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CN102417911A - Over-expression of cabbage type rape BnLAS gene to improve plant drought resistance - Google Patents

Over-expression of cabbage type rape BnLAS gene to improve plant drought resistance Download PDF

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CN102417911A
CN102417911A CN2010106235055A CN201010623505A CN102417911A CN 102417911 A CN102417911 A CN 102417911A CN 2010106235055 A CN2010106235055 A CN 2010106235055A CN 201010623505 A CN201010623505 A CN 201010623505A CN 102417911 A CN102417911 A CN 102417911A
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plant
gene
bnlas
drought resistance
expression
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CN102417911B (en
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周永明
杨明贵
杨庆勇
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Huazhong Agricultural University
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Huazhong Agricultural University
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Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to cloning of a cabbage type rape BnLAS gene with drought resistance and obtaining an arabidopsis transformation plant with high drought resistance by utilizing overexpression of the gene in arabidopsis. The method comprises the following steps of using the nucleotide sequence shown as SEQ ID NO: 2 and 3, determining that the sequence of the amplified DNA fragment is a homologous sequence with an LAS gene in arabidopsis thaliana, and is named as BnLAS, wherein the nucleotide sequence of the BnLAS is shown as a sequence table SEQ ID NO: 1 is shown. The expression quantity detection is carried out by extracting RNA of different tissue parts of the cabbage type rape and adopting RT-PCR, and the expression difference of the gene in the cabbage type rape at any moment is found. An overexpression vector is constructed by using BnLAS genes and a CaMV35S promoter, the overexpression vector is transformed into wild type arabidopsis through an agrobacterium-mediated genetic transformation method, the overexpression of the BnLAS genes in BnLAS gene positive transformation plants is identified through an RT-PCR technology, and drought-tolerant arabidopsis transformation plants are obtained through combination of field morphology identification.

Description

Cross expression swede type rape BnLAS gene and improve plant drought resistance
Technical field
The invention belongs to the plant molecular breeding technical field, be specifically related to the clone of swede type rape BnLAS gene and utilize this gene in plant, to cross expression, caused that the expression plant drought resistance improved, thereby cultivate the plant that drought resistance significantly increases.
Background technology
Arid mainly shows four aspects to the harm of growth and development of plants: 1) thus the disappearance of moisture reduces the movable growth and development of plant that suppresses of plant metabolism in the plant materials; 2) the following influence of drought condition owing to stomatal closure, photosynthesis of plant decrease in efficiency even generation light restraining effect; 3) plant respiration strengthens under the drought condition, thereby accelerates the decomposition of plant substance in vivo; 4) generation of plant interior free yl and the system of removing balance are broken under the drought stress, thereby the aggravation peroxidization causes oxidative damage to plant.According to statistics, 1/3rd of world land area is arid or semiarid zone, and it is the arid and semi-arid area that there is 1/2nd land area in China.Constantly increase at population, cultivated area reduces day by day and the serious deficient scarce condition of Freshwater resources under, effectively reduce Freshwater resources consumption and improve the contradiction of agricultural production efficiency, be one of key subjects that press for both at home and abroad solution.
Under drought condition, plant can change the normal growth of keeping plant through self morphological structure and grow, diminish like plant type, and well developed root system, pore sink, stomatal closure, rising area minimizing etc.In addition, plant also can strengthen the ability that drought stress is to external world restrained oneself through the variation of osmotic potential in the cell, as producing proline(Pro) and trimethyl-glycine, oozing the material of transferring element etc. to have raising plant osmotic adjustment ability.In adapting to the drought stress process, the arid system of replying is a controlled by multiple genes on the interior molecular level of plant materials.These genes mainly show two aspects: one type is the gene of abduction delivering quantitative changeization under drought stress, like osmoregulation gene (P5CS), Lea albumen, aquaporin (PM28A), anti-oxidative damage system etc.; Another kind of is to regulate transcription factor such as the DREB that the drought resisting functional gene is expressed.The molecular biology mechanism research of current plant drought resistance deepens continuously, and transgenic technology increasingly mature is for a new approach has been created in the plant drought resistance breeding.
The GRAS protein family is one type and extensively is present in distinctive transcription factor in the higher plant; This family gene principal feature is on protein level, to have not conservative N end; The leucine Tumor-necrosis factor glycoproteins of VHIID die body and both sides; PFYRE and SAW die body (Pysh LD, Wysocka-Diller J, Camilleri C; Bouchez D, Benfey PN (1999) The GRAS gene family in Arabidopsis:sequence characterization and basic expression analysis of the SCARECROW-LIKE genes.Plant J 18:111-119; Bolle C (2004) The role of GRAS proteins in plant signal transduction and development.Planta; 218:683-692) 33 GRAS members (TAIR, http://www.arabidopsis.org have been found in the arabidopsis gene group at present; Lee et al.2008).
Though GRAS family has found 33 members at present in Arabidopis thaliana, have only member's function of 1/3rd to know its function through the research of two mutants.Find that in these members research GRAS albumen relates to the growth of root on the different cells level, meristematic tissue is kept and critical function such as GA signal transduction.The LAS gene is GRAS transcription factor family member, and this gene only has an exons structure.This gene is conservative at rape plant camber such as Arabidopis thaliana, swede type rapes, and the LAS gene ORF zone of different plant species has the homology of 75-90% in belonging to.
Summary of the invention
The BnLAS gene that the objective of the invention is in the overexpression swede type rape improves plant drought resistance, has good drought resistance thereby cultivate, and the plant of ability genetic stability.
The present invention realizes through following technical scheme:
The present invention has at first cloned the LAS homologous gene in the swede type rape, and the applicant is its called after BnLAS gene, through at CaMV35S promotor control overexpression BnLAS gene in Arabidopis thaliana down, causes the plant drought resistance enhancing.
The applicant obtains a kind of BnLAS gene that swede type rape has drought resistance that derives from through separation and cloning process, and its nucleotide sequence is shown in sequence table SEQ ID NO:1.
