CN108570470A

CN108570470A - Sedum lineare resistant gene of salt SLTRSA and its application

Info

Publication number: CN108570470A
Application number: CN201710142171.1A
Authority: CN
Inventors: 王洁华; 宋秋芳; 杨少辉; 宋英今
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2017-03-10
Filing date: 2017-03-10
Publication date: 2018-09-25

Abstract

The present invention relates to a kind of sedum lineare resistant gene of salt SLTRSA and its application, the sedum lineare resistant gene of salt SLTRSA is nucleotide sequence shown in SEQ ID NO.1 in sequence table, it is demonstrated experimentally that sedum lineare resistant gene of salt SLTRSA enhancing arabidopsis or willow salt resistant character.It can make the host plant including arabidopsis, willow that there is preferable saline-alkaline tolerance.

Description

Sedum lineare resistant gene of salt SLTRSA and its application

Technical field

The present invention relates to a kind of sedum lineare (Sedum lineare Thunb) resistant gene of salt and its applications, particular, it is found that one The new resistant gene of salt SLTRSA of kind, belongs to molecular biology and biotechnology.

Background technology

Environment-stress has become an important factor for restricting China's agricultural and forest development, wherein with salt stress and doing especially Drought stress is the most serious.The Efficiency in Buildings in Tianjin Area Bohai Sea Pin Lin, level of ground water is shallow, and salt-affected soil area proportion is larger.And soil is transformed Earth is higher with the economic cost for adapting to biological growth, therefore, new resistant gene of salt is found and detached by genetic engineering, to cultivate Salt-resistant plant provides a kind of highly effective method, to improvement coastal saline-alkali soil, improves the ecosystem in salt-soda soil, improves soil Ground productivity, the profound significance with reality；Also there is great application to the balance and sustainable development that maintain the ecosystem Value.

Invention content

The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of sedum lineare resistant gene of salt.

Second object of the present invention is to provide the cloning vector containing sedum lineare resistant gene of salt.

Third object of the present invention is to provide the host cells containing above-mentioned cloning vector.

Fourth object of the present invention is to provide the expression vector containing sedum lineare resistant gene of salt.

Fifth object of the present invention is to provide the host cells containing above-mentioned expression vector.

Sixth object of the present invention is to provide the purposes of sedum lineare resistant gene of salt enhancing arabidopsis or willow salt resistant character.

Technical scheme of the present invention is summarized as follows：

Sedum lineare resistant gene of salt SLTRSA is nucleotide sequence shown in SEQ ID NO.1 in sequence table.

The cloning vector pJET1.2_SLTRSA of the SLTRSA of resistant gene of salt containing sedum lineare.

Host cell containing cloning vector pJET1.2_SLTRSA.

The expression vector pBI121_SLTRSA of the SLTRSA of resistant gene of salt containing sedum lineare.

Host cell containing expression vector pB121_SLTRSA.

The purposes of sedum lineare resistant gene of salt SLTRSA enhancing arabidopsis or willow salt resistant character.

Advantages of the present invention：

It is demonstrated experimentally that sedum lineare resistant gene of salt SLTRSA enhancing arabidopsis or willow salt resistant character.Can make include arabidopsis, Host plant including willow has preferable saline-alkaline tolerance.

Description of the drawings

Fig. 1 is SLTRSA gene cloning electrophoresis schematic diagrames.

Fig. 2 is that SLTRSA is inserted into schematic diagram after expression vector

Fig. 3 is transformant Genomic PCR the selection result after pBI121_SLTRSA arabidopsis thaliana transformations.

Fig. 4 is T3 homozygote semiquantitive PCRs measurement expression result after pBI121_SLTRSA arabidopsis thaliana transformations.

Fig. 5 is SLTRSA transgenic arabidopsis T3 homozygote salt resistance experiment effect photos.

Fig. 6 is SLTRSA transgenic poplar salt resistance experiment effect photos.

Specific implementation mode

With reference to specific embodiment, the present invention is further illustrated.

Test method without specific conditions in embodiment, the item usually according to normal condition and described in handbook Part, or according to the normal condition proposed by manufacturer.

