CN109402139A - Sedum lineare resistant gene of salt SLLAZY1 and its application - Google Patents
Sedum lineare resistant gene of salt SLLAZY1 and its application Download PDFInfo
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- CN109402139A CN109402139A CN201710712881.3A CN201710712881A CN109402139A CN 109402139 A CN109402139 A CN 109402139A CN 201710712881 A CN201710712881 A CN 201710712881A CN 109402139 A CN109402139 A CN 109402139A
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Abstract
The invention discloses sedum lineare resistant gene of salt SLLAZY1 and its applications, sedum lineare resistant gene of salt SLLAZY1, it is nucleotide sequence shown in SEQ ID NO.1 in sequence table, it is demonstrated experimentally that the salt resistant character of the host plants such as arabidopsis or tobacco can be enhanced in sedum lineare resistant gene of salt SLLAZY1.
Description
Technical field
The present invention relates to a kind of sedum lineare (Sedum lineare Thunb, abbreviation SlT) resistant gene of salt and its applications, belong to
In molecular biology and field of biotechnology.
Background technique
Currently, saline Land is the serious problems faced in urban garden and green space system greening, therefore salt-tolerant plant is selected to build
If salt-resistant type greenery patches, the Landscape benefit and ecological benefits of urban afforestation are effectively played, is to realize that water-saving, inexpensive garden greenland is built
And if one of the means of maintenance target.Sedum lineare (Sedumlineare) belongs to the perennial meat herbaceous plant of Crassulaceae, adaptability
It is extremely strong, there is heat-resisting, drought-enduring, cold-resistant, resistance to lean characteristic, be mainly born on hill meadow bog and rock, be excellent ground cover plant,
Artificial cultivation is in flower garden, garden greenland center.Therefore, it selects sedum lineare for research object, understands the physiological foundation of plant salt tolerance
With the approach of plant anti-salt, the signal pass through mechanism of Salt Strees Condition is recognized, understand fully the function and effect of resistant gene of salt, inquire into salt tolerant
The progress of plant election effects is accelerated saline-alkali land vegetation and is restored with important for further pushing salt-tolerant plant election effects
Meaning.Simultaneously rationally to carry out garden greenland using sedum lineare, beautifies the environment, provide theoretical foundation for construction urban green space.
Summary of the invention
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 SLLAZY1.
A second object of the present invention is to provide the cloning vectors containing sedum lineare resistant gene of salt SLLAZY1.
Third object of the present invention is to provide the host cells for containing above-mentioned cloning vector.
Fourth object of the present invention is to provide the expression vector containing sedum lineare resistant gene of salt SLLAZY1.
Fifth object of the present invention is to provide the host cells for containing above-mentioned expression vector.
Sixth object of the present invention is to provide sedum lineare resistant gene of salt SLLAZY1 enhancing arabidopsis or Tobacco Salt performances
Purposes.
Technical solution of the present invention is summarized as follows:
Sedum lineare resistant gene of salt SLLAZY1 is nucleotide sequence shown in SEQ ID NO.1 in sequence table.
The cloning vector pJET1.2_SLLAZY1 of the SLLAZY1 of resistant gene of salt containing sedum lineare.
Host cell containing cloning vector pJET1.2_SLLAZY1.
The expression vector pBI121_SLLAZY1 of the SLLAZY1 of resistant gene of salt containing sedum lineare.
Host cell containing expression vector pBI121_SLLAZY1.
Sedum lineare resistant gene of salt SLLAZY1 enhances the purposes of arabidopsis or Tobacco Salt performance.
Advantages of the present invention:
It is demonstrated experimentally that sedum lineare resistant gene of salt SLLAZY1 enhancing arabidopsis or Tobacco Salt performance.
Detailed description of the invention
Fig. 1 is SLLAZY1 gene cloning electrophoresis schematic diagram.
Fig. 2 is that SLLAZY1 is inserted into schematic diagram after expression vector.
Fig. 3 is transformant Genomic PCR the selection result after pBI121_SLLAZY1 arabidopsis thaliana transformation.
