CN110128514A - Rise's boot period cold resistance GAP-associated protein GAP CTB4b and encoding gene and application - Google Patents

Abstract

The invention discloses a kind of Rise's boot period cold resistance GAP-associated protein GAP CTB4b and encoding gene and applications.The present invention provides following any albumen: 1) albumen shown in SEQ ID No.2；2) by SEQ ID No.2 by the substitution of one or several amino acid residues, deletion and/or addition and the albumen for having identical function；1) or 2) 3) have 99%, 95%, 90%, 85% or 80% or more homology with the sequence limited and have the albumen of identical function；4) 1) -3) fusion protein that obtains after the N-terminal and/or C-terminal connection label of the albumen of any restriction.CTB4b gene provided by the present invention can high Rise's boot period cold resistance, the Cold Tolerance at Booting Stage of rice varieties can be improved using the gene, to expand the planting area of rice, ensure the yield stability of the late season rice in China and its paddy rice in cold region planting area.

Description

Rise's boot period cold resistance GAP-associated protein GAP CTB4b and encoding gene and application

Technical field

The present invention relates to field of biotechnology more particularly to a kind of Rise's boot period cold resistance GAP-associated protein GAP CTB4b and volumes Code gene and application.

Background technique

Rice is a kind of important cereal crops, and the whole world has more than the people of more than half using it as staple food.Rice is again A kind of low-temperature sensitive crop originating from subtropical and tropical zones.With the continuous expansion of Monitoring of Paddy Rice Plant Area, low temperature becomes Limit one of the principal element of Rice Cropping.In China, the Northeast is averaged in 3-4 will be one year by chilling injury. The rice of the middle and lower reach of Yangtze River and South China will receive the influence of " cold dew wind " and " cold spell in later spring ".The Northeast, China in 2009 Consecutive low temperature rainy weather, causes crop growthing development to postpone, and Seed-Setting Percentage in Rice reduces, and seriously affects yield.Almost in In all plantation areas of state, rice is all subject to damage to plants caused by sudden drop in temperature influence in each growth period, thus can cause every year The grain drop in production that ten thousand tons of 300-500.

Analysis of Rice Chilling Injury refers mainly to rice and meets with the continuous or short-term minimum critical-temperature of low temperature low temperature effect below, from And postpone Rice Growing, nutrition is also caused when serious or reproductive organs is destroyed, and is unable to normal development so as to cause rice And the underproduction.Rice was all easy to meet with chilling injury in the time of infertility.According to low temperature occur period, can be divided into sprouting stage damage to plants caused by sudden drop in temperature, Seedling stage damages to plants caused by sudden drop in temperature, boot stage damages to plants caused by sudden drop in temperature and damages to plants caused by sudden drop in temperature with florescence.Boot stage damage to plants caused by sudden drop in temperature refer to rice since reproductive growth the full heading time to Between by chilling injury, eventually lead to the abnormal pollination that then influences normally to bloom of pollen development and form empty grain.Florescence damages to plants caused by sudden drop in temperature Refer to and encounter low temperature in rice anthesis, causing anther that cannot normally split loose powder, the pollen that is scattering on column cap cannot be normal Fertilization is sprouted on ground, directly affects fertilization, and the one kind for generating empty grain damages to plants caused by sudden drop in temperature, due to this kind of generation period and boot stage damaged to plants caused by sudden drop in temperature Damage to plants caused by sudden drop in temperature very close, production is sometimes more difficult in practice to be strictly separated the two to come, and the two is often collectively referred to as booting florescence and is damaged to plants caused by sudden drop in temperature. Damaging to plants caused by sudden drop in temperature for meeting with when boot stage among these will cause yield to show particularly important due to irreversible loss.

Therefore, it is the period for influencing rice yield most serious that boot stage, which damages to plants caused by sudden drop in temperature, is had to the cold-resistant Journal of Sex Research of Rise's boot period Highly important meaning.But utilize the traditional breeding way cultivation cold-tolerant rice period long, it is in progress slower, and utilize Technique for gene engineering can the direct genetic background of plant modification at the genetic level, the inhereditary feature of directional transformation plant.Hair Digging Rise's boot period cold tolerance gene has highly important theory and practice meaning to cold resistant paddy rice new varieties are cultivated.

