CN103665128A - Protein related with heat resistance of plants as well as encoding gene and application of protein - Google Patents

Protein related with heat resistance of plants as well as encoding gene and application of protein Download PDF

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CN103665128A
CN103665128A CN201310698347.3A CN201310698347A CN103665128A CN 103665128 A CN103665128 A CN 103665128A CN 201310698347 A CN201310698347 A CN 201310698347A CN 103665128 A CN103665128 A CN 103665128A
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protein
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wheat
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CN103665128B (en
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孙其信
梁荣奇
倪中福
李保云
尤明山
彭惠茹
解超杰
秦丽燕
王树斌
姚颖垠
杜金昆
刘志勇
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China Agricultural University
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    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance

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Abstract

The invention discloses protein related with the heat resistance of plants as well as an encoding gene and an application of the protein. The protein related with the heat resistance of the plants is the following (a) or (b): (a) protein consisting of amino acid sequences represented in sequence 1 in a sequence table; (b) protein formed by performing substitution and/or absence and/or addition of one or more amino acid residues on the amino acid sequences represented by the sequence 1, derived from the sequence 1 and related to the heat resistance of the plants. Experiments prove that after heat stress on transgenic plants, the content of malondialdehyde in plant blades and the relative electrical conductivity are obviously lower than those of receptor plants, which proves that the heat stability and the heat resistance of cell membranes of transgenic lines are enhanced. Thus it can be seen that the protein and the encoding gene of the protein have important theoretical and practical significance in cultivating wheat with enhanced heat resistance and other new varieties of plants and have wide application prospect.

Description

A kind of albumen relevant to plant heat resistance property and encoding gene and application
Technical field
The invention belongs to biological technical field, relate to a kind of albumen relevant to plant heat resistance property and encoding gene and application, particularly a kind ofly derive from black rice bean, the albumen relevant to plant heat resistance property and encoding gene thereof and application.
Background technology
Wheat is the third-largest food crop of China, and its yield and quality is directly connected to daily life.Meanwhile, wheat is again a kind of cool C3 crop of liking, poor to the adaptability of high temperature.In the main wheat belt of China, in the filling stage of wheat grain, often meet with more than 30 ℃ high temperature, cause the significantly underproduction of wheat.China's Huang, Huaihe River, Haihe basin and Xinjiang Mai Qu, high temperature also often follows arid to occur, forms hot dry wind, and hazard area can reach 2/3 of this region wheat planting area, makes wheat yield 10%～20%; In northern China and middle and lower reach of Yangtze River Mai Qu, also often there is " high temperature is forced ripe " disaster.Along with the continuous aggravation of Greenhouse effect, global temperature on average constantly raises in recent years, has badly influenced the production of wheat.It is estimated, within crop growing season, 1 ℃ of the every rising of temperature, output 10% left and right that will decline.
Therefore, formulate out the wheat germplasm that thermotolerance strengthens and there is very important using value.
Summary of the invention
The object of this invention is to provide a kind of albumen relevant to Heat Resistance of Plant and encoding gene and application.
Protein provided by the present invention, name is called Vefer, derives from black rice bean (Vigna eylindrica), is following (a) or (b):
(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;
(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to plant heat resistance property through one or several amino-acid residue by the aminoacid sequence of sequence 1.
Wherein, in sequence table, sequence 1 is comprised of 254 amino-acid residues.
For the ease of the purifying of Vefer albumen, the N-terminal of the protein that can form at the amino acid residue sequence of sequence in sequence table 1 or C-terminal connect label as shown in the table.
Table: the sequence of label
Label Residue Sequence
Poly-Arg 5-6(is generally 5) RRRRR
Poly-His 2-10(is generally 6) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
Vefer albumen in above-mentioned (a) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the Vefer albumen in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table at its 5 ' end and/or 3 ' end obtains.
The nucleic acid molecule of described Vefer albumen of encoding also belongs to protection scope of the present invention.
Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, described nucleic acid molecule is specially the gene (called after Vefer) of the described Vefer albumen of coding; Described Vefer gene is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in the 58-822 position of sequence 2 in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) DNA molecule hybridize that limits and the DNA molecular of the described Vefer albumen of encoding;
4) with 1) or 2) or 3) DNA molecular limiting has the DNA molecular of 90% above homology and the described Vefer albumen of encoding.
Above-mentioned stringent condition can be with 6 * SSC, the solution of 0.5%SDS, and at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.
Wherein, sequence 2 is comprised of 878 Nucleotide, and 58-822 position is ORF, the Vefer albumen in code sequence list shown in sequence 1.
The recombinant vectors that contains above-mentioned nucleic acid molecule, expression cassette, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.
Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
In one embodiment of the invention, described recombinant cloning vector is specially the recombinant cloning vector that described nucleic acid molecule (gene) is connected into pGEM-Teasy carrier gained.
Described recombinant expression vector can be used existing plant expression vector construction.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter rd29A etc., they can be used alone or are combined with other plant promoter; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process recombinant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor, have the antibiotic marker thing of resistance or anti-chemical reagent marker gene etc.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.
In an embodiment of the present invention, the promotor that starts described Vefer genetic transcription in described recombinant expression vector is specially Ubi promotor.
More specifically, described recombinant expression vector for inserting the recombinant plasmid that described Vefer gene obtains in the restriction enzyme site of pBAC47P-ubi carrier.Described restriction enzyme site is specially BamH I and Kpn I.