Fig. 4 is T3 homozygote semiquantitive PCR measurement expression result after pBI121_SLLAZY1 arabidopsis thaliana transformation.
Fig. 5 is SLLAZY1 transgenic arabidopsis T3 homozygote salt resistance experiment effect photo.
After Fig. 6 pBI121_SLLAZY1 transformation of tobacco, transformant Genomic PCR the selection result.
After Fig. 7 pBI121_SLLAZY1 transformation of tobacco, semiquantitive PCR measures expression result.
Fig. 8 SLLAZY1 transgene tobacco salt resistance experiment effect.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
Test method without specific conditions in embodiment, usually according to normal condition and item described in handbook
Part, or according to the normal condition proposed by manufacturer.
Carrier pJET1.2:Thermo, Clone JET PCR Cloning Kit#K1231
Carrier pBIl21 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) SLLAZY1 gene
From the sedum lineare (being derived from Pests in Tianjin Binhai New Area) that 150mM NaCl aqueous solution is handled, plant RNeasy is used
Plant Mini Kit (Transgene Code#E101-0150rxns) extracts total serum IgE, and utilizes EasyScript
Frist-Strand cDNA SynSgesis SuperMix (Transgene Code#AE301-03100rxns) reverse transcription goes out
cDNA.3 ' the end sequences that high-flux sequence (source Nuo Hezhi company carries out high-flux sequence) obtains SLLAZY1 gene are carried out to cDNA
Column are obtained amplification using the full length cDNA sequence that RACE technology (Takara-RACE kit) obtains SLLAZY1 gene
SLLAZY1 gene carries out sequencing analysis, and obtaining complete SLLAZY1 full length gene is 921bp.When constructing overexpression 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 builds.The specific steps of which are as follows:
1) synthesis of the first chain of .cDNA
With reverse transcription reagent box TaKaRaRNAPCR Kit (AMV) Ver.3.0, using total serum IgE as template, Oligo (dT) is
Primer synthesizes the first chain of cDNA under the action of AMV reverse transcriptase, and reverse transcription system is as follows:
Reaction condition: 42 DEG C of 60min, 99 DEG C of 5min.
2) sedum lineare SLLAZY1 gene 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 system is as follows:
Reaction condition: 94 DEG C of 3min；94 DEG C of 30s, 40 DEG C of 30s, 72 DEG C of 50s, 35cycles；72℃5min.
3) sedum lineare SLLAZY1 genetic fragment PCR amplification
Sequencing analysis is carried out using the SLLAZY1 gene that Takara RACE kit is expanded, obtains complete sedum lineare
SLLAZY1 full length gene is 921bp (SEQ ID No.1).The protein encoded by sedum lineare SLLAZY1 gene, is SEQ ID
Amino acid sequence shown in No.2.Primer software design SLLAZY1 gene upstream and downstream primer is utilized according to known cDNA sequence:
SEQ ID No.7:5'-ATGAAGTTACTAGGTTGGATGC-3',
SEQ ID No.8:5'-TTATGCTGTGCTCTCTGGATG-3', PCR reaction system is as follows:
PCR reaction condition are as follows: 95 DEG C, 5min；95 DEG C, 30s, 58 DEG C, 30s, 72 DEG C, 90s, 35cycles；72 DEG C,
10min；4 DEG C, ∞.
PCR after reaction, takes 1 μ LPCR product to carry out 1.0% agarose gel electrophoresis, detects the quality of PCR product
(see Fig. 1), remaining is used as the purification and recovery of product.
4) constructs the cloning vector containing sedum lineare SLLAZY1 gene
Construct the carrier pJET1.2_SLLAZY1 containing sedum lineare SLLAZY1 gene
Sedum lineare SLLAZY1 gene target fragment after glue recovery purifying utilizes Clone JET PCR Cloning Kit
In (pJET1.2:Thermo, Clone JET PCR Cloning Kit#K1231) recombination to carrier pJET1.2,
Obtain carrier pJET1.2_SLLAZY1.