Summary of the invention

The object of the present invention is to provide a kind of Rise's boot period cold resistance GAP-associated protein GAP CTB4b and encoding gene and applications.

In a first aspect, a kind of claimed protein.

Present invention protein source claimed is named as CTB4b, specifically in rice (Oryza sativa L.) Can be following any:

(a1) protein shown in SEQ ID No.2；

(a2) by amino acid sequence shown in SEQ ID No.2 by one or several amino acid residues substitution and/or Deletion and/or addition and protein with the same function；

(a3) there is 99% or more, 95% or more, 90% or more, 85% with amino acid sequence defined by (a1) or (a2) Above or 80% or more homology and protein with the same function；

(a4) fusion obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (a1)-(a3) Albumen.

Further, the label is including but not limited to as follows:

Table: the sequence of label

Label	Residue	Sequence
			Poly-Arg	5-6 (usually 5)	RRRRR
Poly-His	2-10 (usually 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Second aspect, the nucleic acid molecules of claimed code for said proteins.

The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules are also possible to RNA, such as mRNA, hnRNA or tRNA.

In the present invention, the nucleic acid molecules are specially the gene of code for said proteins；The gene is specially as follows DNA molecular shown in any:

(b1) DNA molecular shown in SEQ ID No.1；

(b2) hybridize and the DNA molecular of code for said proteins with (b1) DNA molecular limited under strict conditions；

(b3) DNA sequence dna limited with (b1) or (b2) has 99% or more, 95% or more, 90% or more, 85% or more Or 80% or more homology and code for said proteins DNA molecular.

The third aspect, the claimed recombinant vector containing above-mentioned nucleic acid molecules, expression cassette or recombinant bacterium.

The recombinant vector can be recombinant expression carrier, can also be recombinant cloning vector.

The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture Bacillus carrier and the carrier etc. that can be used for plant micropellet bombardment, as pMDC32, pCAMBIA3301, pCAMBIA1300, pBI121, The derivative plant expression vector of pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other.The plant expression vector is also It may include 3 ' end untranslated regions of foreign gene, i.e., comprising polyadenylation signals and any other participation mRNA processing or gene The DNA fragmentation of expression.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor.Use the gene It, can be plus any enhanced, composing type, organizing specific type before its transcription initiation nucleotide when constructing recombinant expression carrier Or inducible promoter, such as cauliflower mosaic virus (CaMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi), stress induced promoter rd29A etc., they can be used alone or are used in combination with other plant promoters；This When outside, using gene constructed recombinant expression carrier of the invention, enhancer, including translational enhancer or transcription enhancing also can be used Son, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must be with coded sequence Reading frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon is extensive , can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.For Convenient for transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be processed, such as plus The coding that entering can express in plant can produce the enzyme of color change or the gene of luminophor, resistant antibiotic mark Remember object or anti-chemical reagent marker gene etc..Any selected marker can also be not added, conversion is directly screened with adverse circumstance and is planted Strain.

The expression cassette is by that can start the promoter of the gene expression, the gene and transcription terminator group At.

In the present invention, the promoter for starting the gene expression in the recombinant vector is 35S promoter.It is more specific , in one embodiment of the invention, the recombinant vector is in pMDC32 vector multiple cloning site (such as Kpn I and Pac I the recombinant plasmid obtained after the insertion gene (SEQ ID No.1) in).

Fourth aspect, the claimed protein or the nucleic acid molecules or the expression cassette or the recombination The application of carrier or the recombinant bacterium in regulation plant frigostabile.

In the application, the lower temperature resistance of the protein more high plant of activity in the plant is stronger；Institute State protein in the plant activity it is lower, the lower temperature resistance of the plant is weaker.

5th aspect, claimed following method I or method II:

Method I: a method of plant that cultivating lower temperature resistance enhancing, specifically may include making albumen described in recipient plant The step of expression quantity and/or activity of matter increase.

Method II: a method of the plant that lower temperature resistance weakens is cultivated, specifically may include making egg described in recipient plant The step of expression quantity and/or activity of white matter reduce.

In the method I, the expression quantity for making protein described in recipient plant and/or activity increase be pass through to The nucleic acid molecules are imported in the recipient plant to realize.

Further, it can be realized by any technological means that can be realized this purpose.Such as pass through the shape of recombinant vector Formula (recombinant vector as previously described) imports the nucleic acid molecules in the recipient plant.

