CN106967724B

CN106967724B - Afriocan agapanthus SK3Type dehydrin protein and its encoding gene and probe

Info

Publication number: CN106967724B
Application number: CN201710184546.0A
Authority: CN
Inventors: 杨舟; 张荻; 申晓辉
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2017-03-24
Filing date: 2017-03-24
Publication date: 2019-10-08
Anticipated expiration: 2037-03-24
Also published as: CN106967724A

Abstract

The present invention relates to a kind of Afriocan agapanthus (Agapanthus praecox) SK₃Type dehydrin protein and its encoding gene and probe, protein of the albumen including following (a) or (b): (a) protein being made of the amino acid sequence as shown in SEQ ID NO.4；(b) amino acid sequence shown in SEQ ID NO.4 is by replacing, lacking or add one or several amino acid and have Afriocan agapanthus SK₃The active protein as derived from (a) of type dehydrin protein.The present invention also provides a kind of nucleic acid sequences for encoding above-mentioned protein, and the probe of the above-mentioned nucleic acid sequence of detection.The present invention provides foundation to improve the anti-adversity ability of Afriocan agapanthus and all kinds of ornamental flowers；Theoretical basis has been established for ornamental plant marker assisted selection and Germ-plasma resources protection, there is very big application value.

Description

Afriocan agapanthus SK3Type dehydrin protein and its encoding gene and probe

Technical field

The present invention relates to the important protected protein SK of one of Afriocan agapanthus environment stress response process₃Type dehydrin protein its Encoding gene and probe, and in particular to a kind of Afriocan agapanthus SK₃Type dehydrin protein and its encoding gene and probe.

Background technique

Dehydrins (Dehydrin) belong to cell stage development advanced stage abundance protein second family (LEAII), can be in plant It great expression and plays a significant role in Late Embryogenesis and plant in the adverse circumstances such as arid, low temperature, saline and alkaline, there is height The characteristics of hydrophily, randomness and inoxidizability.A large amount of plants can be long-pending during embryonic development is formed and under stress reaction Tired Dehydrins, to cope with such as arid, high temperature severe cold and abiotic stress environment with high salt.Many researchs confirm, in the abiotic side of body Under compeling, there is positive correlations between the expression and accumulation and stress resistance of plant of plant dehydration element.

Afriocan agapanthus (Agapanthus praecox), unifacial leaf perennial herb flowers originate in African south, and alias is blue Lily, Afric lilium.Its plant is tall and straight, leaf beautiful, and flower amount is big, the florescence is long, ornamental value is high.It is by the favorite of people Common landscape flower is chiefly used in garden cultivation and cut-flower production, has the good reputation of " agapanthus " in European and American areas.

In recent years in the physiological molecular studies of plant stress-resistance, wheat, barley, rice, corn and soybean, cotton, Dehydrin gene is separated and cloned in the various crops such as loquat, pear tree, Oak Tree and fruit tree.But for ornamental plant especially ball The clone of Dehydrins, expression pattern and protein sequence are unclear in root flowers.Currently, not there is any and Afriocan agapanthus Dehydrins egg White structure and its relevant document report of coding gene sequence.

Summary of the invention

In view of the drawbacks of the prior art, present invention aims at fill up Afriocan agapanthus SK₃The clone of dehydrin gene, expression mould Formula analysis and Afriocan agapanthus SK₃The blank of type dehydrin protein provides a kind of Afriocan agapanthus SK₃Type dehydrin protein and its coding Gene and probe.The invention discloses Afriocan agapanthus SK₃Physiological effect and expression pattern after genetic transformation arabidopsis are sharp from now on With technique for gene engineering to SK₃The space-time characterisation of gene expression is regulated and controled, to improve the anti-of Afriocan agapanthus and all kinds of ornamental flowers Inverse ability provides foundation, to provide theoretical foundation to improve the anti-adversity ability of ornamental flower and molecular breeding work, has There is very big application value.

In the early-stage study of this experiment, the experiment of Afriocan agapanthus cells,primordial cryopreservation is carried out, and pass through transcript profile The comparative analysis data with protein science are learned, SK is screened₃Type dehydrin protein is compound in face of ultralow temperature with albumin layer in transcription Adverse circumstance all has positive response.Speculating has important regulation to cell activity of the protection plant in compound upper adverse circumstance Effect.

The purpose of the present invention is what is be achieved through the following technical solutions:

In a first aspect, the present invention provides a kind of Afriocan agapanthus SK₃Type dehydrin protein, albumen including following (a) or (b) Matter:

(a) protein being made of the amino acid sequence as shown in SEQ ID NO.4；

(b) amino acid sequence shown in SEQ ID NO.4 through substitution, lack or add one or several amino acid and With Afriocan agapanthus SK₃The active protein as derived from (a) of type dehydrin protein.

