CN103740750B - A kind of method of cultivating dwarfing, increasing the transgenic plant of drought resistance of tillering, improve - Google Patents

Abstract

The invention provides a kind of interference plant sterol methyltransgerase SMT1(cycloartenol-C24-Methyltransferases1) gene, cultivate the method downgrading, increase the transgenic plant of drought resistance of tillering, improve, belong to plant genetic engineering field.The invention provides a kind of interference carrier of sterol methyl tramsferase SMT1 gene, this interference carrier is after the plant of transfection paddy rice preparation interference SMT1 genetic expression, control the expression amount of individual gene by RNAi interference technique, thus make dwarfing plants, tiller number increases and drought resistance improves.Present method process is simple, and can create decorative plant and the agricultural prods with new commercial value, and therefore present method is significant especially in agricultural and Horticulture field.

Description

A kind of method of cultivating dwarfing, increasing the transgenic plant of drought resistance of tillering, improve

Technical field

The invention belongs to plant genetic engineering field, specifically provide a kind of interference plant sterol methyltransgerase SMT1(cycloartenol-C24-Methyltransferases1) gene, cultivate the method downgrading, increase the transgenic plant of drought resistance of tillering, improve.

Background technology

Dwarfing is a kind of important character of plant, and plant plant height proterties both controlled by internal gene, is also subject to the impact of various hormone and external environmental factor.Dwarfing plants is the result of key-gene expressional function of short stem, is also subject to the impact of modifying factor and suppressor gene simultaneously.By breeding wheat for semidwarfness, reduce plant height, not only make crop fertilizer resistance anti-lodging, and change plant type, can harvest index be improved.Abiotic stress in environment, such as arid, saline and alkaline, to damage to plants caused by sudden drop in temperature and heat evil etc. affects the g and D of plant, cause the underproduction of farm crop, also affect forest, fruit tree, flowers, the normal growth of gardening ornamental plant and quality, cultivating resistance to inverse plant variety is one of major objective of plant husbandry.So cultivate not only downgrade, resistance but also high plant variety be so very necessary.

RNA (RNAi) technology of interfering is the expression that the double-stranded RNA utilizing some little carrys out efficiently, blocks specifically specific gene in body, and impels mRNA to degrade, thus entice cell shows the phenotype of specific gene disappearance.This technology has been widely used in plant gene function research.Compared with other technologies, RNA interference technique has high specificity, silence efficiency advantages of higher.

Sitosterol, Stigmasterol, campesterol and cholesterol are the main sterols of plant materials, and Sitosterol (Sitosterol) and campesterol (Campesterol) are the intermediate products in plant hormone brassinolide biosynthetic pathway.Many physiological processs that discovered in recent years plant sterol involved in plant grows, and these effects do not rely on brassinolide (Schaller, 2004; Carlandetal., 2010).A common feature is exactly the growth of regulating plant, but whether plant sterol regulates and controls drought resistance does not also report.The biosynthesizing of plant sterol is for initial compounds with ring Ah enol (cycloartenol), under SMT1 catalysis, generate methylene radical cholestenol (24-methylenecycloartenol), therefore, SMT1 is an enzyme foremost in sterol biosynthesis approach, also be key enzyme (Dieneretal., 2000 of the various phytosterol bio synthesis of regulation and control; Clouseetal., 2002).Arabidopis thaliana smt1 mutant has short petiole and less and mellow and full leaf, and compared with SMT1 wild-type, they are compacter, shorter, but spends normal (Dieneretal., 2000), proves that SMT1 is closely related with dwarfing proterties.But also do not find the report of smt1 mutant or T-DNA insertion mutation body in unifacial leaf, can SMT1 regulate and control monocotyledons and downgrade, tiller and drought resistance, has no report.

Summary of the invention

The method of horn of plenty dwarfing plants, Drought-resistant Breeding, primary and foremost purpose of the present invention is the interference carrier providing a kind of sterol methyl tramsferase SMT1 gene.

Another object of the present invention is to the interference carrier preparation method that above-mentioned sterol methyl tramsferase SMT1 gene is provided.

Another object of the present invention is that providing a kind of interferes plant sterol methyltransgerase SMT1 gene, cultivates the method downgrading, increase the transgenic plant of drought resistance of tillering, improve.

Object of the present invention is achieved through the following technical solutions: a kind of interference carrier of sterol methyl tramsferase SMT1 gene, is specially and builds with plant interference vector the restructuring interference carrier containing rice Os SMT1 Gene interfere fragment;

Described rice Os SMT1 Gene interfere fragment, select the gene fragment that specificity is higher, called after OsSMT1-1S by the Nucleotide of rice Os SMT1 and amino acid Multiple sequence alignments, its sequence (SEQIDNO1) is as follows:

atggttggggtgaatccttccactttgctcacagatggaatggagaatccctacgtgaaagcattaagcgacatgagcactttcttgcccttcagcttggagtgaaaccaggaatgaaggttttggacgtcggttgtggaataggcggaccactaagagaaattgccaaatttagcttggcttcagtcactggattgaacaacaatgagtaccagataactaggggaaaggagcttaatcgggtagcaggagttagtggaacttgcgactttgtgaaggcagacttcatgaagatgccgttttctgataacacttttgatgctgtctatgccattgaggcaacatgccacgcacctgatccggttggctgctataaggagatctatcgtgtattgaagc；

