CN101503692A - Method for cultivating drought-resistant sugarcane variety by DREB2B gene - Google Patents

Abstract

The invention provides a method for culturing a drought-tolerant sugarcane variety by utilizing a DREB2B gene. The method comprises the steps of constructing a plant expression vector containing the DREB2B gene, culturing a drought-tolerant transgenic DREB2B gene material, screening and identifying a novel drought-tolerant transgenic sugarcane material. According to the culturing method, sugarcane induces the expression of a drought-tolerant gene under drought stress, thus the drought tolerance of the sugarcane is obviously superior to a receptor variety, and stability among different asexual generations can be basically maintained. Under a droughty condition, the growth of the sugarcane variety is faster than that of the receptor variety; the height of plants increases by over 20 percent; the number of effective stalks increases by over 30 percent; the yield of sugarcane stalks per hectare increases by 30 percent; and the sugar content (absolute value) of the sugarcane increases by over 1.5 times.

Description

Utilize the DREB2B gene to cultivate the method for drought-resistant sugarcane variety

Technical field

The present invention relates to a kind of method of using genetic engineering technique to cultivate sugar cane breed, particularly utilize the DREB2B gene to cultivate the method for drought-resistant sugarcane variety, belong to the plant gene engineering technology field.

Technical background

Sugarcane is the most important sugar crop in the China and even the whole world, also is ideal renewable energy source crop and transgenosis safe plants.China sugarcane is planted in the nonirrigated farmland, hills more than 85%, arid becomes the key constraints of restriction China sugarcane industry development.Therefore design, seed selection and the popularization of drought resisting good quality and high output new variety of sugarcane have broad application prospect.

Earlier 1990s, take to the research of resisting abiotic stress gene both at home and abroad, the research of resistant transgenic has obtained remarkable progress, be transformed in tobacco and the potato as the choline dehydrogenase gene BetA that will encode, anti-salt and low temperature resistant transgenic line (Lilius etal, 1996) have been obtained; By the content of trimethyl-glycine in the transgenosis means increase tobacco, can obviously improve tobacco to salt and the cold ability of restraining oneself (Nakamura et al., 1997) of coercing; The Mn-SOD gene of tobacco is changed among the M.saliva, make output and the survival rate of M.saliva under drought stress that raising (McKersie et al., 1999) all be arranged.But by the quantitative character of controlled by multiple genes, plant is not often depended on certain term single gene to the power of doing morning, high salt and low-temperature resistance to the resistance of plant, but is subjected to the common regulation and control of many genes often.Drought resisting signal key factor can be regulated and control many correlation function expression of gene (Liu Qiang, 2000), therefore, can make the resistance of plant obtain improvement comprehensive, essence by the effect that strengthens some drought resisting signal key factor, improve the traditional method of certain resistance with importing or improvement discrete function gene and compare, importing drought resisting signal factor gene will be more efficiently method and the approach that improves crop anti-adversity.

DREB transcription factor (Dehydration responsive element binding protein) is a species specific transcription factor, when plant under high salt, low temperature and arid environment stress, the DREB transcription factor is by abduction delivering, then with DRE element specific combination, starting the downstream functional gene relevant with resistance expresses, regulate various biochemical reactions in the plant materials, plant is improved the resistance of environment-stress.The DREB transcription factor can activate a series of resistant genes with DRE cis-acting elements, as rd29A, and corl5a, erdl0, kin1, rd17, rd22 etc.These expression of gene products are being brought into play different functions in the plant stress-resistance reaction, thereby make the resistance of plant obtain to improve (Jaglo-ottosen etc., 1998; Liu etc., 1998; Gilmour etc., 2000; Jaglo-Ottosen etc., 2001; Park etc., 2001; Dubouzet etc., 2003; Kasuga etc., 2004).Therefore, DREB serves as the role of " master switch " in the adverse circumstance answering of plant: the overexpression of a transcription factor can impel a plurality of functional genes to play a role, and makes the plant resistance of reverse obtain comprehensive improvement.Degeneration-resistant associated transcription factor DREB and cell development (Drews, 1991), hormone (Ohme, 1995), disease-resistant (Zhou, 1997), anti-low temperature (Kasuga, 1999) and arid, high salt (Liu, the 1998) signal of etc.ing transmit relevantly, in the range gene expression, play important regulating and controlling effect.Dreb gene is imported in the Arabidopis thaliana by the Agrobacterium-mediated Transformation method from Stockinger (1997), now obtained many degeneration-resistant commentaries on classics dreb gene plants.