Preferably, the protein be SEQ ID NO.4 shown in amino acid sequence by 1~50 amino acid missing, Insertion and/or substitution, or sequence obtained from amino acid within 1~20 is added in C-terminal and/or N-terminal.

It is further preferred that the protein is 1~10 amino acid in amino acid sequence shown in SEQ ID NO.4 by property The sequence that matter is similar or similar amino acid is replaced and is formed.

Second aspect, the present invention provides a kind of above-mentioned Afriocan agapanthus SK of coding₃The nucleic acid sequence of type dehydrin protein.

Preferably, the nucleic acid sequence specifically:

(a) base sequence such as SEQ ID NO.3 the 1st~648；

Or (b) with the nucleic acid of SEQ ID NO.3 the 1st~648 have at least 70% homology sequence；

Or the sequence that can (c) be hybridized with the nucleic acid of SEQ ID NO.3 the 1st~648.

Preferably, the nucleic acid sequence is specially 1~90 nucleotide in the nucleic acid sequence of SEQ ID NO.3 the 1st~648 Missing, insertion and/or substitution, and 5 ' and/or 3 ' end addition 60 with inner nucleotide formed sequences.

The third aspect, the present invention also provides a kind of above-mentioned Afriocan agapanthus SK of detection₃The spy of type dehydrin protein nucleic acid sequence Needle, the probe are the nucleic acid molecules with above-mentioned 8~100 continuous nucleotides of nucleic acid sequence, which can be used for detecting sample With the presence or absence of coding Afriocan agapanthus SK in product₃The relevant nucleic acid molecules of type Dehydrins.

Fourth aspect, a kind of amplification Afriocan agapanthus SK₃The specific primer pair of the nucleic acid sequence of type dehydrin protein, institute It is as follows to state primer pair:

ORF-S:5 '-ATGGCAGAGGAGAATGTGGA-3 ',

ORF-A:5 '-CTAATGAGCCTTCTCGGTCTC-3 '.

5th aspect, the present invention also provides above-mentioned Afriocan agapanthus SK₃The application of type dehydrin protein encoding gene, the gene Base sequence as shown in SEQ ID NO.3 the 1st~648, the described application includes improving plant stress-resistance ability.

In the present invention, " isolated DNA ", " DNA of purifying " refer to that the DNA or segment are located under native state It is separated in the sequence of its two sides, also refers to that the DNA or segment are separated with the component under native state with nucleic acid, and Separated with the protein to accompany in cell.

In the present invention, term " Afriocan agapanthus SK₃Type dehydrin protein coded sequence " refers to that coding has Afriocan agapanthus SK₃Type is de- The nucleotide sequence of the active polypeptide of water fibroin, the 1st~648 acid sequence and its degenerate sequence as shown in SEQ ID NO.3. The degenerate sequence refers to, is located in the 1st~648 acid shown in SEQ ID NO.3, has one or more codons to be encoded identical The sequence generated after replaced the degenerate codon of amino acid.Due to the degeneracy of codon, so with SEQ ID NO.3 institute The 1st~648 acid sequence homology shown can also encode out sequence shown in SEQ ID NO.4 down to about 70% degenerate sequence. The term further includes the nucleotide sequence with the homology at least 70% of nucleotide sequence shown in SEQ ID NO.3.

The term further includes that can encode natural Afriocan agapanthus SK₃Shown in the identical function of type dehydrin protein, SEQ ID NO.3 The variant form of sequence.These variant forms include (but being not limited to): be usually 1~90 nucleotide missing, insertion and/ Or replace, and be added to 60 at 5 ' and/or 3 ' ends with inner nucleotide.

In the present invention, term " Afriocan agapanthus SK₃Type Dehydrins " refer to Afriocan agapanthus SK₃Type dehydrin protein is active The polypeptide of sequence shown in SEQ ID NO.4.The term further includes having and natural Afriocan agapanthus Dehydrins SK₃Albumen identical function , the variant form of SEQ ID NO.4 sequence.These variant forms include (but being not limited to): being usually 1~50 amino Missing, insertion and/or the substitution of acid, and in C-terminal and/or N-terminal addition one or be amino acid within 20.For example, In the art, when being substituted with similar nature or similar amino acid, the function of protein is not usually changed.Compare again Such as, the function of protein will not be changed by adding one or several amino acid generally also in C-terminal and/or N-terminal.The term also wraps Include Afriocan agapanthus SK₃The active fragment and reactive derivative of type dehydrin protein.

Afriocan agapanthus SK of the invention₃The variant form of type Dehydrins includes: homologous sequence, conservative variant, equipotential change Allosome, natural mutation, induced mutants, can be with Afriocan agapanthus SK under high or low high stringency conditions₃Type Dehydrins correlation DNA The encoded albumen of the DNA of hybridization and utilization Afriocan agapanthus SK₃More peptide or proteins that the antiserum of type dehydrin protein obtains.

