CN103146701B - Salt mustard salt resistance control mi ribonucleic acid (RNA)-tsa-mi R n 1927 and application thereof - Google Patents

Abstract

The invention relates to salt mustard salt resistance control mi ribonucleic acid (RNA)-tsa-mi R n 1927 and application of the salt mustard salt resistance control mi RNA-tsa-mi R n 1927. Salt mustard salt resistance control mi RNA and nucleotide sequence are shown as sequencer (SEQ) identification (ID) number 1. The invention further relates to a precursor of the salt mustard salt resistance control mi RNA, a deoxyribonucleic acid (DNA) fragment of the precursor of the salt mustard salt resistance control mi RNA and the application of the salt mustard salt resistance control mi RNA in cultivating salt resistance transgenic crops. The salt mustard salt resistance control mi RNA-tsa-mi R n 1927 plays an important role in plant stress resistance, especially in plant salt resistance, and has potential application value in cultivating salt resistance crops.

Description

A kind of salt mustard salt tolerance regulation and control miRNA-tsa-miRn1927 and application thereof

Technical field

The present invention relates to a kind of salt mustard salt tolerance regulation and control miRNA-tsa-miRn1927 and application thereof, belong to technical field of biotechnology.

Background technology

The soil salinization has become an important factor that affects crop growthing development, yield and quality, is the one of the main reasons of crop production reduction.At present, approximately there are in the world 20% arable land and at least 40% sewage farming to there is salinification in various degree, along with sharp increase and the industrialized high speed development of population, arable area sharply declines, aggravation and the fertilizer application of unreasonable irrigation, industrial pollution are improper, cause the secondary salinization in a large amount of good farmlands, expected the year two thousand fifty whole world salinification is in various degree occurred in the arable land that has 50%.Occurring in nature, plant has obvious difference to the adaptation of salt, and subject range is very extensive, and existing salt-sensitive glycophyte has again the halophytes of salt tolerant.Because most of farm crop are salt-sensitive glycophyte, salt is still the key constraints that affects crop yield.At present, to the existing certain progress of the research of plant salt tolerance mechanism, the discovery of for example Arabidopis thaliana SOS1, SOS2, SOS3 approach and Mechanism of Salt-tolerant thereof illustrate (Zhu J.K.Salt and drought stress signal transduction in plants.Annu.Rev.Plant Biol., 2002,53:247-273.), impact (the Apse M.P that Blumwald laboratory overexpression AtNHX1 produces for raising plant salt endurance, Blumwald E, etal.Salt Tolerance conferred by Overexpression of a Vacuolar Na ⁺/ H ⁺antiporter in Arabidopsis.Science.1999,285:1256-1258.) etc.But the research to plant salt tolerance mechanism in the world at present, mainly concentrates on glycophyte, thereby also has some limitations for the understanding of plant salt endurance mechanism.

Salt mustard (Thellungiella salsuginea) is and the new model system of the plant salt tolerance molecule genetics research of the nearly edge of Arabidopis thaliana, the gene order-checking of salt mustard will be completed in the end of the year 2011 by USDA, and its genome sequence website is referring to http://www.phytozome.net/thellungiella.php.Salt mustard and Arabidopis thaliana are all cress, and in cDNA level, both exist the similarity of 90%-95%.But with respect to glycophyte Arabidopis thaliana, salt mustard is a kind of typical halophytes, the natural environment that is adapted to salt stress of energy also can complete its growth history even if the short period of time is exposed to 500mM NaCl.Along with completing of salt mustard gene order-checking, the salt mustard functional genomics especially function of salt-resistant related gene has become the emphasis of its research work.