Its response procedures is as follows:
24 DEG C of reaction condition, 10min static 30min on ice, 42 DEG C of heat shock 1min30s static 2min30s on ice,
It is transferred to competent cell DH5 α, 37 DEG C, 180rpm, 45min, bacterium solution is coated onto LB after this EP (end of program) and (is added anti-
Raw element Amp100uM) in solid 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
Row bacterium colony PCR verifying screens positive bacterium colony sequencing, obtains the host cell containing cloning vector pJET1.2_SLLAZY1.
Note: pJET1.2:Thermo, Clone JET PCR Cloning Kit#K1231 carrier are purchased from invitrogen；
Escherichia coli used are DH5 α competent cell, TIANGEN, CB101-2.
5) constructs the expression vector containing sedum lineare SLLAZY1 gene
Construct the expression vector pBI121_SLLAZY1 containing sedum lineare SLLAZY1 gene
When constructing overexpression vector, then pBI121 recombination sites are added at 5 ' ends 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'-ACGGGGGACTCTAGAGGATCCATGAAGTTACTAGGTTGGATGC-3'
SEQ ID No.10:5'-CGATCGGGGAAATTCGAGCTCTTATGCTGTGCTCTCTGGATG-3'
The plasmid that correct pJET1.2- gene is sequenced is extracted, as template, using containing recombination site SEQ ID
No.9, SEQ ID No.10 are that primer carries out PCR amplification, and reaction system is as follows
PCR reaction condition are as follows: 95 DEG C, 5min；95 DEG C, 30s, 58 DEG C, 30s, 72 DEG C, 90s, 35cycles；72 DEG C,
10min；4 DEG C, ∞.
PCR after reaction, takes 1 μ LPCR product to carry out 1.0% agarose gel electrophoresis, detects the quality of PCR product
(see Fig. 1), remaining is used as the purification and recovery of product.
PBI121 plasmid is extracted, Vector map (see Fig. 2) carries out double digestion linearisation to it, and program is as follows:
Reaction condition: 37 DEG C, 12h, 80 DEG C 20min inactivations.
Gene and the pBI121 plasmid of linearisation are used into Clone Express Entry One Step Cloning
Kit kit carries out recombination to construct, and response procedures are as follows:
Response procedures: 37 DEG C, 30min, on ice 5min, 42 DEG C of heat shock 1min30s static 2min30s on ice are transferred to impression
State cell DH5 α, is coated onto LB (addition antibiotic kan 50uM) solid for bacterium solution after this EP (end of program) and trains by 37 DEG C, 180r, 45min
It supports in base, 37 DEG C are incubated overnight.
The upstream and downstream primer (see SEQ ID No.9 and SEQ ID No.10) of carrier and target fragment is utilized respectively to same
A bacterium colony carries out bacterium colony PCR double verification, and screening positive bacteria drops into row sequencing (see SEQ ID No.11), contains Fo Jia to obtain
The expression vector pBI121_SLLAZY1 of careless resistant gene of salt SLLAZY1.
Note: this step is purchased from vazyme using Clon Express Entry One Step Cloning Kit,
6) contains the recombinant vector conversion Agrobacterium competent cell of sedum lineare SLLAZY1 gene
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), and helper plasmid is anti-with gentamicin
Property (Gen).
Using electric shock Agrobacterium-mediated Transformation method, by the coli expression carrier pBI121_ containing sedum lineare SLLAZY1 gene
SLLAZY1 is transformed into Agrobacterium strain C58 (pMP90) competent cell, 28 DEG C, cultivates 36h, bacterium colony PCR selects positive colony
Bacterium colony.
Embodiment 2
1. arabidopsis thaliana transformation
(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 medium that (addition antibiotic Gen makes concentration
30mg/L, addition antibiotic Rift make concentration 25mg/L and antibiotic Sp make concentration 50mg/L) culture 15 hours or so
(to OD600=0.8 or so), 180rpm, 28 DEG C.
