CN108841837A - Application of the encoding gene of arabidopsis splicing factor SR45a spliceosome in negative regulation plant salt stress response - Google Patents

Abstract

The invention discloses application of the encoding gene of arabidopsis splicing factor SR45a spliceosome in negative regulation plant salt stress response, or the application in the plant that preparation/cultivation has anti-salt property；The encoding gene is AT1G07350.1, and AT1G07350.2, AT1G07350.3, one of AT1G07350.4, nucleotide sequence is successively as shown in SEQ ID NO.1,2,3,4.The present invention has cloned four kinds of spliceosome encoding genes of SR45a from arabidopsis, and it studies and demonstrates four kinds of spliceosome overexpression strains in the effect of Their Seed Germinating Period, seedling development early stage and seedling stage negative regulation plant salt resistance, through testing, by overexpressing tetra- kinds of spliceosome encoding genes of SR45a, certain salt stress hypersensitivity is shown compared with wild type in plantlet stage.The present invention provides theoretical foundation and gene source to cultivate the crops of salt stress-resistant.

Description

The encoding gene of arabidopsis splicing factor SR45a spliceosome is coerced in negative regulation plant salt Compel the application in response

Technical field

The present invention relates to tetra- kinds of spliceosomes of arabidopsis splicing factor SR45a and its encoding gene in negative regulation plant salt stress Application in response, belongs to molecular biology and field of biotechnology.

Background technique

Plant in nature can be broadly divided into biotic and Fei Sheng by various stress in growth and development process Object stress, it mainly includes low that wherein abiotic stress, which mainly covers various environment-stress as a kind of stress widest in area, Temperature, high temperature, Saline Alkali Stress and drought stress etc.；Wherein influence of the salt stress to plant growth is very serious, and the whole world is about There are 13,000,000,000 hm²Land, wherein 3,000,000,000 hm²For salt-soda soil.China is with industrialized fast-developing, organic chemical fertilizer and agriculture at present The reasons such as the abuse of medicine cause the salinization of soil in arable land to be on the rise, and have constituted larger threat to production, how to improve plant Salt resistance ability have very important significance to the development of China's economy agricultural.

Salt stress be mainly by the soil exist be higher than the be resistant to concentration of plant normal level soluble-salt and The abiotic stress of formation.Many salt ions present in soil can all damage plant, and sodium salt is used as and causes salt stress Most important salt, it is most representative in the research of plant reply salt stress.The salt of soil middle and high concentration, such as Na⁺And Cl^-Meeting It causes the flow of water in soil to decline, so as to cause plant moisture missing or by osmotic stress, and then leads to plant itself system The growth and development process of column is impaired, among these mainly includes the following aspects：(1) it causes plant growth and development slow, influences The normal development of the histoorgans such as plant roots, stem, leaf；(2) inhibit the normal photosynthesis of plant, salinity is by reducing plant Photosynthetic area and the reduction for causing plant carbon assimilation quantity, influence the synthesis of photosynthetic pigments in plant leaf；(3) inhibit protein and The synthesis of lipid causes the lipid component in the plasma membrane of plant root to change under condition of salt stress, reduces plant to stress Tolerance；(4) activity and synthesis of antioxidase in plant are influenced, SOD, POD, CAT activity are by salt stress in plant Influence so that in plant Scavenger of ROS ability decline.It is influenced caused by plant for salt stress, research plant rings It answers the molecular mechanism of salt stress to be very important, salt tolerance genetically modified plants is obtained by Protocols in Molecular Biology and are promoted Into industrial crops.

During responding environment stress, its own gene can be adjusted plant by transcriptional level and translation skill Control, and alternative splicing is exactly a kind of mode regulated and controled on post-transcriptional level.The deep shadow of the alternative splicing of stress-inducing The expression of loud expression and the gene for encoding spliceosome component with stress-related genes.There is 61% introne to contain in arabidopsis There is gene to undergo alternative splicing (Marquez et al.2012), and transcript profile sequencing data analysis finds that abiotic stress can Significantly change the intracorporal alternative splicing events of plant (Filichkin et al.2010；Thatcher et al.2016；Zhang et al.2016；Zhu et al.2017).For example, salt stress can make in arabidopsis, the variable of a gene is cut more than 6000 Part of taking over a job changes (Feng et al.2015)；And high temperature stress can change in grape more than 1000 a gene can Become montage event (Jiang et al.2017).Research finds that these abiotic stress (salt stress, ABA) can promote gene The selection of the upper non-classical splice site of pre-mRNA, changing for this splice site selection may be by the multiplicity of promotion transcript profile Property cope with stress, and there is research to have been proven that the key regulator in some stress response approach passes through alternative splicing Functional and non-functional transcript is generated, two kinds of transcripts adjust the table of related gene by adjusting the ratio of itself Up to so that plant responding is coerced.And the alternative splicing process after genetic transcription is then mainly that splicing complex mediates completion, montage Complex mainly forms (Koncz et by 5 kinds of micronuclear ribonucleoproteins (snRNPs) and non-micronuclear ribonucleoprotein Al.2012), the SR albumen and SR-like protein family rather than in micronuclear ribonucleoprotein are then main as montage regulatory factor It ensure that the correct identification of the correct assembly and splice site of splicing complex.Montage regulatory factor is in addition in alternative splicing process In play a significant role outer, more and more evidences show that these montage regulatory factors also play in plant responding stress procedure Very important effect.The alternative splicing existing defects of IRT1 gene are in splicing factor defect mutant sr34b to reduce The resistance (Zhang et al.2014) that sr34b coerces Cd；The deletion mutant of splicing factor RS40 and RS41 are shown The hypersensitivity of ABA and salt stress, and the alternative splicing of many stress-related genes changes in rs40, rs41, thus Guess that RS40, RS41 have participated in the alternative splicing (Chen et al.2013) of gene under Abiotic stress conditions；SNW/ The identification that Ski interaction Protein S KIP and montage regulatory factor SR45 interaction passes through the adjusting splice site of related gene 5 ' and 3 ' Or montage adjusts the alternative splicing of related gene in turn, makes plant responding stress response (Wang et al.2012).In quasi- south There is also alternative splicings for splicing factor having in mustard itself, and the alternative splicing of itself changes and then influences after by stress-inducing The alternative splicing function of itself bringing into normal play, changes the alternative splicing of downstream target gene.Such as two kinds of spliceosomes of SR45 in sr45 The ratio of both SR45.1 and SR45.2 is significantly lower than the ratio both in wild type, and this difference is existing for the glucose In the case of significantly increase, research it has also been found that SR45 regulation inositol polyphosphate enzyme 5PTase13 gene alternative splicing, 5PTase13 The stability that ABA is just adjusting factor S nRK1 can be combined and adjust, therefore SR45 participates in having regulated and controled ABA signal pathway again, most terminates Fruit proves that SR45 can be by inhibiting the ABA accumulation of glucose induction to adjust Sugar signal access (Carvalho et come negative al.2016)。