Described plant interference vector includes but not limited to carrier for unifacial leaf via Particle Bombardment Transformation and double base agrobacterium vector; Described double base agrobacterium vector comprises pRNAi-Ubi and pRNAi-35S serial carrier;

Described plant interference carrier is preferably pYLRNAi;

The preparation method of the interference carrier of above-mentioned sterol methyl tramsferase SMT1 gene, comprises the following steps:

(1) loading of forward fragment: with paddy rice nucleotide sequence OsSMT1(gene numbering AAP21419) full length sequence is for template, and design is respectively with the upstream and downstream primer amplification OsSMT1-1 gene fragment of BamHI and HindIII restriction enzyme site, and primer sequence is as follows:

ZG1375：GAGT GGATCCATGGTTGGGGT

ZG1376：TGGCC AAGCTTCAATACACGATAG；

Object fragment is obtained for template and primer ZG1375 and ZG1376 carry out pcr amplification to spend the cDNA of 11 blades in paddy rice, amplification object fragment length is about 400bp, pGEM-Teasy carrier is connected into after PCR primer OsSMT1-1S is purified, transformation of E. coli sequencing analysis obtain pGEM-OsSMT1-1S, with BamHI and HindIII respectively enzyme cut the pGEM-OsSMT1-1S vector plasmid after pYLRNAi empty carrier and restructuring; Reclaim object fragment OsSMT1-1S to be respectively connected with after pYLRNAi, transformation of E. coli Top10F ', detect recon, extracting plasmid, check order errorless acquisition recombinant plasmid pYLRNAi-OsSMT1-1S.Then the loading of reverse fragment is carried out;

(2) loading of reverse fragment: recon plasmid process in step (1) verified is template, utilize universal primer, MluI(CACCCTGACGCGTGGTGTTACTTCTGAAGAGG) and PstI(ACTAGAACTGCAGCCTCAGATCTACCATGGTCG) amplification obtains reverse fragment, with MluI and PstI double digestion after purifying, the positive colony plasmid of synchronous MluI and PstI double digestion pYLRNAi-OsSMT1-1S, reclaim object fragment, after connecting, after transformation of E. coli DH5 α, obtain interference carrier pYLRNAi-OsSMT1-1-Ri.

The interference carrier of above-mentioned sterol methyl tramsferase SMT1 gene is by the application of preparation transgenic plant in reduction plant strain senior middle school.

The interference carrier of above-mentioned sterol methyl tramsferase SMT1 gene is applied in increase plant tillering number by preparation transgenic plant.

The interference carrier of above-mentioned sterol methyl tramsferase SMT1 gene is applied in raising plant drought resistance by preparation transgenic plant.

A kind of interference plant sterol methyltransgerase SMT1 gene, cultivate the method downgrading, increase the transgenic plant of drought resistance of tillering, improve, be specially:

1) interference carrier (pYLRNAi-OsSMT1-1-Ri) of sterol methyl tramsferase SMT1 gene imports in agrobacterium tumefaciens EHA105 by the method that electricity consumption turns, and obtains containing the positive bacterium colony of interference vector (pYLRNAi-OsSMT1-1-Ri) Agrobacterium;

2) the Agrobacterium EHA105 containing interference vector (pYLRNAi-OsSMT1-1-Ri) of activation is contaminated plant callus, through Dual culture and antibiotic-screening, obtain the transgenic plant downgrading, increase drought resistance of tillering, improve.

Step 2) described in plant optimization be monocotyledons;

Described monocotyledons is preferably paddy rice.

The present invention has following advantage and effect relative to prior art:

The invention provides a kind of interference carrier of sterol methyl tramsferase SMT1 gene, this interference carrier is after the rice plant of transfection paddy rice preparation interference SMT1 genetic expression, control the expression amount of individual gene by RNAi interference technique, thus make dwarfing plants, tiller number increases and drought resistance improves.Present method process is simple, and can create decorative plant and the agricultural prods with new commercial value, and therefore present method is significant especially in agricultural and Horticulture field.

Accompanying drawing explanation

Fig. 1 is the expression cassette schematic diagram of plant interference vector pYLRNAi-OsSMT1-1-Ri.

Fig. 2 is the pcr amplification agarose gel electrophoresis figure of OsSMT1-1 gene fragment of the present invention, and wherein, M is stranded DNA molecule; 1 is the amplified fragments of OsSMT1-1 gene.

Cleavage map is verified in Fig. 3 pYLRNAi-OsSMT1-1-Ri interference vector building process, wherein, (A): pYLRNAi-OsSMT1-1-Ri interference vector first round enzyme cuts qualification, M is MarkerDL5000; Carrier segments after 1:BamH I and HindIII double digestion (on) and OsSMT1-1 gene fragment (under).(B): pYLRNAi-OsSMT1-1-Ri interference vector second is taken turns enzyme and cut qualification, M is MarkerDL5000; 2:HindIII and BamHI double digestion is identified; 3:HindIII single endonuclease digestion is identified.