The expansion culture of positive colony bacterium: (the addition of suitable antibiotic is added in the YEB fluid nutrient medium of fresh 10ml
Antibiotic Gen concentration is 30mg/L, antibiotic Rift concentration is 25mg/L and antibiotic Sp concentration is 50mg/L), then connect
The appropriate positive colony bacterium solution of kind is cultivated into YEB fluid nutrient medium, 180rpm, in 28 DEG C of cultures to OD600=0.6.
2. converting
Supernatant is abandoned into bacterium solution centrifugation (3000rpm, 15 DEG C, 10min) afterwards, with the mass concentration of twice the taken bacterium solution of volume
Thallus (slowly operation is to guarantee thallus vigor) is resuspended for 5% aqueous sucrose solution, so that thallus 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, grows under the condition of culture that relative humidity is 70%, until
Seed is mature.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
By the T1 collected for seed after disinfection, it is placed on 4 DEG C of refrigerator three days, then on the super-clean bench by transgenosis
Arabidopsis seed is uniformly sowed on the 1/2MS solid screening and culturing medium containing 50 μ g/mL kanamycins, in 1800Lux, light week
Phase 16h illumination/8h is dark, grows 8-10 days, and leaf is that bottle green is the T1 of transgenic arabidopsis for positive transformant.Work as T1
When growing to 3-4 piece true leaf for positive transformant plant, being transplanted to soil, (German import peat soil 422#, is purchased from
Klasmann-Deilmann GmbH, Germany, http://www.klasmann-deilmann.com) in, at 25 DEG C of temperature,
1800Lux, photoperiod 16h illumination/8h is dark, continued growth 14 days under the condition of culture that relative humidity is 70%, first makees positive
The identification (see Fig. 3) of transformant, then first the expression of its transgenosis is identified (see Fig. 4) by semiquantitive PCR, it chooses
The low independent transformation strain of expression 6 and high independent transformation strain 7 of expression.Continue to give birth under the above conditions
Long, it is T2 for transformed the seed that seed is collected about after one and a half months.Repeat the above steps to obtain No. 6 and No. 7 T3 for homozygote
Seed.(3) salt stress processing is carried out to transgenic arabidopsis
No. 6 T3 are planted in the soil for homozygote seed, No. 7 T3 for homozygote seed, wild arabidopsis seed respectively,
At 25 DEG C of temperature, 1800Lux, photoperiod 16h illumination/8h is dark, grows 21 days under the condition of culture that relative humidity is 70%, often
Kind of plant retains 21 plants of consistent seedling of growing way, 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 solution).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
1. transformation of tobacco
Tobacco for positive colony bacterium conversion is NC89 (6855-2 × 6772) tissue-cultured seedling.
(1) concrete operation step of transformation of tobacco:
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 medium that (addition antibiotic Gen concentration is
30mg/L, antibiotic Rift concentration are 25mg/L and antibiotic Sp concentration is 50mg/L) 15 hours or so are cultivated (to OD600
=0.8 or so), 180rpm, 28 DEG C.
The expansion culture of positive colony bacterium: (the addition of suitable antibiotic is added in fresh 10Ml YEB fluid nutrient medium
Antibiotic Gen concentration is 30mg/L, antibiotic Rift concentration is 25mg/L and antibiotic Sp concentration is 50mg/L), then connect
The appropriate positive colony bacterium solution of kind is cultivated into YEB fluid nutrient medium, 180rpm, in 28 DEG C of cultures to OD600=0.6.
2. converting
Supernatant is abandoned into bacterium solution centrifugation (3000rpm, 15 DEG C, 10min) afterwards, with the mass concentration of twice the taken bacterium solution of volume
Thallus (slowly operation is to guarantee thallus vigor) is resuspended for 5% aqueous sucrose solution, so that thallus is scattered, adjusts OD600=0.8.