In the present invention, " Afriocan agapanthus SK₃Type Dehydrins conservative variation's polypeptides " refer to and amino shown in SEQ ID NO.4 Acid sequence is compared, and has at most 10 amino acid to be replaced by amino acid with similar or analogous properties and form polypeptide.These are conservative Property Variant polypeptides are replaced preferably based on table 1 and are generated.

Table 1

Initial residue	Representative substitution	It is preferred to replace
			Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
			Asn(N)	Gln；His；Lys；Arg	Gln
Asp(D)	Glu	Glu
			Cys(C)	Ser	Ser
Gln(Q)	Asn	Asn
			Glu(E)	Asp	Asp
Gly(G)	Pro；Ala	Ala
			His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu；Val；Met；Ala；Phe	Leu
			Leu(L)	Ile；Val；Met；Ala；Phe	Ile
Lys(K)	Arg；Gln；Asn	Arg
			Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Leu；Val；Ile；Ala；Tyr	Leu
			Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
			Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
			Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu；Met；Phe；Ala	Leu

The invention also includes Afriocan agapanthus SK₃The analog of type dehydrin protein or polypeptide.These analogs and Afriocan agapanthus SK₃ The difference of type Dehydrins related polypeptide can be the difference on amino acid sequence, be also possible on the modified forms for not influencing sequence Difference, or have both at the same time.These polypeptides include natural or induction genetic variant.Induce variant can be by various Technology obtains, and such as generates random mutagenesis by radiating or being exposed to mutagens, can also be by known to site-directed mutagenesis or other The technology of molecular biology.Analog further includes with the similar of the residue (such as D- amino acid) different from natural L-amino acids Object, and the analog with non-naturally occurring or synthesis amino acid (such as β, gamma-amino acid).It should be understood that of the invention Polypeptide is not limited to the above-mentioned representative polypeptide enumerated.

Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetyl of internal or external polypeptide Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia Acid, phosphoserine, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimization The polypeptide of solubility property.

In the present invention, Afriocan agapanthus SK can be analyzed with the method for real-time fluorescence quantitative PCR₃Dehydrin gene is in arabidopsis Physiological effect expression pattern, i.e., analysis Afriocan agapanthus SK₃MRNA transcript of the dehydrin gene in transgenic arabidopsis is deposited Whether and quantity.

It whether there is Afriocan agapanthus SK in test sample of the present invention₃The detection method of type Dehydrins related nucleotide sequences, packet It includes and is hybridized with above-mentioned probe with sample, then whether detection probe has occurred combination.The sample is the production after PCR amplification Object, wherein PCR amplification primer corresponds to Afriocan agapanthus SK₃Type Dehydrins related nucleosides coding sequences, and the code sequence can be located at The two sides or centre of column.Primer length is generally 15~50 nucleotide.

In addition, Afriocan agapanthus SK according to the present invention₃Type Dehydrins nucleotide sequence and amino acid sequence, can be same in nucleic acid On the homology basis of source property or expression protein, Afriocan agapanthus SK is screened₃Type Dehydrins associated homologous gene or homologous protein.

In order to obtain with Afriocan agapanthus SK₃The dot matrix of type Dehydrins related gene can screen Afriocan agapanthus with DNA probe CDNA library, these probes are used under low high stringency conditions³²P is to Afriocan agapanthus SK₃The relevant all or part of type Dehydrins is put It penetrates obtained by activity mark.The cDNA library for being suitable for screening is the library from Afriocan agapanthus.Building comes from interested cell Or the method for the cDNA library of tissue is that molecular biology field is well-known.In addition, many such cDNA libraries It is commercially available, such as purchased from Clontech, Stratagene, Palo Alto, Cal..This screening technique can identify with Afriocan agapanthus SK₃The nucleotide sequence of the relevant gene family of type Dehydrins

Afriocan agapanthus SK of the invention₃Type Dehydrins associated nucleotide full length sequence or its segment can usually use PCR amplification Method, recombination method or artificial synthesized method obtain.It, can disclosed related nucleotides sequence according to the present invention for PCR amplification method Column, especially open reading frame sequence carry out design primer, and with the commercially available library cDNA or by well known by persons skilled in the art normal The library cDNA prepared by rule method expands as template and obtains related sequence.When sequence is longer, it is often necessary to carry out twice or Then the segment that each time amplifies is stitched together by multiple PCR amplification by proper order again.

After obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually by it It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, mutation can be also introduced into protein sequence of the present invention by chemical synthesis.

Other than being generated with recombination method, solid phase technique is also can be used in the segment of albumen of the present invention, passes through direct synthetic peptide Produced (Stewart et al., (1969) Solid phase peptide synthssis, WH Freeman Co., San Francisco； Merrifield J.(1963)J.Am Chem.Soc 85:2149-2154).In vitro synthetic proteins matter can by hand or from It is dynamic to carry out.For example, dynamic circuit connector can be come from the 431A type peptide synthesizer (Foster City, CA) of Applied Biosystems At peptide.Each segment of chemical synthesis albumen of the present invention can be distinguished, then chemically connected to generate point of overall length Son.