Fig. 4 is the PCR qualification result agarose gel electrophoresis figure of the transgenic paddy rice importing OsSMT1-1 gene interference fragment, and wherein, W represents wild-type; PI represents pYLRNAi-OsSMT1-1-Ri carrier, is positive control; Numbering I1, I2, I3, I4, I5, I6, I7, I8, I9, I10, I11, I12 represent different and turn the transgenic paddy rice sample after OsSMT1-1 Gene interfere fragment.

Fig. 5 is Southern hybridization and the real-time quantitative PCR that four OsSMT1-1 interfere rice plant, and wherein, NT represents negative plant; Numbering R2, R4 and R5 represent that different transgenosiss interferes rice strain; Figure (A) is Southern hybridization, and figure (B) is that real-time quantitative PCR detects; Alphabetical a, b, c and d above post represent the significant difference (P≤0.05) between differing materials.

Fig. 6 is that 55 days wild-types and OsSMT1-1 interfere plant forms to be observed, and wherein WT is wild-type; Numbering R2, R4 and R5 represent that different transgenosiss interferes rice strain; Figure (A) is the outside drawing of different rice strain, and figure (B) is that 55 days wild-types and OsSMT1-1 interfere plant plant height, and figure (C) is that 55 days wild-types and OsSMT1-1 interfere plant tillering number statistic data; Alphabetical a, b and c above post represent the significant difference (P≤0.05) between differing materials.

Fig. 7 leads OsSMT1-1 to interfere the drought resistance of plant to detect, and wherein scheme the moisture determination figure that (A) is each plant after Osmotic treatment, figure (B) is that the relative conductivity of each plant after Osmotic treatment measures; Alphabetical a, b and c above post represent the significant difference (P≤0.05) between differing materials.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Embodiment

Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Rice paddy seed: kind is middle colored 11(84-213), rice 1989016 is examined in Tianjin, this laboratory propagation and preservation.

Agrobacterium tumefaciens (Agrobacteriumtumefaciens) EHA105: purchased from sky, Beijing bounties Gene Tech. Company Limited.

PYLRNAi interference vector: Agricultural University Of South China doctor Liu Yaoguang is so kind as to give.

Escherichia coli DH5a: purchased from Chao Yan bio tech ltd, Shanghai.

The clone of embodiment 1OsSMT1-1 gene fragment

One, rice cDNA Template preparation

By rice paddy seed (in spend 11) volume fraction 70% alcohol disinfecting 2min, soak seed in 28 DEG C of water 1d, 30 DEG C of vernalization 3d, and 28 DEG C of sowings, by the described method nursery such as Jiang Dean (1987), management.The mature leaf of water intaking rice, extracts total serum IgE by Trizol method, and obtain cDNA template with M-MLV Reverse Transcriptase kit (Promega company) reverse transcription ,-20 DEG C save backup.

Two, design amplification OsSMT1-1 gene primer

NCBI carries out download rice Os SMT1 gene order (gene numbering AAP21419), by this sequence signature of DNAMAN software compare of analysis, finds out the OsSMT1-1S Gene interfere fragment that specificity is higher, according to this fragment design primer:

OsSMT1-1S gene fragment amplification upstream primer ZG1375:GAGT gGATCCaTGGTTGGGGT;

OsSMT1-1S gene fragment amplification upstream primer ZG1376:TGGCC aAGCTTcAATACACGATAG;

Three, amplification obtains OsSMT1-1 gene fragment and carrier construction

The primer of the template prepared by above-mentioned steps one and step 2 design carries out pcr amplification.

PCR reaction system (50 μ L): KOD-Plus-DNA polysaccharase (TOYOBO company, 1U μ L ^-1) 1 μ l, 10 × buffer5 μ l, dNTPs(10mmolL ^-1) 5 μ l, MgSO ₄(25mmolL ^-1) 2 μ l, upstream primer (10 μm of olL ^-1) 1.5 μ l, downstream primer (10 μm of olL ^-1) 1.5 μ l, reverse transcription first chain cDNA2 μ l, ddH ₂o complements to 50 μ l.

PCR response procedures: PCR response procedures: 94 DEG C 3 minutes; 94 DEG C 0.5 minute, 55 DEG C 0.5 minute, 72 DEG C 0.5 minute, 35 circulations; 72 DEG C 10 minutes.Amplified production OsSMT1-1S Gene interfere fragment detects (Fig. 2) with the agarose gel electrophoresis of 0.8%.

Obtain the PCR fragment (Fig. 2) that OsSMT1-1S Gene interfere fragment is consistent with expection fragment length (~ 400bp).Carry out end further by the reaction of Taq enzyme catalysis after the PCR primer DNA gel of acquisition being reclaimed test kit (Qiagen company) recovery and add A, reaction system is as follows: 2 μ l10 × buffer, 1 μ l4mMdATP, 11 μ gPCR reclaim fragment, 0.1 μ lTaqDNA polysaccharase, adding aseptic deionized water cumulative volume is 20 μ l, and 70 DEG C are reacted 20 minutes.