Described pBAC47P-ubi carrier be by pBAC202 carrier (referring to document " Gao Zefa; Chen Xuqing, Yang Fengping, Liang Rongqi; Zhang Liquan; Zhang Xiaodong. expression and the anti-aphid effect research thereof of synthetic gna gene in wheat, Journal of Agricultural Biotechnology, 2006; 14 (4): 559-564 ") with Hind III enzyme, cut, remove " rbcs-sGNA-NOS " about 1.92KB section, reclaim 5.26Kb carrier segments, the carrier obtaining after connecting with T4DNA ligase enzyme.
Described expression cassette is by the promotor that can start described Vefer genetic expression, described Vefer gene, and transcription termination sequence forms.
Described Vefer albumen, or described nucleic acid molecule, or described recombinant expression vector, expression cassette or recombinant bacterium are at following a1) or a2) in application also belong to protection scope of the present invention:
A1) thermotolerance of regulating plant;
A2) seed selection thermophytes kind.
In one embodiment of the invention, described regulation and control plant thermotolerance is specially the thermotolerance that improves plant.
The method of described seed selection thermophytes kind, specifically can comprise the step that the higher plant of described Vefer expressing quantity is hybridized as parent.
Another object of the present invention is to provide a kind of method of cultivating the transgenic plant of thermotolerance raising.
The method comprises and will in the gene importing object plant of the described Vefer albumen of coding, obtain the step of transgenic plant; Described transgenic plant are compared with described object plant, and thermotolerance improves.
In the present invention, described thermotolerance improves, and is embodied in following (a) and/or (b):
(a) after heat stress, in plant tissue, the content of mda reduces;
(b) after heat stress, the relative conductivity of plant tissue reduces.
More concrete, be (a): after heat stress (as processed 24h in 34 ℃ of two one heart stages of leaf, processing 2.5h for 45 ℃), the mda content in described transgenic plant tissue (as blade) with as described in object plant compare reduction; (b) be: after heat stress (as processed 1 day in 34 ℃ of two one heart stages of leaf), the relative conductivity of described transgenic plant tissue (as blade) with as described in object plant compare reduction.
Described relative conductivity document " Ma Xiaodi, Wang Li, the mensuration of Peng Huiru .Siete Cerros * Seri-82 recombinant inbred strain Heat Tolerance of Wheat Cultivars. wheat crops journal, 2006,26(1): 126-128 " in disclosed.
In one embodiment of the invention, described relative conductivity calculates according to the method comprising the steps: plant tissue to be measured is placed in to deionized water, after sealing, in 45 ℃ of constant temperature, process 1h, take out 25 ℃ standing 24 hours, measure specific conductivity T1, proceed to again 125 ℃ of processing (all oozing out by ionogen to kill all cells) taking-up in 15 minutes and be cooled to after 25 ℃, measure specific conductivity T2; Described relative conductivity is T1/T2.
The expression amount of described Vefer albumen in described transgenic plant is higher than described object plant; The gene (being Vefer gene) of described Vefer albumen of encoding is that described gene is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in the 58-822 position of sequence 2 in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) DNA molecule hybridize that limits and the DNA molecular of the described Vefer albumen of encoding;
4) with 1) or 2) or 3) DNA molecular limiting has the DNA molecular of 90% above homology and the described Vefer albumen of encoding.
Above-mentioned stringent condition can be with 6 * SSC, the solution of 0.5%SDS, and at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.
Described Vefer gene specifically can import in described object plant by above-mentioned arbitrary described recombinant expression vector, obtains described transgenic plant.Specifically can be by using the conventional biological method such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, agriculture bacillus mediated, particle gun by described recombinant expression vector transformed plant cells or tissue, and the plant tissue of conversion is cultivated into plant.The agriculture bacillus mediated biological method that waits is transformed in vegetable cell or tissue.
Described plant can be monocotyledons, also can be dicotyledons, and described unifacial leaf plant is as wheat.
In one embodiment of the invention, described plant is specially wheat (Triticum aestivum L.) kind Zhongyou9507 or protects rich 104.
The primer pair of described Vefer full length gene or its arbitrary fragment of increasing also belongs to protection scope of the present invention.
In one embodiment of the invention, described primer pair is specific as follows:
5’- GGATCCTTTCCTTTCCCTCTTTCTCG-3’；
5’- GGTACCCCCAGAACTTAATACAAGAGCAA-3’。
Experiment showed, before the expression amount that carries out finding after 45 ℃ of thermal treatment 2.5h the Vefer of transfer-gen plant to turning Vefer gene plant is processed and increase, and plant leaf mda (MDA) content is significantly with extremely significantly lower than check variety Zhongyou9507; The relative conductivity of transgenic line is all significantly protected rich 104, Zhongyou9507 lower than thermo-labile kind China spring, acceptor kind, and TAM107 is similar with heat-stable material, illustrates that thermostability and the thermotolerance of the cytolemma of transgenic line strengthens.Black rice bean Vefer gene is expressed in wheat, can improve Heat Tolerance of Wheat Cultivars.Visible Vefer albumen and encoding gene thereof have important theoretical and practical significance for wheat and other neies variety of plant of cultivating thermotolerance raising, have broad application prospects.
Accompanying drawing explanation
Fig. 1 is the plasmid map of recombinant expression vector pBAC47P-ubi-fer and carrier pBAC202.Wherein, A is the plasmid map of recombinant expression vector pBAC47P-ubi-fer; B is the plasmid map of carrier pBAC202.