MiRNA is the non-coding RNAs that is extensively present in about 21-24nt in eukaryote, the modulins such as the most target transcription factor of Mirnas of plant s, thereby the central position in control of plant gene expression.And environment stress (salt, sulphur or phosphorus scarcity and oxidative stress etc.) not only can inducing plant protein coding gene expression, the expression of also inducing some nonprotein encoding gene miRNA.Mirnas of plant s is the focus of current international research, but only there is a few experiments chamber to pay close attention to the relevant miRNAs of salt tolerant, and be mainly taking model plant Arabidopis thaliana and paddy rice etc. as examination material, for example, in the species such as Arabidopis thaliana, paddy rice and willow, be cloned into and the relevant miRNAs such as coerce to salt, phosphorus shortage, sulphur shortage, oxidation and mechanics; Result shows the negative regulatory factor of miRNAs target protein, and its regulatory function is not only development of plants, also in to processes such as replying of the environment stresses such as salt, serves as important regulating and controlling effect.

Become one of principal element of restriction agriculture production in view of the soil salinization, Physiology and biochemistry and the Molecular Biology Mechanism of coercing by research plant salt tolerance, excavate resistant gene of salt resource, and use genetic engineering means to cultivate salt tolerant crop, have important practical significance for the output that improves farm crop.Thereby, taking the new model system of this salt tolerant molecule genetics research of salt mustard as examination material, carry out its miRNAs and the research with salt tolerance relation thereof, there is certain perspective and novelty.In addition, in searchable prior art, the research of salt mustard miRNAs be have not been reported.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, the regulation and control of miRNA-tsa-miRn1927 salt mustard salt tolerance miRNA, its precursor sequence and the application thereof that derive from salt mustard are provided.

For solving the problems of the technologies described above, the technical solution used in the present invention is as follows:

A kind of salt mustard salt tolerance regulation and control miRNA, nucleotide sequence is as shown in SEQ ID NO.1.

The precursor of above-mentioned salt mustard salt tolerance regulation and control miRNA, nucleotide sequence is as shown in SEQ ID NO.2.

The above-mentioned salt mustard salt tolerance of encoding regulates and controls the DNA fragmentation of the precursor of miRNA, and nucleotide sequence is as shown in SEQ ID NO.3.

The precursor of above-mentioned salt mustard salt tolerance regulation and control miRNA, salt mustard salt tolerance regulation and control miRNA is in the application of cultivating in salt tolerant genetically modified crops.

As a kind of optimal technical scheme of above-mentioned application, step is as follows: the precursor of crossing the salt mustard salt tolerance regulation and control miRNA shown in salt mustard salt tolerance regulation and control miRNA or the SEQ ID NO.2 expressing shown in SEQ ID NO.1 in crop, or the precursor of the salt mustard salt tolerance regulation and control miRNA shown in inhibition SEQ ID NO.1 or the salt mustard salt tolerance regulation and control miRNA shown in SEQ ID NO.2, cultivate the genetically modified crops with high-salt tolerance.

Above-mentioned crop is wheat, paddy rice, corn or cotton.

Experiment Solexa sequencing result of the present invention proves, tsa-miRn1927 is subject to the adjusting of salt stress, illustrates that it plays an important role in the high salt patience mechanism of salt mustard.Given this, can utilize this gene to carry out the cultivation of salt tolerant genetically modified crops, and then there is potential using value for the output that improves crop.

Beneficial effect

The present invention adopts the technology of Solexa high throughput sequencing technologies and bioinformatic analysis subsequently and real-time quantitative PCR, from salt mustard, identify tsa-miRn1927 first, tsa-miRn1927 lowers after salt is processed, salt is processed its counting 0 in little RNAs storehouse, and be 2363 in contrast, tsa-miRn1927 expresses and is subject to severe inhibition after salt is processed; Empirical tests, tsa-miRn1927 plays an important role in plant stress tolerance especially salt tolerance, and salt tolerant crop cultivate in the potential using value of tool.