The OsSMT1-1S Gene interfere fragment that test kit reclaims above-mentioned acquisition is reclaimed by PCR primer.Use T ₄pCR is reclaimed fragment and is connected with pGEM-Teasy carrier (Promega company) by DNA ligase (TaKaRa company), and reaction system is as follows: 1 μ l10 × T ₄ligase enzyme damping fluid, reclaims object fragment 60ng, 1 μ lT ₄ligase enzyme, 2 μ lpGEM-Teasy carriers (10ng), adding sterilized water to cumulative volume is 10 μ l, and 16 DEG C of connections are spent the night.

The preparation of competent escherichia coli cell: go bail for transfering loop and be stored in the intestinal bacteria DH10B bacterium liquid of-80 DEG C of Ultralow Temperature Freezers, SOB solid medium draws plate, overnight incubation in 37 DEG C of incubators; The mono-colony inoculation of picking intestinal bacteria DH10B, in the SOB liquid nutrient medium of 1ml, 37 DEG C of constant-temperature tables is about 6h with 200rpm shaking culture, is inoculated in 200mlSOB liquid nutrient medium, on 37 DEG C of constant-temperature tables with 200rpm shaking culture to OD ₅₅₀=0.6-0.8.Within centrifugal 10 minutes, collect thalline with 2500rpm, add 10% glycerine washing of precooling, collected by centrifugation bacterium.Repeat with 10% glycerine washing, collected by centrifugation bacterium.Finally by bacterium packing, save backup in-70 DEG C.

Connect product Electroporation intestinal bacteria: above-mentioned OsSMT1-1S Gene interfere fragment is connected with pGEMT-vector product 1/3 TE(1MNaCl, 10mMTris-HClpH8.0,1mMEDTA) in dialyse 30 minutes.Get 20 μ l competent escherichia coli cells to pole cup (aperture 1mm), add the connection product of 1 μ l through dialysis, with electric exciter (MicroPulser, Bio-RAD company) Electroporation.Electric shock parameter is: resistance 200 Ω, 1800V, 25 μ F.Thalline after electric shock proceeds in 1mlSOC liquid, with 200rpm shaking culture 1 hour on 37 DEG C of constant-temperature tables.Get on LB solid (containing 100 μ g/mLAmp) substratum that 100 μ l bacterium liquid coat containing IPTG and X-gal, in 37 DEG C of incubators, be inverted overnight incubation.

The screening of recombinant plasmid pGEM-OsSMT1-1S, purifying and order-checking:

Bacterium colony in locus coeruleus is not containing Insert Fragment, and only picking is the bacterium colony of hickie, is bacterium colony PCR and detects.The positive bacterium colony of picking PCR, is inoculated in 2ml containing in the LB liquid nutrient medium of 100 μ g/ml penbritins, spends the night on 37 DEG C of constant-temperature tables with 200rpm shaking culture.Draw 2ml bacterium liquid, with plasmid DNA purification test kit (Qiagen company) upgrading grain, 4 DEG C save backup.To the plasmid BamH I/HindIII double digestion obtained, the size of electrophoresis detection Insert Fragment, determines positive colony further.The recombinant plasmid called after pGEM-OsSMT1-1S of purifying, deliver Shanghai Sangon Biological Engineering Technology And Service Co., Ltd and check order, sequencing result is as follows:

TGAAATAGAGGAAGCAATAACATCCACAACAAAAAGAGTGGATCC A TGGTTGGGGTGAATCCTTCCACTTTGCTCACAGATGGAATGGAGAATCCCT ACGTGAAAGCATTAAGCGACATGAGCACTTTCTTGCCCTTCAGCTTGGAGT GAAACCAGGAATGAAGGTTTTGGACGTCGGTTGTGGAATAGGCGGACCAC TAAGAGAAATTGCCAAATTTAGCTTGGCTTCAGTCACTGGATTGAACAACA ATGAGTACCAGATAACTAGGGGAAAGGAGCTTAATCGGGTAGCAGGAGTT AGTGGAACTTGCGACTTTGTGAAGGCAGACTTCATGAAGATGCCGTTTTCT GATAACACTTTTGATGCTGTCTATGCCATTGAGGCAACATGCCACGCACCT GATCCGGTTGGCTGCTATAAGGAGATCTATCGTGTATTGAAGCTTGGCCAGATTAAGGTCCTCAAGACACAAATGCCTG

Sequencing result carries out sequence alignment at NCBI, proves that the PCR primer amplified is OsSMT1-1 gene fragment (OsSMT1-1S), as underscore annotate portions in above-mentioned sequencing sequence.This gene fragment is positioned at the 191bp-589bp of OsSMT1 open reading frame.

The plant interference vector (pYLRNAi-OsSMT1-1-Ri) of embodiment 2OsSMT1-1 gene builds

According to embodiment 1 obtain pGEM-OsSMT1-1S with BamH I/HindIII restriction enzyme site, shake bacterium and expand numerous extraction plasmid.Then use restriction enzyme BamH I (TaKaRa company) and HindIII(TaKaRa company) double digestion pGEM-OsSMT1-1S plasmid, purifying reclaim amplification obtain enzyme cut after OsSMT1-1S Gene interfere fragment.