The special primer of wherein cloning said BnLAS gene is YM113 and YM114, its nucleotide sequence (seeing sequence table SEQ IDNO:2,3) as follows:
YM113:5′-CAGTGTCGACAACAATGCTTACTTCCTTCAAA-3′;
YM114:5′-CAGTGAGCTCTCATTTCCACGACGAAAC-3′。
The applicant has set up the method that a kind of BnLAS of utilization gene improves plant Arabidopis thaliana drought resistance, and it comprises the steps:
1) the described primer of design claim 1, the clone has the BnLAS of drought resistance gene is arranged from the swede type rape genome, and its nucleotide sequence is shown in SEQ ID NO:1;
2) make up BnLAS gene overexpression carrier, and this carrier is transformed in the Agrobacterium;
3) through the agrobacterium mediation method arabidopsis thaliana transformation, screening obtains BnLAS gene overexpression transformed plant;
4) adopt molecular detecting method that the expression transformed plant of crossing of step 3) is carried out the drought resistance evaluation;
5) further identified blade surface wax layer, chlorophyll content and the blade construction of expressing transformed plant in the field.
4, the application of the described gene of claim 1 in improving plant drought resistance.
5, the application of the described primer of claim 2 in improving plant drought resistance.
6, the described gene of claim 1 improves the application in the plant drought resistance rape genus GRAS gene family member.
More detailed technical scheme is described below:
1, the clone of BnLAS gene:
According to LAS sequence (http://www.arabidopsis.org/servlets/TairObject in the Arabidopis thaliana? Type=gene&id=431571) the design primer carries out PCR in swede type rape China two No. 5 (this kind is the open cabbage type rape varieties of promoting for China); After amplification checks order; Utilize known array design TAIL-PCR (Liu YG; Misukawa N, Oosumi T and Whittier R F.Efficient isolation and mapping of Arabidopsis thaliana T-DNA insertjunctions by thermal asymmetric interlaced PCR.1995.The Plant Journal 8:457-463.) primer separates the unknown flanking sequence of this known array.After confirming through information biology behind the above acquisition sequence assembly is that a complete opening is read frame.Carrying out confirming that the LAS gene is a homology sequence in this sequence and the Arabidopis thaliana after BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) analyzes, is the BnLAS gene with the LAS dna homolog property unnamed gene of swede type rape respectively according to its source.
2, the BnLAS gene is expressed checking in swede type rape:
In order further to verify the BnLAS gene, carry out the detection of expression amount through RT-PCR through the RNA that extracts the two No. 5 different tissues positions of cabbage type rape variety China.
3, cross the structure of expression vector:
Additional enzyme cut-grafting head in BnLAS gene two ends is connected in the PGEM-T carrier (available from Promega company),, connects pBI121 carrier (available from Clontech company), through CaCl through double digestion 2Chemical process be converted in the intestinal bacteria.Extract positive colony plasmid in the intestinal bacteria, (with reference to J. Sa nurse Brooker, EF is the Ritchie not through chemical cold shock method with it; T Manny A Disi work, Huang Peitang, Wang Jiaxi etc. translate; Molecular cloning experiment guide (third edition), Science Press, 2002 editions) be converted in the Agrobacterium GV3101 fungus strain.
4, the conversion of 35S::BnLAS plant expression vector:
Adopt the mode of vacuum infestation just to transform arabidopsis thaliana inflorescence the Agrobacterium bacterium liquid of crossing expression vector that builds at the florescence.Treat in the 1/2MS substratum, to carry out the resistance separation behind the fruit maturation; There is resistant plant to transfer to (the basic nutrition soil that contains paddy growth in the rice nutrition soil of aseptic routine report; As be added with conventional nitrogenous fertilizer, phosphate fertilizer and potash fertilizer and the soil in vegetable garden, other nutrition are not had particular requirement).Obtaining positive transformed plant existence and overexpression through round pcr and this gene of RT-PCR technical evaluation.
5, cross the evaluation of expressing the plant drought resistance:
Above-mentioned steps 4 is obtained positive plant through the mensuration of excised leaf percentage of water loss and drought resisting evaluation under greenhouse earth culture growth conditions, confirm that this plant has than the tangible drought resisting phenotype of wild-type Arabidopis thaliana plant.
6, the analysis of causes of drought resistance Arabidopis thaliana transformed plant
Through importing the Arabidopis thaliana transformed plant that the acquisition of swede type rape BnLAS foreign gene has good drought resistance; Express the reason that plant produces drought resistance for further analyzing the mistake that the present invention obtained, the applicant has carried out crossing wax content and the blade construction observation of expressing the observation of BnLAS gene plant leaf color, measuring chlorophyll content, blade surface.
More detailed scheme is of " embodiment ".
Positively effect of the present invention
The present invention causes Arabidopis thaliana survival rate under lack of water is handled to improve through importing swede type rape BnLAS foreign gene.Its drought resistance mechanism is that the reducing of transformed plant stomatal aperture, blade surface wax layer increase, and slows down thereby cause the plant leaf surface-moisture to scatter and disappear, and finally reaches the effect of drought resisting; The present invention provides a kind of new thinking and feasible method for improving plant drought resistance, is having wide practical use aspect the drought resistance breeding of crop.
Description of drawings
Sequence table SEQ ID NO:1 is the BnLAS gene that the present invention clones, and the sequence total length is 1326bp.
Sequence table SEQ ID NO:2 and 3 is a pair of primers that the present invention clones the BnLAS gene, respectively called after primer YM113 and primer YM114.
Fig. 1: be techniqueflow chart of the present invention.
Fig. 2: be BnLAS gene different tissues expression analysis figure in swede type rape.
Fig. 3: the plant eukaryotic expression vector pBnLAS that is one of them embodiment of the present invention makes up.A is a pBI121 carrier synoptic diagram; B is a pBnLAS carrier synoptic diagram.
Fig. 4: be transfer-gen plant BnLAS overexpression detected result, swimming lane 1 is a wild-type Arabidopis thaliana plant, and 2-5 is different transgenic lines, 6 negative contrasts.Among the figure: the BnLAS amplimer is to being HY225/HY226 (primer sequence is seen table 3); BnActin2 is confidential reference items, and amplimer is to being Actin2F/Actin2R (primer sequence is seen table 3).