Utilize Afriocan agapanthus SK of the invention₃Type Dehydrins can be filtered out and Afriocan agapanthus by various conventional screening assays SK₃The substance or inhibitor and antagonist etc. that type Dehydrins correlation interacts.

Afriocan agapanthus ornamental value is high, is widely used, and is excellent fresh-cut flower variety, in addition most can table again in addition to rose Up to the agapanthus of love, the market demand is also increasing.

Compared with prior art, the present invention have it is following the utility model has the advantages that

The present invention clones SK in Afriocan agapanthus plant for the first time₃The coded sequence of type Dehydrins, and it is transformed into mode plant In object arabidopsis, SK is analyzed using the method for fluorescence real-time quantitative PCR₃Physiological effect of the type dehydrin gene in arabidopsis and Expression pattern, to regulate and control SK using technique for gene engineering from now on₃The spatial and temporal expression of type dehydrin gene, for improvement Afriocan agapanthus and respectively The anti-adversity ability of class ornamental flower provides foundation, has established theory for ornamental plant marker assisted selection and Germ-plasma resources protection Basis has very big application value to provide theoretical foundation in terms of for the fast numerous, breeding of new variety of body embryo.

Detailed description of the invention

Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention, Objects and advantages will become more apparent upon:

Fig. 1 is Afriocan agapanthus SK of the invention₃Type dehydrin gene and plum blossom (Prunus mume) dehydrin COR47- Homology search (GAP) result of the nucleotide sequence of like gene mRNA；

Fig. 2 is Afriocan agapanthus SK of the invention₃Type dehydrin protein and Musa acuminata (Musa acuminata subsp. Malaccensis) Homology search (FASTA) result of the amino acid sequence of dehydrin COR410 albumen, wherein identical Amino acid is marked between two sequences with amino acid monocase.

Fig. 3 is wild type (WT) and SK₃Transgenic Arabidopsis plants Phenotypic Observation under salt stress；

Fig. 4 is wild type (WT) and SK₃Transgenic Arabidopsis plants root length histogram under salt stress；

Fig. 5 is wild type (WT) and SK₃Transgenic Arabidopsis plants Weight per plant amount histogram under salt stress；

Fig. 6 is wild type (WT) and SK₃Transgenic Arabidopsis plants Phenotypic Observation under infiltration temperature stress；

Fig. 7 is wild type (WT) and SK₃Transgenic Arabidopsis plants lotus throne size histogram under osmotic stress；

Fig. 8 is wild type (WT) and SK₃Transgenic Arabidopsis plants Weight per plant amount histogram under osmotic stress；

Fig. 9 is wild type (WT) and SK₃Transgenic Arabidopsis plants Phenotypic Observation under low temperature stress；

Figure 10 is wild type (WT) and SK₃Transgenic Arabidopsis plants Phenotypic Observation under drought stress；

Figure 11 is wild type (WT) and SK₃Phenotypic Observation under gene Arabidopsis plant normal growing conditions；

Figure 12 is wild type (WT) and SK₃Transgenic arabidopsis SK₃Quantitative analysis of gene expression.

Specific embodiment

Present invention will be further explained below with reference to specific examples.These embodiments are merely to illustrate the present invention and do not have to In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as The molecular clonings such as Sambrook: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.It should be pointed out that the ordinary skill of this field For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.

Embodiment 1Afriocan agapanthus SK₃The clone of gene

1. the acquisition of vegetable material

Afriocan agapanthus (Agapanthus praecox) leaf tissue is taken, for extracting RNA.

The extracting of 2.RNA

With " RNA prep pure plant total RNA extraction reagent box " extracted total RNA (Trizol:Invitrogen), use 1% agarose electrophoresis detects the integrality of RNA, then in spectrophotometer (Thermo Scientific NANODROP The purity and concentration of RNA are measured on 1000Spectrophotometer).

3. the full-length clone of gene

The protein function annotation of (RNA-seq) is sequenced as a result, obtaining Afriocan agapanthus SK according to Afriocan agapanthus transcript profile₃Gene core Segment.It is complete that cDNA is carried out using RACE method (SMARTerTMRACE cDNA Amplification Kit:Clonetech) Long clone, divides three phases to carry out:

(1) PCR obtains gene intermediate segment

By gene core segment by carrying out BLAST (http://blast.ncbi.nlm.nih.gov/) in the website NCBI Existing database (GenBank) is compared, knows its nucleic acid sequence and coding albumen and known two fringe false bromegrass, without the hidden son grass of awns It is very high with the homology of the dehydrin gene of barley, it was initially believed that it is a dehydrin gene.