Carrier pJET1.2 pJET1.2:Thermo,Clone JET PCR Cloning Kit#K1231

Carrier pBI121 is purchased from Chinese plasmid vector strain cell pnca gene collection, http:// biovector.blog.163.com/

Embodiment 1

The clone of sedum lineare 1. (Sedum lineare Thunb, abbreviation SlT) SLTRSA genes

Experiment sedum lineare used is derived from Pests in Tianjin Binhai New Area, sedum lineare growth conditions：25 DEG C, 1800Lux of temperature, light week Phase 16h illumination/8h is dark, and relative humidity 70% carries out sedum lineare 30 days by a definite date salt treatment.It is molten with 150mM NaCl Liquid respectively poured it at the 1st, 2,3,4 week of experiment.To the end of salt treatment is tested after two weeks, it is good to choose upgrowth situation Good sedum lineare plant, is carried using plant RNeasy Plant Mini Kit (Transgene Code#E101-0150rxns) Total serum IgE is taken, and utilizes EasyScript Frist-Strand cDNA SynSgesis SuperMix (Transgene Code#AE301-03100rxns) reverse transcription goes out cDNA.High-flux sequence (sources Nuo Hezhi company) is carried out to cDNA to obtain The 3' terminal sequences of SLTRSA genes obtain the full-length cDNA sequence of SLTRSA genes using RACE technologies (Takara-RACE kit) Row, the SLTRSA genes that amplification is obtained carry out sequencing analysis, and it is 1326bp to obtain complete SLTRSA full length genes.Structure is super When expression vector, then respectively in 5 ' end addition pBI121 recombination sites of specific primer, upstream 5 '- ACGGGGGACTCTAGAGGATCC-3 ' (SEQ ID No.3), downstream 5 '-CGATCGGGGAAATTCGAGCTC-3 ' (SEQ ID No.4), in favor of the structure of later stage experssion carrier.

It is as follows：

1) synthesis of the first chains of cDNA

With reverse transcription reagent box TaKaRaRNAPCRKit (AMV) Ver.3.0, using total serum IgE as template, Oligo (dT) is to draw Object, synthesizes the first chains of cDNA under the action of AMV reverse transcriptases, and reverse transcription system is as follows：

Reaction condition：42 DEG C of 60min, 99 DEG C of 5min.

2) sedum lineares SLTRSA genes reverse transcription quality PCR amplification detects

With sedum lineare Actin gene specific primer SEQ ID No.5:5'–GAACTTACTAGCCGACTG-3',SEQ ID No.6：5'-CCTCAAGCCTTATACGCAA-3', PCR amplification, to verify reverse transcription reaction and RNA mass.PCR reaction systems It is as follows：

Reaction condition：94℃3min；94 DEG C of 30s, 40 DEG C of 30s, 72 DEG C of 50s, 35cycles；72℃5min.

3) sedum lineare SLTRSA genetic fragments PCR amplification

Sequencing analysis is carried out using the SLTRSA genes that Takara RACE kit are expanded, obtains complete sedum lineare SLTRSA full length genes are 1326bp (SEQ ID No.1).It is SEQ ID by the protein of sedum lineare SLTRSA gene codes Amino acid sequence shown in No.2.Primer Software for Design SLTRSA gene upstream and downstream primers are utilized according to known cDNA sequence SEQ ID No.7:CGACCGTAACATGTGTCATTTG-3' and SEQ ID No.8:5'- CGGGCCTATCCGCAACGAATTTA-3', PCR reaction system and program are as follows：

PCR after reaction, takes 1 μ LPCR products to carry out 1.0% agarose gel electrophoresis, detects the quality of PCR product (see Fig. 1), remaining purifying for being used as product are recycled.

4) cloning vector containing sedum lineare SLTRSA genes is built

Build the carrier pJET1.2_SLTRSA containing sedum lineare SLTRSA genes

Sedum lineare SLTRSA gene target fragments after glue recovery purifying utilize Clone JET PCR Cloning Kit (pJET1.2:Thermo, Clone JET PCR Cloning Kit#K1231) it recombinates onto carrier pJET1.2, it is cloned Carrier pJET1.2_SLTRSA.

Its reaction system is as follows：

24 DEG C, 10min of reaction condition, static 30min on ice, 42 DEG C of heat shock 1min30s, static 2min30s, is transferred on ice Competent cell DH5 α, 37 DEG C, 180r, 45min, LB (addition antibiotic Amp100uM) is coated onto admittedly after this EP (end of program) by bacterium solution In body culture medium, 37 DEG C are incubated overnight.