In the method II, the expression quantity for making protein described in recipient plant and/or activity reduction are to pass through Nucleic acid molecules described in the recipient plant are knocked out or inhibit to express to realize.

Further, it can be realized by any technological means that can be realized this purpose, such as pass through sequence specific nucleic acid Enzyme (such as CRISPR/Cas9 nuclease) carries out specific editor to the nucleic acid molecules, to knock out it in the recipient plant In expression, or inhibition expression is carried out to the nucleic acid molecules by RNAi means.

In the method I and the method II, by the recombinant vector for carrying the nucleic acid molecules or it is used for Nucleic acid molecules described in the recipient plant are knocked out or inhibit when expression the gene editing tool that uses import it is described by Body plant, concretely: by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, The conventional biology methods such as mediated by agriculture bacillus convert plant cell or tissue, and the plant tissue of conversion is cultivated into plant.

In method described in application described in fourth aspect and the 5th aspect, the lower temperature resistance concretely boot stage To the tolerance of low temperature.In one embodiment of the invention, the lower temperature resistance enhancing is embodied as being transferred to the albumen After boot stage is by low temperature stress, setting percentage is higher than without transgenosis the plant of the encoding gene (CTB4b gene) of matter Recipient plant.

In method described in application and the 5th aspect described in fourth aspect, the plant can be monocotyledon, It can be dicotyledon.

Further, the monocotyledon can be gramineae plant.

Further, the gramineae plant can be rice.

More specifically, in one embodiment of the invention, the rice is rice varieties Towada.

CTB4b gene provided by the present invention can high Rise's boot period cold resistance, can be to rice product using the gene The Cold Tolerance at Booting Stage of kind is improved, to expand the planting area of rice, ensures the late season rice in China and its paddy rice in cold region kind The yield stability in growing area domain.

Detailed description of the invention

Fig. 1 is parent KMXBG, Towada and near isogenic lines NIL1913 in Beijing (normal temperature) and Yunnan (breeding time Low temperature, CS-HAA) phenotypic map.

Fig. 2 is parent KMXBG, Towada and near isogenic lines NIL1913 in Beijing (normal temperature) and Yunnan (breeding time Low temperature, CS-HAA) setting percentage statistical chart.

Fig. 3 is expression pattern analysis figure of the CTB4b in NIL1913 and Towada different tissues.Internal reference is OsActin1 Gene.Stem is stem, and Leaf is mature blade, and Young leaf is spire, and Sheath is leaf sheath, and Root is root, and Node is Stipes, YP10 are 10cm fringe, and YP15-20 is 15-20cm fringe.

Fig. 4 is CTB4b expression pattern analysis chart under NIL1913 and Towada fringe portion cold-induction.Internal reference is OsActin1 Gene.

Fig. 5 is that PCR identifies that T0 is overexpressed plant for CTB4b.

Fig. 6 is that CTB4b is overexpressed plant difference transgenic line real-time fluorescence quantitative PCR testing result.

Fig. 7 is that different cold treatment modes indicates to scheme.

Fig. 8 is that CTB4b is overexpressed field and fringe portion phenotypic map after the processing of plant boot stage natural low temperature.

Fig. 9 is that CTB4b is overexpressed plant and adjoining tree in high altitude localities plantation, cold water irrigation processing, artificial climate Setting percentage statistical chart after the cold treatment of room.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

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

Rice varieties KMXBG: document: the heredity of the Yunnan Rice Kunmingxiaobaigu cold resistance index character such as garden Dai Lu point Rice in China science is analysed, 1999,13 (2): the 73-76. public can obtain from China Agricultural University.

Rice varieties Towada: document: the heredity of the Yunnan Rice Kunmingxiaobaigu cold resistance index character such as garden Dai Lu point Rice in China science is analysed, 1999,13 (2): the 73-76. public can obtain from China Agricultural University.

Rice genotype NIL1913: the building of this laboratory and preservation.Document: Zhanying Zhang^*,Jinjie Li^*,Yinghua Pan^*,Jilong Li^*,Lei Zhou^*,Hongli Shi^*,Yawen Zeng,Haifeng Guo, Shuming Yang,Weiwei Zheng,Jianping Yu,Xingming Sun,Gangling Li,Yanglin Ding, Liang Ma,Shiquan Shen,Luyuan Dai,Hongliang Zhang,Shuhua Yang,Yan Guo,Zichao Li^#.Natural variation in CTB4a enhances rice adaptation to cold Habitats.Nature Communications, the 2017,8:14788. public can obtain from China Agricultural University.