SR45a is distinctive a kind of splicing factor in plant, belongs to SR-like protein family member (Tanabe et al.2007).SR protein family (Lopato et al.1996 in arabidopsis containing 19 seed types；Golovkin and Reddy 1998；Golovkin and Reddy 1999；Lopato et al.2002；Lorkovic and Barta 2002； Ali et al.2003；Tillemans et al.2005), there is SR protein family member one or two RNA to identify base Sequence (RRM) is located at N-terminal, and then contains the RS structural domain (Zuo for being rich in serine/arginine (S/R) repetitive sequence in C-terminal and Manley1994；Manley and Tacke 1996；Graveley 2001；Reddy 2004), in RNA spliceosome (Manley and Tacke 1996 is played an important role during assembling and alternative splicing；Valcarcel and Green 1996).Most of SR albumen is the necessary factor of existence, passes through its RS structural domain and distinctive other structures Domain realizes that the correct of splice site is completed in the interaction with the specific sequence or other splicing factors of Pre-mRNA, collaboration Selection or the formation (Graveley and Maniatis 1998) for promoting spliceosome, and SR albumen is plant response environment condition Key regulator.SR albumen generally has three point features in mammals：(1) it can be identified by monoclonal antibody mAb104； (2) all there are two types of design features altogether：Motif (RRM) is identified containing one or more RNA；Contain a C-terminal RS domain；Most of albumen play a significant role in RNA montage, assembly and metabolism.And there are two types of SR-like albumen generally has Feature is different from SR albumen：(1) generally lack some design features in SR albumen, for example do not identified by mAb104, lack spy Fixed RRM structural domain；(2) though containing RS domain, this domain not only enrichment thread Serine/Arginine (S/R), Often it is enriched with arginine/glutamic acid (R/E), aspartic acid/arginine (D/R) dipeptide structure (Fu 1995；Neugebauer et al.1995；Manley and Tacke 1996；Blencowe et al.1999).SR45a is as a kind of typical case in arabidopsis SR-like albumen (Tanabe et al.2007), respectively contain a RS structural domain in N-terminal and C-terminal and centre tied by RRM Structure domain separates, and contains 6 kinds of spliceosomes (atSR45a-1a-e and atSR45a-2) according to it is recorded on TAIR, wherein RS structural domain (Tanabe et al.2007) (Tanabe et of these four spliceosomes of atSR45a-1b-e shortage C-terminal al.2009).It is compound with montage respectively to have been reported that an important component for having been proven that SR45a as splicing complex passes through Body core factor U2AF³⁵B, U1-70K interacts, and then influences montage efficiency (the Tanabe et of pre-mRNA al.2009).And SR albumen equally plays a very important role during plant responding environment stress.The response of SR albumen The expression of gene can be adjusted by influencing the selection of gene splicing site in stress procedure, itself can pass through alternative splicing again Generate the alternative splicing that a variety of spliceosomes play corresponding function controlling target gene.

Currently, studies have found that the induction that SR45a is coerced by bloom, the alternative splicing by regulating and controlling related gene participate in high Light stress response process (Yoshimura et al.2011).And research discovery SR45a aba2-1 in ABA defect mutant Middle expression quantity obviously raises, and contains ABA response element in the upstream SR45a, thus it is speculated that SR45a probably participates in regulation ABA Signal path (Cruz et al.2014).And research is the absence of so far for SR45a response salt stress response, and for Between salt stress and alternative splicing connection this respect research or it is fewer；Therefore the salt side of body is responded by research SR45a The process of compeling, alternative splicing and salt stress are connected, and the vacancy for making up this respect research is very important.

Summary of the invention

For the above-mentioned prior art, the present invention provides four kinds of spliceosomes of arabidopsis splicing factor SR45a and its codings A kind of new application of gene --- the application in negative regulation plant salt stress response, the present invention also provides its expression vectors.

The present invention is achieved by the following technical solutions：

The encoding gene of four kinds of spliceosomes of arabidopsis splicing factor SR45a：AT1G07350.1, AT1G07350.2, AT1G07350.3, AT1G07350.4, nucleotide sequence is successively as shown in SEQ ID NO.1,2,3,4.

Four kinds of spliceosomes of arabidopsis splicing factor SR45a, amino acid sequence is successively such as the institute of SEQ ID NO.5,6,7,8 Show.

Four kinds of montages of the encoding gene, arabidopsis splicing factor SR45a of arabidopsis splicing factor tetra- kinds of spliceosomes of SR45a Application of the body in negative regulation plant salt stress response, the application in the plant that preparation/cultivation has anti-salt property.The present invention By the study found that tetra- kinds of spliceosomes of arabidopsis splicing factor SR45a overexpress the sprouting of strain seedling under condition of salt stress Rate is slower than wild type, and seedling stage shows the big phenotype of trophosome compared with wild type, and after salt stress processing It shows to salt stress hypersensitivity.The present invention provides the support of gene and technology for crops especially salt tolerance breeding.

A kind of plant expression vector, in the encoding gene containing tetra- kinds of spliceosomes of above-mentioned arabidopsis splicing factor SR45a At least one.Preferably, plasmid used in the plant expression vector is PBI121.

A kind of genetically engineered host cell, it is quasi- containing being inserted in above-mentioned plant expression vector or its genome At least one of the encoding gene of southern tetra- kinds of spliceosomes of mustard splicing factor SR45a.

The construction method of the genetically engineered host cell is conventional technical means, and plant expression vector is imported place Chief cell (can be by the conventional biology methods such as mediated by agriculture bacillus, Ti-plasmids, plant viral vector, microinjection, particle gun Any one or more method combination, be integrated into it in arabidopsis gene group, pass through breeding and obtain homozygous transgenosis Overexpress strain), make at least one in plant expression vector/arabidopsis splicing factor tetra- kinds of spliceosomes of SR45a encoding gene Kind effective expression in host cell.

Above-mentioned plant expression vector, genetically engineered host cell are in the plant that preparation/cultivation has anti-salt property Application in application.The plant includes arabidopsis.

The present invention passes through the gene that biochip technology combination RT-PCR screening alternative splicing is regulated and controled by salt stress first, It was found that the alternative splicing of SR45a responds salt stress.The present invention extracts arabidopsis total serum IgE, reverse transcription by synthesis arabidopsis cDNA First chain cDNA is obtained, using arabidopsis cDNA as template, according to tetra- kinds of spliceosome coding gene sequence design primers of SR45a (SEQ ID NO.9,10 shown in) carries out PCR amplification, recycling and purifying pcr amplification product, and is sequenced.Obtain arabidopsis SR45a The encoding gene of four kinds of spliceosomes.The nucleotides sequence of tetra- kinds of spliceosomes of SR45a is listed in overexpression in arabidopsis by the present invention, knot Fruit shows that the transgenic line expression quantity of four kinds of spliceosomes is above wild type.Pass through tetra- kinds of spliceosome overexpressions of observation SR45a Growing state of the strain seedling under condition of salt stress finds that its sprouting rate of the overexpression strain of four kinds of spliceosomes is slower than open country Raw type, and seedling stage shows the big phenotype of trophosome compared with wild type, and shows to coerce salt after salt stress processing Compel hypersensitivity.The present invention provides the support of gene and technology for crops especially salt tolerance breeding.

All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and Phrase is with well known to a person skilled in the art general senses.The term and phrase referred to if any inconsistent with common art-recognized meanings, The meaning that the present invention of being subject to is stated.

Detailed description of the invention

Fig. 1：Expand the PCR products electrophoresis map of tetra- kinds of spliceosome encoding genes of SR45a.

Fig. 2：Tetra- kinds of spliceosomes of SR45a overexpression strain seeds and wildtype Arabidopsis thaliana seed normal 1/2MS culture medium, Sprouting situation (culture 7 days) on 200mM NaCl 1/2MS culture medium, wherein A：Normal 1/2MS culture medium；B：200mM NaCl1/2MS culture medium.WT represents wildtype Arabidopsis thaliana seed, and AS1, AS2, AS3, AS4 respectively represent tetra- kinds of spliceosomes of SR45a Overexpress strain.