Fig. 5: be that expression Arabidopis thaliana plant and wild-type arabidopsis ' chlorophyll content compare
Among the figure, A, B represent wild-type Arabidopis thaliana WT (A) respectively and cross the seedling that expression Arabidopis thaliana plant OL18 (B) grew 16 days on the 1/2MS substratum.C is wild-type Arabidopis thaliana WT and crosses expression Arabidopis thaliana plant OL14, OL18, OL10, OL50 chlorophyll content relatively; Asterisk (* *) represented that expression Arabidopis thaliana plant chlorophyll content was with significantly (the t test of wild-type Arabidopis thaliana WT comparing difference; In the P=0.01 level, n=5).
Fig. 6: transfer-gen plant (OL14 and OL18) and wild-type plant excised leaf dehydration rate-of-loss of coolant are relatively.A is 3 hours and 6 hours stripped dehydration pictures of blade, and a left side is followed successively by the contrast of wild-type plant, crosses expression plant family OL14, crosses expression plant family OL18 to the right side.B is the stripped rate-of-loss of coolant graphic representation of blade, and * (OL14) or # (OL18) and * * or ## represent the significant difference of transfer-gen plant with wild-type t test under P<0.05 and P<0.01 level respectively.
Fig. 7 was that expression Arabidopis thaliana plant and wild-type Arabidopis thaliana plant drought resistance compare.Among the figure: (A) wild at plant strain growth in the time of 30 days, keep every basin living weight to equate after, fill water and carry out drought resisting again and handle, figure below is 15 days pictures after the drought resisting.Experiment is carried out three times and is repeated.WT: wild-type Arabidopis thaliana, OL18: transfer-gen plant, las: two mutants plant.(B) drought is coerced transfer-gen plant and the wild-type plant after the recovery.Plant strain growth is filled water and is carried out drought and coerce processing after 30 days.The plant rehydration was counted after 5 days, experiment repetition 3 times, and each repeats 15-20 plant.(C) wild-type, cross the expression plant (OL14, OL18) with las two mutants rehydration after survival rate.Error line is a standard deviation.* represent respectively that with * * transgenic is with wild-type plant t test significant difference under P<0.05 and P<0.01 level.
Fig. 8: be that the wild-type Arabidopis thaliana is expressed abaxial surface pore form and the distribution of family OL18 with crossing under SEM and the opticmicroscope.Compare with wild-type plant (A), the epidermic cell of crossing expression family OL18 (B) diminishes and closes stoma number and increases.(C) wild-type plant and expression fresh blade of plant OL18 and the total stomatal number of stator blades unit surface and open pores number calculating excessively under the same terms.Error line is a standard error, * * represent transgenic with the wild-type plant through the significant difference under t test P<0.01 level.(D) cross expression plant OL18 and wild-type plant stator blades and fresh blade stomatal aperture.Error line is a standard error, and * * represented that the expression plant passed through t test significant difference (P<0.01) with the wild-type plant.
Fig. 9: cross the surface wax layer increase of expression BnLAS gene Arabidopis thaliana plant leaf and show that through the chlorophyll leaching blade epidermis perviousness changes.Compare with wild-type Arabidopsis leaf surface (A), cross expression plant OL18 (B) epicutile wax content and rise.(C) wild-type plant and expression plant OL14 excessively, OL18 lotus throne leaf chlorophyll leaches to be analyzed.Data from three revision tests.Error line is represented standard error.* (OL14) or # (OL18) and * * or ## represented respectively that the expression plant was with wild-type t test significant difference under P<0.05 and P<0.01 level.
Embodiment
Embodiment 1 swede type rape BnLAS gene isolation
The clone of BnLAS gene: in the present embodiment; Nucleotide sequence design primer YM113 according to Arabidopis thaliana LAS gene ORF two ends; In two No. 5 of swede type rape China, carry out pcr amplification with primer YM114 (this primer sequence see specification sheets table 1 with sequence table SEQ ID NO:2 and 3); Reaction system 100 μ l, reaction system is following: 1 * PCR damping fluid (contains MgCl 2, available from MBI Fermentas, Lithuania company); 200 μ M dNTPs, 1U Pfu polysaccharase, (primer is that Shanghai living worker's biotechnology ltd is synthetic to 0.25 μ M primer; All the other threes are available from MBIFermentas, Lithuania company), add distilled water to 100 μ l.The pcr amplification appearance is PTC-100 (available from a Bio-Rad company).Reaction conditions is following: 94 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 1min20s, 29 circulations; 72 ℃ of 4min.Reaction product is concentrated, operate as follows: add rearmounted-20 ℃ of placement 30min of 2 times of volume absolute ethyl alcohols after adding 1/9 volume 3M sodium-acetate, whizzer (available from eppendorf company) high speed centrifugation 10min removes supernatant, dries up 10min and adds 50 μ l distilled waters.Carry out end and add A, reaction system is following: 1 * PCR damping fluid, 1.5mMMgCl 2, 200 μ M dATP add PCR product to 50 μ l.Reaction conditions is following: 94 ℃ of 3min, 72 ℃ of 30min.Adopt UNIQ-10 pillar DNA glue to reclaim test kit (worker's biotechnology ltd is given birth in Shanghai) after electrophoresis finishes and cut the glue recovery.Reclaimer is explained according to test kit: cut out the centrifuge tube that target fragment is put into 1.5ml with blade, add binding buffer liquid, place 55 ℃ of water-baths to heat 10min, every 2min mixing once; Be transferred to after glue melts fully in the UNIQ-10 post that is enclosed within the collection tube, room temperature is placed 2min; 8,000rpm, centrifugal 1min; Outwell the waste liquid in the collection tube, add 500 μ l washing fluids, 8, the centrifugal 1min of 000rpm room temperature repeats this step once; Outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, 12, the centrifugal 15sec of 000rpm; The UNIQ-10 post is put into the centrifuge tube of a new 1.5ml, add the distilled water of 30 μ l in pillar film central authorities, room temperature is placed 5min; 12, the centrifugal 1min of 000rpm, the liquid in the centrifuge tube is the dna fragmentation of recovery.The target fragment that reclaims is connected pGEM