Brief description of the drawings

The secondary structure figure of Fig. 1 tsa-miRn1927 precursor sequence;

Wherein: dash area is ripe miRNA position;

The column schematic diagram of the gene expression abundance of tsa-miRn1927 under contrast and salt processing in Fig. 2 Solexa order-checking;

Wherein: CL is the expression level of normal growth salt mustard tsa-miRn1927, TL is the expression level that 200mM NaCl processes lower salt mustard tsa-miRn1927;

The mRNA degraded schematic diagram of Fig. 3 tsa-miRn1927 to target gene Ninja-family albumin A FP4, transcription factor PHOX2/ARIX and serine/threonine protein matter phosphatase gene;

Fig. 4 is the column schematic diagram of transgenic saline mustard and wild salt mustard fresh dry weight under different concns condition of salt stress;

Wherein: the column schematic diagram that A figure is fresh weight, M3-14 is that transgenic saline mustard M3-14, M5-3 are that transgenic saline mustard M5-3, WT are wild-type salt mustard;

B figure is the column schematic diagram of dry weight, and M3-14 is that transgenic saline mustard M3-14, M5-3 are that transgenic saline mustard M5-3, WT are wild-type salt mustard.

Specific embodiments

Below in conjunction with embodiment, technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.

Experimental technique in following embodiment, if no special instructions, is this area ordinary method.

Embodiment 1: salt canola seed gathers and plantation

Collect the seed of wild-type salt mustard (Thellungiella salsuginea) at Dongying city Saline-alkaline Soils of Yellow River Delta, dry, process surface sterilization: the alcohol that volume percent is 70%, process 1min, 1wt%NaClO solution, vortex 15min, aseptic double-distilled water is seeded in MS substratum after rinsing, the Temperature Setting of illumination box: 25 DEG C of daytimes, 16h, 22 DEG C of nights, 8h.After growing 2 true leaves, transfer on Hogland liquid nutrient medium, cultivate after 5 weeks, divide into groups.NaCl treatment group is in Hogland nutritive medium, to add NaCl to make its final concentration to be 200mM, to cultivate 24 hours; Control group is the Hogland nutritive medium that does not contain NaCl, cultivates 24 hours.

MS nutrient media components is as follows: macroelement 50ml/L, calcium salt soln 50ml/L, trace element solution 5ml/L, iron salt solutions 10ml/L, vitamin solution 10ml/L, sucrose 30g/L, agar powder 7.5g/L.

Wherein, macroelement is 20 times of mother liquors, and component is: KNO ₃38g/L, NH ₄nO ₃33g/L, MgSO ₄﹒ 7H ₂o7.4g/L, KH ₂pO ₄3.4g/L;

Calcium salt soln is 20 times of mother liquors, and component is: CaCl ₂﹒ 2H ₂o8.8g/L;

Trace element solution is 200 times of mother liquors, and component is: KI0.166g/L, H ₃bO ₃1.24g/L, MnSO ₄﹒ 4H ₂o4.46g/L, ZnSO ₄﹒ 7H ₂o1.72g/L, Na ₂moO ₄﹒ 2H ₂o0.05g/L, CuSO ₄﹒ 5H ₂o0.005g/L, CoCl ₂﹒ 6H ₂o0.005g/L;

Iron salt solutions is that 100 times of mother liquor components are: FeSO ₄-EDTA3.67g/L;

Vitamin solution is 100 times of mother liquors, and component is: nicotinic acid 0.05g/L, vitamin B6 0.05g/L, vitaminB10 .01g/L, glycine 0.2g/L, inositol 10g/L.

Every liter of component of Hogland nutritive medium is as follows: Ca (NO ₃) ₂945mg, KNO ₃607mg, (NH ₃) ₃pO ₃ammonium phosphate 115mg, MgSO ₄493mg, iron salt solutions 10ml, trace element solution 5ml, regulates pH value to 6.0.

Wherein iron salt solutions is 100 times of mother liquors, and component is: FeSO ₄-EDTA3.67g/L;

Trace element solution is 200 times of mother liquors, and component is: KI0.166g/L, H ₃bO ₃1.24g/L, MnSO ₄﹒ 4H ₂o4.46g/L, ZnSO ₄﹒ 7H ₂o1.72g/L, Na ₂moO ₄﹒ 2H ₂o0.05g/L, CuSO ₄﹒ 5H ₂o0.005g/L, CoCl ₂﹒ 6H ₂o0.005g/L.