Fig. 3：Tetra- kinds of spliceosomes of SR45a overexpression strain seeds and wildtype Arabidopsis thaliana seed normal 1/2MS culture medium, The statistical conditions (culture 7 days) sprouted on 200mM NaCl 1/2MS culture medium, wherein A：Normal 1/2MS culture medium；B： 200mM NaCl 1/2MS culture medium.WT represents wildtype Arabidopsis thaliana seed, and AS1, AS2, AS3, AS4 respectively represent SR45a tetra- Kind spliceosome overexpresses strain.

Fig. 4：Tetra- kinds of spliceosomes of SR45a overexpression strain seeds and wildtype Arabidopsis thaliana seed normal 1/2MS culture medium, Sprouting situation (culture 10 days) on 300mM mannitol 1/2MS culture medium, wherein A：Normal 1/2MS culture medium；B：200mM NaCl 1/2MS culture medium.WT represents wildtype Arabidopsis thaliana seed, and AS1, AS2, AS3, AS4 respectively represent tetra- kinds of montages of SR45a Body overexpresses strain.

Fig. 5：Tetra- kinds of spliceosomes of SR45a overexpression strain seeds and wildtype Arabidopsis thaliana seed normal 1/2MS culture medium, The statistical conditions (culture 10 days) sprouted on 300mM mannitol 1/2MS culture medium, wherein A：Normal 1/2MS culture medium；B： 200mM NaCl 1/2MS culture medium.WT represents wildtype Arabidopsis thaliana seed, and AS1, AS2, AS3, AS4 respectively represent SR45a tetra- Kind spliceosome overexpresses strain.

Fig. 6：Tetra- kinds of spliceosome overexpression strain seeds of SR45a and wildtype Arabidopsis thaliana two weeks big seedlings are through 200mM NaCl The comparison photo of survival rate after processing, wherein A：Before pouring salt water；B：After pouring salt water.WT represents wildtype Arabidopsis thaliana seed, AS1, AS2, AS3, AS4 respectively represent tetra- kinds of spliceosome overexpression strains of SR45a.

Fig. 7：Tetra- kinds of spliceosome overexpression strain seeds of SR45a and wildtype Arabidopsis thaliana two weeks big seedlings are through 200mM NaCl The comparison of survival rate after processing, wherein A：Before pouring salt water；B：After pouring salt water.WT represents wildtype Arabidopsis thaliana seed, AS1, AS2, AS3, AS4 respectively represent tetra- kinds of spliceosome overexpression strains of SR45a.

Fig. 8：Tetra- kinds of spliceosomes of SR45a overexpress strain seeds compared with wildtype Arabidopsis thaliana seedling trophosome size, In, WT represents wildtype Arabidopsis thaliana seed, and AS1, AS2, AS3, AS4 respectively represent tetra- kinds of spliceosome overexpression strains of SR45a.

Fig. 9：Tetra- kinds of spliceosome overexpression strain seeds of SR45a and wildtype Arabidopsis thaliana seedling trophosome fresh weight count, In, WT represents wildtype Arabidopsis thaliana seed, and AS1, AS2, AS3, AS4 respectively represent tetra- kinds of spliceosome overexpression strains of SR45a.

Specific embodiment

Below with reference to embodiment, the present invention is further illustrated.However, the scope of the present invention is not limited to following realities Apply example.One of skill in the art, can be to the present invention it is understood that under the premise of without departing substantially from the spirit and scope of the present invention Carry out various change and modification.

The present invention carries out general and/or specific description to the material and test method arrived used in test.Though So many materials and operating method used in purpose are it is known in the art that still the present invention is still herein to realize the present invention It is described in detail as far as possible.

Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..

Biological material source：

Seed of Colombia's type arabidopsis from this laboratory long-term preservation.

Escherichia coli (E.coli) TOP10 is purchased from TransGen Biotech company.

Carrier PBI121 is purchased from Clontech company.

Reagent source：

Cloning vector pEasy-Blunt simple Cloning kit is purchased from the golden biology (TransGen of the full formula in Beijing Biotech) Technology Co., Ltd..

DNA gel QIAquick Gel Extraction Kit is purchased from U.S. Omega Bio-Tek company.

Plasmid extraction kit, TRIzol reagent (9109), reverse transcription reagent box (RR047A), DNA Marker DL2502+2504 is purchased from Shanghai JaRa (Generay) bioengineering Co., Ltd.

EVO archaeal dna polymerase only praises (Vazyme) Biotechnology Co., Ltd purchased from Nanjing promise.

T4DNA ligase is purchased from silent winged generation that (Thermo fisher) Science and Technology Ltd. of match.

Description of equipment：

Agarose gel electrophoresis uses UVP GelDoc-It gel analysis system, is purchased from U.S. UVP company.

PCR reaction uses 1124310193LabCycler gene-amplificative instrament, is purchased from SensoQuest GmbH company of Germany.

Agarose gel electrophoresis uses DYY-60 type electrophoresis apparatus, is purchased from Beijing Liuyi Instrument Factory.

Embodiment 1:The clone of arabidopsis splicing factor SR45a tetra- kinds of splicing factor encoding genes AS1, AS2, AS3, AS4

(1) synthesis of Arabidopsis thaliana Seedlings cDNA：Arabidopsis thaliana Seedlings total serum IgE is extracted, reverse transcription obtains first chain cDNA；

(2) PCR amplification of tetra- kinds of splicing factor AS1, AS2, AS3, AS4 genes of SR45a：Using Arabidopsis thaliana Seedlings cDNA as mould Plate carries out PCR amplification, recycles and purify and is of corresponding size according to the gene order design primer of tetra- kinds of splicing factors of SR45a Pcr amplification product, and be sequenced.Primer is

AS forward primer:5'-GGATCCATGGGGAAACGTGAAATTCA-3'(SEQ ID NO.9).

AS reverse primer：5'-GTCGACTAGAGACTGTTATGGGCTGA-3'(SEQ ID NO.10).

PCR reaction system and amplification condition such as table 1.

Table 1

PCR program setting is:95 DEG C of initial denaturation 5min；95 DEG C of denaturation 15s, 55 DEG C of annealing 15s, 72 DEG C of extension 30s, 35 Circulation；Extend 5min, 16 DEG C of preservations after 72 DEG C.

Recycling and the concrete operations of purifying pcr amplification product are as follows：After gel electrophoresis (as shown in Figure 1), purpose will be met The target fragment of gene band size scales off respectively, is put into centrifuge tube, contains target gene with plastic recovery kit recycling Gel.

Embodiment 2：Tetra- kinds of splicing factor encoding gene AS1, AS2, AS3, AS4 overexpressions of arabidopsis splicing factor SR45a The building of carrier and the acquisition of transgenic plant

(1) glue target fragment after the recovery pEasy-Blunt simple Cloning kit is connected into cloning vector And it rotates into Escherichia coli TOP10 competent cell, linked system such as table 2.

Table 2

Connection product, is transferred in 50 μ L TOP10, ice bath 30min by 25 DEG C of connection 10min after the completion of connection, and 1mL is added After culture medium LB (-), the competence TOP10 for being transferred to connection product is placed in 37 DEG C of shaking tables, 1h30min is cultivated；It then will training Thallus centrifugation 7000rpm 1min after supporting is collected, and is applied on the solid medium containing corresponding resistant, and clone's connection will be contained The solid medium of product thallus, which is put into 37 DEG C of incubators, is inverted culture 12h.