Over-express vector pMDC32: document: Mark D.Curtis and Ueli Grossniklaus.A Gateway Cloning Vector Set for High-Throughput.Plant Physiology,October 2003,Vol.133, Pp.462-469. public can obtain from China Agricultural University.

Agrobacterium tumefaciems EHA105: document: the such as high generation force influence Agrobacterium tumefaciems EHA105 competent cell transformation efficiency The torrid zone research biological journal .2012 the 1st phase of volume 3 March of factor, the public can obtain from China Agricultural University.

Embodiment 1, the positioning of CTB4b gene and clone

1, the acquisition of CTB4b gene

The cold-resistant near isogenic lines (NIL1913) constructed using cold-resistant kind KMXBG and cold sensitive varieties Towada, into And segregating population is constructed near isogenic lines NIL1913 and Towada, parent is investigated in the setting percentage in Beijing and Kunming, as fixed The phenotypic data (Fig. 1, Fig. 2) of position.Meanwhile it being screened from 1000 pairs of SSR markers being evenly distributed on 12 chromosomes of rice Polymorphism mark out carries out Genotyping to target group.Linkage mapping is marked using Mapmaker 3.0, Kosambi method calculates genetic distance.QTL scanning uses QTL IciMapping 3.0 (Wang Jiankang etc., 2009), with LOD value 3.0 as threshold value existing for QTL, while calculating additivity and dominant effect.Finally minor effect QTL can be detected in two environment QCTB4-1, the QTL explain that the contribution rate of phenotype is respectively 0.9% and 3.93% between RM518-RM6770.Then sharp Table is carried out to 3102 single plant BC6F2 mass screenings, and using F3 family with the two sides qCTB4-1 label RM2146 and RM6770 Type determines.Finally target gene is located between label RM16349 and STS12, physical distance 134.8kb.In the section 4 candidate genes are shared, wherein gene Os04g0131900 encodes a UDP-glucose sterol glucityl transport protein.The base Because there is SNP difference at 2 in the code area only in NIL1913 and Towada, being possible candidate gene, being named as CTB4b。

The nucleotides sequence of CTB4b gene is classified as SEQ ID No.1, and the albumen of coding is CTB4b, the amino acid of the albumen Sequence is SEQ ID No.2.

2, the expression pattern of endogenous CTB4b gene

Normal processing: the sample of different tissues under the normal planting conditions of NIL1913 and Towada, including root, stem, stem are taken Section, spire, climax leaves, leaf sheath, 10cm fringe, 15-20cm fringe.Liquid nitrogen flash freezer is used after sampling, -80 DEG C save backup.

Low temperature stress processing: the NIL of grown under normal conditions to boot stage and Towada are transplanted to 18 degree of artificial gas Indoor, blade and fringe portion sample when taking processing 0h, 1h, 2h, 4h, 8h, 12h, 16h, 1d, 2d, 3d, 4d, 5d, 6d, 7d respectively are waited, Liquid nitrogen flash freezer, -80 DEG C save backup.

The sample for extracting above-mentioned various processing takes total serum IgE, synthesizes the first chain of cDNA using reverse transcriptase M-MLV reverse transcription, Using first chain of cDNA as template, real-time quantitative analysis is carried out using primer, using OsActin1 as reference gene；

CTB4b amplimer:

CTB4b-RT42-3F:5 '-CTGGTATTCCTTTCAAAGTGGAT-3 '；

CTB4b-RT42-3R:5 '-CCAAGCCAAATCATAGAGTCAAC-3 '.

OsActin1 amplimer:

Actin1-F:5 '-ACAGGTATTGTGTTGGACTCTGG-3 '；

Actin1-R:5 '-AGTAACCACGCTCCGTCAGG-3 '.

CTB4b tissue expression pattern such as Fig. 3, it can be seen that CTB4b gene is the base expressed in different tissues Cause, the gene expression abundance highest especially in root, stipes and fringe.