SK-S (SEQ ID NO.1): ATCAAGGAAAAGCTCGGC

SK-A (SEQ ID NO.2): TCATCGTGGCTAGCACTCT

With above-mentioned primer pair SK₃Gene core segment is expanded, and 284bp segment is obtained.It recycles and is connected to pMD18-T On Simple vector carrier, and use SK-S and SK-A as sequencing primer pair, using termination object fluorescent marker (Big- Dye, Perkin-Elmer, USA) method, be sequenced on ABI377 sequenator (Perkin-Elmer, USA).

(2)3′RACE

Using 3 ' RACE ready cDNA as template, two wheel nest-type PRCs complete the amplification of 3 ' end sequences.

The first round: UPM+3 '-GSP1 (SEQ ID NO.5):

5′-CCGCCGTGGTAACCGAACAGGA-3′

Second wheel: NUP+3 '-GSP2 (SEQ ID NO.6):

5′-CCCCGGCCACAACAAGAAGGAGG-3′

UPM and NUP provide for kit.3 ' RACE obtain Afriocan agapanthus SK₃3 ' the end sequences (505bp) of gene, recycling, It is sequenced after being connected to pMD18-T Simple vector carrier with the method for same step (1) equally.

(3)5′RACE

Using 5 ' RACE ready cDNA as template, the amplification of 5 ' end sequences is completed by two wheel nest-type PRCs,

The first round: UPM+5 '-GSP1 (SEQ ID NO.7):

5′-CGACCACGTCCTGTTCGGTTACCAC-3′

Second wheel: NUP+5 '-GSP2 (SEQ ID NO.8):

5′-GCGGCGGTTTCTTCTTCTTTCTCGT-3′

UPM and NUP provide for kit.5 ' RACE obtain Afriocan agapanthus SK₃5 ' the end sequences (444bp) of gene, recycling It is sequenced after connection with the method for same step (1) equally.

The sequencing result for the sequence that 1-3 method through the above steps obtains is spliced, splicing sequence is submitted into BLAST Analysis, as a result proves that the Dehydrin gene newly obtained from Afriocan agapanthus is the relevant gene of a dehydrin protein really, will Be sequenced splicing result combination NCBI ORF (Open Reading Frame) Finder (https: // Www.ncbi.nlm.nih.gov/orffinder/ it) predicts, it was found that Afriocan agapanthus SK₃The initiation codon of gene and termination are close Numeral, and Afriocan agapanthus SK has been determined₃The area ORF of gene.According to the sequence of acquisition, respectively from initiation codon and terminator codon Place's design specific primer, expands the area ORF:

ORF-S (SEQ ID NO.9): 5 '-ATGGCAGAGGAGAATGTGGA-3 ',

ORF-A (SEQ ID NO.10): 5 '-CTAATGAGCCTTCTCGGTCTC-3 '.

PCR is carried out by template of Afriocan agapanthus cDNA, amplification obtains 648bp coding Afriocan agapanthus SK₃Type dehydrin protein it is complete Long code sequence (SEQ ID NO.3).

Embodiment 2, Afriocan agapanthus SK₃The sequence information and homology analysis of gene

Afriocan agapanthus SK of the invention₃Full length gene opening code-reading frame sequence is 648bp, and detailed sequence is shown in SEQ ID NO.3 Shown sequence.Afriocan agapanthus SK is derived according to opening code-reading frame sequence₃The amino acid sequence of type dehydrin protein, totally 215 ammonia Base acid residue, molecular weight 23.7232kDa, isoelectric point (pI) are 4.79, and detailed sequence is shown in sequence shown in SEQ ID NO.4；

By Afriocan agapanthus SK₃The opening code-reading frame sequence of gene and its amino acid sequence blast program for encoding albumen exist Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+ Nucleotide and protein homology search are carried out in PDB+SwissProt+Superdate+PIR database, as a result, it has been found that its On nucleotide level with plum blossom (Prunus mume) dehydrin COR47-like gene (accession number: XM_ 008222942.1) there is 84% consistency, as shown in Figure 1 (Query: Afriocan agapanthus SK₃Coding gene sequence；Sbjct: plum Flower dehydrin COR47-like gene mRNA sequence)；On amino acid levels, with fruitlet open country any of several broadleaf plants COR410 (accession number: XP_009382058.1), consistency 56%, as shown in Figure 2 (Query: Afriocan agapanthus SK₃Amino acid sequence；Sbjct: fruitlet is wild Any of several broadleaf plants COR410 protein amino acid sequence).

ClustalX compares that show that each sequence in Y/S/K guards homology except domain lower, but inside conservative domain then Higher, the Y/S/K function fragment for showing dehydrin protein has the conservative of height.

It can be seen that Afriocan agapanthus SK₃The Dehydrin gene of gene and other known species is on nucleic acid and protein level All there is higher homology.