(2) next day is observed and grows bacterial plaque on the culture medium being incubated overnight, on the selective LB culture medium that picking is incubated overnight 12 different single colonies, carry out PCR amplification with target fragment primer and whether be successfully connected to clone with testing goal segment Carrier.PCR amplification system such as table 3.

Table 3

PCR program setting is:94 DEG C of initial denaturation 10min；94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 35 A circulation；Extend 5min, 16 DEG C of preservations after 72 DEG C.PCR product carries out gel electrophoresis, observes electrophoresis result, and it is clear to choose band And the bacterium solution without miscellaneous band carries out shaking bacterium, places 37 DEG C of shaking tables and is incubated overnight.

(3) next day, in super-clean bench, a part is sucked out by corresponding ratio with pipettor in the bacterium that will be cultivated in 37 DEG C of shaking tables Example, is saved with sterile glycerol, is placed in -80 DEG C of refrigerator long-term preservations；Then about 100 μ L bacterium solutions are sucked out respectively is placed in new centrifuge tube In, for being sequenced." client's sample presentation step on based on table " will be filled in as required by the bacterium sample of sequencing, send to Qingdao Sheng Gong biotech firm and survey Sequence；The remaining bacterium sample plasmid extraction kit shaken in tube extracts plasmid.After having extracted plasmid, in restricted accordingly Enzyme cutting digested plasmid carries out gel electrophoresis after digestion, observe electrophoresis result, the plasmid that digestion has purpose stripe size is just success Connect the plasmid of cloning vector.

(4) purpose band after digestion is recycled with plastic recovery kit, product after the recovery connects expression vector, and turns Enter in Escherichia coli TOP10 competent cell, linked system such as table 4.

Table 4

Connection product, is transferred in 50 μ L TOP10, ice bath 30min by 22 DEG C of connection 60min after the completion of connection, and 1mL is added After culture medium LB (-), the competence TOP10 for being transferred to connection product is placed in 37 DEG C of shaking tables, 1h30min is cultivated；It then will training Thallus centrifugation 7000rpm 1min after supporting is collected, and is applied on selective LB solid medium, and clone connection product bacterium will be contained The solid medium of body, which is put into 37 DEG C of incubators, is inverted culture 12h.Bacterial plaque is grown on the culture medium that next day observation is incubated overnight, 12 different single colonies on the LB culture medium that picking is incubated overnight carry out PCR amplification with target fragment primer to detect mesh Segment whether be successfully connected to expression vector.PCR product carries out gel electrophoresis, observes electrophoresis result, choose band it is clear and The bacterium solution of no miscellaneous band carries out shaking bacterium, places 37 DEG C of shaking tables and is incubated overnight.Next day, in super-clean bench, by what is cultivated in 37 DEG C of shaking tables Bacterium is sucked out a part in corresponding ratio with pipettor, is saved with sterile glycerol, be placed in -80 DEG C of refrigerator long-term preservations；It is remaining The bacterium sample plasmid extraction kit shaken in tube extracts plasmid.After having extracted plasmid, with corresponding digestion with restriction enzyme Plasmid carries out gel electrophoresis after digestion, observe electrophoresis result, the plasmid that digestion has purpose stripe size is just successfully to connect expression The plasmid of carrier.

(5) 1 μ g (200ng/ μ L) purpose plasmid is added in 50 μ L competence Agrobacteriums, is placed on ice after mixing 30min is put into liquid nitrogen frozen 1min, is then removed from liquid nitrogen, and is put into 5min in 37 DEG C of water-baths, then places on ice 2min, is added 1ml LB liquid medium, and bacterium solution collection is finally applied to selective plating medium by 28 DEG C of shaking table culture 3h On, 48h is cultivated in 28 DEG C of inversions.

(6) the positive monoclonal Agrobacterium bacterium colony that picking converts is into 5ml selectivity LB liquid medium, at 28 DEG C Shaking table is incubated overnight, and is terminated when bacterium solution OD=0.5.Above-mentioned Agrobacterium bacterium solution is centrifuged 5min at 7000rmp, is discarded supernatant Afterwards, precipitating is resuspended with 5% sucrose of 100ml and 0.5%silwet L-77 mixed solution, obtains Agrobacterium-mediated Transformation solution；It will intend The inflorescence that southern mustard blooms immerses in Agrobacterium-mediated Transformation solution, stands 12s, wraps up processed plant with black plastic bag, is protected from light For 24 hours, polybag is then removed, is cultivated under normal operation to maturation, harvests seed.It is trained in the 1/2MS of the resistance containing respective carrier It supports screening on base and obtains T1 for transgenic positive seedling, T1 is further selfed for material, can be determined by T2 for Resistant segregation ratio Whether it is single copy insertion, then obtains homozygous transgenic line in T3 generation.4 overexpression strains are obtained, are respectively designated as AS1, AS2, AS3, AS4 respectively correspond four kinds of spliceosomes of SR45a.

Embodiment 3：Tetra- kinds of spliceosome overexpression strain expression quantity detections of SR45a and Seedling Stage salt stress phenotypic evaluation

(1) overexpression strain expression quantity detection：

Tetra- kinds of SR45a be sieved to homozygous spliceosome overexpression strain seeds are spread to cultivate 7 days on 1/2MS culture medium, Then materials extract RNA, and RT-PCR detects expression quantity, select the high strain of expression quantity and carry out next phenotype experiment.

(2) strain Seedling Stage salt stress phenotypic evaluation is overexpressed：

By the overexpression strain of tetra- kinds of spliceosomes of SR45a and arabidopsis wild type seeds point of the same period in 200mM chlorination On sodium 1/2MS culture medium and 1/2MS (-) culture medium, and multiple repeating groups are done, the culture dish for having put seed is sealed into film black Plastic bag gets up to be placed in 4 DEG C of refrigerator laminations 3 days.Culture dish is taken out after lamination and is placed in 22 DEG C of illumination boxs, is observed and is counted On 200mM sodium chloride 1/2MS culture medium and 1/2MS (-) culture medium the sprouting rate of seed and sprout after cotyledon be unfolded situation；See Fig. 2, Fig. 3, the sprouting rate of tetra- kinds of spliceosomes overexpression strain seeds of SR45a and situation is unfolded in cotyledon after sprouting under normal condition No difference compared with WT, and tetra- kinds of spliceosome overexpression strain seeds of SR45a on 200mM sodium chloride 1/2MS culture medium It sprouts rate and cotyledon deployment rate is slower than WT.These results illustrate the salt stress answering of SR45a negative regulation plant.

(3) overexpression strain Seedling Stage salt stress phenotype reason identification：

It is since infiltration is coerced to further analyze four kinds of spliceosomes overexpression strain Seedling Stage salt density value phenotype of SR45a Compel caused by or Ion toxicity caused by, so again respectively observation counted tetra- kinds of spliceosomes of SR45a overexpression strain and Sprouting rate of the arabidopsis wild type seeds of the same period on the 1/2MS culture medium of 12mM LiCl and 300mM Man；See figure 4, on Fig. 5,12mM LiCl 1/2MS culture medium after the sprouting rate and sprouting of tetra- kinds of spliceosome overexpression strain seeds of SR45a Situation simultaneously indifference with WT compared with, and tetra- kinds of super tables of spliceosome of SR45a on the 1/2MS culture medium of 300mM Man are unfolded in cotyledon Sprouting rate up to strain seed will be slower than WT.These results illustrate that the overexpression strain of tetra- kinds of spliced bodies of SR45a is sprouted in seed Hair phase high-salt stress sensitivity is due to caused by osmotic stress.