CTB4b cold-induction expression analysis such as Fig. 4, it can be seen that all by low temperature induction in NIL1913 and Towada, Illustrate that CTB4b gene is a gene relevant to low temperature stress.

The functional verification of embodiment 2, CTB4b gene

One, CTB4b is overexpressed the building of recombinant vector

The RNA of rice varieties KMXBG and reverse transcription are extracted into cDNA, then using the cDNA as template, with CTB4b-1F and CTB4b-1R carries out PCR amplification, obtains the PCR product of 1830bp, and by sequencing, which is CTB4b gene C DS sequence, For shown in sequence 1.

(underscore part is restriction enzyme to CTB4b-1F:5 '-CGGGGTACCATGGCCTCCGCCGCCGAGG-3 ' The identification sequence of KpnI)；

CTB4b-1R:5 '-CCTTAATTAA(underscore part is in restricted to CTATGAGCAACCAAGACATTTA-3 ' The identification sequence of enzyme cutting PacI).

DNA fragmentation shown in SEQ ID No.1 is replaced to the piece on expression vector pMDC32 between KpnI and PacI restriction enzyme site Section obtains recombinant vector 35S::CTB4b, and as CTB4b is overexpressed recombinant vector, and wherein CTB4b gene is inserted into double Tobacco mosaics The downstream viral promotors 35S.

Two, turn the acquisition of CTB4b rice

1, recombinant bacterium

The recombinant vector 35S::CTB4b freeze-thaw method that above-mentioned one is prepared converts Agrobacterium tumefaciems EHA105, obtains recombinant bacterium EHA105/35S::CTB4b, for infecting the callus of transgene receptor kind.

2, turn CTB4b rice

The callus of classical mediated by agriculture bacillus is taken to infect method, the specific steps are as follows:

(1) it the acquisition of embryo callus subculture: will be used after mature Towada seed (hereinafter also referred to wild rice) decladding 75% alcohol disinfecting 2min, then 40min is sterilized with 20%NaClO, rinsed with sterile water 3-5 times air-dries.It is inoculated in Fiber differentiation Base, 28 DEG C dark culture 2 weeks, embryo callus subculture is peeled, subculture to new induced medium, squamous subculture 2 weeks (subculture is twice).

(2) it prepares infected liquid: drawing the 20 μ l of agrobacterium liquid of preservation, being applied to YEP, (rifampin containing 20mg/L resists with corresponding Raw element) on solid medium, 28 DEG C of inversion dark culture 2d scrape a small amount of Agrobacterium (acetyl containing 10mM into AAM fluid nutrient medium Syringone), bacterial concentration OD600 is about 0.3.

(3) it co-cultures: selecting nature dispersion, graininess callus that color cadmium yellow, diameter are about 3~5mm to triangle In bottle, the infected liquid prepared is added, infects 10min, extra infected liquid is drawn with aseptic filter paper, is placed in and is covered with one layer of filter paper Co-culture medium on, 19 DEG C of co-cultivation 2-3d.

(4) screening of resistant calli: the callus after co-cultivation is taken out, and cleaning 5 is quickly shaken with sterile water ~6 times, then with the sterile water wash 20min of cephalosporin containing 250mg/L and 250mg/L carbenicillin, be finally placed in sterile 3h is drained on filter paper.Then it moves on on screening and culturing medium.Biweekly, it screens twice.

(5) break up: the resistant calli that screening is obtained accesses in pre- differential medium, and 26 DEG C, dark culture 2 weeks, so After be transferred on differential medium, illumination cultivation 2-3 weeks, obtain regeneration of transgenic seedling plants.

(6) seedling is moved on root media, after seedling takes root and grows up to, removes culture bottle, cleans the culture on root Base, hardening 7d move to crop field plantation, until mature, obtaining T0 generation turns CTB4b rice.

Used medium formula is as follows in above-mentioned transgenic protocol:

(1) embryo callus subculture induction and subculture medium: NB minimal medium (2,4-D containing 2mg/L).

(2) co-culture medium: NB minimal medium (contains 2mg/L 2,4-D, 10g/L glucose, 10mM acetyl cloves Ketone, pH 5.6).

(3) screening and culturing medium: NB minimal medium (contain 2mg/L 2,4-D, 400mg/L Ticarcillin/Clavulanate Acid and 50mg/L hygromycin, pH 5.8)。

(4) pre- differential medium: NB minimal medium (6-BA containing 1mg/L, 2mg/L NAA, 5mg/L ABA, 50mg/L Hygromycin, pH 5.8).