Fig. 2 is that the enzyme of recombinant expression vector pBAC47P-ubi-fer is cut qualification result.Wherein, swimming lane M1 is DNA molecular amount standard DL2000; Swimming lane M2 is DNA molecular amount standard 1KB ladder; Swimming lane 1 and swimming lane 2 are BamH I and the Kpn I double digestion qualification result of the recombinant expression vector pBAC47P-ubi-fer of two repetitions.
Fig. 3 is part T 0in generation, turns the PCR detected result of Vefer gene plant.Wherein, swimming lane M is DNA molecular amount standard DL2000; Swimming lane+be is usingd the positive control of pBPC47P-ubi-fer plasmid as template; Swimming lane 0 is with ddH 2o is as the blank of template; Swimming lane-negative contrast (not genetically modified acceptor plant, CK); Swimming lane 1～72 is transfer-gen plant, amplifies the positive plant of object band (arrow indication), does not amplify the negative plant of object band.In figure, the acceptor plant in A and B is Zhongyou9507; Acceptor plant in C is for protecting rich 104.
Fig. 4 is part T 0in generation, turns the RT-PCR detected result of Vefer gene plant.In figure, A is Vefer amplification; B is the amplification of the reference gene β-actin of corresponding transfer-gen plant.In A and B, swimming lane M is DNA molecular amount standard DL2000; Swimming lane 0 is with ddH 2o is as the blank of template.Two swimming lanes in A+be are usingd the positive control of pBPC47P-ubi-fer plasmid as template; Two swimming lanes in B+be respectively are usingd acceptor kind Zhongyou9507 and the Bao Feng 104 wheat cdna group DNA contrast as template.In A and B, it is the transfer-gen plant of acceptor that swimming lane 1～12 is to protect rich 104, swimming lane 13～24 is take the transfer-gen plant that Zhongyou9507 is acceptor, amplifies the positive plant of object band (arrow indication) in A, does not amplify the negative plant of object band in A.
Fig. 5 is part T 1in generation, turns Vefer DNA triticum Real-time PCR after being subject to heat stress and detects Vefer gene expression amount.* represents and before heat stress, compares utmost point significant difference, and * represents to compare significant difference with before heat stress.
Fig. 6 is T 2in generation, turns the assay of mda (MDA) in Vefer DNA triticum leaf tissue.* represents to compare utmost point significant difference with unconverted acceptor wheat Zhongyou9507, and * represents to compare significant difference with unconverted acceptor wheat Zhongyou9507.
Fig. 7 is T 2in generation, turns the mensuration of Vefer DNA triticum leaf tissue relative conductivity.Zy9507 represents Zhongyou9507; CS represents China spring; Bf104 represents to protect rich 104.In A, to represent to compare difference with Zhongyou9507 extremely remarkable for * *; In B, * represents and protects rich 104 and compare significant difference.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
Black rice bean (Vigna eylindrica), Papilionaceae plant, have another name called black rice bean, a kind of in rice bean (cow gram), be purchased from supermarket, trade name: the black rice bean of Bama of Guangxi special product, commodity brand: centenarian, Bama of Guangxi elderly person for whom a birthday celebration is being held's health industry at the age of one hundred years old company limited product, commodity article No.: ZG000223.
Wheat (Triticum aestivum L.) kind Zhongyou9507: the high-quality wheat variety of being cultivated by Institute of Crop Science, Chinese Academy of Agricultural Science.Document " Zhongyou9507 high quality bread wheat. Hebei Agriculture science and technology, 08 phase in 2000, the 36th page " in disclose, the acquisition of public Ke Cong China Agricultural University.
Wheat (Triticum aestivum L.) kind protects rich 104: the wheat commercial variety that is the Tianjin authorization of Plant Protection institute, Chinese Academy of Agricultral Sciences's Powdery Mildew group cultivation; document " Zhou Yilin; Duan Xiayu; Sheng Baoqin; the department power people. high-yield disease resisting New Winter Wheat Variety---protect rich 104. wheat crops journals; 2003,23 (4): 147 " in disclose, the acquisition of public Ke Cong China Agricultural University.
Thermo-labile wheat (Triticum aestivum L.) kind China spring: as the wheat standard Cultivar of RESEARCH ON CELL-BIOLOGY, earlier 1800s is introduced west from Sichuan Province China.As thermo-labile check variety, this material and thermotolerance thereof are at document " Ma Xiaodi, Wang Li, Peng Huiru in the present invention.The mensuration of Siete Cerros * Seri-82 recombinant inbred strain Heat Tolerance of Wheat Cultivars.Wheat crops journal, 2006,26(1): 126-128 ", disclosed, public Ke Cong China Agricultural University obtains.
Heat-resisting wheat (Triticum aestivum L.) kind TAM107: in the present invention as heat-resisting check variety, this material and thermotolerance thereof are at document " Ma Xiaodi, Wang Li, Peng Huiru.The mensuration of Siete Cerros * Seri-82 recombinant inbred strain Heat Tolerance of Wheat Cultivars.Wheat crops journal, 2006,26(1): 126-128 ", disclosed, public Ke Cong China Agricultural University obtains.
The acquisition of embodiment 1, black rice bean ferritin gene (Vefer)
One, the extraction of the total RNA of black rice bean
The black rice bean plant root of water planting 10d is placed in to the Fe that concentration is 100mM 2+in-EDTA the aqueous solution, process 24h, getting its young root, to be placed in immediately liquid nitrogen freezing standby.The black rice bean young root that 100mg is fully ground through liquid nitrogen extracts total RNA with Trizol test kit, and concrete operations are referring to test kit specification sheets.