Embodiment 4：Tetra- kinds of spliceosome overexpression strain seedling stage salt stress phenotypic evaluations of SR45a and the measurement of trophosome fresh weight

(1) strain seedling stage salt stress phenotypic evaluation is overexpressed：

Tetra- kinds of spliceosome overexpression strains of SR45a and arabidopsis wild type seeds are spread on 1/2MS plating medium, 4 DEG C refrigerator dark lamination 3 days.Culture dish is taken out after 3 days be placed in 22 DEG C of illumination boxs cultivate, it, will after seedling grows to 6 days Tetra- kinds of spliceosome overexpression strains of SR45a and WT are moved to simultaneously in the black surround for filling matrix, move 8 basins.After 10 days, first by every basin Seedling photographs to record, and then pours the salt water containing 200mM NaCl, pours primary brine every three days, pours altogether three times.See Fig. 6, Fig. 7, Before pouring salt water, tetra- kinds of spliceosome overexpression strain trophosomes of SR45a are significantly greater than wild type WT；After salt stress processing, with WT phase Yellow more obvious than tetra- kinds of spliceosome overexpression strains of SR45a shows the super quick phenotype of salt stress, this illustrates SR45a in seedling stage Salt stress regulation process is participated in.

(2) strain seedling stage trophosome phenotypic evaluation is overexpressed：

Tetra- kinds of spliceosome overexpression strains of SR45a and arabidopsis wild type seeds are spread on 1/2MS plating medium, 4 DEG C refrigerator dark lamination 3 days.Culture dish is taken out after 3 days be placed in 22 DEG C of illumination boxs cultivate, it, will after seedling grows to 6 days Tetra- kinds of spliceosomes of SR45a overexpression strains and WT are moved to simultaneously in the black alms bowl for filling matrix, every kind of spliceosome overexpression strain with WT respectively moves two groups of repetitions various 60 to partly moving on in a basin matrix.After plant grows to 10 days, photograph to record；Then cut off Lotus throne leaf weighs fresh weight, and every 20 are a repetition, does 3 repeating groups.See that Fig. 8, Fig. 9, tetra- kinds of spliceosomes of SR45a overexpress strain It is that trophosome is significantly greater than wild type WT, and fresh weight is apparently higher than WT.These results illustrate that four kinds of spliceosomes of SR45a also assist in The growth and development process of plant is regulated and controled.

Above-described embodiment is provided to those skilled in the art, how to implement and use to be advocated with full disclosure and description Embodiment, rather than for limiting range disclosed herein.Obvious modification will to those skilled in the art Within the scope of the appended claims.The all publications, patents and patent applications of this specification citation are incorporated by reference into this Text, as these publications, patents and patent applications respectively show particularly and individually to be incorporated herein by reference.

Sequence table

<110>Shandong Agricultural University

<120>Application of the encoding gene of arabidopsis splicing factor SR45a spliceosome in negative regulation plant salt stress response