Embodiment 2: the structure in salt mustard miRNA library and Solexa order-checking

Under the normal growth condition that embodiment 1 is obtained-control group (CL) and 200mM NaCl process under-the salt mustard sample for the treatment of group (TL) is sent to the constructed miRNA of Shenzhen Hua Da gene studies library, carries out Solexa order-checking.After order-checking, remove 3 ' joint, polluted sequence, be less than after 18nt sequence and polyA sequence, in CLmiRNA library, altogether obtain 12010658 of high quality small RNA fragments (sRNA), in TL miRNA library, altogether obtain 12330771 of high quality small RNA fragments (sRNA).Bioinformatics Prediction result shows that the ripe body sequence of tsa-miRn1927 is:

CAAGGCAGAAGAAGGCUGUUU（SEQ?ID?NO.1）。

Tsa-miRn1927 precursor sequence is:

GCAAAGUGAUCAAGGCAGAAGAAGGCUGUUUUGGCAAUAGGAUUAGCGCUGAUCCUAUUGCCAAAACAGCCUCUUCUUCCUUGUUUACUUUGA（SEQ?ID?NO.2），

It is set at genome meta: scaffold_19:2418388:2418480:+.According to the precursor of tsa-miRn1927 and ripe body sequence, obtain the secondary structure of tsa-miRn1927 precursor with RNAstructure software, as shown in Figure 1, dash area is ripe miRNA position, its precursor sequence is folded into a kind of stable loop-stem structure, and Energy value is-63.1.Belong to the typical secondary structure of miRNA precursor, meet the constitutional features of miRNA precursor.

Sequencing result shows that tsa-miRn1927 order-checking abundance is under normal operation 2363, and salt order-checking abundance after treatment is 0, shows that the expression of tsa-miRn1927 is being subject to suppressed after salt stress (Fig. 2).Indicating that tsa-miRn1927 adapts to play an important role in high-salt stress mechanism in salt mustard.Such as, excessive or suppress the expression of tsa-miRn1927 in farm crop (corn, wheat, paddy rice, rape, soybean etc.), will affect the adaptive faculty of crop to salt stress.

Checking and the functional analysis of embodiment 3:tsa-miRn1927 target gene

Utilize the complementarity of miRNA and target gene, predict the target gene of tsa-miRn1927 by the method for information biology in the full genomic level of salt mustard.According to method (the Schwab R et al. of Schwab etc., Specific effects of microRNAs on plant transscriptome.Dev.Cell, 2005) use following rule: the mispairing between sRNA and target gene must not exceed 4 (G-U pairings think 0.5 mispairing); In miRNA/ target gene complex body, must not there is the mispairing that exceedes 2 places and occur adjacent site; In miRNA/ target gene complex body, from the 5 ' end of miRNA, 2-12 site must not have adjacent site that mispairing all occurs; Must not there is mispairing in 10-11 site of miRNA/ target gene complex body; In miRNA/ target gene complex body, from the 5 ' end of miRNA, 1-12 site must not have and exceed 2.5 mispairing; The minimum free energy (MFE) of miRNA/ target gene complex body should be not less than this miRNA in the time that its best complement is combined MFE 75%.With Mireap software at the full genomic level of salt mustard prediction target gene.