Two, reverse transcription obtains cDNA
At the 0.2mL PCR pipe without RNase, add following system: total RNA2 μ g, Primer T 15(10 μ M) 1 μ L, uses DEPC ddH 2o complements to 10 μ L.
Low speed is of short duration is centrifugally concentrated to the PCR pipe end by system all the components.On PCR instrument, 70 ℃ of sex change 5min, are placed on ice immediately, are formulated as follows reverse transcription reaction system: above-mentioned reaction soln 10 μ L, and 5 * M-MLV buffer4.0 μ L, RNA enzyme inhibitors 0.5 μ L, dNTPs1.0 μ L, M-MLV1.0 μ L, uses DEPC ddH 2o complements to 20 μ L.
Low speed is of short duration centrifugal, and reaction system all the components is concentrated to the PCR pipe end.42 ℃ of water-bath 1h; 75 ℃ of sex change 5min, are placed on ice immediately, obtain cDNA.
Three, pcr amplification obtains black rice bean ferritin gene Vefer
1, design of primers
Be designed for the upstream and downstream primer of the black rice bean ferritin gene Vefer of amplification, as follows:
Vc.Fer-F:5 '- gGATCCtTTCCTTTCCCTCTTTCTCG-3 ' (underscore is partly the recognition site of restriction enzyme site BamH I, and sequence is thereafter the 1-20 position of sequence 2);
Vc.Fer-R:5 '- gGTACCcCCAGAACTTAATACAAGAGCAA-3 ' (underscore is partly the recognition site of restriction enzyme site Kpn I, and sequence is thereafter the reverse complementary sequence of the 856-878 position of sequence 2)
2, pcr amplification
PCR reaction system (50 μ L): 10 * PCR Buffer5.0 μ L, primer Vc.Fer-F(10 μ M) 1.0 μ L, primer Vc.Fer-R(10 μ M) 1.0 μ L, dNTPs0.5 μ L, Taq enzyme 2.0U, template cDNA2.0 μ L, uses ddH 2o complements to 50 μ L.
Amplification program: before amplification,, at 94 ℃ of initial sex change 5min, pcr amplification is 35 circulations, and cycling program is: 94 ℃ of sex change 45s, 61 ℃ of annealing 45s, 72 ℃ are extended 90s, after last loop ends, at 72 ℃, extend 10min.4 ℃ of preservations.
Get PCR product electrophoresis on 1% sepharose.The complete object band that cuts under ultraviolet lamp of electrophoresis, reclaims test kit with sepharose DNA and purifies.
Reclaim fragment and pGEM-Teasy(promega company) be connected.In PCR pipe, add successively following reagent: reclaim fragment 3 μ L, 2 * T 4ligase enzyme damping fluid 5 μ L, pGEM-Teasy carrier (50ng/ μ L) 1 μ L, T 4dNA ligase (3U/ μ L) 1 μ L, cumulative volume 10 μ L.In 4 ℃ of connections, spend the night.
By recon called after pTE-fer.Deliver order-checking company sequence verification.Sequencing result demonstration, the exogenous gene sequence in recon pTE-fer is " GGATCC+ sequence 2+GGTACC ".By unnamed gene shown in sequence 2, be Vefer, wherein the 58-822 position of sequence 2 is its open reading frame, the protein shown in sequence 1 in code sequence list (called after Vefer).
The structure of embodiment 2, recombinant expression vector pBAC47P-ubi-fer and evaluation
One, the structure of recombinant expression vector pBAC47P-ubi-fer
With BamH I and Kpn I enzyme, cut the recombinant plasmid pTE-fer that embodiment 1 obtains, reclaim the object band that size is about 880bp, it is connected with the skeleton large fragment of the pBAC47P-ubi carrier (construction process sees below) of the same double digestion of process, obtain recombinant plasmid, by its called after pBAC47P-ubi-fer(plasmid map as shown in A in Fig. 1).
Wherein, to cut system (20 μ L) as follows for enzyme: plasmid DNA 3 μ L, 10 * L buffer2 μ L, BamH I 1 μ L, Kpn I 1 μ L, H 2o13 μ L.37 ℃ of enzymes are cut 2h.
The construction process of described pBAC47P-ubi carrier and follow-up Screening and Identification process are referring to document " Sambrook J; Fritsch E F; Maniatis T.Molecular cloning – A laboratory Mauual; 2nd ed.; New York:Cold Spring Harbor Laboratory Press, 1989. ".Specific as follows:
PBAC202 carrier by plasmid map as shown in B in Fig. 1 is (referring to document " Gao Zefa, Chen Xuqing, Yang Fengping, Liang Rongqi, Zhang Liquan, Zhang Xiaodong.Expression and the anti-aphid effect research thereof of synthetic gna gene in wheat, Journal of Agricultural Biotechnology, 2006,14 (4): 559-564 ") with Hind III enzyme, cut; remove " rbcs-sGNA-NOS " about 1.92KB section; reclaim 5.26Kb carrier segments, with T4DNA ligase enzyme, from connecting, obtain described pBAC47P-ubi carrier.