Be utilized respectively the upstream and downstream primer (SEQ ID No.7 and SEQ ID No.8) of target fragment, to different bacterium colonies into The PCR verifications of row bacterium colony screen positive bacterium colony and are sequenced to obtain the host cell containing cloning vector pJET1.2_SLTRSA.

Note：pJET1.2:Thermo, Clone JET PCR Cloning Kit#K1231 carriers are purchased from invitrogen； Escherichia coli used are DH5a competent cells, TIANGEN, CB101-2.

6) expression vector containing sedum lineare SLTRSA genes is built

Build the carrier pBI121_SLTRSA containing sedum lineare SLTRSA genes

When building overexpression vector, then pBI121 recombination sites are added at the ends 5' of specific primer and 3' respectively,

SEQ ID No.3:5’-ACGGGGGACTCTAGAGGATCC-3’,

SEQ ID No.4:5’-CGATCGGGGAAATTCGAGCTC-3’

It obtains：

SEQ ID No.9:5'-ACGGGGGACTCTAGAGGATCCCGACCGTAACATGTGTCATTTG-3'

SEQ ID No.10:5-CGATCGGGGAAATTCGAGCTCCGGGCCTATCCGCAACGAATTTA-3'

The plasmid of correct pJET1.2- genes is sequenced in extraction, as masterplate, using containing recombination site SEQ ID No.9, SEQ ID No.10 are that primer carries out PCR amplification, and reaction system and program are as follows

PCR after reaction, takes 1 μ LPCR products to carry out 1.0% agarose gel electrophoresis, detects the quality of PCR product, Remaining purifying for being used as product is recycled.

PBI121 plasmids are extracted, Vector map (see Fig. 2) carries out double digestion linearisation to it, and reaction system is as follows：

Reaction condition：37 DEG C, 80 DEG C of 20min inactivations after 12h.

Gene and the pBI121 plasmids of linearisation are tried using ClonExpress Entry OneStep Cloning Kit Agent box carries out recombination to construct, and reaction system is as follows：

Response procedures：37 DEG C, 30min, on ice 5min, 42 DEG C of heat shock 1min30s, static 2min30s, is transferred to impression on ice State cell DH5 α, are coated onto LB (addition antibiotic kan 50uM) solid by bacterium solution after this EP (end of program) and train by 37 DEG C, 180r, 45min It supports in base, 37 DEG C are incubated overnight.

The upstream and downstream primer (SEQ ID No.9 and SEQ ID No.10) of carrier and target fragment is utilized respectively to same Bacterium colony carries out bacterium colony PCR double verifications, screens positive bacterium colony sequencing (SEQ ID No.11).

Note：This step is purchased from vazyme using ClonExpress Entry OneStep Cloning Kit,

7) recombinant vector containing sedum lineare SLTRSA genes converts Agrobacterium competent cell

Experiment agrobacterium strains used be C58 (be purchased from Chinese plasmid vector strain cell pnca gene collection, http://biovector.blog.163.com/), C58 has rifampicin resistance (Rif), helper plasmid anti-with gentamicin Property (Gen).

Using electric shock Agrobacterium-mediated Transformation method, by the coli expression carrier pBI121_ containing sedum lineare SLTRSA genes SLTRSA is transformed into Agrobacterium strain C58 (pMP90) competent cell, 28 DEG C, cultivates 36h, bacterium colony PCR selects positive colony bacterium It falls.

2 arabidopsis thaliana transformation of embodiment

(1) arabidopsis thaliana transformation.

The concrete operation step of arabidopsis thaliana transformation：

1. the activation of positive colony bacterium colony and expansion that embodiment 1 obtains are cultivated

Activation：The positive colony bacterium colony for choosing preservation is placed in 3mLYEB fluid nutrient mediums (addition Gen, Rift, Sp antibiosis Element, it is respectively 30mg/L to make concentration, 25mg/L, 50mg/L) culture 15 hours or so (to OD₆₀₀=0.8 or so), 180rpm, 28℃。

The expansion culture of positive colony bacterium：Be added in fresh 10mlYEB fluid nutrient mediums suitable antibiotic (Gen, Rift, Sp antibiotic, concentration are respectively 30mg/L, 25mg/L, 50mg/L), appropriate positive colony bacterium solution is then inoculated with to YEB liquid It is cultivated in body culture medium, 180rpm, is cultivated to OD at 28 DEG C₆₀₀=0.6.