Embodiment 3, Afriocan agapanthus SK₃Genetic transformation model plant arabidopsis

1. (the Afriocan agapanthus SK containing target gene₃Gene) expression vector building

According to Afriocan agapanthus SK₃Full length gene coded sequence (SEQ ID NO.3), design amplification is in completely coding reading frame Primer, and introduce restriction endonuclease sites (depending on the carrier selected) respectively in upstream and downstream primer, it is carried with building expression Body.Using the amplified production obtained in embodiment 1 as template, after PCR amplification, by Afriocan agapanthus SK₃The coding region sequence of gene connects It is connected in intermediate vector (such as pMD19-T) and is sequenced, then correct Afriocan agapanthus SK will be sequenced₃The coding region sequence of gene is into one Step is cloned into expression vector (such as pHB), is transferred in Agrobacterium tumefaciems (such as under the premise of identifying that reading frame is correct GV3101 PCR identification), and to the Agrobacterium after conversion is carried out, to guarantee to contain Afriocan agapanthus SK₃The plant expression vector of gene at Function is transformed into Agrobacterium tumefaciems.

2. Agrobacterium-Mediated Transformation arabidopsis

(1) shake Agrobacterium in advance: choose positive monoclonal to 25ml kanamycins containing 50mg/L, 50mg/L gentamicin, In the YEP fluid nutrient medium of 25mg/L rifampin, 28 DEG C, 200rpm shakes bacterium for 24 hours；

(2) spread cultivation Agrobacterium: the Agrobacterium bacterium solution shaken in advance is spread cultivation with 1:100 to the YEP of kalamycin resistance containing 400mL In culture medium, 28 DEG C, 200rpm, 13~16h is cultivated, culture reaches to absorbance OD600 receives bacterium between 1.5-2.0, receive bacterium item Part is 23 DEG C, 5000rpm, 8min；

(3) transformed plant: (need before conversion one day or the conversion same day cut off silique all on plant and it is in full bloom with And the little Hua to show money or valuables one carries unintentionally) the 1/2MS solution that 500mL contains 5% sucrose is prepared, and the 6BA (100mg/L) and 200 μ of 50 μ L is added LSilwet L-77 forms Agrobacterium-mediated Transformation buffer.The Agrobacterium for taking appropriate conversion buffer solution to collect step (2) is heavy Shallow lake hangs, and after shaking up, base of the plant and inflorescence are impregnated 1min in the bacterium solution, takes out drain bacterium solution later, use is lighttight Black plastic bag wraps up plant, is protected from light culture and takes out plant afterwards for 24 hours, normal to cultivate, and can be infected again after a week.

3. the screening of transgenic positive strain

Sowing after the whole maturations of plant silique after to be transformed, is placed at room temperature for one in the desiccation culture ware for being lined with filter paper Week keep seed all dry, sieve filter seed with the stainless steel of 50 mesh later, remove silique, collection transgenosis T0 for seed simultaneously It is seeded in hole tray, carries out Resistance of Seedling screening with 0.05% (v/v) glyphosate, obtain T1 for transgenic plant, lasting screening Until obtaining T2 for transgenic plant.

4. transgenic Arabidopsis plants SK₃Gene expression difference

Arabidopsis wild type and T2 are sheared for SK₃The blade 0.2g of transgenic plant extracts RNA, preparation cDNA and carries out Real-time PCR Analysis.SK in Real-time PCR₃The specific primer of gene quantification analysis are as follows:

RtSK-S (SEQ ID NO.11): 5 '-AAGAGCCAAGAGGAGGTT-3 ',

RtSK-A (SEQ ID NO.12): 5 '-CTTCTTCTCGCCGTCTTC-3 '.

Reference gene is arabidopsis UBQ5 gene, primer are as follows:

UBQ5-F (SEQ ID NO.13): 5 '-GACGCTTCATCTCGTCC-3 ',

UBQ5-R (SEQ ID NO.14): 5 '-CCACAGGTTGCGTTAG-3 '.

It is carried out by primer pair wild type and transgenic arabidopsis cDNA of SEQ ID NO.11 and SEQ ID NO.12 After RT-PCR, using 2^-△△CtMethod makees relative quantitative assay, the results showed that SK in transgenic arabidopsis₃Expression quantity it is higher, be interior 7.10 times for joining gene UBQ5, the SK in wild type₃(Figure 12) is not expressed.

Embodiment 4, arabidopsis SK₃Transgenic plant environment stress Phenotypic Observation

By wild type and T2 for SK₃Transgenic Arabidopsis plant is transferred to respectively after growing 3d on 1/2MS culture medium Added with the salt stress culture medium of 100mM, 150mM, 200mM sodium chloride, and added with the 1/ of 300mM, 400mM, 500mM mannitol On 2MS Thief zone coercing cultivation base, salt stress and osmotic stress processing are carried out respectively.22 DEG C of temperature, 6000 lux of light intensity, After growing in the dark growth cabinet in 16 hours photoperiods, illumination/8 hour, photograph taking and Relevant phenotype index are carried out Measurement statistics.Wild type and T2 are sowed in the soil simultaneously for SK₃Transgenic arabidopsis carries out arid and low temperature stress processing Comparison and observe phenotype.