The prediction of table 1tsa-miRn1927 target gene

Predict the outcome in table 1, tsa-miRn1927 predicts 3 target genes---and Ninja-family albumin A FP4(encoding gene nucleotide sequence is as shown in SEQ ID NO.4, aminoacid sequence is as shown in SEQ ID NO.5), transcription factor PHOX2/ARIX gene (nucleotide sequence is as shown in SEQ ID NO.6) and serine/threonine protein matter phosphatase gene (nucleotide sequence is as shown in SEQ ID NO.7), tsa-miRn1927 lays respectively at Ninja-family albumin A FP4 to the cleavage site of target gene, (Fig. 3: tsa-miRn1927 is to target gene Ninja-family albumin A FP4 in the coding region of transcription factor PHOX2/ARIX and serine/threonine protein matter phosphatase gene, the schematic diagram of the mRNA degraded of transcription factor PHOX2/ARIX and serine/threonine protein matter Phosphoric acid esterase).Serine/threonine protein Phosphoric acid esterase comprises ABI1 and the ABI2 of homology, well-known ABI1 and ABI2 are the attemperators of ABA signal pathway, Gosti etc. (1999) find that the cryptic mutant abi1 on ABI1 site has susceptibility to ABA in seed germination and growth of seedling, shows that ABI1 is a negative regulation person of ABA signal.Correlative study show ABI1 and ABI2 for the multiple abiotic stress of plant comprise salt, arid and cold patience all most important, serine/threonine Phosphoric acid esterase also regulates MAPK cascade system in addition.And the also called after ABI5-binding albumen 4 of Ninja-family albumin A FP4 illustrates that it also belongs to serine/threonine protein matter phosphatase family, its negative regulator person as ABA signal participates in salt stress responsing reaction.Tsa-miRn1927 target Ninja-family albumin A FP4, transcription factor PHOX2/ARIX and serine/threonine protein matter phosphatase gene, indicating that tsa-miRn1927 plays a very important role at aspect tools such as stress response such as salt mustard salt tolerants, thereby thering is biological significance and application prospect widely.

Embodiment 4:tsa-miRn1927 transforms salt mustard checking miRNA function

The acquisition of tsa-miRn1927 precursor sequence: according to salt mustard genome sequence information design primer amplification tsa-miRn1927 precursor sequence, forward primer contains EcoRI restriction enzyme site: CCGGAATTC TGGGCAATAACTGTCTTCG, reverse primer contains BamHI restriction enzyme site: CGCGGATCC ATCAAACCGGCAACAAAC.

The method of extracting salt thaliana genomic dna is as follows:

(1) get the salt mustard young leaflet tablet of 1g, become powder by liquid nitrogen grinding, be placed in the Eppendof pipe of Liquid nitrogen precooler;

(2) preheating CTAB extracting solution (2%CTAB, 1.4mol/L NaCl, 20mmo/L EDTA (pH8.0), 100mmol/L Tris-HCl (pH8.0), 2%pvp-40) in 65 DEG C of water-baths;

(3) quality of estimation sample tissue, adds the CTAB extracting solution of 700 μ L preheatings in every 200mg sample, mixes rapidly, bathes 10～30min 65 DEG C of temperature, mixes during this time 2～5 times;

(4) add the phenol/chloroform/primary isoamyl alcohol (volume ratio 12:12:1) of 1 times of volume, mix;

(5) the centrifugal 10min of 12000rpm room temperature;

(6) supernatant is transferred in a new centrifuge tube;

(7) repeat (4)～(6) step with chloroform/primary isoamyl alcohol (volume ratio 24:1);

(8) add the Virahol of 0.7 times of volume-20 DEG C precooling, put upside down and mix, room temperature is placed 10min;

(9) the centrifugal 15min of 12000rpm room temperature;

(10) outwell supernatant, wash and precipitate 2～3 times with 70% ethanol of 500 μ l-20 DEG C precoolings;

(11) precipitation dry after, with 50 μ l deionized waters or TE dissolving DNA, be placed in-20 DEG C for subsequent use.

(12) draw the DNA that 5 μ l dissolve, add 45 μ l deionized waters, mix that to make the genomic dna of salt mustard stand-by.