Application number is that the patent of invention of 200710008719.X " utilizes the pmi gene to obtain the method for transgenic sugarcane fast ", introduced and a kind ofly contained the plant expression vector of pmi gene, utilized seminose to obtain the method for transgenic plant as selective agent, dichlorophenol sulfonphthalein method rapid detection resistant plant by structure.

The present invention is exactly at above background technology, the plant expression vector that contains the DREB2B gene by structure, utilize the method for particle gun transduction, by screening of Totomycin (hygromycin) or seminose and transgenosis detection technique fast and efficiently, increase substantially sugarcane drought resisting breeding efficiency, for the good technique basis is established in the cultivation of sugarcane drought-resistant variety.

Summary of the invention

The purpose of this invention is to provide a kind of DREB2B of utilization gene and cultivate the method for drought resisting sugar cane breed.The technical problem that is solved is a kind of method of utilizing the drought resisting of transgenic technology improvement sugarcane.Specifically, utilization has the carrier of Totomycin or phosphomannose isomerase selection markers, structure contains the plant expression vector of DREB2B gene, utilize the particle gun transduction to be inserted in the genome of sugar cane breed good fortune farming 95-1702, obtain the resistant transgenic sugarcane through transgenosis screening system and molecular detection technology.

The present invention utilizes the DREB2B gene to cultivate the method for drought resisting sugar cane breed, the cultivation of DREB2B genetic material and the Screening and Identification of drought resisting transgenic sugarcane novel material are changeed in the structure, the drought resisting that comprise the plant expression vector that contains the DREB2B gene, it is characterized in that may further comprise the steps:

1, make up plant expression vector, described plant expression vector is pRD29A-DREB-Hyg and pRD29A-DREB-pmi; Described pRD29A-DREB-Hyg is to be goal gene with DREB2B, and RD29A is a promotor, and Nos-PolyA is a terminator, and Hyg is the plant expression vector of selection markers gene; Described pRD29A-DREB-pmi is to be goal gene with DREB2B, and RD29A is a promotor, and Nos-PolyA is a terminator, and pmi is the plant expression vector of selection markers gene;

2, the method by the particle gun transduction changes the above-mentioned carrier that contains pRD29A-DREB-Hyg and pRD29A-DREB-pmi in the sugarcane explant over to;

3, through in the substratum that contains Totomycin or seminose subculture, the acquisition resistant plant breaks up, takes root; The described substratum that contains Totomycin or seminose promptly adds 10～50gL respectively in subculture medium, division culture medium and root media ^-1Totomycin, the seminose that perhaps adds 20～70% (w/v) substitutes sucrose; Described succeeding transfer culture based component is: MS+2,4-D 2.0mgL ^-1, regulate pH5.8; Described differentiation culture based component is: MS+6-BA 1.0mgL ^-1+ NAA 0.1mgL ^-1+ KT2.0mgL ^-1+ sucrose 30gL ^-1+ agar 7gL ^-1, regulate pH5.8; Described short root medium component is: 1/2MS+NAA 3.0mgL ^-1+ 6-BA 0.1mgL ^-1+ sucrose 40gL ^-1, regulate pH5.8.

4, detect the sugarcane plant that obtains to change the DREB2B gene by PCR, southern dot-blotting.

According to the commentaries on classics DREB2B gene drought-resistant variety that above-mentioned method of cultivation obtains, its drought resistance, photosynthetic capacity, output, sugar part all are better than sugarcane acceptor kind good fortune farming 95-1702 and group training offspring thereof.Acquired commentaries on classics DREB2B gene sugar cane breed is respectively DR ₄, DR ₁₃, DR ₁₅And DR ₄₄

Transgenic sugarcane kind of the present invention has the following advantages: sugarcane is induced the expression of anti-drought gene under drought stress, its drought resistance obviously is better than the acceptor kind, and kept stable between different asexual generation; Under drought condition, growth is faster than the acceptor kind, and plant height increases more than 20%, and number of productive tiller increases more than 30%, and the per hectare cane yield increases by 30%, and sucrose part (absolute value) is improved more than 1.5%.