Figure BSA00000413701400051
-T carrier (available from Promega company).The method that schedule of operation provides by test kit: in the centrifuge tube of 0.5ml, set up following ligation system: 2 * fast connect damping fluid (available from MBI Fermentas, Lithuania company), 2.5 μ l; PGEM -T carrier (50ng/ μ l), 0.5 μ l; T4 dna ligase (3U/ μ l) 0.5 μ l; PCR reclaims product 1.5 μ l (T4 dna ligase and 2 * be connected damping fluid fast all available from MBI Fermentas, Lithuania company).Add to accomplish flick behind the above composition moving mixing centrifugal after, place 4 ℃ of connections of spending the night.To connect product then and be converted into intestinal bacteria: method for transformation adopts CaCl 2Method (with reference to J. Sa nurse Brooker, FF is the Ritchie not, T Manny A Disi work, and Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions), step is following: take out the DH5 α CaCl for preparing in-70 ℃ of refrigerators 2Competent cell is placed on ice, and 5min treats that it thaws; Get 2 μ l ligation liquid and 50 μ l competent cells add the aseptic centrifuge tube of 1.5ml respectively, flick mixing, be placed on 30min on ice with finger; Heat shock 90s in 42 ℃ of water-baths takes out and places 2-3min on ice; The LB liquid nutrient medium that adds 400 μ l is at 37 ℃ of shaking culture 1h (30-50rpm/min); Conversion fluid 200 μ l after the absorption shaking culture are coated on the aseptic LB solid medium that contains ammonia benzyl mycin resistance, place 16-24hrs at 37 ℃; Select positive colony to 10 μ l 0.02N NaOH and place the laggard performing PCR of 10min and detect, with the clone of test positive choose put LB liquid nutrient medium that 5ml contains 100mg/L ammonia benzyl mycin resistance 37 ℃ of shaking tables vibrate (150rpm) cultivate 12-18hrs and cultivate; Is that mixing in 1: 1 is saved in the aseptic centrifuge tube of 1.5ml with bacterium liquid with 50% aseptic glycerine volume ratio, checks order respectively.Show that through sequencing result this mrna length is that 1326bp (referring to sequence table SEQ ID NO:1) possesses a complete ORF reading frame, contains 441 aminoacid sequences.This gene shows that with LAS (At1g55580) the gene comparison of Arabidopis thaliana homology is 96.6%, so with this gene fragment called after BnLAS gene.
Be used to clone the sequence table of swede type rape BnLAS gene the primer among table 1 the present invention
Figure BSA00000413701400061
For the BnLAS gene two terminal sequence amino acid levels of verifying acquisition do not change and this fragment is the complete genome fragment.Carry out TAIL-PCP technology (Liu YG; Mitsukawa N; Oosumi T, Whittier RF.Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR.1995.Plant J 8:457-463) sequence at BnLAS two ends is carried out the flank separation.The BnLAS gene 5 ' end design primer (MGp03, primer sequence is seen table 1 for MGp01, MGp02) and 3 ' end design primer (MGp06, primer sequence is seen table 1 for MGp04, MGp05).The following first set reaction system of TAIL-PCR step is following: 1 * PCR damping fluid (available from MBI Fermentas, Lithuania company), 1.5mM MgCl 2, 200 μ M dNTPs, 0.2 μ M primer sp1 (MGp01 or MGp04, primer sequence is seen table 1), 2 μ M merger property primer (A, A2, A3, primer sequence is seen table 1), 0.75U polysaccharase, 20ng DNA.In pcr amplification appearance (available from Bio-Rad company), react first set reaction in reaction conditions such as the table 2.Get 50 times of 2 μ l reaction product dilutions after reaction finishes, get 1 μ l as secondary pcr template.Reaction system is following for the second time: 1 * PCR damping fluid (available from MBI Fermentas, Lithuania company), 1.5mM MgCl 2, 200 μ M dNTPs, 0.2 μ M primer MGp02 or MGp05 (primer sequence is seen table 1), 1.5-2 μ M merger property primer (A, A2, A3, primer sequence is seen table 1), 0.75U polysaccharase, 1 μ l first round PCR cut back.Reaction for the second time in response procedures such as the table 2.Get second and take turns amplified production 2 μ l and add 98 μ l distilled waters dilutions, preserve or be used for the next round reaction for-20 ℃.Reaction system is with the reaction system second time for the third time, and primer is respectively MGp03 or MGp06 (primer sequence is seen table 1).Program is reacted with table 2 for the third time.BnLAS gene 5 ' end is annexed second of property primer A1 third round amplified production and A2 take turns amplified production, and BnLAS gene 3 ' end A3 third round amplified production check order (order-checking is by the completion of the big-and-middle living Science and Technology Ltd. of Beijing China).Confirm YM113, the YM114 extension increasing sequence is the complete ORF sequence of this gene order, and any variation does not take place two terminal amino acid sequences.This sequence is further analyzed through BLAST, learnt that this candidate gene reaches 90.0% with the homology of LAS gene on dna level, reach 87.8% with the homology consistence of LAS gene on the protein level and reach 96.8% with similarity.Promptly this gene is the homologous genes of LAS, and the applicant is with its called after BnLAS.