(5) differential medium: (6-BA containing 2mg/L, 1mg/L NAA, 1mg/L KT, 50mg/L tide are mould for NB minimal medium Element, pH 5.8).

(6) root media: 1/2MS culture medium basis (NAA containing 0.5mg/L, 0.25mg/L paclobutrazol, pH 5.8)。

(7) NB culture medium basis includes N6 a great number of elements, B5 microelement, B5 organic principle, 150mg/L inositol, 300mg/L caseinhydrolysate, 500mg/L glutamine, 600mg/L proline, 30g/L sucrose, 3g/L plant gel.

Empty carrier pMDC32 is transferred in wild rice using same method, it is (empty for pMDC32 rice is turned to obtain T3 Carry control).

3, Molecular Identification

T0 is extracted for CTB4b oryza sativa genomic dna is turned, is 5 '-AGGACCAGAAGGCAAGAATC-3 ' and 5 '-with primer GATGTGCTGCAAGGCGATTA-3 ' carry out PCR amplification, the PCR product (target gene Hyg) for obtaining 1583bp is positive T0 generation Turn CTB4b rice, the plant without containing target fragment is feminine gender, the qualification result of part sample (OE-234, OE- as shown in Figure 5 235、OE-236、OE-237、OE-238)。

Positive T0 withholds kind, sowing, obtains T3 generation.

T3 is extracted for CTB4b rice strain RNA is turned, reverse transcription obtains cDNA, with CTB4b amplimer

CTB4b-RT42-3F:5 '-CTGGTATTCCTTTCAAAGTGGAT-3 '；

CTB4b-RT42-3R:5 '-CCAAGCCAAATCATAGAGTCAAC-3 '.

It is expanded, using OsActin1 as reference gene.OsActin1 amplimer:

Actin1-F:5 '-ACAGGTATTGTGTTGGACTCTGG-3 '；

Actin1-R:5 '-AGTAACCACGCTCCGTCAGG-3 '.

As a result as shown in Figure 6, it can be seen that compared with wild rice (Towada), in OE236, OE237 strain CTB4b gene expression amount improves 10 times or more.

Subsequent selection expresses higher T3 and carries out cold-resistant identification experiment for CTB4b rice strain OE236 and OE237 is turned.

Three, turn the Identification of Cold Tolerance of CTB4b rice

Identification of Cold Tolerance uses three kinds of methods, is respectively boot stage crop field (CS-DW) 50cm deep cooling pond (18 DEG C) 7 days, 18 DEG C of boot stage phjytotron (CS-PT) handle the methods of 7 days and Yunnan natural (CS-HAA) identification multi-party verification CTB4b's Boot stage cold-resistant function (Fig. 7).

Choose wild rice (cold sensitive varieties Towada), T3 generation turns CTB4b rice strain OE236, OE237 and T3 generation It is listed to turn the main fringe of pMDC32 rice (zero load control) in boot stage, does mark.After stress, will participate in the experiment material multiple cropping To crop field, restores paddy growth, the setting percentage for the main fringe that is listed is investigated after mature, be control with the plant setting percentage of normal growth, Relative setting rate (setting percentage * 100% under relative setting rate=treated setting percentage/normal condition) is calculated, as cold resistance Identification of indicator.Yunnan natural appraisal is then in Yunnan Heilongtang area, and 1916 meters of height above sea level, 19-22 DEG C of long-term temperature, from sowing to pregnant Ear period is under natural low temperature state and grows, and investigates main fringe setting percentage after mature.3 repetitions are arranged in above-mentioned each material, often 10 plants of a repetition.

Result is as shown in Figure 8 and Figure 9 under the natural low temperature processing in the Yunnan time of infertility, and the setting percentage for being overexpressed strain is equal Significantly beyond Towada.

The 7 days results in boot stage crop field 50cm deep cooling pond (18 DEG C) are as shown in Figure 9, it can be seen that the cold water irrigation 7 in crop field Under it stress, the setting percentage of strain is overexpressed significantly beyond Towada.

18 DEG C of boot stage phjytotron processing 7 days results as shown in figure 9, Towada relative setting rate be 39.40%, The relative setting rate of overexpression is 69.80% and 65.42%, is also significantly higher than Towada.