<141> 2018-06-28

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1149

<212> DNA

<213> Arabidopsis thaliana

<400> 1

atggggaaac gtgaaattca ttttactccg gtgggtcgtc aggtgcagag agttctggaa 60

tatccattgc gcttggagaa tcgttcgccg atgtcttact caagaaggtc aagatactct 120

ccttcactat ctccttatga caagcgtcgt ggaaggtctg tgtcaaggtc attgtctaga 180

agcccgacga ggagtgtctc aagcgatgct gagaatcctg gtaacagttt atatgtaact 240

ggattgtctc accgggttac tgaaagagat ctggaggatc atttcgctaa agaaggaaag 300

gtaactgatg ttcaccttgt cctggaccca tggactagag aatctcgcgg atttggtttt 360

atctctatga aaagtgttgg tgatgctaac cgttgcatca gatctctaga tcactctgtt 420

ctgcagggcc gcgtcatcac tgttgagaag gcaagacgtc gtagaggacg tactccaact 480

ccaggaaagt acttggggct gagaactgct cgaggacgac ataagtctcc tagctactct 540

ccccgcaggt ctgttagctg ctctcgtagt cgtagtcgaa gctactcatc tgatcggggc 600

agatcttatt ctccaagcta tgggagaaga ggaaggtcat cctcgtactc acccttctat 660

cgacgacgca gattctactc tccttcgaga tctccttcac ctgatgatcg ttacaacagg 720

agacgcgaca gatcatactc accttactac aggcggaggg accggtccag atcctactca 780

cgtaactgta gagcacggga cagatcacct tactacatgc ggaggtacag gtccagatcc 840

aggtcatact cgcctcgcta cagagcacgt gaccgatcat gctcacccta ctacagggga 900

agagaccggt cttattcacc ccactaccaa gggagagaca gatcctactc acctgaaagt 960

cgttactaca gaaggcacag gtcggtatcg ggaagcgtaa gccctggagg gagaagcatg 1020

tcacgtagca tatccccaag gaagggaagg aaagagagca gaagcaagtc tcggaggcac 1080

gacaggcaat cttcaatgtg tcattcgagg agcgcaagat caagcacctc cagatccgtc 1140

agcccataa 1149

<210> 2

<211> 1245

<212> DNA

<213> Arabidopsis thaliana

<400> 2

atggggaaac gtgaaattca ttttactccg gtgggtcgtc aggtgcagag agttctggaa 60

tatccattgc gcttggagaa tcgttcgccg atgtcttact caagaaggtc aagatactct 120

ccttcactat ctccttatga caagcgtcgt ggaaggtctg tgtcaaggtc attgtctaga 180

agcccgacga ggagtgtctc aagcgatgct gagaatcctg gtaacagttt atatgtaact 240

ggattgtctc accgggttac tgaaagagat ctggaggatc atttcgctaa agaaggaaag 300

gtaactgatg ttcaccttgt cctggaccca tggactagag aatctcgcgg atttggtttt 360

atctctatga aaagtgttgg tgatgctaac cgttgcatca gatctctaga tcactctgtt 420

ctgcagggcc gcgtcatcac tgttgagaag tttctgtggc agcaggtctg ctgtttgtag 480

cagcagtgct tcaccaaata gcaaacgttg caacagttca aacacatcaa gtttcagaca 540

tttagggcaa gacgtcgtag aggacgtact ccaactccag gaaagtactt ggggctgaga 600

actgctcgag gacgacataa gtctcctagc tactctcccc gcaggtctgt tagctgctct 660

cgtagtcgta gtcgaagcta ctcatctgat cggggcagat cttattctcc aagctatggg 720

agaagaggaa ggtcatcctc gtactcaccc ttctatcgac gacgcagatt ctactctcct 780

tcgagatctc cttcacctga tgatcgttac aacaggagac gcgacagatc atactcacct 840

tactacaggc ggagggaccg gtccagatcc tactcacgta actgtagagc acgggacaga 900

tcaccttact acatgcggag gtacaggtcc agatccaggt catactcgcc tcgctacaga 960

gcacgtgacc gatcatgctc accctactac aggggaagag accggtctta ttcaccccac 1020

taccaaggga gagacagatc ctactcacct gaaagtcgtt actacagaag gcacaggtcg 1080

gtatcgggaa gcgtaagccc tggagggaga agcatgtcac gtagcatatc cccaaggaag 1140

ggaaggaaag agagcagaag caagtctcgg aggcacgaca ggcaatcttc aatgtgtcat 1200

tcgaggagcg caagatcaag cacctccaga tccgtcagcc cataa 1245

<210> 3

<211> 1401

<212> DNA

<213> Arabidopsis thaliana

<400> 3

atggggaaac gtgaaattca ttttactccg gtgggtcgtc aggtgcagag agttctggaa 60

tatccattgc gcttggagaa tcgttcgccg atgtcttact caagaaggtc aagatactct 120

ccttcactat ctccttatga caagcgtcgt ggaaggtctg tgtcaaggtc attgtctaga 180

agcccgacga ggagtgtctc aagcgatgct gagaatcctg gtaacagttt atatgtaact 240

ggattgtctc accgggttac tgaaagagat ctggaggatc atttcgctaa agaaggaaag 300

gtaactgatg ttcaccttgt cctggacccg tggactagag aatctcgcgg atttggtttt 360

atctctatga aaagtgttgg tgatgctaac cgttgcatca gatctctaga tcactctgtt 420

ctgcagggcc gcgtcatcac tgttgagaag taaaaagact aatcactagt gattccttct 480

atagaaccat aaagaatatg atctggccga agctggtatt gatatatgca attactaggc 540

actttgatat tgattttttc ttctctttta aaatatttaa gttgtttgct tgtttccact 600

ttcaagtttc tgtggcagca ggtctgctgt ttgtagcagc agtgcttcac caaatagcaa 660

acgttgcaac agttcaaaca catcaagttt cagacattta gggcaagacg tcgtagagga 720

cgtactccaa ctccaggaaa gtacttgggg ctgagaactg ctcgaggacg acataagtct 780

cctagctact ctccccgcag gtctgttagc tgctctcgta gtcgtagtcg aagctactca 840

tctgatcggg gcagatctta ttctccaagc tatgggagaa gaggaaggtc atcctcgtac 900

tcacccttct atcgacgacg cagattctac tctccttcga gatctccttc acctgatgat 960

cgttacaaca ggagacgcga cagatcatac tcaccttact acaggcggag ggaccggtcc 1020

agatcctact cacgtaactg tagagcacgg gacagatcac cttactacat gcggaggtac 1080

aggtccagat ccaggtcata ctcgcctcgc tacagagcac gtgaccgatc atgctcaccc 1140

tactacaggg gaagagaccg gtcttattca ccccactacc aagggagaga cagatcctac 1200

tcacctgaaa gtcgttacta cagaaggcac aggtcggtat cgggaagcgt aagccctgga 1260

gggagaagca tgtcacgtag catatcccca aggaagggaa ggaaagagag cagaagcaag 1320

tctcggaggc acgacaggca atcttcaatg tgtcattcga ggagcgcaag atcaagcacc 1380

tccagatccg tcagcccata a 1401

<210> 4

<211> 1548

<212> DNA

<213> Arabidopsis thaliana

<400> 4

atggggaaac gtgaaattca ttttactccg gtgggtcgtc aggtgcagag agttctggaa 60

tatccattgc gcttggagaa tcgttcgccg atgtcttact caagaaggtc aagatactct 120

ccttcactat ctccttatga caagcgtcgt ggaaggtctg tgtcaaggtc attgtctaga 180

agcccgacga ggagtgtctc aagcgatgct gagaatcctg gtaacagttt atatgtaact 240

ggattgtctc accgggttac tgaaagagat ctggaggatc atttcgctaa agaaggaaag 300

gtaactgatg ttcaccttgt cctggaccca tggactagag aatctcgcgg atttggtttt 360

atctctatga aaagtgttgg tgatgctaac cgttgcatca gatctctaga tcactctgtt 420

ctgcagggcc gcgtcatcac tgttgagaag taaaaagact aatcactagt gattccttct 480

atagaaccat aaagaatatg atctggccga agctgatatt gatatatgca attactaggc 540

actttgatat tgattttttc ttctctttta aaatatttaa gttgtttgct tgtttccact 600

ttcaagtttc tgtggcagca ggtctgctgt ttgtagcagc agtgcttcac caaatagcaa 660

acgttgcaac agttcaaaca catcaagttt cagacattta gggttgtatg ccctgcattt 720

ctctctttct tgaacaattt aatattccgt cctttagtag cttcaatata ggaatgttgt 780

ttttgtcgtg tttcctgttt cattgatgaa tgtctgacag atctgtgata tctctggttt 840

tctgttcagg caagacgtcg tagaggacgt actccaactc caggaaagta cttggggctg 900

agaactgctc gaggacgaca taagtctcct agctactctc cccgcaggtc tgttagctgc 960

tctcgtagtc gtagtcgaag ctactcatct gatcggggca gatcttattc tccaagctat 1020