Reaction system and the response procedures of pcr amplification are as follows: taking the genomic dna of above-mentioned salt mustard as template, taking the forward primer that contains BamHI restriction enzyme site and the reverse primer that contains PstI restriction enzyme site as primer, carry out DNA cloning at PCR instrument (TaKaRa TP600) with high-fidelity enzyme, PCR response procedures is: 95 DEG C of 5min; 94 DEG C of 1min again, 51 DEG C of 1min, 72 DEG C of 1min, 30 circulations; 72 DEG C of 7min finish reaction.Get 5 μ L PCR products through 1% agarose gel electrophoresis, observations under ultraviolet lamp, gel reclaims the band of 400bp left and right, adds A reaction with adding A reaction kit (purchased from the biochemical limited scientific & technical corporation of sky root).Learnt from else's experience and added the PCR recovery product 4 μ l of A, added pMD18-T simple carrier 1.0 μ l(purchased from TaKaRa company) mix, then add 5.0 μ l Solution I Butter, mix 16 DEG C of incubated overnight.

The preparation of competent cell: the E.coli DH5 α (purchased from the biochemical limited scientific & technical corporation of sky root) preserving with aseptic inoculation ring picking-70 DEG C glycerine, the method for diluting by line obtains single bacterium colony of DH5 α after 37 DEG C are cultivated 16～20h in flat board; Choose a single bacterium colony in the LB of 5ml liquid nutrient medium, 180rpm shaking culture 12h; Get 1ml DH5 α LB bacterium liquid in the LB of 100ml liquid nutrient medium, 180rpm shaking culture is 0.4 to OD value; Place on ice 15 minutes, pour in 50ml Beckman centrifuge tube under aseptic condition, 4 DEG C, the centrifugal 10min of 3500rpm, abandons supernatant; The 0.1M CaCl of 30ml ice precooling for precipitation ₂solution is resuspended; 4 DEG C, the centrifugal 10min of 3500rpm, carefully pours out supernatant liquor; Precipitation be resuspended in the precooling of 2ml ice containing 15%(volume percent) the 0.1M CaCl of glycerine ₂in solution; These competent cells are distributed into competent cell liquid by every part of 100 μ l.

Colibacillary conversion: the connecting fluid of above-mentioned 10 μ l is added in 100 μ l competent cell liquid and mixed, 30min on ice, 42 DEG C of heat shock 90s, 2 minutes on ice, then add 600 μ l liquid LB(NaCl10g/L, Tryptone10g/L, yeast powder 5g/L), 37 DEG C of 120rpm shaking culture 1.5h, the centrifugal 1min of 5000rpm, is evenly applied to intestinal bacteria (NaCl10g/L on the solid LB substratum of the penbritin that contains 50 μ g/ml, Tryptones 10g/L, yeast powder 5g/L, agar powder 15g/L), 37 DEG C of overnight incubation.Picking list bacterium colony is in 4mL liquid LB, and 37 DEG C are shaken bacterium 8～10h, by DNA extraction test kit (purchased from Omega company) extraction plasmid DNA, positive colony is carried out to bacterium liquid PCR checking and sequence verification.Response procedures and the reaction system of bacterium liquid PCR, template changes into uses bacterium liquid, and other program is with reaction system and the response procedures of above-mentioned pcr amplification.Extraction is connected with the escherichia coli plasmid of tsa-miRn1927.

Build pRT101-tsa-miRn1927 intermediate carrier:

By through sequence verification, the plasmid and pRT101 plasmid (purchased from Biovector company) EcoRI restriction endonuclease and the BamHI restriction endonuclease that are connected with tsa-miRn1927 carry out double digestion, reclaim respectively tsa-miRn1927 precursor sequence and carrier sequence, both are connected with T4DNA ligase enzyme: 10 × T4DNA ligase buffer1.0 μ l, tsa-miRn1927 precursor sequence 6.0 μ l, pRT1012.0 μ l, T4DNA ligase1.0 μ l, 16 DEG C of connections are spent the night, make and connect product pRT101-tsa-miRn1927 intermediate carrier, connect product and transform bacillus coli DH 5 alpha.Picking colony carries out PCR checking, and the expression cassette framework that contains 35S promoter-tsa-miRn1927-polyA terminator has been built.