For above-mentioned each method, the setting percentage that T3 generation turns pMDC32 rice (zero load control) is almost the same with wild type, No difference of science of statistics.

The above results show that boot stage meets with low temperature and mainly affects the height of setting percentage, at different location, different modes Under reason mode, the main fringe setting percentage for being overexpressed strain is all significantly higher than cold sensitive parent Towada, illustrates that gene C BT4b has and mentions Low temperature resistant function of high boot stage is the gene for playing positive long-acting, can be in cold stress decline low temperature to solid The influence of rate.

<110>China Agricultural University

<120>Rise's boot period cold resistance GAP-associated protein GAP CTB4b and encoding gene and application

<130> GNCLN180362

<160> 2

<170> PatentIn version 3.5

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<211> 609

<212> PRT

<213>rice (Oryza sativa L.)

<400> 2

Met Ala Ser Ala Ala Glu Glu Ala Lys Gly Val Glu Glu Gly Gly Gly

1 5 10 15

Gly Ala Ser Ala Asn Gly Gly Asp Asn Pro Ala Thr Ala Thr Ala Ser

20 25 30

Ala Ser Ala Ala Ala Ala Ala Ser Ser Ser Ser Pro Ala Asp Asp Arg

35 40 45

Gly Leu Pro Arg Ser Ser Thr Met Pro Gly Gly Ile Asn Asn Val Glu

50 55 60

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

65 70 75 80

Glu Arg Arg Arg Gln Asn Asn Pro Ala Asp Asp Pro Thr Lys Gln Leu

85 90 95

Phe Asp Asp Lys Ile Ser Leu Lys Lys Lys Leu Lys Met Ile Asn Arg

100 105 110

Ile Ala Thr Val Lys His Asp Gly Thr Val Val Val Asp Val Pro Ser

115 120 125

Ser Leu Glu Thr Ser Thr Thr Asp Gly Val Ala Tyr Asp Gly Tyr Ser

130 135 140

Asp Val Thr Val Glu Glu Pro Leu Asp Gly Ala Asp Ile Pro Val Arg

145 150 155 160

Pro Pro Met Gln Ile Val Ile Leu Ile Val Gly Thr Arg Gly Asp Val

165 170 175

Gln Pro Phe Ile Ala Ile Gly Lys Arg Leu Gln Asp Tyr Gly His Arg

180 185 190

Val Arg Leu Ala Thr His Ala Asn Phe Lys Glu Phe Val Leu Thr Ala

195 200 205

Gly Leu Glu Phe Phe Pro Leu Gly Gly Asp Pro Lys Ile Leu Ala Glu

210 215 220

Tyr Met Val Lys Asn Lys Gly Phe Leu Pro Ser Gly Pro Ser Glu Ile

225 230 235 240

Pro Ile Gln Arg Lys Gln Met Lys Glu Ile Ile Phe Ser Leu Leu Pro

245 250 255

Ala Cys Lys Glu Pro Asp Pro Asp Thr Gly Ile Pro Phe Lys Val Asp

260 265 270

Ala Ile Ile Ala Asn Pro Pro Ala Tyr Gly His Thr His Val Ala Glu

275 280 285

Ala Leu Lys Val Pro Ile His Ile Phe Phe Thr Met Pro Trp Thr Pro

290 295 300

Thr Ser Glu Phe Pro His Pro Leu Ser Arg Val Lys Gln Ala Ala Gly

305 310 315 320

Tyr Arg Leu Ser Tyr Gln Ile Val Asp Ser Met Ile Trp Leu Gly Ile

325 330 335

Arg Asp Met Ile Asn Glu Phe Arg Lys Lys Lys Leu Lys Leu Arg Pro

340 345 350

Val Thr Tyr Leu Ser Gly Ala Gln Gly Ser Gly Asn Asp Ile Pro His

355 360 365

Gly Tyr Ile Trp Ser Pro His Leu Val Pro Lys Pro Lys Asp Trp Gly

370 375 380

Pro Lys Ile Asp Val Val Gly Phe Cys Phe Leu Asp Leu Ala Ser Asn

385 390 395 400

Tyr Val Pro Pro Glu Pro Leu Ile Lys Trp Leu Glu Ala Gly Asp Lys

405 410 415

Pro Ile Tyr Val Gly Phe Gly Ser Leu Pro Val Gln Asp Pro Ala Lys

420 425 430

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

435 440 445

Ile Ile Asn Lys Gly Trp Gly Gly Leu Gly Thr Leu Ala Glu Pro Lys

450 455 460

Asp Phe Val Tyr Leu Leu Asp Asn Cys Pro His Asp Trp Leu Phe Leu

465 470 475 480

Gln Cys Lys Ala Val Val His His Gly Gly Ala Gly Thr Thr Ala Ala

485 490 495

Gly Leu Lys Ala Ala Cys Pro Thr Thr Ile Val Pro Phe Phe Gly Asp

500 505 510

Gln Pro Phe Trp Gly Asp Arg Val His Ala Arg Gly Val Gly Pro Leu

515 520 525