gggagaagag gaaggtcatc ctcgtactca cccttctatc gacgacgcag attctactct 1080

ccttcgagat ctccttcacc tgatgatcgt tacaacagga gacgcgacag atcatactca 1140

ccttactaca ggcggaggga ccggtccaga tcctactcac gtaactgtag agcacgggac 1200

agatcacctt actacatgcg gaggtacagg tccagatcca ggtcatactc gcctcgctac 1260

agagcacgtg accgatcatg ctcaccctac tacaggggaa gagaccggtc ttattcaccc 1320

cactaccaag ggagagacag atcctactca cctgaaagtc gttactacag aaggcacagg 1380

tcggtatcgg gaagcgtaag ccctggaggg agaagcatgt cacgtagcat atccccaagg 1440

aagggaagga aagagagcag aagcaagtct cggaggcacg acaggcaatc ttcaatgtgt 1500

cattcgagga gcgcaagatc aagcacctcc agatccgtca gcccataa 1548

<210> 5

<211> 383

<212> PRT

<213> Arabidopsis thaliana

<400> 5

Met Gly Lys Arg Glu Ile His Phe Thr Pro Val Gly Arg Gln Val Gln

1 5 10 15

Arg Val Leu Glu Tyr Pro Leu Arg Leu Glu Asn Arg Ser Pro Met Ser

20 25 30

Tyr Ser Arg Arg Ser Arg Tyr Ser Pro Ser Leu Ser Pro Tyr Asp Lys

35 40 45

Arg Arg Gly Arg Ser Val Ser Arg Ser Leu Ser Arg Ser Pro Thr Arg

50 55 60

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

65 70 75 80

Gly Leu Ser His Arg Val Thr Glu Arg Asp Leu Glu Asp His Phe Ala

85 90 95

Lys Glu Gly Lys Val Thr Asp Val His Leu Val Leu Asp Pro Trp Thr

100 105 110

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

115 120 125

Ala Asn Arg Cys Ile Arg Ser Leu Asp His Ser Val Leu Gln Gly Arg

130 135 140

Val Ile Thr Val Glu Lys Ala Arg Arg Arg Arg Gly Arg Thr Pro Thr

145 150 155 160

Pro Gly Lys Tyr Leu Gly Leu Arg Thr Ala Arg Gly Arg His Lys Ser

165 170 175

Pro Ser Tyr Ser Pro Arg Arg Ser Val Ser Cys Ser Arg Ser Arg Ser

180 185 190

Arg Ser Tyr Ser Ser Asp Arg Gly Arg Ser Tyr Ser Pro Ser Tyr Gly

195 200 205

Arg Arg Gly Arg Ser Ser Ser Tyr Ser Pro Phe Tyr Arg Arg Arg Arg

210 215 220

Phe Tyr Ser Pro Ser Arg Ser Pro Ser Pro Asp Asp Arg Tyr Asn Arg

225 230 235 240

Arg Arg Asp Arg Ser Tyr Ser Pro Tyr Tyr Arg Arg Arg Asp Arg Ser

245 250 255

Arg Ser Tyr Ser Arg Asn Cys Arg Ala Arg Asp Arg Ser Pro Tyr Tyr

260 265 270

Met Arg Arg Tyr Arg Ser Arg Ser Arg Ser Tyr Ser Pro Arg Tyr Arg

275 280 285

Ala Arg Asp Arg Ser Cys Ser Pro Tyr Tyr Arg Gly Arg Asp Arg Ser

290 295 300

Tyr Ser Pro His Tyr Gln Gly Arg Asp Arg Ser Tyr Ser Pro Glu Ser

305 310 315 320

Arg Tyr Tyr Arg Arg His Arg Ser Val Ser Gly Ser Val Ser Pro Gly

325 330 335

Gly Arg Ser Met Ser Arg Ser Ile Ser Pro Arg Lys Gly Arg Lys Glu

340 345 350

Ser Arg Ser Lys Ser Arg Arg His Asp Arg Gln Ser Ser Met Cys His

355 360 365

Ser Arg Ser Ala Arg Ser Ser Thr Ser Arg Ser Val Ser Pro Glx

370 375 380

<210> 6

<211> 415

<212> PRT

<213> Arabidopsis thaliana

<400> 6

Met Gly Lys Arg Glu Ile His Phe Thr Pro Val Gly Arg Gln Val Gln

1 5 10 15

Arg Val Leu Glu Tyr Pro Leu Arg Leu Glu Asn Arg Ser Pro Met Ser

20 25 30

Tyr Ser Arg Arg Ser Arg Tyr Ser Pro Ser Leu Ser Pro Tyr Asp Lys

35 40 45

Arg Arg Gly Arg Ser Val Ser Arg Ser Leu Ser Arg Ser Pro Thr Arg

50 55 60

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

65 70 75 80

Gly Leu Ser His Arg Val Thr Glu Arg Asp Leu Glu Asp His Phe Ala

85 90 95

Lys Glu Gly Lys Val Thr Asp Val His Leu Val Leu Asp Pro Trp Thr

100 105 110

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

115 120 125

Ala Asn Arg Cys Ile Arg Ser Leu Asp His Ser Val Leu Gln Gly Arg

130 135 140

Val Ile Thr Val Glu Lys Phe Leu Trp Gln Gln Val Cys Cys Leu Glx

145 150 155 160

Gln Gln Cys Phe Thr Lys Glx Gln Thr Leu Gln Gln Phe Lys His Ile

165 170 175

Lys Phe Gln Thr Phe Arg Ala Arg Arg Arg Arg Gly Arg Thr Pro Thr

180 185 190

Pro Gly Lys Tyr Leu Gly Leu Arg Thr Ala Arg Gly Arg His Lys Ser

195 200 205

Pro Ser Tyr Ser Pro Arg Arg Ser Val Ser Cys Ser Arg Ser Arg Ser

210 215 220

Arg Ser Tyr Ser Ser Asp Arg Gly Arg Ser Tyr Ser Pro Ser Tyr Gly

225 230 235 240

Arg Arg Gly Arg Ser Ser Ser Tyr Ser Pro Phe Tyr Arg Arg Arg Arg

245 250 255

Phe Tyr Ser Pro Ser Arg Ser Pro Ser Pro Asp Asp Arg Tyr Asn Arg

260 265 270

Arg Arg Asp Arg Ser Tyr Ser Pro Tyr Tyr Arg Arg Arg Asp Arg Ser

275 280 285

Arg Ser Tyr Ser Arg Asn Cys Arg Ala Arg Asp Arg Ser Pro Tyr Tyr

290 295 300

Met Arg Arg Tyr Arg Ser Arg Ser Arg Ser Tyr Ser Pro Arg Tyr Arg

305 310 315 320

Ala Arg Asp Arg Ser Cys Ser Pro Tyr Tyr Arg Gly Arg Asp Arg Ser

325 330 335

Tyr Ser Pro His Tyr Gln Gly Arg Asp Arg Ser Tyr Ser Pro Glu Ser

340 345 350

Arg Tyr Tyr Arg Arg His Arg Ser Val Ser Gly Ser Val Ser Pro Gly

355 360 365

Gly Arg Ser Met Ser Arg Ser Ile Ser Pro Arg Lys Gly Arg Lys Glu

370 375 380

Ser Arg Ser Lys Ser Arg Arg His Asp Arg Gln Ser Ser Met Cys His

385 390 395 400

Ser Arg Ser Ala Arg Ser Ser Thr Ser Arg Ser Val Ser Pro Glx

405 410 415

<210> 7

<211> 467

<212> PRT

<213> Arabidopsis thaliana

<400> 7

Met Gly Lys Arg Glu Ile His Phe Thr Pro Val Gly Arg Gln Val Gln

1 5 10 15

Arg Val Leu Glu Tyr Pro Leu Arg Leu Glu Asn Arg Ser Pro Met Ser

20 25 30

Tyr Ser Arg Arg Ser Arg Tyr Ser Pro Ser Leu Ser Pro Tyr Asp Lys

35 40 45

Arg Arg Gly Arg Ser Val Ser Arg Ser Leu Ser Arg Ser Pro Thr Arg

50 55 60

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

65 70 75 80

Gly Leu Ser His Arg Val Thr Glu Arg Asp Leu Glu Asp His Phe Ala

85 90 95

Lys Glu Gly Lys Val Thr Asp Val His Leu Val Leu Asp Pro Trp Thr

100 105 110

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

115 120 125

Ala Asn Arg Cys Ile Arg Ser Leu Asp His Ser Val Leu Gln Gly Arg

130 135 140

Val Ile Thr Val Glu Lys Glx Lys Asp Glx Ser Leu Val Ile Pro Ser

145 150 155 160

Ile Glu Pro Glx Arg Ile Glx Ser Gly Arg Ser Trp Tyr Glx Tyr Met

165 170 175

Gln Leu Leu Gly Thr Leu Ile Leu Ile Phe Ser Ser Leu Leu Lys Tyr

180 185 190

Leu Ser Cys Leu Leu Val Ser Thr Phe Lys Phe Leu Trp Gln Gln Val

195 200 205

Cys Cys Leu Glx Gln Gln Cys Phe Thr Lys Glx Gln Thr Leu Gln Gln

210 215 220

Phe Lys His Ile Lys Phe Gln Thr Phe Arg Ala Arg Arg Arg Arg Gly

225 230 235 240

Arg Thr Pro Thr Pro Gly Lys Tyr Leu Gly Leu Arg Thr Ala Arg Gly

245 250 255

Arg His Lys Ser Pro Ser Tyr Ser Pro Arg Arg Ser Val Ser Cys Ser

260 265 270

Arg Ser Arg Ser Arg Ser Tyr Ser Ser Asp Arg Gly Arg Ser Tyr Ser

275 280 285