Embodiment

Embodiment is in order to explain that in more detail the present invention utilizes the DREB2B gene to cultivate the method for drought-resistant sugarcane variety, but the present invention is not limited to this.

The key instrument equipment that the present invention is used:

(1) AB204N electronic analytical balance: Switzerland Mei Tele company;

(2) EPS 301 electrophoresis apparatuses: peace Pharmacia company;

(3) Ultrospec 2100 type nucleic acid-protein determinators: Sweden APBiotech company;

(4) Biohazard Safety Equipment: Shanghai Li Xin company limited;

(5) particle gun (PDS-1000He): U.S. Bio-Rad company;

(6) Master Cyclergradient 96 PCR instrument: German Eppendorf company;

(7) BIO-PROFIL gel imaging and analytical system: French VL company;

(8) Model 400 types hybridization case: the Robbin company of the U.S.;

(9) the UV-crosslinked instrument of UVC500: U.S. Hoefer company;

(10) 5810R type desk type high speed refrigerated centrifuge: German Eppendorf company;

(11) RC-5C PLUS flooring high speed freezing centrifuge: U.S. Kendro company;

(12) traditional vacuum concentration systems: German Eppendorf company.

Restriction endonuclease commonly used and test kit:

Restriction enzyme, Access RT-PCR System test kit are available from Promega company;

M-MLV reversed transcriptive enzyme, Trizol test kit are available from Gibico-BRL company;

The Southern hybridization kit is available from Clontech company;

The PCR fragment reclaims test kit, Ex Tag archaeal dna polymerase available from precious biological (Dalian) company;

Primer, examining order are given birth to worker Engineering Co., Ltd by last marine life and are finished.

Chemical reagent commonly used:

Nylon membrane is available from Millipore company;

RNaseA is available from Beijing Huamei Bio-Engrg Co.;

CaCl ₂2H ₂O (C-7902), spermidine (S-0266) are available from Sigma company

IPTG, X-Gal, etc. available from precious biotechnology (Dalian) company limited;

Microbiotic such as Totomycin, seminose is glad through Bioisystech Co., Ltd of section available from Beijing;

DNA Marker, dNTPs mixture, DEPC are available from Shanghai biotechnology company limited;

Other reagent is given birth to worker biotechnology company limited or homemade analytical reagent available from Shanghai.

Embodiment: a kind of method of utilizing the DREB2B gene to cultivate drought-resistant sugarcane variety may further comprise the steps:

One, the structure of DREB2B gene plant expression vector is changeed in drought resisting

With NcoI and SphI double digestion PC-hyg, the no terminator that downcuts is inserted in the TDREB carrier of using NcoI and SphI double digestion, obtain the TDREB-NOS intermediate carrier.With EcoRI and SacI double digestion pGreen-hyg, the 35s-hyg-polyA structural unit that downcuts is inserted in the T-Prd29a plasmid of using EcoRI and SacI double digestion, obtain the rd29a-hyg intermediate carrier.Use PstI and SphI double digestion TDREB-NOS intermediate carrier then, the DREB-Nos that downcuts is inserted in the rd29a-hyg intermediate carrier of using PstI and SphI double digestion, obtain the rd29a-dreb-hyg plant expression vector, identify this recombinant chou with PstI and the single double digestion of SphI.Related plasmid extracts in the operating process, enzyme is cut, reclaim, the enzyme of connection, cell transformation, recombinant plasmid is cut and identified and PCR is accredited as molecular biological ordinary method.

Two, be used for the acquisition of genetic transformation acceptor

Experimental cultivar is the new variety of sugarcane good fortune farming 95-1702 that sugarcane institute of University Of Agriculture and Forestry In Fujian cultivates.Eugonic good fortune farming 95-1702 taper 30cm is chosen at first disinfection, removes its blade and top 5cm.Behind 70% alcohol surface sterilization, 2～3min, peel off its outermost layer on the Bechtop, again with peelling off time skin behind 70% alcohol surface sterilization, 1～2min, carefully cutting diameter then is 0.4～0.8cm, thick is the young leaves of 1～2mm, be inoculated on the inducing culture, cultivate in the darkroom, culture temperature is (25 ± 1 ℃).Subculture was once in subculture medium in per 15 days.Choose well-grown, be rich in gloss, the color cadmium yellow, the embryo callus subculture of tissue block particle densification is the acceptor material of genetic transformation.