PYLRNAi interference vector is also used restriction enzyme BamH I (TaKaRa company) and HindIII(TaKaRa company) double digestion, reclaim enzyme respectively and cut rear carrier large fragment, after OsSMT1-1S Gene interfere fragment after cutting through with the enzyme of above-mentioned acquisition is afterwards connected, transform Top10F ' competent cell, go out positive colony through BamH I and HindIII double digestion evaluation and screening.Result, as shown in figure (Fig. 3 A), shows the successful connection of forward fragment.OsSMT1-1S Gene interfere fragment is inserted between the BamH I of the multiple clone site in interference vector pYLRNAi and HindIII, will build the restructuring interference vector called after pYLRNAi-OsSMT1-1S obtained.

With the positive colony plasmid of pYLRNAi-OsSMT1-1S for template, by universal primer MluI-F and the reverse fragment of PstI-R specific amplified.

MluI-F（CACCCTGACGCGTGGTGTTACTTCTGAAGAGG）

PstI-R（ACTAGAACTGCAGCCTCAGATCTACCATGGTCG）

This reverse fragment is reclaimed rear MluI and PstI double digestion, the positive colony plasmid of synchronous MluI and PstI double digestion pYLRNAi-OsSMT1-1S, reclaims object fragment, transformation of E. coli after connecting, two kinds of enzymatic cleavage methods qualifications are used to recon.As shown in Figure 3 B, HindIII single endonuclease digestion can cut out the reverse fragment of forward fragment+intron+ to result, about 1kb, and double digestion can cut out carrier segments and interfere fragment, and result shows that interference vector successfully constructs, carrier called after pYLRNAi-OsSMT1-1-Ri.

The expression cassette schematic diagram of described plant interference vector pYLRNAi-OsSMT1-1-Ri as shown in Figure 1.

The generation of embodiment 3 transgenic paddy rice and Molecular Detection

One, the generation of transgenic paddy rice

1. pYLRNAi-OsSMT1-1-Ri is imported agrobacterium tumefaciens EHA105

By agrobacterium tumefaciens (Agrobacteriumtumefaciens) EHA105 competent cell, (method with reference to J. Pehanorm Brooker (2002) is also improved, get EHA105 bacterial classification in the flat lining out of MYB, cultivate 48 hours for 28 DEG C, picking list bacterium colony, is inoculated in 50mL liquid SOC, 28 DEG C of overnight incubation, draw 0.5mL bacterium liquid, be inoculated in 500mL liquid SOC, cultivate 6 ~ 8h, to OD for 28 DEG C ₆₀₀be about 0.6, ice bath cooling 10min, pours in the 200mL centrifuge tube of sterilizing, balance latter 4 DEG C, 4000rpm, centrifugal 10min, collect thalline, remove SOC, be inverted on paper handkerchief, control solid carbon dioxide divides, and adds 50mL10% glycerine, in shaking on ice, suspension thalline, 4 DEG C, 4000rpm, centrifugal 15min, collects thalline, abandons 10% glycerine, repeated washing once, add 2mL10% glycerine, suspension thalline, packing, 20 ~ 25 μ L/ manage, after adding liquid nitrogen flash freezer, obtain agrobacterium tumefaciens EHA105 competent cell, in-80 DEG C of Refrigerator stores.Be placed in and thaw on ice, the electric shock cup of 0.2cm internal diameter is placed in precooling on ice.In Bechtop, respectively 1.5 μ lpYLRNAi-OsSMT1-1-Ri plasmids (20ng/ μ l) are added in the competent cell that 20 μ l thaw, flick tube wall mixing, ice bath is after 1 minute, be transferred in electric shock cup, between the electrode being placed in electric shock instrument (MicroPulser, Bio-RAD company), selection procedure Agr, shocks by electricity.After electric shock terminates, in Bechtop, pour 1mlYEB liquid nutrient medium into electric shock cup rapidly, then be transferred to liquid-transfering gun and shake in tube, 28 DEG C of gentle agitation cultivate 2 hours.Draw 0.3ml bacterium liquid, be coated on YEB flat board (containing the paraxin of 35mg/l and the kantlex of 50mg/l).Cultivation 48 hours is inverted in 28 DEG C of incubators.

2. containing the qualification of the positive bacterium colony of interference vector pYLRNAi-OsSMT1-1-Ri Agrobacterium

Single bacterium colony on picking flat board carries out bacterium colony PCR detection, and carries out mark.Further picking PCR positive bacteria drops down onto in 3mlYEB liquid nutrient medium (containing the paraxin of 35mg/l and the kantlex of 50mg/l), 28 DEG C of shaking culture 40 hours.Get 2ml bacterium liquid alkaline lysis extracting plasmid, with restriction enzyme BamH I (TaKaRa company) and HindIII(TaKaRa company) carry out enzyme and cut detection, determine in positive colony containing plant interference vector pYLRNAi-OsSMT1-1-Ri.Get 0.8ml agrobacterium liquid, add 0.2ml80% glycerine, be stored in-80 DEG C of Ultralow Temperature Freezers after mixing for subsequent use.