As shown in Fig. 3-5 the result shows that, SK₃Transgenic arabidopsis shows preferable under NaCl Stress processing Anti-adversity ability.Under the conditions of Different stress, transgenosis and each 20 plants of progress phenotype index determining of wildtype Arabidopsis thaliana are taken respectively.It is real Test the result shows that, after Stress treatment 10d, under 150mM NaCl stress, transgenic arabidopsis root length (Fig. 4) and single Plant weight amount (Fig. 5) is obviously improved compared with WT lines；After Stress treatment 5d, under 200mM NaCl salt stress, transgenic Arabidopsis survival rate (40%) is higher than wild type (10%)；As shown in Fig. 6-8 the result shows that, under mannitol Stress treatment, Transgenic plant phenotype is equally better than WT lines.Under the conditions of Different stress, transgenosis and wildtype Arabidopsis thaliana are taken respectively Each 20 plants of progress phenotype index determining.The experimental results showed that after Stress treatment 10d, under 300mM mannitol stress, transgenosis Type arabidopsis lotus throne size (Fig. 7) and single plant weight (Fig. 8) are obviously improved compared with WT lines.

For low temperature stress, transgenic and wildtype Arabidopsis thaliana after normal growth 4 weeks, are put into 8000 Le gram in the soil This light intensity grows 9d (Fig. 9) in 4 DEG C of low temperature incubators.From the result of Fig. 9 as it can be seen that compared with wildtype Arabidopsis thaliana, SK₃Turn base It is stronger to the tolerance of low temperature environment because arabidopsis growing way is preferable.

For drought stress, transgenic and wildtype Arabidopsis thaliana in the soil normal growth cut off the water supply after 4 weeks 7d, 10d, Then 20d carries out rehydration (Figure 10).From the result of Figure 10 it is found that stress group is compared with the control group normally to water, wild type is planted Strain shows apparent stress damage, and seedling development is suppressed.SK₃Transgenic arabidopsis and wildtype Arabidopsis thaliana phase Than drought-resistant ability is obviously improved, and compared with the control group of normal irrigation, growth situation is influenced smaller by drought stress.Table Bright SK₃Gene has positive effect for the resistance promoted under arabidopsis drought stress.

Meanwhile in normal circumstances (as shown in figure 11), SK₃Transgenic arabidopsis grows speed compared with wildtype Arabidopsis thaliana Degree is obviously improved, and shows apparent early floral formation.

Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned Particular implementation, those skilled in the art can make a variety of changes or modify within the scope of the claims, this not shadow Ring substantive content of the invention.In the absence of conflict, the feature in embodiments herein and embodiment can any phase Mutually combination.

SEQUENCE LISTING

<110>Shanghai Communications University

<120>Afriocan agapanthus SK3 type dehydrin protein and its encoding gene and probe

