacatg 1500

gttgaatttg tgagaagcat ggcagaaggt ggccggaaca gaagtctttc agagtgcgac 1560

tctaccggaa gcgacctctt tgttcgtttt atcaaagaga agaaagtcga gaaaaccaag 1620

gaaactgttc ttgtgtag 1638

<210> 2

<211> 545

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 2

Met Thr Leu Thr Leu Arg Ser Ser Thr Ser Phe Val Asn Leu Lys Asp

1 5 10 15

His Glu Gly Leu Lys Thr Ser Gly Asp Ser Pro Gly Thr Ile Cys Phe

20 25 30

Ala Gln Ile Lys Pro Ser Cys Cys Val Arg Ala Lys Ser Ser Met Gln

35 40 45

Glu Thr Arg Leu Leu His Glu Asn Leu Met Thr Ile Ser Asp Gly Arg

50 55 60

Arg Glu Leu Leu His Ala Leu Pro Gly Ile Ser His Thr Ser Ser Asp

65 70 75 80

Asn Lys Val Pro Val Tyr Val Met Leu Pro Leu Asp Thr Val Ser His

85 90 95

Gly Gly His Leu Asn Lys Pro Arg Ala Met Asn Ala Ser Leu Met Ala

100 105 110

Leu Lys Gln Ala Gly Val Glu Gly Val Met Val Asp Val Trp Trp Gly

115 120 125

Leu Val Glu Glu Asp Gly Pro Ser Lys Tyr Asn Trp Glu Gly Tyr Ala

130 135 140

Glu Leu Val Gln Met Val Gln Lys His Gly Met Lys Ile Gln Val Val

145 150 155 160

Met Ser Phe His Gln Cys Gly Gly Asn Val Gly Asp Ser Cys Ser Ile

165 170 175

Pro Leu Pro Pro Trp Val Leu Glu Glu Val Ser Lys Asn Pro Asp Leu

180 185 190

Val Tyr Thr Asp Lys Ser Gly Arg Arg Asn Pro Glu Tyr Ile Ser Leu

195 200 205

Gly Cys Asp Ser Leu Pro Val Leu Gly Gly Arg Thr Pro Ile Gln Val

210 215 220

Tyr Thr Asp Tyr Met Arg Ser Phe His Asp Arg Phe Ile Asp Tyr Leu

225 230 235 240

Gly Asp Val Ile Val Glu Ile Gln Val Gly Met Gly Pro Cys Gly Glu

245 250 255

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

260 265 270

Gly Ile Gly Glu Phe Gln Cys Tyr Asp Lys Tyr Met Arg Ala Ser Leu

275 280 285

Glu Ala Ser Ala Glu Ala Leu Gly Lys Arg Asp Trp Gly Arg Ser Gly

290 295 300

Pro His Asp Ser Gly Gln Tyr Asn Gln Phe Pro Glu Asp Thr Gly Phe

305 310 315 320

Phe Lys Arg Glu Gly Thr Trp Asn Thr Glu Tyr Gly Gln Phe Phe Leu

325 330 335

Glu Trp Tyr Ser Gly Lys Leu Leu Arg His Gly Asp Arg Ile Leu Ser

340 345 350

Ala Ala Lys Gly Val Phe Gln Gly Ser Gly Ala Lys Leu Ser Gly Lys

355 360 365

Ile Ala Gly Ile Tyr Trp His Tyr Gly Ser Arg Ser His Ala Ala Glu

370 375 380

Leu Thr Ala Gly Tyr Tyr Asn Thr Arg His Arg Asp Gly Tyr Val Pro

385 390 395 400

Thr Ala Met Met Phe Ser Lys Asn Met Val Val Leu Asn Phe Thr Cys

405 410 415

Met Glu Met Lys Asp Ser Glu Gln Pro Glu His Ala Asn Cys Ser Pro

420 425 430

Glu Gly Leu Val Arg Gln Val Arg Met Ala Thr Lys Ser Ala Gly Ile

435 440 445

Asp Leu Ala Gly Glu Asn Ala Leu Glu Arg Tyr Asp Thr Gly Ala Phe

450 455 460

Ala Gln Val Leu Ala Thr Ser Arg Ser Asp Ser Gly Asn Ser Leu Ser

465 470 475 480

Ala Phe Thr Tyr Leu Arg Met Asn Lys Arg Leu Phe Glu Gly Asp Asn

485 490 495

Trp Arg Asn Met Val Glu Phe Val Arg Ser Met Ala Glu Gly Gly Arg

500 505 510

Asn Arg Ser Leu Ser Glu Cys Asp Ser Thr Gly Ser Asp Leu Phe Val

515 520 525

Arg Phe Ile Lys Glu Lys Lys Val Glu Lys Thr Lys Glu Thr Val Leu

530 535 540

Val

545

<210> 3

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

atgacgttaa ctctccgttc ttcaac 26

<210> 4

<211> 25

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cacaagaaca gtttccttgg ttttc 25

<210> 5

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

tgggctttgc tcctcttac 19

<210> 6

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

ccttcgtgct catcttacc 19

<210> 7

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gctctagaat gacgttaact ctccgttctt caac 34

<210> 8

<211> 34

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

tcccccgggc acaagaacag tttccttggt tttc 34

<210> 9

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

tcctcggatt ccattgccca gc 22

Claims

1. a kind of pears Cold resistant genes PbrBAM3, which is characterized in that the pears Cold resistant genes PbrBAM3 has SEQ IDNo.1 institute The nucleotide sequence shown.

2. the protein of pears Cold resistant genes PbrBAM3 coding described in claim 1 has amino acid shown in SEQ ID No.2 Sequence.

3. protein as claimed in claim 2 is improving the application in plant cold resistance ability.

4. the acquisition methods of pears Cold resistant genes PbrBAM3 described in claim 1, comprising the following steps:

The pears Cold resistant genes primer pair includes pears Cold resistant genes upstream primer and pears Cold resistant genes downstream primer, the pears cold-resistant Upstream region of gene primer has nucleotide sequence shown in SEQ ID No.3, and the pears Cold resistant genes downstream primer has SEQ ID Nucleotide sequence shown in No.4.

5. a kind of expression vector containing pears Cold resistant genes PbrBAM3 described in claim 1, which is characterized in that the expression Carrier includes pears Cold resistant genes PbrBAM3 described in claim 1.

6. expression vector according to claim 5, which is characterized in that the expression vector is to be inserted into plasmid pBI121 Pears Cold resistant genes PbrBAM3 described in claim 1 is built-up.

7. the pears cold-resistant that acquisition methods described in pears Cold resistant genes PbrBAM3 described in claim 1 or claim 3 obtain Gene PbrBAM3 is improving the application in plant cold resistance ability.

8. application according to claim 7, which is characterized in that the method for the cold tolerance for improving plant includes following Step:

The recombinational agrobacterium is infected into plant.

9. application according to claim 7 or 8, which is characterized in that the plant includes tobacco or pears.

10. expression vector described in claim 5 or 6 is improving the application in plant cold resistance ability.