The TAIL-PCR response procedures that table 2 the present invention adopts
Figure BSA00000413701400071
The specific expressed analysis of embodiment 2BnLAS gene organization
The total RNA of swede type rape extracts and adopts Trizol reagent to extract (reagent is available from Invitrogen company), and extraction procedure carries out according to the specification sheets of this reagent, and concrete steps are following: the swede type rape root; Stem; Leaf, flower, bud and stem top are fixed in the liquid nitrogen rapidly after organizing clip; Taking out the 100mg material grinds with liquid nitrogen in the precooling mortar fast; Grind the back material and add like concuss in the 1.5ml centrifuge tube that contains 1ml Trizol evenly rapidly, room temperature is placed 5min; Add the 0.2ml chloroform, vibration 15s; The centrifugal 10min of 2,000 * g; Get 500-600 μ l supernatant in new no RNA enzyme 1.5ml centrifuge tube; Adding equivalent volumes does not have the Virahol of RNA enzyme, turns upside down for several times, leaves standstill 10-30min on ice; The centrifugal 10min of 12,000 * g; Outwell supernatant, this moment, RNA was visible; Add 1ml 75% no RNA enzyme ethanol, outwell after the flushing, dry up; Add 30-50 μ l0.05%DEPC and handle the distilled water dissolving.Carry out the RT-PCR reaction after RNA detects, (this test kit is available from Fermentas company, and MBI), schedule of operation is carried out according to the specification sheets of test kit by a commercial reagents box completion in this reaction.Concrete steps are following: following composition is joined 0.5ml not to be had in the RNA enzyme centrifuge tube, the total RNA of 10ng-5 μ g, 1 μ l (dT) 18Primer (0.5 μ g/ μ l), DEPC handles distilled water and is settled to 12 μ g; With of short duration centrifugal behind the above composition mixing; Be placed on 70 ℃ of reactions of PTC-100PCR amplification appearance (available from Bio-Rad company) 5min, take out cooled on ice, centrifugal; In centrifuge tube, continue to add following composition: 4 μ l, 5 * damping fluid, 1 μ l RNA enzyme inhibitors (20U/ μ l), 2 μ l 10mM dNTP are centrifugal behind the mixing; Be seeded in 37 ℃ of 5min; Add 1 μ l RevertAid TMH Minus M-MuLV ThermoScript II (200U/ μ l); Mixture is seeded to 42 ℃ of reaction 60min; 70 ℃ of reaction 10min termination reactions; Preserve or use with PCR and detect for-20 ℃.Use HY225, HY226 primer (primer sequence is seen table 3) carries out, and the PCR reaction conditions is following: 94 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 30s, 31 circulations; 72 ℃ of 4min.Detected result shows that the BnLAS gene is expressed the strongest at the root of swede type rape, next bud and stem top, and stem has faint expression, and blade is not expressed.
Swede type rape genetic expression and transfer-gen plant detect the primer sequence table among table 3 the present invention
Primer title primer sequence (5 '-3 ')
HY225 GAAGATGCCACCGAGAA
HY226 AGTGTTTTCAGACAAGCC
Actin2F AGCGCTGAGGCTGATGATATTCAAC
Actin2R TCTAGAAACATTTTCTGTGAACGATTC
NPT3 GAGGCTATTCGGCTATGACTG
NPT4 ATCGGGAGCGGCGATACCGTA
YM14 AAGACCGGCAACAGGATTCA
The structure of embodiment 3 plant expression vectors and Arabidopis thaliana genetic transformation
1. the structure of plant expression vector
The pGEM that will comprise the BnLAS gene among the embodiment 1
Figure BSA00000413701400081
-T vector plasmid pV5 carrier and pBI121 carrier (available from Clontech company) are carried out Sac I and Sal I double digestion (the carrier details are seen shown in Figure 3) respectively; The endonuclease reaction system is following: 1 * Tango damping fluid; 1U Sac I restriction endonuclease; 1U Sal I restriction endonuclease is (all available from MBI Fermentas; Lithuania company), about 2 μ g DNA add distilled water and are settled to 50 μ l.The centrifugal back 37 ℃ of constant temperature 4h of mixing cut glue and reclaim.It is following that the BnLAS gene fragment that reclaims is connected to pBI21 carrier reaction system: 10 * T4DNAl connects damping fluid 1 μ l; Enzyme is cut pBI121 carrier DNA 5 μ l; BnLAS gene fragment 3 μ l; T4 dna ligase (5U/ μ l) 1 μ l, TV are 10 μ l (10 * T4 dna ligase damping fluid and T4 dna ligases all available from MBI Fermentas, Lithuania company).Mix and connect product transformed into escherichia coli bacterial strain DH5 α after back 4 ℃ of connections are spent the night.Screening positive clone in the LB solid plate that contains 50 μ g/ml kantlex; The extracting plasmid carries out that enzyme is cut and PCR identifies (seeing embodiment 1); Order-checking confirms not have the reading frame sudden change, obtains to contain to insert the segmental recombinant clone of purpose, with this carrier called after pBnLAS.The application freeze-thaw method (with reference to J. Sa nurse Brooker, EF is the Ritchie not, T Manny A Disi work, and Huang Peitang, Wang Jiaxi etc. translate, molecular cloning experiment guide (third edition), Science Press, 2002 editions) described carrier pBnLAS is imported among the Agrobacterium GV3101.It is following to be converted into the GV3101 concrete steps: get the GV3101 competent cell 50 μ l that melt on ice and add respectively in the aseptic centrifuge tube of 1.5ml with the vector plasmid that builds, pipettor is inhaled gently and is beaten mixing; Place 30min on ice; The liquid nitrogen middling speed is frozen into solid and takes out 37 ℃ of water-baths placement 4min; 28 ℃ of 130-150rpm shake 2hrs after adding 1ml liquid LB mixing; Centrifugal bacterium liquid precipitate is outwelled the supernatant bottom and is stayed 50-100 μ l, mixed bacteria liquid approximately to managing at the end; Shop LB (containing qingfengmeisu qiong 25 μ g/ml and kantlex 50 μ g/ml) dries up back 28 ℃ of anti-2-3 days; The picking bacterial plaque is carried out PCR and is detected, and positive colony shakes bacterium packing-70 ℃ preservation or directly is used for Plant Transformation.