3. the acquisition of transgenic rice plant

(1) induction of Rice Callus and subculture

Choose in full health and spend 11 mature seed peelings, with 70% alcohol-pickled 1min, distillation washing 1 time, then mercuric chloride (HgCl solution) the sealing process 15min using 0.1%, period constantly shakes on shaking table.Then in Bechtop, outwell mercuric chloride, aseptic distillation washing 4-5 time, the large filter paper being placed on 3 sterilizings dries.Finally, sterilizing seed evenly lies against on NB inducing culture, evoked callus under 25 DEG C of dark conditions.The seed light culture about 15-25 days of induction, when seeing after faint yellow particulate state callus grows, being connected to callus on subculture medium and continuing callus induction.

(2) activation of Agrobacterium and suspension

Get the Agrobacterium EHA105 stock solution containing expression vector pYLRNAi-OsSMT1-1-Ri, in the upper line of YEB flat board (containing the paraxin of 35mg/l and the kantlex of 50mg/l), 28 DEG C of cultivations.Choose and be separated good single bacterium colony, be inoculated in 2ml liquid YEB(containing the paraxin of 35mg/l and the kantlex of 50mg/l) in, 28 DEG C of vibration light culture 24-48 hour.Draw bacterium liquid 20-50 μ l, coat containing on same antibiotic YEB flat board, be inverted light culture 24-36 hour for 28 DEG C.The Agrobacterium newly grown is scraped off, suspends with MS liquid nutrient medium and dilute, make OD ₆₀₀be 0.06 ~ 0.08.

(3) infect, the generation of Dual culture and transgenic seedling

In Bechtop, in advance dried callus and Agrobacterium are infected liquid mixing (ensureing that Agrobacterium is infected liquid and floods callus), then adds Syringylethanone, final concentration is 100 μMs, and then on shaking table, 150rpm, 28 DEG C of dark infect 20min.In Bechtop, pour out and infect liquid, drying about 5-10min on little plate callus being placed in multilayer sterilizing filter paper, being transferred to by callus haves three layers on the large plate of sterilizing filter paper again, in blower fan blowing down, drying is 1.5-2h about, is then received by callus on the solidified co-cultivation medium of an Amoxcillin filter paper, in blower fan blowing down, drier 2-3h.The callus of drying process, in Bechtop, after blower fan blowing down 0.5-1h, accesses screening culture medium respectively, then seals, light culture 2 weeks at 25 DEG C, screens 2 times altogether.

The pre-differentiation of kanamycin-resistant callus tissue and differentiation: select the good kanamycin-resistant callus tissue of growth conditions and access pre-division culture medium (NB substratum+0.5g/LL-glutamine+0.5g/LL-proline(Pro)+0.3g/L acid hydrolysis casein food grade+30g/L sucrose+20g/LD-sorbyl alcohol+1mg/LNAA+5mg/L6-BA+2.5mg/LCuSO4.5H2O+5mg/LABA+50mg/L hygromycin B+200mg/L cephamycin+200mg/L Pyocianil+10g/L agar powder pH5.8), in 25 DEG C, cultivate under alternation of light and darkness, until grow new callus, pre-differentiation is about 3-4 week.Differentiation and seedling emergence is continued by there being the callus of green point to move in division culture medium.

(4) growth of transgenic paddy rice

In time breaking up seedling and grow to 3-5cm, it transferred in the triangular flask Rooting and hardening-off culture base of 100mL from division culture medium, 25 DEG C of alternation of light and darkness cultivate 3-4 week, until grow new root.If seedling well-grown, new root is looked than comparatively fast, then can shift to an earlier date transplant and root.When seedling root grows new long root, shifted out by seedling from substratum, and the substratum of root is rinsed well, a point individual plant is transplanted in basin.With Kimura B nutritive medium, cultivate 2-3 week at natural lighting strength condition.Finally move in soil and carry out, outside greenhouse, natural condition are cultivated.

The PCR of two transgenic paddy rices detects

1. micromethod DNA rapid extraction

Get the blade of size as 1.5mL centrifuge tube lid in 1.5mL centrifuge tube, add the extracting solution (200mMTris-HClpH8.0 that 400 μ L are preheating to 80 DEG C, 250mMNaCl, 25mMEDTA, massfraction 0.5%SDS), fully grind in 1.5mL centrifuge tube with the little hammer that grinds, 70 DEG C of water-bath 10min, then place 2min on ice.The centrifugal 1min of 13000rpm, gets 300 μ L supernatant liquors, adds equivalent Virahol, and mixing, room temperature leaves standstill 5min.The centrifugal 2min of 13000rpm, abandons most supernatant liquor.50 μ LTE(1MNaCl are dissolved in, 10mMTris-HClpH8.0,1mMEDTA after DNA precipitation is air-dry), be stored in-20 DEG C of refrigerators.

2. the PCR of transgenic paddy rice detects

Due to infect plant carrier pYLRNAi in its right boundary district also with homomycin(HPT) resistant gene, therefore using can the primer ZG599(CGAAATTGCCGTCAACCAAGCTCT of this gene of specific recognition) and ZG600(CAGCGTCTCCGACCTGATGCAGCT) carry out testing goal gene and whether be successfully transferred to Plant Genome.Take out the DNA that obtains for template carry out pcr amplification, with pYLRNAi-OsSMT1-1-Ri recombinant plasmid for positive control so that previous step is micro-.Reaction system (20 μ L): Taq DNA polymerase 0.1 μ L, PCR10 × buffer2 μ L, dNTP(10mM) 1.6 μ L, ZG599(10 μM) 0.5 μ L, ZG600(10 μM) 0.5 μ L, consumption in DNA1 μ L(positive control be pYLRNAi-OsSMT1-1-Ri plasmid 20ng), ddH ₂o14 μ L.PCR response procedures: 94 DEG C of 2min; 94 DEG C of 0.5min, 57 DEG C of 0.5min, 72 DEG C of 30s, 35 circulations; 72 DEG C of 5min.PCR primer is detected with 1% agarose gel electrophoresis.