Pro Ile Pro Val Asp Gln Phe Ser Leu Gln Lys Leu Val Asp Ala Ile

530 535 540

Asn Phe Met Met Glu Pro Lys Val Lys Asp Asn Ala Val Glu Leu Ala

545 550 555 560

Lys Ala Met Glu Ser Glu Asp Gly Val Ser Gly Ala Val Arg Ala Phe

565 570 575

Leu Arg His Leu Pro Ser Arg Ala Glu Glu Thr Ala Pro Gln Gln Thr

580 585 590

Ser Ser Phe Leu Glu Phe Leu Gly Pro Val Ser Lys Cys Leu Gly Cys

595 600 605

Ser

Claims (10)

1. protein is following any:

(a1) protein shown in SEQ ID No.2；

(a2) amino acid sequence shown in SEQ ID No.2 is passed through to the substitution and/or missing of one or several amino acid residues And/or addition and protein with the same function；

(a3) have 99% or more, 95% or more, 90% or more, 85% or more with amino acid sequence defined by (a1) or (a2) Or 80% or more homology and protein with the same function；

(a4) fusion protein obtained after N-terminal and/or C-terminal the connection label of protein defined by any in (a1)-(a3).

2. encoding the nucleic acid molecules of protein described in claim 1.

3. nucleic acid molecules according to claim 2, it is characterised in that: the nucleic acid molecules are the base of code for said proteins Cause；The gene is following any shown DNA molecular:

(b1) DNA molecular shown in SEQ ID No.1；

(b2) hybridize and the DNA molecular of code for said proteins with (b1) DNA molecular limited under strict conditions；

(b3) with (b1) or (b2) limit DNA sequence dna have 99% or more, 95% or more, 90% or more, 85% or more or The DNA molecular of 80% or more homology and code for said proteins.

4. expression cassette, recombinant vector or recombinant bacterium containing nucleic acid molecules described in Claims 2 or 3.

5. expression cassette described in protein described in claim 1 or nucleic acid molecules described in claim 2 or 3 or claim 4, The application of recombinant vector or recombinant bacterium in regulation plant frigostabile.

6. method is method I or method II:

Method I: a method of the plant of lower temperature resistance enhancing is cultivated, including makes albumen described in claim 1 in recipient plant The step of expression quantity and/or activity of matter increase；

Method II: a method of the plant that lower temperature resistance weakens is cultivated, including makes albumen described in claim 1 in recipient plant The step of expression quantity and/or activity of matter reduce.

7. described to want right in recipient plant according to the method described in claim 6, it is characterized by: in the method I Asking the expression quantity of 1 protein and/or activity to increase is by importing described in Claims 2 or 3 into the recipient plant Nucleic acid molecules are realized；

Further, the nucleic acid molecules are imported in the recipient plant by recombinant vector as claimed in claim 4；

It is described that the expression quantity of protein described in claim 1 and/or activity reduction in recipient plant is made to be in the method II It is realized by being knocked out to nucleic acid molecules described in Claims 2 or 3 in the recipient plant or inhibiting to express.

8. method described in application according to claim 5 or claim 6 or 7, it is characterised in that: the lower temperature resistance It is boot stage to the tolerance of low temperature.

9. according to the application or method any in claim 5-9, it is characterised in that: the plant be monocotyledon or Dicotyledon.

10. application according to claim 9 or method, it is characterised in that: the monocotyledon is gramineae plant；

Further, the gramineae plant is rice.