2. converting

Supernatant is abandoned after bacterium solution is centrifuged (3000rpm, 15 DEG C, 10min), with the mass concentration of twice the taken bacterium solution of volume Thalline is resuspended for 5% aqueous sucrose solution, so that thalline is scattered, adjusts OD₆₀₀=0.8.

The wildtype Arabidopsis thaliana for choosing 3-4 weeks bolting 5-7cm of culture, is inverted in the container equipped with conversion fluid, makes entire Inflorescence is immersed in bacterium solution 15 seconds, is taken out arabidopsis and is couched in pallet, covers moisturizing, and dark treatment 12h with plastic film, make Arabidopsis stands upright on 25 DEG C of temperature, and photoperiod 16h illumination/8h is dark, is grown under the condition of culture that relative humidity is 70%, until Seed maturity.It is put into 37 DEG C of baking ovens after seed collection to dry two weeks, in case follow-up test uses.

(2) the homozygotic screening of transgenic arabidopsis positive transformant

The T1 collected is placed on 4 DEG C of refrigerator three days for seed after disinfection, then by transgenosis on super-clean bench Uniformly sowing is on the 1/2MS solid screening and culturing mediums containing 50 μ g/ml kanamycins for arabidopsis seed, in 1800Lux, light week Phase 16h illumination/8h is dark, grows 8-10 days, and leaf is the T1 of transgenic arabidopsis for positive transformant for bottle green.Work as T1 When growing to 3-4 piece true leaves for positive transformant plant, it is transplanted to soil and (is purchased from EPAGMA, Dutch, http:// Www.epagma.eu/ in), in 25 DEG C, 1800Lux of temperature, photoperiod 16h illumination/8h is dark, the training that relative humidity is 70% Continued growth 14 days, first makees the identification (see Fig. 3) of positive transformant, then by semiquantitive PCR first to its transgenosis under the conditions of supporting Expression identified (see Fig. 4), choose expression high No. 4 independences low with expression of independent transformation strain turn Change strain 5.Continued growth under these conditions, collection seed is T2 for transformed the seed about after one and a half months.It repeats above-mentioned Step obtains No. 8 and No. 2 T3 for homozygote seed.

(3) transgenic arabidopsis carries out salt stress processing

No. 8 T3 are planted in the soil for homozygote seed, No. 2 T3 for homozygote seed, wild arabidopsis seed respectively, In 25 DEG C, 1800Lux of temperature, photoperiod 16h illumination/8h is dark, grows 21 days under the condition of culture that relative humidity is 70%, often Kind of plant retains the consistent seedling of 21 plants of growing ways, is randomly divided into three groups of parallel laboratory tests, every group of each 7 plants of different types of plant, Pouring processing is carried out with salinity treatment fluid (150mM NaCl aqueous solutions).It pours within 3 days once, each irrigation amount is soil quality 0.5 times, to keep constant, the plant photograph after coprocessing 15 days for the treatment of fluid concentration in basin (see Fig. 5).

Embodiment 3 converts willow

Willow for positive colony bacterium conversion is trembling poplar × white poplar (Populus tremula × P.albaINRA Clone N7171-B4, hereinafter referred to as 717 willows) tissue-cultured seedling.

(1) 717 willow axillary-bud or top-buds are placed in successive propagation on minimal medium, cultivate 6 weeks and obtains tissue-cultured seedling；It cuts The tissue-cultured seedling 1cm without axillary bud stem section, scratch mouth after under 24 DEG C of dark conditions preculture 3 days；