Two, the evaluation of recombinant expression vector pBAC47P-ubi-fer
By recombinant expression vector pBAC47P-ubi-fer, after inscribe restriction enzyme BamH I and Kpn I enzyme are cut, sepharose detected result as shown in Figure 2.As can be seen from the figure, pBAC47P-ubi-fer carrier obtains two fragments after double digestion, and large fragment is carrier segments, the Vefer gene fragment of small segment.The size of two fragments all, with to obtain before clip size consistent, illustrates that pBAC47P-ubi-fer carrier successfully constructs.
Further, to recombinant expression vector, pBAC47P-ubi-fer checks order, result shows, the structrual description of recombinant expression vector pBAC47P-ubi-fer is as follows: between the restriction enzyme site BamH I of recombinant expression vector and Kpn I, inserted the recombinant plasmid obtaining after sequence 2 in sequence table.
In recombinant expression vector pBAC47P-ubi-fer, the promotor that starts described Vefer gene (sequence 2) patent is Ubi promotor.
Embodiment 3, the acquisition that turns Vefer DNA triticum and evaluation
One, turn the acquisition of Vefer DNA triticum
Callus inducing medium SD 2: MS minimum medium (phyto Technology Laboratories tMcompany, article No. M524)+VB 11mg/L+ asparagine 150mg/L+2,4-D2mg/L+ sucrose 30g/L+ plant gel 2.8g/L, pH5.8.Each concentration is the final concentration of respective components in substratum.
Height oozes substratum: callus inducing medium SD 2+ sorbyl alcohol 200mM+ N.F,USP MANNITOL 200mM.Each concentration is the final concentration of respective components in substratum.
Recovery media SD 0: MS minimum medium (phyto Technology Laboratories tMcompany, article No. M524)+VB11mg/L+ asparagine 150mg/L+ sucrose 30g/L+ plant gel 2.8g/L, pH5.8.Each concentration is the final concentration of respective components in substratum.
Screening division culture medium: 1/2MS substratum+0.5mg/L VB1+0.25mg/L VB6+0.25mg/L nicotinic acid+1mg/L Gly+50mg/L inositol+sucrose 20g/L, adds zeatin 5mg/L, selective agent Basta2～5mg/L, plant gel 2.8g/L; PH5.8.Each concentration is the final concentration of respective components in substratum.
Rooting and hardening-off culture base: 1/2MS+0.5mg/L VB1+0.25mg/L VB6+0.25mg/L nicotinic acid+1mg/L Gly+50mg/L inositol+sucrose 20g/L, 0.3～0.5mg/L IAA, 0.5mg/L MET+ plant gel 2.8g/L; PH5.8.Each concentration is the final concentration of respective components in substratum.
Wheat breed as transformation receptor: Zhongyou9507 and Bao Feng 104.
In the present embodiment, obtain and turn Vefer DNA triticum as follows:
Get the prematurity wheat grain of the rear 10-12d of pollination, with 70% alcohol rinsing 2-3min, with 15%(15g/100mL) clorox sterilization 15min, sterilized water washing 4～5 times is chosen rataria scultellum and is inoculated in callus inducing medium SD upward under aseptic condition 2on.25 ℃ of dark culturing 4～7d evoked callus, for via Particle Bombardment Transformation.
Before particle gun bombardment, callus being transferred to height oozes and on substratum, carries out height and ooze and process 4～6h.Callus after particle gun bombardment (1100psi, plasmid pBAC47P-ubi-fer1 μ g/ rifle, bronze 60 μ g/ rifles) continues to ooze on substratum and cultivate after 16～18h at height, transfers to recovery media SD 0on.At recovery media SD 0after upper 25 ℃ of dark culturing 7d, transfer to callus inducing medium SD 225 ℃ of dark culturing of upper continuation 7 days, totally two time-of-week induced embryonic callus.
Embryo callus is transferred on screening division culture medium, and under 25 ℃ of illumination conditions, differentiation screening is 2～3 times.
After growing stem, differentiation seedling moved on Rooting and hardening-off culture base; 25 ℃ of illumination condition lower strong sprouts, treat that seedling grows 2 young leaves, after 3～4 main roots, clean substratum, be transplanted to basin and be placed in the covered with plastic film booth vernalization of outdoor solarium.
In May, 2010, via Particle Bombardment Transformation obtained more than 600 strain Basta screening T altogether 0transfer-gen plant, wherein transplant survival 465 strains.
Experiment arranges the transgenosis contrast that proceeds to pBAC47P-ubi empty carrier simultaneously.
Two, turn the evaluation of Vefer DNA triticum
1, the PCR of transgenic wheat identifies
Get the T that step 1 obtains 0for Transgenic plant of wheat blade, after liquid nitrogen grinding, adopt CTAB method to extract genomic dna as template, adopt and carry out pcr amplification for following primer pair UHD-5/UHD-3.
UHD-5:5 '-TCAACTGTGCCTCTTACTGGG-3 ' (the 202-222 position of sequence 2);
UHD-3:5 '-CGTGACCCTTTCCAACCATTC-3 ' (reverse complementary sequence of the 752-772 position of sequence 2).
Reaction system: 10 * PCR Buffer2.0 μ L, primer UHD-5 (10 μ M) 1.0 μ L, primer UHD-3 (10 μ M) 1.0 μ L, dNTPs0.5 μ L, Taq enzyme 1.0U, template DNA 300～500ng, ddH 2o complements to 20 μ L.
Response procedures: 94 ℃ of denaturation 5min → 94 ℃ sex change 30s, 57 ℃ of annealing 45s, 72 ℃ are extended 45s, → 72 ℃ of extension 10min → 4 ℃ preservations of 35 circulations.