<130> DAG28909

<160> 14

<170> PatentIn version 3.3

<210> 1

<211> 18

<212> DNA

<213> artificial sequence

<400> 1

atcaaggaaa agctcggc 18

<210> 2

<211> 19

<212> DNA

<213> artificial sequence

<400> 2

tcatcgtggc tagcactct 19

<210> 3

<211> 1057

<212> DNA

<213> artificial sequence

<400> 3

acatggggat caattattgt ttccatcagc ttaatttgtg ttgagagaga ggaaagtttg 60

gcttatattt ttttcattga agtttataat taattaaaaa aatggcagag gagaatgtgg 120

aggtgagtga gagagggttg tttggtttcg tggggaagaa ggaagagaaa gaggagaaga 180

gccaagagga ggttctcgtc gccggagtcg agagcttgaa agttgaggag gcgaagaaag 240

aagaagttaa caaggagggg ctttttgata aattgcaccg atctcatagc tctagctctt 300

cgagcgacga agaggaagtg ggcgaagacg gcgagaagaa gaagaagaag aagaagaagg 360

gaataaagga gaagatcaag gaaaagctcg gccgcgacga gaaagaagaa gaaaccgccg 420

ccgccgtggt aaccgaacag gacgtggtcg tcgcggccgc ggccgccgaa gcggaagata 480

cgaccgtcgt cgtggagaag atcgaggaga ccgtcgtcgt ggagaagatc gaggaggaag 540

agaagaaagg gttcctcgat aagatcaaac agaagctccc cggccacaac aagaaggagg 600

ctgccgccgc cgccgaagtc gaggcgccgg cggcgaagga gagtgctagc cacgatgaag 660

gaggggagaa gaagggcatt ttcggaaaga tcatggacaa gataccaggg tatcacaagg 720

aggacaagga gaccgagaag gctcattaga ggaggttatt aattatgtgt cgctgtttat 780

tttatgtgtg tatgttttga atattaaatg tttgtgttga tcgagtgagt gctttggtta 840

ctgttttgtt tttgatttgt tagggttgtt tctttagtaa gttgagcatg caggatgtgt 900

atggagcttg cttttttgtg cgtggcaaca atcattttgt gttttaaaat gatgagagat 960

ggagtttgga gctaggttat gaatgaatgg ctggtgttga tgtatttttc catcgtggat 1020

aaatgtttta caaaaaaaaa aaaaaaaaaa aaaaaaa 1057

<210> 4

<211> 215

<212> PRT

<213> artificial sequence

<400> 4

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

1 5 10 15

Val Gly Lys Lys Glu Glu Lys Glu Glu Lys Ser Gln Glu Glu Val Leu

20 25 30

Val Ala Gly Val Glu Ser Leu Lys Val Glu Glu Ala Lys Lys Glu Glu

35 40 45

Val Asn Lys Glu Gly Leu Phe Asp Lys Leu His Arg Ser His Ser Ser

50 55 60

Ser Ser Ser Ser Asp Glu Glu Glu Val Gly Glu Asp Gly Glu Lys Lys

65 70 75 80

Lys Lys Lys Lys Lys Lys Gly Ile Lys Glu Lys Ile Lys Glu Lys Leu

85 90 95

Gly Arg Asp Glu Lys Glu Glu Glu Thr Ala Ala Ala Val Val Thr Glu

100 105 110

Gln Asp Val Val Val Ala Ala Ala Ala Ala Glu Ala Glu Asp Thr Thr

115 120 125

Val Val Val Glu Lys Ile Glu Glu Thr Val Val Val Glu Lys Ile Glu

130 135 140

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

145 150 155 160

Gly His Asn Lys Lys Glu Ala Ala Ala Ala Ala Glu Val Glu Ala Pro

165 170 175

Ala Ala Lys Glu Ser Ala Ser His Asp Glu Gly Gly Glu Lys Lys Gly

180 185 190

Ile Phe Gly Lys Ile Met Asp Lys Ile Pro Gly Tyr His Lys Glu Asp

195 200 205

Lys Glu Thr Glu Lys Ala His

210 215

<210> 5

<211> 22

<212> DNA

<213> artificial sequence

<400> 5

ccgccgtggt aaccgaacag ga 22

<210> 6

<211> 23

<212> DNA

<213> artificial sequence

<400> 6

ccccggccac aacaagaagg agg 23

<210> 7

<211> 25

<212> DNA

<213> artificial sequence

<400> 7

cgaccacgtc ctgttcggtt accac 25

<210> 8

<211> 25

<212> DNA

<213> artificial sequence

<400> 8

gcggcggttt cttcttcttt ctcgt 25

<210> 9

<211> 20

<212> DNA

<213> artificial sequence

<400> 9

atggcagagg agaatgtgga 20

<210> 10

<211> 21

<212> DNA

<213> artificial sequence

<400> 10

ctaatgagcc ttctcggtct c 21

<210> 11

<211> 18

<212> DNA

<213> artificial sequence

<400> 11

aagagccaag aggaggtt 18

<210> 12

<211> 18

<212> DNA

<213> artificial sequence

<400> 12

cttcttctcg ccgtcttc 18

<210> 13

<211> 17

<212> DNA

<213> artificial sequence

<400> 13

gacgcttcat ctcgtcc 17

<210> 14

<211> 16

<212> DNA

<213> artificial sequence

<400> 14

ccacaggttg cgttag 16

Claims

1. a kind of Afriocan agapanthus SK₃Type dehydrin protein, characterized in that the protein including following (a):

(a) protein being made of the amino acid sequence as shown in SEQ ID NO.4.

2. Afriocan agapanthus SK described in a kind of coding claim 1₃The nucleic acid sequence of type dehydrin protein.

3. encoding the Afriocan agapanthus SK as claimed in claim 2₃The nucleic acid sequence of type dehydrin protein, characterized in that the core Acid sequence specifically:

(a) base sequence is as shown in SEQ ID NO.3 the 1st~648；

Or (b) with SEQ ID NO.3 the 1st~648 shown in nucleic acid have at least 70% homology sequence.

4. a kind of amplification Afriocan agapanthus SK as claimed in claim 2₃The specific primer pair of the nucleic acid sequence of type dehydrin protein, It is characterized in, the primer pair is as follows:

ORF-S (SEQ ID NO.9): 5 '-ATGGCAGAGGAGAATGTGGA-3 ',

ORF-A (SEQ ID NO.10): 5 '-CTAATGAGCCTTCTCGGTCTC-3 '.

5. a kind of Afriocan agapanthus SK as described in claim 1₃The application of type dehydrin protein encoding gene, which is characterized in that described For the base sequence of gene as shown in SEQ ID NO.3 the 1st~648, the application includes improving plant stress-resistance ability.