2. the genetic transformation of Arabidopis thaliana
After the pBnLAS carrier of above-mentioned structure is converted into Agrobacterium GV3101; The vacuum method for transformation (Clough SJ and BentAF.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.1998.Plant J 16:735-743) that adopts is transformed in the wild-type Arabidopis thaliana plant; Concrete steps are following: the mono-clonal bacterial plaque on the picking flat board is to 6mlLB (containing qingfengmeisu qiong 25 μ g/ml and kantlex 50 μ g/ml); 28 ℃, 250rpm spends the night; Mix bacterium liquid 1ml and join 250ml and contain in the LB liquid nutrient medium of 25 μ g/ml qingfengmeisu qiongs and 50 μ g/ml kalamycin resistances shaking, shaken overnight OD600 reaches between the 1.2-1.4; Take out bacterium liquid to the 250ml centrifugal bottle, the centrifugal 10min of (available from the Beckman company) 6000rpm of Beckman super-magnum centrifuge behind the adjustment; After carefully outwelling supernatant, add a small amount of 1/2MS liquid nutrient medium bacterium liquid is mixed, add the 1/2MS liquid nutrient medium, add 0.01%silwet-77 (a kind of tensio-active agent is available from U.S. GE company) before the conversion to 400ml; The bacterium liquid of cultivating that contains the pBnLAS carrier is poured in the basin, vacuumized 4min in the Arabidopis thaliana plant handstand basin that bolting is bloomed; The Arabidopis thaliana plant that transformed secretly cultivated to put under the normal condition behind the 24h grow, collect fruit and do down the step and analyze.
3. transformed plant resistance screening
Transformed plant is collected to dry behind the fruit and is carried out positive seedling screening, and step is following: transformed the seed is assigned in the spiral mouth 1.5ml centrifuge tube, added 1ml95% ethanol sterilization 1min; Outwell ethanol, add 1ml 1/284 thimerosal, sterilization 10min; The centrifugal 10s of 6000rpm, pipettor is drawn supernatant, adds aseptic washing 3min, repeats twice; Add 1ml 0.01% agarose suspension seed; With the seed 1/2MS solid medium (containing kantlex 50 μ g/ml, Timtin 100 μ g/ml, sucrose 0.75%) that tiles, Bechtop dries up, and in the illumination cultivation chamber, grows after sealing film phonograph seal; After treating plant strain growth 2-3 week, the young plant that can distinguish resistance (has the resistance young plant to be green and ability normal growth; Non-resistant young plant yellowing can not normal growth), will have resistant plant and be transferred in the soil and grow.
4, the positive molecular Biological Detection of transformed plant
The arabidopsis thaliana transformation plant that obtains carries out the PCR positive identification, and NPT3/NPT4 (this primer sequence is seen table 3) detects the carrier resistance marker, and YM113/YM14 (primer sequence is seen table 1 and 3) detects and inserts BnLAS gene fragment integrity.Specific procedure is following: the total DNA extraction of the transformed plant of (1) Arabidopis thaliana adopts the Edwards method to extract (Edwards K; Johnstone C and Thompson C.A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.1991.Nucleic Acids Research; 19:1349-1350); Concrete steps are following: the blade (the about 4mm of blade tip * 4mm size) of getting young tender Arabidopis thaliana transformed plant; Add 400 μ l Edwards damping fluids and grind sample, room temperature was placed 1 hour; 13, the centrifugal 1min of 000rpm gets in 300 μ l supernatants to the new centrifuge tube, adds the equal-volume Virahol, puts 10-20min on ice; 13, the centrifugal 5min of 000rpm, supernatant drains; Add 50 μ l TE dissolving, preserve or directly be used for the PCR reaction for-20 ℃.(2) PCR reaction system: 1 * PCR damping fluid (available from MBI Fermentas, Lithuania company), 1.5mM MgCl 2, 200 μ M dNTPs, 1U Taq polysaccharase, 0.25 μ M primer (it is synthetic that primer is that worker's biotechnology ltd is given birth in Shanghai, and all the other threes are available from MBI Fermentas, Lithuania company) adds distilled water to 20 μ l.React in the PTC-100 amplification appearance (Bio-Rad) and carry out, it is following that resistance marker detects the PCR response procedures: 94 ℃ of 3min; 94 ℃ of 30s, 58 ℃ of 45s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 4min.BnLAS gene sheet degree integrity PCR response procedures is following: 94 ℃ of 3min; 94 ℃ of 30s, 56 ℃ of 45s, 72 ℃ of 1min20sec, 30 circulations; 72 ℃ of 4min.Amplified production used 1 * TAE damping fluid after pcr amplification was accomplished on horizontal strip electrophoresis groove (production of Beijing 61 instrument companies), voltage 3V/cm, and about electrophoresis 30min, 0.8% sepharose (containing EB) electrophoretic separation is utilized GeneRuler TMDNA Marker (MBI Fermentas, Lithuania company) estimation amplified production size.Electrophoresis finishes, in gel imaging system (UVP) preservation of taking pictures, and the record result.
Crossing expression plant exogenous gene expression detects: because T0 is relatively more consistent for the most phenotype of plant, show as that plant strain growth speed delays, plant type diminishes, the leaf look deepens etc.Select some representative positive plants to carry out the detection on the rna level.T1 gets its blade 100mg for plant, and (Invitrogen USA) carries out RNA and extracts TP reference reagent specification sheets to use Trizol.Actin2F/Actin2FR is as internal standard gene Action2 primer (primer sequence is seen table 3), and HY225/HY226 (primer sequence is seen table 3) carries out detection of expression (accompanying drawing 4) as foreign gene BnLAS Auele Specific Primer.The result shows in the transfer-gen plant that the exogenous gene expression amount significantly rises.