1% agarose electrophoresis detects PCR product.Expanding fragment length is about 0.55Kb.
Unconverted acceptor wheat plant is set as negative control simultaneously, usings plasmid pBAC47P-ubi-fer alternate template as positive control, with H 2o alternate template is as blank, and the transgenic wheat that proceeds to pBAC47P-ubi empty carrier of take is empty carrier contrast.
The detected result of part transfer-gen plant as shown in Figure 3, as can be seen from the figure, all amplifies as pBAC47P-ubi-fer and the part transfer-gen plant of positive control the object band that size is about 0.55Kb, and not transgenosis adjoining tree, ddH 2o blank and empty carrier contrast all do not amplify band, and the transfer-gen plant explanation Vefer gene that amplifies object band has proceeded to acceptor kind.104 two acceptor kinds of the Zhongyou9507 of this conversion and Bao Feng, detect and obtain altogether the positive T of 205 strain PCR through PCR 0for transfer-gen plant.
2, the RT-PCR of transgenic wheat detects
(1) extraction of total RNA
The PCR of the step 1 of learning from else's experience is accredited as positive T 0for transgenic wheat blade, use immediately liquid nitrogen freezing and grind, with TRIzol test kit, extract the total RNA of wheat.RNA sample with DEPC, process without RNase water dissolution, and be stored in-80 ℃.Get the RNA40 μ L slightly carrying, add each 5 μ L of 10 * RNA-Free Dnase buffer and RNA-Free Dnase, after 37 ℃ of water-bath 30min, add 1 μ L RQ 1dnase stop solution, 65 ℃ of water-bath 10min termination reactions, standby.
(2) RT-PCR detects
To being accredited as positive T through step 1PCR 0for Vefer gene in the blade of transgenic wheat, further in mRNA level, whether express and detect.Specific as follows: to get total RNA that 2 μ g steps (1) are extracted, with Olig (dT) 15for primer, M-MLV ThermoScript II is carried out reverse transcription and is obtained cDNA, and use primer UHD-5 and UHD-3(sequence are the same), PCR detects the expression of Vefer gene in wheat leaf blade.
Reaction system: 10 * PCR Buffer2.0 μ L, primer UHD-5 (10 μ M) 1.0 μ L, primer UHD-3 (10 μ M) 1.0 μ L, dNTPs0.5 μ L, Taq enzyme 1.0U, template cDNA300～500ng, ddH 2o complements to 20 μ L.
Response procedures: 94 ℃ of denaturation 5min → 94 ℃ sex change 30s, 57 ℃ of annealing 45s, 72 ℃ are extended 45s, → 72 ℃ of extension 10min → 4 ℃ preservations of 35 circulations.
Wheat β-actin gene of take is internal reference.During amplification β-actin gene, across intron, design primer (β-actin-F and β-actin-R), use acceptor kind wheat cdna group DNA as template, whether have the pollution of genomic dna in can the total RNA leaching process of comparison and detection.
β-actin-F：5’-AAACCTTCAGTTGCCCAGC-3’；
β-actin-R：5’-TCACACCATCACCAGAGTCG-3’。
Unconverted acceptor wheat plant is set as negative control simultaneously, usings plasmid pBAC47P-ubi-fer alternate template as positive control, with H 2o alternate template is as blank, and the transgenic wheat that proceeds to pBAC47P-ubi empty carrier of take is empty carrier contrast.
As shown in Figure 4, as can be seen from the figure, the amplified production band of wheat β-actin is clear for the detected result of part transfer-gen plant, illustrates that RNA extracts quality and reverse transcription effect is better.As the pBPC47P-ubi-fer of positive control and part transfer-gen plant, all increase and obtained 0.55kb left and right target product, and not transgenosis adjoining tree, ddH 2o blank and empty carrier contrast all do not have band, illustrate that the Vefer gene in these transfer-gen plants is expressed.This detects 53 strain RT-PCR positive plants altogether, wherein transforms Zhongyou9507 acceptor and obtains the plant be numbered the 31 strain RT-PCR test positive such as 21,51 and 61, protects rich 104 acceptors and obtains the plant that is numbered 72 22 strain RT-PCR test positive such as grade.
3, the mensuration of transgenic wheat Vefer gene expression amount after being subject to heat stress
Experiment material: will be accredited as positive T through above step 2RT-PCR 0for Transgenic plant of wheat selfing, bear seeds and plant into plant (T 1), from plant, select 21-119,21-23,51-120,61-53,72-46 to carry out heat stress processing.Wherein, the transformation receptor of 21-119,21-23,51-120,61-53 is Zhongyou9507, and the transformation receptor of 72-46 is Zhongyou9507.
Above each wheat plant as experiment material is planted in filling Nutrition Soil: in basin alms bowl vermiculite=3:1(volume ratio), in illumination box, under 22 ℃/18 ℃ (daytime/night) illumination, cultivate about 10d, photoperiod 12h/12h(daytime/night), grow to two leaves wholeheartedly.Give 34 ℃ of forge hot refining 24h, 45 ℃ of thermal treatment 2.5h get respectively the Real-time pcr analysis that blade carries out Vefer gene expression amount before forge hot refining with after thermal treatment.
Real-time pcr analysis is specific as follows:
According to the cDNA sequences Design gene-specific primer of Vefer gene, primer sequence is as follows:
Vefer-4L:5 '-GCCCTCGCTCCTTCTAAAGT-3 ' (the 61-80 position of sequence 2);
Vefer-154R:5 '-GCACAGTTGAGGCAGAAACA-3 ' (reverse complementary sequence of the 192-211 position of sequence 2).