The tobacco tissue-cultured seedling for choosing growth 30 days in order, selects thick and solid blade, subtracts limb edge, blade is cut into
The explant fragment of 1cm × 1cm.The blade material sheared is immersed to the sucrose water of the mass concentration 5% containing Agrobacterium thallus
In solution, shake culture 10min under the conditions of 24 DEG C.After infecting, be placed on after explant is blotted on MS culture medium (MS salt 4.4g,
Sucrose 30g is settled to 1L, pH=5.7, agar 7.2g), dark culturing 3 days.By the explant Jing Guo dark culture with sterilized
Distilled water cleans 2-3 times, and after being blotted with filter paper, explant is placed on MS salt 4.4g on Selective agar medium, sucrose 30g, auxin
NAA1.86mg, basic element of cell division 2ip1.02mg, kanamycins 500mg, cephalosporin 500mg, agar 7.2g are settled to 1L,
PH=5.7；Condition of culture: cultivating 14 days under 2000Lux light, and illumination/dark is 16h/8h)
(2) pass through Selective agar medium, when explant grows small young plant, whole small young plant is cut, root restriction is erected
Straight cutting is into culture medium on MS culture medium, root induction.After the completion of under growth root, positive identification is carried out to each independent transformation
(see Fig. 6).
The independent transformation strain 6 of high expression level and the independent transformants of low expression level are chosen by semiquantitive PCR
It is No. 8 progress salt resistance experiments (see Fig. 7)
(3) tobacco is subjected to salt treatment
By the uniform transgenosis height expression tobacco of the growing way for growing 7 days, low expression transgene tobacco, wild-type tobacco transplanting
Into native basin, after native basin seedling is grown 15 days, every kind of plant retains 21 plants of consistent seedling of growing way, is randomly divided into three groups, every group
Each 7 plants of different types of plant carries out pouring processing with salinity treatment fluid (150mM NaCl aqueous solution aqueous solution).Pour one within 3 days
Secondary, each irrigation amount is 0.5 times of soil quality, to keep constant, the observation plant after coprocessing 30 days for the treatment of fluid concentration in basin
Strain is simultaneously taken a picture (see Fig. 8).
SEQUENCE LISTING
<110>University Of Tianjin
<120>sedum lineare resistant gene of salt SLLAZY1 and its application
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<170> PatentIn version 3.3
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ctgaagctca tcagcgatga acttgaaaag gtcctggcag ccgaggcaat ggaagacgac 420
tgtaatttct catctggtag aaacagtcat gtcagcaatg ggagaaacag tcatgtcagc 480
aatgggagaa acagttatgt aagcaatggg agaagcagca atggtagcac cattaccttg 540
agtggaagag cactagaaag cacagaggct gctggaaatg gaaacgttat ctgtcccctc 600
cagggttacc tttttgggtc agcccttgaa gaacaaccaa gtactatgcc aaagagagac 660
cacagaacat cgcttggaga gttgtttcaa aatagcaaag cggttgagga ctactacaag 720
gaaaagtctg ggaagcgaga aacagacaag tcagccatcc aactgatgaa gaaacttctg 780
aagaaaacaa agattcacac atcatcaaag aacaaaagta catccactgg aggcacagaa 840
gatactactt ttactgatac caaattggct aagattttac aaatgtttca gagaaaagtt 900
catccagaga gcacagcata a 921
Claims (6)
1. sedum lineare resistant gene of salt SLLAZY1, it is characterized in that nucleotide sequence shown in SEQ ID NO.1 in sequence table.
2. the cloning vector pJET1.2_SLLAZY1 of the sedum lineare resistant gene of salt SLLAZY1 containing claim 1.
3. the host cell containing cloning vector pJET1.2_SLLAZY1.
4. the expression vector pBI121_SLLAZY1 of the sedum lineare resistant gene of salt SLLAZY1 containing claim 1.
5. the host cell containing expression vector pBI121_SLLAZY1.
6. the purposes that sedum lineare resistant gene of salt SLLAZY1 enhances arabidopsis or Tobacco Salt performance.
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| CN116790628A (en) * | 2023-08-05 | 2023-09-22 | 中国农业科学院油料作物研究所 | Application of rape BnLAZY1.A03 gene and allelic variation site |
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| CN116790628B (en) * | 2023-08-05 | 2024-02-06 | 中国农业科学院油料作物研究所 | Application of rape BnLAZY1.A03 gene and allelic variation site |
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