Pro Ser Tyr Gly Arg Arg Gly Arg Ser Ser Ser Tyr Ser Pro Phe Tyr

290 295 300

Arg Arg Arg Arg Phe Tyr Ser Pro Ser Arg Ser Pro Ser Pro Asp Asp

305 310 315 320

Arg Tyr Asn Arg Arg Arg Asp Arg Ser Tyr Ser Pro Tyr Tyr Arg Arg

325 330 335

Arg Asp Arg Ser Arg Ser Tyr Ser Arg Asn Cys Arg Ala Arg Asp Arg

340 345 350

Ser Pro Tyr Tyr Met Arg Arg Tyr Arg Ser Arg Ser Arg Ser Tyr Ser

355 360 365

Pro Arg Tyr Arg Ala Arg Asp Arg Ser Cys Ser Pro Tyr Tyr Arg Gly

370 375 380

Arg Asp Arg Ser Tyr Ser Pro His Tyr Gln Gly Arg Asp Arg Ser Tyr

385 390 395 400

Ser Pro Glu Ser Arg Tyr Tyr Arg Arg His Arg Ser Val Ser Gly Ser

405 410 415

Val Ser Pro Gly Gly Arg Ser Met Ser Arg Ser Ile Ser Pro Arg Lys

420 425 430

Gly Arg Lys Glu Ser Arg Ser Lys Ser Arg Arg His Asp Arg Gln Ser

435 440 445

Ser Met Cys His Ser Arg Ser Ala Arg Ser Ser Thr Ser Arg Ser Val

450 455 460

Ser Pro Glx

465

<210> 8

<211> 516

<212> PRT

<213> Arabidopsis thaliana

<400> 8

Met Gly Lys Arg Glu Ile His Phe Thr Pro Val Gly Arg Gln Val Gln

1 5 10 15

Arg Val Leu Glu Tyr Pro Leu Arg Leu Glu Asn Arg Ser Pro Met Ser

20 25 30

Tyr Ser Arg Arg Ser Arg Tyr Ser Pro Ser Leu Ser Pro Tyr Asp Lys

35 40 45

Arg Arg Gly Arg Ser Val Ser Arg Ser Leu Ser Arg Ser Pro Thr Arg

50 55 60

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

65 70 75 80

Gly Leu Ser His Arg Val Thr Glu Arg Asp Leu Glu Asp His Phe Ala

85 90 95

Lys Glu Gly Lys Val Thr Asp Val His Leu Val Leu Asp Pro Trp Thr

100 105 110

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

115 120 125

Ala Asn Arg Cys Ile Arg Ser Leu Asp His Ser Val Leu Gln Gly Arg

130 135 140

Val Ile Thr Val Glu Lys Glx Lys Asp Glx Ser Leu Val Ile Pro Ser

145 150 155 160

Ile Glu Pro Glx Arg Ile Glx Ser Gly Arg Ser Glx Tyr Glx Tyr Met

165 170 175

Gln Leu Leu Gly Thr Leu Ile Leu Ile Phe Ser Ser Leu Leu Lys Tyr

180 185 190

Leu Ser Cys Leu Leu Val Ser Thr Phe Lys Phe Leu Trp Gln Gln Val

195 200 205

Cys Cys Leu Glx Gln Gln Cys Phe Thr Lys Glx Gln Thr Leu Gln Gln

210 215 220

Phe Lys His Ile Lys Phe Gln Thr Phe Arg Val Val Cys Pro Ala Phe

225 230 235 240

Leu Ser Phe Leu Asn Asn Leu Ile Phe Arg Pro Leu Val Ala Ser Ile

245 250 255

Glx Glu Cys Cys Phe Cys Arg Val Ser Cys Phe Ile Asp Glu Cys Leu

260 265 270

Thr Asp Leu Glx Tyr Leu Trp Phe Ser Val Gln Ala Arg Arg Arg Arg

275 280 285

Gly Arg Thr Pro Thr Pro Gly Lys Tyr Leu Gly Leu Arg Thr Ala Arg

290 295 300

Gly Arg His Lys Ser Pro Ser Tyr Ser Pro Arg Arg Ser Val Ser Cys

305 310 315 320

Ser Arg Ser Arg Ser Arg Ser Tyr Ser Ser Asp Arg Gly Arg Ser Tyr

325 330 335

Ser Pro Ser Tyr Gly Arg Arg Gly Arg Ser Ser Ser Tyr Ser Pro Phe

340 345 350

Tyr Arg Arg Arg Arg Phe Tyr Ser Pro Ser Arg Ser Pro Ser Pro Asp

355 360 365

Asp Arg Tyr Asn Arg Arg Arg Asp Arg Ser Tyr Ser Pro Tyr Tyr Arg

370 375 380

Arg Arg Asp Arg Ser Arg Ser Tyr Ser Arg Asn Cys Arg Ala Arg Asp

385 390 395 400

Arg Ser Pro Tyr Tyr Met Arg Arg Tyr Arg Ser Arg Ser Arg Ser Tyr

405 410 415

Ser Pro Arg Tyr Arg Ala Arg Asp Arg Ser Cys Ser Pro Tyr Tyr Arg

420 425 430

Gly Arg Asp Arg Ser Tyr Ser Pro His Tyr Gln Gly Arg Asp Arg Ser

435 440 445

Tyr Ser Pro Glu Ser Arg Tyr Tyr Arg Arg His Arg Ser Val Ser Gly

450 455 460

Ser Val Ser Pro Gly Gly Arg Ser Met Ser Arg Ser Ile Ser Pro Arg

465 470 475 480

Lys Gly Arg Lys Glu Ser Arg Ser Lys Ser Arg Arg His Asp Arg Gln

485 490 495

Ser Ser Met Cys His Ser Arg Ser Ala Arg Ser Ser Thr Ser Arg Ser

500 505 510

Val Ser Pro Glx

515

<210> 9

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 9

ggatccatgg ggaaacgtga aattca 26

<210> 10

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 10

gtcgactaga gactgttatg ggctga 26

Claims (10)

1. application of the encoding gene of arabidopsis splicing factor SR45a spliceosome in negative regulation plant salt stress response, or Preparation/cultivation has the application in the plant of anti-salt property；The encoding gene be AT1G07350.1, AT1G07350.2, One of AT1G07350.3, AT1G07350.4, nucleotide sequence is successively as shown in SEQ ID NO.1,2,3,4.

2. application of the spliceosome of arabidopsis splicing factor SR45a in negative regulation plant salt stress response, or in preparation/cultivation Application in plant with anti-salt property；The amino acid sequence of the spliceosome, be SEQ ID NO.5, SEQ ID NO.6, One of sequence shown in SEQ ID NO.7, SEQ ID NO.8.

3. application according to claim 1, it is characterised in that：When concrete application, arabidopsis splicing factor SR45a will be contained The plant expression vector of the encoding gene of spliceosome imports plant cell or seed, makes arabidopsis splicing factor SR45a spliceosome Encoding gene overexpression, to obtain the transgenic plant that anti-salt property is weaker than WT lines.

4. plant expression vector, the encoding gene containing arabidopsis splicing factor SR45a spliceosome, the encoding gene are One of AT1G07350.1, AT1G07350.2, AT1G07350.3, AT1G07350.4, nucleotide sequence is successively such as SEQ Shown in ID NO.1,2,3,4.

5. plant expression vector according to claim 4, it is characterised in that：Plasmid used in the plant expression vector is PBI121。

6. a kind of genetically engineered host cell, contains plant expression vector described in claim 4 or 5 or its gene The encoding gene of arabidopsis splicing factor SR45a spliceosome is inserted in group；The encoding gene is AT1G07350.1, One of AT1G07350.2, AT1G07350.3, AT1G07350.4, nucleotide sequence successively as SEQ ID NO.1,2, 3, shown in 4.

7. application of the plant expression vector described in claim 4 or 5 in the plant that preparation/cultivation has anti-salt property.

8. application according to claim 7, it is characterised in that：The anti-salt property of the genetically modified plants is weaker than wild type plant Strain.

9. genetically engineered host cell as claimed in claim 6 answering in the plant that preparation/cultivation has anti-salt property With.

10. application according to claim 8, it is characterised in that：The anti-salt property of the genetically modified plants is weaker than wild type Plant.