Meanwhile, the internal standard gene that the β-actin that adopts wheat is fluorescence real-time quantitative PCR, its primer sequence is as follows:
Ta.β-actin-148F：5’-AAACCTTCAGTTGCCCAGC-3’；
Ta.β-actin-252R：5’-TCACACCATCACCAGAGTCG-3’。
Agents useful for same is Takara company
Figure BDA0000440677630000111
premix Ex Taq tM(Perfect Real Time) test kit, the reagent that 10 μ L reaction systems comprise has: SYBR Premix Ex Taq(2 *) 5 μ L, PCR forward primer (2 μ M) 1 μ L, PCR reverse primer (2 μ M) 1 μ L, cDNA template 1 μ L, dH 2o2 μ L.
PCR reaction conditions: 95 ℃ of 3min; Then 95 ℃ of 10s, 58 ℃ of 10s, 72 ℃ of 20s, 75 ℃ of 1s, read plate, totally 40 circulations; 72 ℃ of 3min, 60 ℃-95 ℃, read plate every 0.2 ℃, do melting curve.Each sample standard deviation is made 3 parallel pipes, and internal standard gene β-actin also makees 3 parallel pipes accordingly.In same reaction system, use ddH 2o does negative control.Whole PCR reaction is carried out in Bio-Rad CFX Manager.
The calculating of net result adopts 2 -△ △ Ctmethod (Ct represents cycle number) is calculated, use Excel2003 software to carry out statistical analysis, specifically referring to " Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the2 -Δ Δ Ctmethod.Methods25 (4): 402～408. " literary composition; use Excel2003 software to carry out statistical analysis, calculate mean value and the variance of every kind of processing, and carry out the analysis (t-test; n=3, P < 0.01 or 0.05) of significant difference.
Experiment repeats 3 times, results averaged.Take mean value (relative expression quantity of Vefer) as ordinate zou drawing.
Result as shown in Figure 5, as can be seen from the figure, T 1for transfer-gen plant 21-119,21-23,51-120,61-53,72-46 Verfer gene before and after thermal treatment, all express, and the expression amount after thermal treatment is improved.Before and after processing, in each transfer-gen plant, Vefer gene expression amount is analyzed through single tail t-test, and plant 21-119 has utmost point significant difference before and after processing, and there were significant differences before and after processing for plant 21-23,51-120,61-53,72-46.
Embodiment 4, turn the Heat tolerance identification of Vefer DNA triticum
For PCR and RT-PCR in examination plant: embodiment 3, be accredited as positive T 1for transfer-gen plant plantation that selfing bears seeds, obtain T 2for plant.Through after PCR screening, called after ufz61, ufz67, ufz74(acceptor are Zhongyou9507 respectively from plant 21-119,51-120 and 61-53, to choose at random three strains respectively).Through after PCR screening, from plant 72-46 choose at random three strains respectively called after ufb21, ufb51 and ufb72(acceptor for protecting rich 104).
One, the mensuration of mda (MDA) content in plant tissue
1) experiment material: by T 2transformation receptor for Transgenic plant of wheat ufz67 and ufz74(is Zhongyou9507), plant in filling Nutrition Soil: in basin alms bowl vermiculite=3:1(volume ratio), in illumination box, under 22 ℃/18 ℃ (daytime/night) illumination, cultivate about 10d, photoperiod 12h/12h(daytime/night), grow to two leaves wholeheartedly.Give 34 ℃ of forge hot refining 24h, 45 ℃ of thermal treatment 2.5h, get wheat leaf blade stand-by.
2) extraction of mda (MDA): the wheat leaf blade in step 1) is shredded, take 0.1g, adding 2mL concentration is 0.1%(w/v) trichoroacetic acid(TCA) (TCA) aqueous solution and a small amount of quartz sand, be ground to homogenate, be transferred in 10mL centrifuge tube, then add 1mL0.1%(w/v) TCA cleaning mortar transfer.Repeat to guarantee for 2 times to shift fully.
3) by the centrifugal 10000g of homogenate, 4 ℃ of centrifugal 20min, supernatant liquor is sample extracting solution.
4) color reaction and mensuration: draw centrifugal supernatant liquor 2mL(contrast and add 2mL dH 2o), add 4mL to contain 0.5%(w/v) 20%(w/v of thiobarbituricacidα-(TBA)) the TCA aqueous solution, homomixture reacts 15min in boiling water bath, rapidly the cooling termination reaction of ice bath.
5) 10000g, 4 ℃, centrifugal 5min.
6) get supernatant liquor and measure the dullness under 532nm, 600nm and 450nm wavelength.
7) calculate content
Two-pack spectrophotometry, 1. directly tries to achieve the concentration of mda in plant sample extracting solution (MDA) by following formula.
C（MDA）=6.45（D 532－D 600）－0.56D 450 ①
Formula 1. in, C(MDA) represent the concentration (unit: μ mol/L) of mda (MDA); D 450, D 532, D 600represent respectively the dullness value under 450nm, 532nm and 600nm wavelength.
With above-mentioned formula, 1. try to achieve the concentration of MDA, according to formula, 2. calculate the content of MDA in plant tissue:
MDA=C（MDA）×V/m ②
Formula 2. in, MDA represents the content (unit: μ mol/g) of MDA in plant tissue; V represents the volume (unit: L) of plant sample extracting solution; Fresh weight (the unit: g) of the plant tissue that m adopts while representing to obtain V volume extracting solution.