Embodiment 4 transgenic arabidopsis plant chlorophyll contents are analyzed
Arabidopis thaliana overexpression BnLAS plant family OL10; OL14; The method that OL18 and OL50 and wild-type plant (contrast) chlorophyll extract is with reference to Lichtenthaler (Lichtenthaler HK.Chlorophylls and carotenoids:pigments of photosynthetic biomembranes.1987.Methods Enzymol 148:350-382) and Laby et al (Laby RJ; Kincaid MS; Kim D, Gibson SI The Arabidopsis sugar-insensitive mutants sis4 and sis5 are defective in abscisic acid synthesis and response.2000.Plant J 23:587-596).Step is following: Arabidopis thaliana seed sterilization back point is cast under the 1/2MS solid medium, place the 16 hours illumination conditions in greenhouse to grow 20 days down; With wild-type arabidopsis thaliana in the petridish, transfer-gen plant (OL14, OL18, OL10, four transgenic lines of OL50) is one according to 3 individual plants respectively and is combined under the balance and weighs, counting; Each combination is done 5 respectively and is repeated to be immersed in and contain in the 4ml 95% alcoholic acid 10ml centrifuge tube; Plant was handled 2-3 hour under 80 ℃ of baking oven half-lights, contain chlorophyllous ethanol everywhere; Add 4ml 95% ethanol again, place again to 80 ℃ of baking oven half-lights and handled 1 hour down, estimate Arabidopsis leaf redgreen chlorophyll.Twice ethanol is mixed, and each centrifuge tube is got 2ml 95% ethanol and is carried out the calculating of chlorophyll content in spectrophotometer UV1601 (Shimadzu Japan) measurement OD664 and OD649 value.The calculation formula of the chlorophyll content of every gram Arabidopis thaliana plant is following: chlorophyll a/fresh sample weight (mg/g)=(13.36 * OD664-5.19 * OD649) * extract volume (ml)/plant weight (g); Chlorophyll b/fresh sample weight (mg/g)=(27.43 * OD649-8.12 * OD664) * extract volume (ml)/plant weight (g); Total chlorophyll/fresh sample weight (mg/g)=(5.24 * OD664-22.24 * OD649) * extract volume (ml)/plant weight (g).The measuring result of chlorophyll content shows four strain systems of transgenic arabidopsis, and (OL18, OL50) chlorophyll total content, chlorophyll a, chlorophyll b are significantly higher than wild-type Arabidopsis leaf chlorophyll content (accompanying drawing 5C) for OL10, OL14.
Embodiment 5 crosses expression BnLAS Arabidopis thaliana plant drought resistance and estimates
(OL14 OL18) has carried out analyzing relatively with the drought resisting ability of coercing of wild-type plant (contrast) the while applicant to crossing the expression family.During Arabidopis thaliana plant strain growth to 6-8 sheet true leaf, get OL14, the lotus throne leaf of OL18 and wild-type plant growth phase of the same race is put in the petridish, puts in the greenhouse 25 ℃ of temperature, carries out the blade dehydration of exsomatizing under humidity 44% condition and identifies.Through whenever weighing once at a distance from one hour, measure preceding 7 hours blade dehydration situation, Arabidopsis leaf rate-of-loss of coolant calculation formula is: blade rate-of-loss of coolant (%)=(start vane weight-dehydration rear blade weight)/start vane weight * 100.This tests triplicate.When dehydration was handled 3 hours, the blade edge that can see wild-type Arabidopis thaliana plant began to occur wilting, and can see that in the time of 6 hours wild-type Arabidopsis leaf wilting degree is obviously than the blade of transfer-gen plant obviously (accompanying drawing 6A).In the data of the stripped dehydration of blade, can see that also the blade rate-of-loss of coolant of transfer-gen plant significantly is slower than wild-type plant leaf rate-of-loss of coolant.
In order to confirm further that the drought-resistant ability of expressing plant improved.Growth is 30 days the time under Arabidopis thaliana plant normal condition, expresses under the condition of the family OL18 living weight equal with the wild-type plant through keeping, the Arabidopis thaliana plant carried out drought coerce processing.After plant stops to water 15 days, can see that wild-type Arabidopis thaliana plant leaf takes place by serious the wilting, and the relative blade performance of transfer-gen plant is full.Explanation is crossed the drought-resistant ability of expressing plan Arabidopis thaliana plant at BnLAS and is superior to the wild-type plant.
Simultaneously through crossing expression family OL14, OL18 and wild-type Arabidopis thaliana and Arabidopis thaliana las two mutants (SALK000896, Hiibara K; Karim MR, Takada S, Taoka K; Furutani M, Aida M, Tasaka M (2006) Arabidopsis CUP_SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation.Plant Cell 18:2946-1957) plant in same alms bowl; Treat that plant strain growth stops after one month watering and carry out drought and coerce processing; Treat the plant leaf back rehydration of here withering, 5 days " Invest, Then Investigate " plant of rehydration mortality ratio (accompanying drawing 7B), this tests triplicate.The Arabidopis thaliana that rehydration after 30 days is coerced in drought resisting plants that transgenic is crossed expression family OL14 and OL18 plant mortality ratio is respectively 71.9% and 91.6%, and the survival rate of the las two mutants plant of wild-type plant is respectively 21.9% and 11% (accompanying drawing 7C).Transgenic crosses expression family OL14 and the OL18 drought-resistant ability significantly is superior to wild-type plant and las two mutants plant.
Embodiment 6BnLAS crosses expression plant pore and wax layer analysis
Get BnLAS gene overexpression family OL18 and wild-type plant contrast CK and carry out light microscopic and the observation of Electronic Speculum condition air holes.BnLAS gene overexpression family OL18 and wild-type plant grew 35 days in 22 ℃ of greenhouses, and first complete blade of clip is cut into to be fixed on immediately behind the fritter of 2 * 2mm to vacuumize simultaneously in 5% the LUTARALDEHYDE and spends the night.Sample is delivered to Hua Zhong Agriculture University's Electronic Speculum platform and is dehydrated.Sample is pasted to aluminium block, carried out gold-plated at EIKO IB-5ION coater (EIKO IB-5ION, Japan) 15min.After directly place under the JSM-3690/LV ESEM (SEM, JSM-6390, Japan) and carry out scanning imagery, the measurement of using software I magJ to carry out stomatal aperture is calculated.Through fixed Arabidopis thaliana plant leaf is carried out scanning discovery under SEM, cross that the stoma number of expressing plant OL18 significantly increases and corresponding epidermic cell size also diminishes (like accompanying drawing 8A, B).Through finding to crossing the quantitative analysis of expressing plant OL18 and contrast wild-type Arabidopis thaliana stoma number that the stoma number of expressing plant had increased by 1.9 times than wild-type plant stoma number; But most pores obviously diminishes and grows undesiredly on its epidermis, has the hole of a lot of pores not have or have only very little hole.Measuring stomatal aperture is to find that the stomatal aperture 0.85 μ m that expresses plant OL18 had only contrast wild-type plant 18% size.