(2) the positive colony bacterium bacterium solution (OD for obtaining the embodiment of selection 1₆₀₀=0.8) it is centrifuged in room temperature, 4000rpm 10min discards supernatant liquid, will precipitate isometric M liquid (M liquid：MS salt 4.4g, sucrose 30g, auxin NAA 1.86mg, carefully Born of the same parents mitogen 2ip 1.02mg, acetosyringone As 19.86mg are settled to 1L, pH=5.7) it is resuspended, 24 DEG C, 100rpm activation 1h obtains infecting liquid；By 40：The ratio of 25mL by the stem section of step (1) preculture be put into it is described infect in liquid, 24 DEG C of conditions Lower 100rpm infects 1h.By co-cultivation (M1 solid mediums：MS salt 4.4g, sucrose 30g, agar 7.2g, auxin NAA 1.86mg, basic element of cell division 2ip 1.02mg, acetosyringone As 19.86mg are settled to 1L, pH=5.7；Condition of culture：26 DEG C, co-cultured 36 hours under dark condition), the delayed selection culture (CIM the delayed selection culture culture mediums：MS salt 4.4g, sucrose 30g, auxin NAA 1.86mg, the basic element of cell division 2ip 1.02mg, cephalosporin 500mg, agar 7.2g are settled to 1L, pH=5.7；Culture Condition：Cultivated 8 days under 800Lux dim lights, illumination/dark is 16h/8h), evoking adventive bud is (in SIM screening and culturing mediums：MS salt 4.4g, sucrose 30g, basic element of cell division TDZ 0.05mg, cephalosporin 500mg, kanamycins 500mg, agar 7.2g are settled to 1L, pH=5.7；Condition of culture：26 DEG C, 2000Lux, illumination/dark be 16h/8h), elongation culture (SEM screening and culturing mediums：MS Salt 4.4g, sucrose 30g, basic element of cell division 2ip 1.02mg, cephalosporin 500mg, kanamycins 500mg, agar 7.2g, constant volume To 1L, pH=5.7；Condition of culture：26 DEG C, 2000Lux illumination, illumination/dark be 16h/8h, cultivate 4 weeks), root induction (RM The group of culture medium becomes：MS salt 2.2g, sucrose 30g, cephalosporin 500mg, kanamycins 500mg, agar 7.2g are settled to 1L, pH=5.7；Condition of culture：26 DEG C, 2000Lux illumination, illumination/dark is 16h/8h) after multistep, growth of poplar to be regenerated After normal, positive identification is carried out to each independent transformation.

By semiquantitive PCR choose the high low independent transformation strain of independent transformation strain and expression of expression into Row salt resistance is tested.

(3) 717 willows are subjected to salt treatment

By the uniform transgenosis height expression willow of 2 months growing ways of growth, low expression transgenic poplar, 717 poplar of wild type In tree transplanting to native basin, after native basin seedling grows 30d, each plant retains the consistent seedling of 21 plants of growing ways, is randomly divided into three groups, Every group of each 7 plants of different types of plant carries out pouring processing with salinity treatment fluid (150mM NaCl aqueous solutions aqueous solution).3 days Pour primary, each irrigation amount is 0.5 times of soil quality, and to keep the constant for the treatment of fluid concentration in basin, coprocessing is seen after 30 days It examines plant and takes a picture.(see Fig. 6).

SEQUENCE LISTING

<110>University Of Tianjin

<120>Sedum lineare resistant gene of salt SLTRSA and its application

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ccggccacca cctttcaggt atccgactat cataaaataa tgtcccaatc aaataaaaca 300

aacccatacc catttcaaac atcaacacat caagtgcttg aggtacaccc caattcgcca 360

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tggaaactgt tgttgttgaa actgtgctta agctcagatc aatcagactt gatggtattg 780

ccaggaaaag ccattcatgt gaggtgtatg aggcatttga ttttgacaat ccagcaaatg 840

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atcccaaaat caattcatta tctttcaagt aatcaggcaa ctccttataa catactaaaa 1080

catgttgttc agtcattgct ttggcagaaa catgtttatt tttctcaaat gcaagtaagc 1140

tcagtagagc ttgtgatgaa gcttgttcat ccaaagtcat atctctctct ccagaaaccc 1200

cagtattgtt ctttacatat agggattgag agactatcga cgtggcgaaa tgaaatgaat 1260

gatgtaaaat atcaagaatt taatgggaag gtatgtgtaa gttttgaagc ttggggatta 1320

aattga 1326

Claims

1. sedum lineare resistant gene of salt SLTRSA, it is characterized in that nucleotide sequence shown in SEQ ID NO.1 in sequence table.

2. the cloning vector pJET1.2_SLTRSA of sedum lineare resistant gene of salt SLTRSA containing claim 1 a kind of.

3. the host cell containing cloning vector pJET1.2_SLTRSA.

4. the expression vector pBI121_SLTRSA of sedum lineare resistant gene of salt SLTRSA containing claim 1 a kind of.

5. the host cell containing expression vector pB121_SLTRSA.

6. the purposes of sedum lineare resistant gene of salt SLTRSA enhancing arabidopsis or willow salt resistant character.