Test in triplicate results averaged.
Unconverted acceptor wheat Zhongyou9507 is set simultaneously and as parent, contrasts, the transgenic wheat (acceptor is wheat Zhongyou9507) that proceeds to pBAC47P-ubi empty carrier of take is empty carrier contrast.
Result as shown in Figure 6, as can be seen from the figure, ufz67, ufz74 compare with unconverted acceptor wheat Zhongyou9507 at 45 ℃ of thermal treatment 2.5h rear blade MDA content, reached respectively remarkable and utmost point significant difference, illustrated that the cell membrane stability of transgenic line ufz67, ufz74 is subject to such an extent that damage is less compared with acceptor contrast strain.
Two, the mensuration of plant tissue relative conductivity
Experiment material: T 2seed for Transgenic plant of wheat ufz61, ufz67, ufz74, ufb21, ufb51, ufb72.Wherein, ufz61, ufz67, ufz74 three's transformation receptor is Zhongyou9507, and ufb21, ufb51, ufb72 three's transformation receptor is protects rich 104.
The wheat seed of each experiment material of imbibition is evenly broadcast in the culture dish soaking at sterilized water, in illumination box, 22 ℃/18 ℃ (daytime/night) illumination cultivation are 10 days, photoperiod 12h/12h(daytime/night), grow to two leaves wholeheartedly.34 ℃ of forge hots are refined 1 day, and every strain seedling is divided into 3 groups, and each group is chosen 6-10 strain seedling, and in the middle of cutting, one section of long blade of 0.5～2cm is put into test tube, uses immediately deionized water rinsing 3～4 times.Finally, the deionized water submergence blade that adds 12ml in test tube.Aluminium foil seals the mouth of pipe, puts into 45 ℃ of thermostat water bath pyroprocessing 1h, takes out room temperature (25 ℃) standing 24 hours, measures specific conductivity T1, test tube is put into inherent 125 ℃ of pressure kettle and process 15 minutes, to kill all cells, by ionogen, all oozes out.After taking out cool to room temperature (25 ℃), test tube is fully vibrated, measure specific conductivity T2.The thermostability that represents cytolemma with relative conductivity (Tl/T2), relative conductivity (Tl/T2) value is lower, represents that genotype thermotolerance is stronger.
Test in triplicate results averaged.
Unconverted acceptor wheat Zhongyou9507 and Bao Feng 104 are set simultaneously as parent contrast, with thermo-labile kind China spring, heat resistant variety TAM107 is as positive and negative contrast, and the transgenic wheat that proceeds to pBAC47P-ubi empty carrier of take is empty carrier contrast.
Result as shown in Figure 7, as can be seen from the figure, the relative conductivity of transgenic line ufz61, ufz67, ufz74 and ufb21, ufb51, ufb72 (Tl/T2) is all significantly protected rich 104, Zhongyou9507 lower than thermo-labile kind China spring, acceptor kind, TAM107 is similar with heat-stable material, illustrates that thermostability and the thermotolerance of the cytolemma of transgenic line obtained enhancing.
Figure IDA0000440677730000011
Figure IDA0000440677730000021
Figure IDA0000440677730000031

Claims (10)

1. protein is following (a) or (b):
(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;
(b) replacement and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to plant heat resistance property through one or several amino-acid residue by the aminoacid sequence of sequence 1.
2. the nucleic acid molecule of protein described in the claim 1 of encoding.
3. nucleic acid molecule according to claim 2, is characterized in that: described nucleic acid molecule is the gene of protein described in coding claim 1; Described gene is following 1) to 4) in arbitrary described DNA molecular:
1) encoding sequence is the DNA molecular shown in the 58-822 position of sequence 2 in sequence table;
2) DNA molecular shown in sequence 2 in sequence table;
3) under stringent condition with 1) or 2) protein DNA molecule described in the DNA molecule hybridize that limits and coding claim 1;
4) with 1) or 2) or 3) DNA molecular that limits there is 90% above homology and the claim 1 of encoding described in protein DNA molecule.
4. the recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain nucleic acid molecule described in claim 2 or 3.
5. recombinant vectors according to claim 4, is characterized in that: described recombinant vectors is recombinant expression vector or recombinant cloning vector.
6. recombinant vectors according to claim 5, is characterized in that: the promotor that starts described genetic transcription in described recombinant expression vector is Ubi promotor.
7. application protein claimed in claim 1, or the nucleic acid molecule described in claim 2 or 3, or recombinant expression vector, expression cassette or recombinant bacterium described in claim 4 or 5 or 6 are at following a1) or a2):
A1) thermotolerance of regulating plant;
A2) seed selection thermophytes kind.
8. cultivate a method for the transgenic plant that thermotolerance improves, comprise will coding claim 1 described in the gene of protein import the step that obtains transgenic plant in object plant; Described transgenic plant are compared with described object plant, and thermotolerance improves.
9. application according to claim 7, or method claimed in claim 8, is characterized in that: described plant is monocotyledons or dicotyledons;
Described monocotyledons is specially wheat.
10. the primer pair of full length gene or its arbitrary fragment described in the claim 3 that increases.
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CN108004248A (en) * 2017-12-07 2018-05-08 华南农业大学 A kind of applications of cucumber calbindin D28K CsCaM in plant heat resistance property is improved
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