Under the Electronic Speculum condition, can form influence to stomatal aperture, the stomatal aperture of fresh blade is implemented under the light microscopic.Aperture observational technique under the light microscopic (with reference to Cominelli E, Galbiati M, Vavasseur A; Conti L; Sala T, Vuylsteke M, Leonhardt N; Dellaporta SL, and Tonelli C.A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance.2005.Curr Biol 15:1196-1200).The 30 days Arabidopsis leaf of growing; Be cut into square epidermis bar about 2cm; Be immersed in the damping fluid (pH 6.5) of 30mM KCl and 10mM MES-KOH composition; Illumination 4h is placed on Nikon ECLIPSE 80i microscope (Tokyo Japan) takes a picture down, and the measurement of using software I magJ to carry out stomatal aperture is calculated under optical condition.Observe after 4 hours carrying out illumination under the MES buffer conditions through the epidermis bar simultaneously.Find that BnLAS crosses expression plant stoma number sum and increased by 1.7 times (accompanying drawing 8C), and stomatal aperture is 40% (the accompanying drawing 8D) of wild-type plant.And wild-type plant and BnLAS cross and express plant open stoma number and no significant difference under two kinds of conditions.Stomatal aperture reduce and can cause Arabidopis thaliana plant drought-resistant ability to improve being proved (with reference to Cominelli E, Galbiati M, VavasseurA; Conti L; Sala T, Vuylsteke M, Leonhardt N; Dellaporta SL, and Tonelli C.A guard-cell-specific MYB transcription factor regulates stomatal movements and plant drought tolerance.2005.Curr Biol 15:1196-1200).
Cross simultaneously and express family OL18 and wild-type plant control scan Electronic Speculum has been carried out the observation of blade epidermis wax layer; The result can find out on the surface of crossing expression family OL18 blade (accompanying drawing 9B) and covered one deck wax, and the wild-type plant leaf is indicated wax layer and not obvious (accompanying drawing 9A).Plant leaf epicutile wax layer thickens the change that causes the stratum corneum penetrating power and causes drought resisting to confirm (Zhang JY with the back of the body; Broeckling CD; Sumner LW; Wang ZY.Herterologous expression of two Medicago truncatula putative ERF transcription factor genes; WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumulation and improved drought tolerance, but differential response in freezing tolerance.2007.Plant Mol Biol 64:265-278).Chlorophyll leaching experiment (Lolle SJ; Berlyn GP; Engstrom EM; Krolikowski KA, Reiter W, Pruitt RE (1997) Developmental regulation of cell interactions in the Arabidopsis fiddlehead-1 mutant:a role for the epidermal cell wall and cuticle.Dev Biol 189:311-321) in crossing expression family OL14, OL18 and the contrast of wild-type plant, carry out.Concrete steps are following: after will removing root with embodiment 6 isometric growth condition Arabidopis thaliana plant, the lotus throne leaf is immersed in 30ml 80% ethanol, places the dark place.Every at a distance from 15min, 30min, 60min, 90min, 120min, 180min take out 3ml solution and carry out wavelength 649 and 664 mensuration.It is following to calculate total chlorophyll content formula: total chlorophyll content (mg/g flesh tissue)=7.93 (A664)+19.53 (A647).The chlorophyll leaching experiment shows, cross expression family OL14, first three hour chlorophyll total amount leaching velocity of OL18 is slow than the wild-type plant.Explain that BnLAS crosses expression plant stratum corneum penetrating power and weakens.
Above-mentioned experimental result has confirmed that the enhancing of BnLAS gene overexpression plant drought-resistant ability is to reduce to thicken realization with wax layer through crossing the stomatal aperture of expressing the Arabidopis thaliana plant.
Figure ISA00000413701600011

Claims (6)

1. one kind isolatingly derives from the BnLAS gene that swede type rape has drought resistance, and its nucleotide sequence is shown in sequence table SEQ ID NO:1, and the sequence total length is 1326bp.
2. clone special primer YM113, the primer YM114 of the said gene of claim 1, its nucleotide sequence is as follows:
YM113:5′-CAGTGTCGACAACAATGCTTACTTCCTTCAAA-3′;
YM114:5′-CAGTGAGCTCTCATTTCCACGACGAAAC-3′。
3. the described BnLAS gene of claim 1 improves the method for plant Arabidopis thaliana drought resistance, and it comprises the steps:
1) the described primer of design claim 1, the clone has the BnLAS of drought resistance gene is arranged from the swede type rape genome, and its nucleotide sequence is shown in SEQ ID NO:1;
2) make up BnLAS gene overexpression carrier, and this carrier is transformed in the Agrobacterium;
3) through the agrobacterium mediation method arabidopsis thaliana transformation, screening obtains BnLAS gene overexpression transformed plant;
4) adopt molecular detecting method that the expression transformed plant of crossing of step 3) is carried out the drought resistance evaluation;
5) further identified blade surface wax layer, chlorophyll content and the blade construction of expressing transformed plant in the field.
4. the application of the described gene of claim 1 in improving plant drought resistance.
5. the application of the described primer of claim 2 in improving plant drought resistance.
6. the described gene of claim 1 improves the application in the plant drought resistance rape genus GRAS gene family member.
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CN110029190A (en) * 2019-05-27 2019-07-19 西南大学 A kind of cabbage type rape Drought-tolerant gene and its molecular labeling and application
CN110804614A (en) * 2019-10-08 2020-02-18 湖南农业大学 Cabbage type rape drought-resistant gene BnatZF1A, primer, expression vector and application thereof, and method for improving drought resistance

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CN110029190A (en) * 2019-05-27 2019-07-19 西南大学 A kind of cabbage type rape Drought-tolerant gene and its molecular labeling and application
CN110804614A (en) * 2019-10-08 2020-02-18 湖南农业大学 Cabbage type rape drought-resistant gene BnatZF1A, primer, expression vector and application thereof, and method for improving drought resistance
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