Build the tsa-miRn1927 overexpression plant vector being driven by 35S promoter:

PRT101-tsa-miRn1927 intermediate carrier and pCAMBIA3301 plasmid (purchased from Biovector company) are carried out to single endonuclease digestion with PstI restriction endonuclease respectively, reclaim the expression Frame sequence and the carrier sequence that contain 35S promoter-tsa-miRn1927-polyA terminator.The pCAMBIA3301 linear carrier of single endonuclease digestion is carried out to dephosphorisation reaction: pCAMBIA330120pmol, 10 × Alkaline Phosphatase Buffer5 μ l, Alkaline Phosphatase1 μ l, 37 DEG C or 50 DEG C are reacted 30 minutes.PCAMBIA3301 linear carrier after purifying dephosphorization, fragment is connected with carrier with T4DNA ligase enzyme: 10 × T4DNA ligase buffer, 1.0 μ l, the expression frame fragment 6.0 μ l of 35S promoter-tsa-miRn1927-polyA terminator, pCAMBIA33012.0 μ l, T4DNA ligase1.0 μ l, 16 DEG C of connections are spent the night, and connect product and transform bacillus coli DH 5 alpha.Picking colony carries out PCR checking and sequence verification.

The competent cell of preparation Agrobacterium EHA105: picking Agrobacterium EHA105(is purchased from sky, Beijing bounties Bioisystech Co., Ltd) single bacterium colony shakes bacterium to 5ml LB substratum (containing Rifampin 50 μ g/ml) and spends the night; Then get 1ml and be inoculated into (containing Rifampin 50 μ g/ml) in 50ml liquid LB substratum, 28 DEG C of enlarged culturing, 220rpm concussion is cultured to OD ₆₀₀=0.5; Ice bath 10min, 4 DEG C of 4500rpm, centrifugal 10min; The 0.1M CaCl of 10ml precooling for thalline ₂resuspended, 4 DEG C of 4500rpm recentrifuge 10min; Precipitation be resuspended in the precooling of 1ml ice containing 10%(volume percent) the 20mM CaCl of glycerine ₂in solution, 80 μ l packing, liquid nitrogen flash freezer is preserved.

Recombinant plasmid transformed Agrobacterium: get the plasmid 2 μ l that above-mentioned sequence verification is correct and join in 80 μ l Agrobacterium competent cells, mix, be put into 45min on ice, 37 DEG C of water-bath 3min, 28 DEG C of 120rpm shaking culture 1.5h, the centrifugal 1min of 5000rpm, is evenly applied on solid LB substratum thalline (containing Rifampin 50 μ g/ml, kantlex 50 μ g/ml), cultivate 2d for 28 DEG C.Picking list bacterium colony is in 4ml liquid LB, and 28 DEG C are shaken bacterium and spend the night, and does bacterium liquid PCR checking, and verification step is the same.After checking is correct, carry out enlarged culturing, draw 1ml bacterium liquid and be added to 100ml with in antibiotic liquid LB, shake bacterium and spend the night, OD value reaches 0.5～0.6.

The genetic transformation of salt mustard:

Salt canola seed is seeded in and fills with in the cultivation cup that the diameter of compost is 7cm, sprouts and moves in 4 DEG C of illumination boxs and place and carry out a vernalization in after 2～3 days, then be put in the tissue culture room of 21 DEG C and grow.When plant grows terminal inflorescence, remove its terminal inflorescence, to stimulate the growth of axillary inflorescence, note avoiding injuring the raw inflorescence of leaf.Treat that axillary inflorescence grows, the flower of its underpart transforms while there is pollination phenomenon.Before conversion, by pollinating, flower and pod are removed.