The Southern hybridization of three transgenic paddy rices

1. the extraction of genomic dna

Get 1g paddy rice tender leaf, put grind into powder in liquid nitrogen, be transferred to 50mL centrifuge tube, add 1.5 × CTAB extracting solution (1.5%CTAB, 75mMTris-HClpH8.0,1MNaCl, 15mMEDTA) that 6mL is preheating to 70 DEG C immediately, vortex vibration 10sec, mixing.70 DEG C of water-bath 1h, are interrupted mixing.Water-bath terminates, and adds 5mL chloroform, screws pipe lid, and turn upside down mixing 10min.The centrifugal 15min of 5000rpm.With cutting off the most advanced and sophisticated careful Aspirate supernatant of 1mL rifle head to 50mL centrifuge tube, and recording volume, add the 10%CTAB solution of 1/10 amount, shake up.Add the chloroform of 4/5 amount, turn upside down mixing 10min.The centrifugal 15min of 5000rpm.With cutting off the most advanced and sophisticated careful Aspirate supernatant of 1mL rifle head to 50mL centrifuge tube, and recording volume, add the precipitated liquid (1%CTAB of equivalent, 50mMTris-HClpH8.0,10mMEDTA), mixing of softly turning upside down, room temperature places 15min(or 55 DEG C of water-bath 10min) precipitate DNA.The centrifugal 10min of 5000rpm, carefully outwells supernatant liquor, the more centrifugal 1min of 6000rpm, exhausts supernatant liquor with liquid-transfering gun.Add 2mL height salt TE(1MNaCl, 10mMTris-HClpH8.0, the RNAase(10mMTris-HClpH7.5 of 1mMEDTA) He 5 μ L10mg/mL and 15mMNaCl solution preparation, through 100 DEG C of water-bath 15min deactivation DNA enzymatic), in 55 DEG C of shaking tables, jog dissolves (30-40min) completely to precipitation.Add the dehydrated alcohol of 2 times of volumes, mixing of gently turning upside down.Go out cotton-shaped DNA with rifle head hook, rinsing in 70% ethanol, then move to 1.5mL centrifuge tube.Of short duration centrifugal, abandon supernatant liquor, suitable air-dry precipitation, with 0.1-0.2mLTE(10mMTris-HCl, pH8.0,1mMEDTA) dissolution precipitation.Get the DNA solution that 2 μ L dilute 10 times, use 0.8% agarose gel electrophoresis, detect the quality of DNA, survey OD260nm and OD280nm, determine purity and the concentration of DNA.

2.DNA enzyme is cut, electrophoresis and transferring film

Concrete operations are with reference to DIGHighPrimeDNALabelingandDetectionStarterKit II (Roche) working instructions.Carry out sex change and neutralization after simply being rinsed by glue after electrophoresis terminates, then use 20 × SSC to adopt the method for kapillary transfer printing to make DNA be transferred to nylon membrane HybondN+(Amersham company) on.Film is placed on on the moistening filter paper of 20 × SSC, carries out UV-crosslinked, 800s, 2 times.Dry with after the simple rinsing of distilled water, wrap with preservative film, carry out mark, be stored in 4 DEG C for subsequent use.

3. the preparation of probe and mark

Be the partial sequence that template amplification goes out hygromycin gene with primer ZG599 and ZG600, pYLRNAi-OsSMT1-1-Ri recombinant plasmid, reclaim the probe template of PCR primer as hybridization with kits, and electrophoresis be quantitative.With DIG-HighPrime, probe is marked.In a reaction tubes, 1 μ g template DNA and autoclaved distilled water is added, final volume 16 μ L with reference to specification sheets method.Boiling water bath 10min, with denatured DNA, then inserts rapidly in mixture of ice and water.Getting 4 μ LDIG-HighPrime joins in denatured DNA, and mixing is also simply centrifugal.37 DEG C of overnight incubation, then 65 DEG C of heating 10min termination reactions.

4.Southern is hybridized

Film be put into use 10 × SSC to soak into Whatman3 qualitative filter paper on.After UV-crosslinked, film put into distilled water and simply rinse.By the hybridization buffer (10mL/100cm of appropriate volume ²filter membrane) be preheating to hybridization temperature (37-42 DEG C) in advance.NC film is put into hybridization buffer at hybrid heater prehybridization 1h.The probe that 3 prepare is put into and puts into mixture of ice and water fast after boiling water bath boils 5min and cool, the digoxin labelled probe of sex change is joined digoxin hybridization buffer (every 100cm of preheating ²film add 3.5mL hybridization buffer) in, fully mixing obtains the mixture of probe hybridization liquid.Pour out prehybridization solution, add the mixture of probe hybridization liquid, overnight incubation in 42 DEG C of hybrid heaters.Wash twice with 2 × SSC, 0.1%SDS continuous oscillation, each 5min, 15-20 DEG C.In 80mL blockades liquid, hatch 45min, in 20mL antibody-solutions, hatch 30min, wash twice with 100mL lavation buffer solution, each 15min, detect in damping fluid at 20mL and balance 2-5min.Lain in by film on preservative film, evenly drip 500 μ LCSPD nitrite ions, layer overlay preservative film, shakeouts nitrite ion from inside to outside gently, and squeezes out unnecessary nitrite ion, and paper using sops up.The film done well is put in darkroom 5min, is placed on lucifuge 1h in 37 DEG C, imaging software scanning imagery.