By the Agrobacterium bacterium liquid 100ml of above-mentioned preparation, centrifugal 15 minutes of room temperature 5000rpm, abandons supernatant liquor, and precipitation is suspended in the infiltration substratum (5wt% sucrose, 200 μ l/L Silwet L-77) of respective volume, the bacterium liquid OD after resuspended ₆₀₀be about 0.8.Agrobacterium suspension is poured in small beaker, and by the long cultivation cup back-off that has a salt mustard, it is upper, and guarantee lotus throne leaf is partially submerged in liquid above, soaks 15 minutes.Take out plant, be disposed across in vinyl disc, seal to keep humidity with preservative film, be placed on thermostatic chamber and cultivate.Within second day, open, vertically cultivate, still keep humidity 3～4 days with preservative film.Cultivate three to surrounding, after seed maturity, collect seed, in moisture eliminator, deposit 2 weeks.The T0 of results is seeded in compost for salt canola seed, in the time that seedling grows 2～3 true leaves, spray 0.2%(volume percent) Basta solution, 24h sprays once, spray after three times, non-transformed seedling yellow also stops growing, and within one week, left and right is withered, and resistance seedling is spraying maintenance green after Basta solution.The salt canola seed of individual plant results resistance seedling is seeded in compost, sprays 0.2%(volume percent) Basta solution, according to the separation case in transgenosis T3 generation, amount to the pure lines that obtain 17 strain transgenic saline mustard.

Choose transgenic saline mustard M3-14, the transgenic saline mustard M5-3 and the wild-type salt mustard (WT) that obtain and be seeded in blending agent, grow after 6 weeks, use respectively 0,300 and 400mM NaCl solution-treated.Process after 2 weeks, the fresh dry weight of wild-type after salt stress and transgenic line is measured, result shows, under 0mM NaCl processes, the growth of transgenic saline mustard and wild-type salt mustard are not significantly distinguished.And under NaCl coerces, the fresh weight of transgenic saline mustard M3-14, transgenic saline mustard M5-3 and dry weight are all starkly lower than wild-type salt mustard (Fig. 4) under different salt concn are processed.Salt stress is mainly reflected in the reduction of plant dry weight and fresh weight on the impact of plant-growth, presentation of results overexpression tsa-miRn1927 has obviously reduced the salt tolerance of salt mustard, and tsa-miRn1927 has regulating and controlling effect to the salt tolerance of salt mustard.

Embodiment 5: the application of salt mustard tsa-miRn1927 in crop germplasm innovation

High-flux sequence shows that tsa-miRn1927 expression amount under salt mustard normal growth is very high, but does not express being subject to after salt stress is processed.Its target gene Ninja-family albumin A FP4, transcription factor PHOX2/ARIX and serine/threonine protein matter phosphatase gene plant grow and Stress response aspect there is vital role.In salt mustard, overexpression tsa-miRn1927 result shows, the phenotype that salt tolerance reduces appears in transgenic saline mustard, and more above-mentioned row experiments all show that tsa-miRn1927 has negative regulation effect for the salt tolerant of salt mustard.In corn, wheat, paddy rice, soybean or peanut and other crops, suppress to express tsa-miRn1927 or its target gene of overexpression, will affect the adaptive faculty of these crops to adverse circumstance, particularly contribute to improve the salt resistance ability of these crops.Thereby tsa-miRn1927 is resistance to contrary for cultivating, particularly the New Crop Varieties of salt tolerant is significant.

Foregoing description only proposes as the enforceable technical scheme of the present invention, not as the Single restriction condition to its technical scheme itself.

Claims (2)

1. a precursor of salt mustard salt tolerance regulation and control miRNA, nucleotide sequence is as shown in SEQ ID NO.2.

2. the DNA fragmentation of the precursor of salt mustard salt tolerance regulation and control miRNA described in coding claim 1, nucleotide sequence is as shown in SEQ ID NO.3.

3 .described in claim 1, the precursor of salt mustard salt tolerance regulation and control miRNA is in the application of cultivating in salt tolerant transgenic saline mustard.