Adopt PCR method to detect (using primer pair ZG599 and ZG600) transgenosis and interfere paddy rice, positive plant can amplify the special band of 500bp, with with plant expression vector pYLRNAi-OsSMT1-1-Ri(positive control) the special band that expands for template is in the same size, and wild type control plants can not expand this special band (Fig. 4), prove that OsSMT1-1 interferes fragment to import in transgenic paddy rice.

Southern results of hybridization shows, the specific hybridization signal of HPT gene is not had in wild rice, transgenosis interferes the specific hybridization signal having HPT gene in rice plant, shows that OsSMT1-1 interferes fragment to be incorporated into transgenosis and interferes in the genome of paddy rice (Fig. 5 A).

Have detected the interference situation of OsSMT1-1 gene with real-time quantitative PCR, result shows, transgenosis interferes the expression level of the OsSMT1-1 gene in paddy rice to be starkly lower than wild rice plant, and this explanation is interfered successfully (Fig. 5 B).

Embodiment 4 transgenosis interferes the morphological analysis of paddy rice

One height and tiller number statistics

When paddy growth was by 55 days, measure root to length when coming into leaves upright most, be plant height (to blade), and tiller number is added up.Wild-type and transgenosis interfere plant respectively to get 25 strains measurements, then get its mean value.Test-results such as Fig. 6 shows, and the expression amount decline of OsSMT1-1 has been downgraded paddy rice and improve the tiller number of paddy rice.

Two drought resistances measure

Transgenosis interfered paddy rice and wild rice seedling thereof after 30 days at warm indoor growing, to start stopping and watering, after 10, collect two blades, measure specific conductivity and relative water content.

Measuring method is:

Blade is put into the triangular flask containing 20ml deionized water, 4 DEG C are spent the night, and measure solution conductivity value (C1) with conductivity meter; Triangular flask is moved on in boiling water bath and be incubated 20 minutes, be cooled to room temperature, again measure solution conductivity value (C2) with conductivity meter.Relative conductivity is calculated by formula (C1/C2) × 100.Each strain plant measures 5 individual plants altogether, calculating mean value.

Claim fresh weight at once after cutting blade, then they are placed in beaker, claim saturated heavy in the dark after placing 24h, in convection oven, under 80 DEG C of constant temperature, dry 48h afterwards, claim dry weight.Repeat, average as the relative water content RWC of this sub-sampling plant leaf for 3 times.Calculation formula: RWC (%)=(fresh weight-dry weight)/(saturated heavy-dry weight) * 100%.

After Osmotic treatment, transgenosis interferes the relative conductivity of rice leaf to be starkly lower than wild rice, and relative water content, apparently higher than wild-type (Fig. 7), shows that the injury that transgenosis interferes paddy rice to be subject under arid is lighter than wild rice, improves drought resistance.

Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (3)

1. the interference carrier of a sterol methyl tramsferase SMT1 gene is applied in increase plant tillering number or raising plant drought resistance by preparing transgenic monocot plant, it is characterized in that: the interference carrier of described sterol methyl tramsferase SMT1 gene, is build with plant interference vector the restructuring interference carrier containing rice Os SMT1 Gene interfere fragment;

The sequence of described rice Os SMT1 Gene interfere fragment is as shown in SEQIDNO1;

Described monocotyledons is paddy rice.

2. a sterol methyl tramsferase SMT1 gene interference carrier cultivation possess dwarfings, increase tiller, the application improved in the transgenic monocot plant of drought resistance, it is characterized in that: the interference carrier of described sterol methyl tramsferase SMT1 gene, is build with plant interference vector the restructuring interference carrier containing rice Os SMT1 Gene interfere fragment;

The sequence of described rice Os SMT1 Gene interfere fragment is as shown in SEQIDNO1;

Described monocotyledons is paddy rice.

3. application according to claim 2, is characterized in that:

By a method of interfering plant sterol methyltransgerase SMT1 gene to cultivate transgenic monocot plant, be specially:

1) interference carrier of sterol methyl tramsferase SMT1 gene imports in agrobacterium tumefaciens EHA105 by the method that electricity consumption turns, and obtains the positive bacterium colony of Agrobacterium containing interference vector;

2) the Agrobacterium EHA105 containing interference vector of activation is contaminated plant callus, through Dual culture and antibiotic-screening, obtain transgenic monocot plant;

Described transgenic monocot plant is the transgenic monocot plant possessing dwarfing, increase drought resistance of tillering, improve.