Three, the cultivation of DREB2B genetic material is changeed in drought resisting

(1) acceptor material pre-treatment: elder generation changes acceptor material in the subculture medium pre-2d of cultivation before the particle gun bombardment, changes over to then to contain permeate agent 0.2molL ^-1N.F,USP MANNITOL and 0.2molL ^-1Pre-treatment 4～6h in the subculture medium of sorbyl alcohol.

(2) particle gun bombardment: the preprepared little culture dish that callus is housed is put on the pallet of particle gun chamber, closed the particle gun door, utilize air pressure 1100psi and shooting distance 6cm directly to bombard the sugarcane embryo callus.

(3) transition is cultivated: the callus after the bombardment is still put back to pre-treatment substratum (MS+2,4-D2mgL ^-1+ Sorbitol 0.2molL ^-1+ Mannitol 0.2molL ^-1) go up and continue to handle 18h, insert MS+2 thereafter, 4-D 2mgL ^-1Substratum on 25 ℃ of dark recoveries cultivate 5～8d.

(4) screening succeeding transfer culture: the callus after the recovery is transferred to subculture screening in the subculture medium, and the 15d subculture once.Subculture screening and culturing based component is: substratum MS+2,4-D 2.0mgL ^-1+ 50mgL ^-1Totomycin or 40% seminose substitute the substratum of sucrose, regulate pH5.8.

(5) screening differentiation culture: the callus of still surviving behind the selection subculture secondary, switching goes into to contain Totomycin (35gL ^-1) or the 30% seminose division culture medium that substitutes sucrose screen differentiation.Screening differentiation culture based component is: MS+6-BA 1.0mgL ^-1+ NAA 0.1mgL ^-1+ KT 2.0mgL ^-1+ sucrose 30gL ^-1+ agar 7gL ^-1+ 35mgL ^-1Totomycin (35gL ^-1) or 30% seminose substitute sucrose, regulate pH5.8.

(6) the short root of screening is cultivated: resistance seedling length changes over to and contains Totomycin (30gL behind 2-4cm ^-1) or 30% seminose substitute screening and root culture on the root media of sucrose.The composition of root media is: 1/2MS+NAA 3.0mgL ^-1+ 6-BA 0.1mgL ^-1+ sucrose 40gL ^-1+ Totomycin 30gL ^-1Or 30% seminose substitutes sucrose, adjusting pH5.8.

(7) hardening and transplanting: when the root of seedling grows to 2.5cm length, move on to outdoor hardening 2d, then seedling is taken out, clean the substratum that adheres to flowing water, be transplanted in the small flower (plant ash: sand: mold=1:1:1 mixes the back autoclaving) regularly spray water the MS inorganic salt liquid of 10 times of dilutions.

(8) Molecular Detection of transfer-gen plant: design the upstream and downstream special primer respectively according to promotor RD29A gene of being transduceed and DREB2B gene, carry out PCR, Southern respectively, transfer-gen plant is carried out the goal gene Molecular Detection.The test kit of the Roche DIG HighPrimer DNA Labeling and Detection Starter Kit I of company, the guide method that operating process provides in strict accordance with test kit are adopted in Southern hybridization.

Four, the Screening and Identification of drought resisting high yield and high sugar transgenic sugarcane novel material

According to national transgenosis security control regulations, the transfer-gen plant that obtains is reached drought resistance and growth, output and sugared part are carried out in contrast accordingly (plant and acceptor kind good fortune farming 95-1702 that the empty carrier bombardment obtains) respectively under potted plant water stress and grown in field condition Screening and Identification, in conjunction with the physiological and biochemical index of drought resisting, volume increase, screening and assessment transgenosis clone.In the transgenosis interim test stage, carry out greenhouse pot culture and land for growing field crops and carry out artificial drought stress evaluation; Carry out the plant evaluation of proterties of workers and peasants in the environmental release test stage; In the evaluation of the production test demonstration phase proterties of carrying out that identify in the larger area field and workers and peasants plant, screen drought resisting transgenic sugarcane new germ plasm material.

(1) drought resistance evaluation

● plant height injury rate detects

Adopt field identification method, 3 repetitions are established in the test site, and the sub-district area is not less than 33.3m ²Rainfall distribution and the damage caused by a drought long-term according to various places, when begin dry season, stem, the leaf speed of growth of per 5 days investigation 10 strain sugarcanes, continuous observation one month.When soil moisture content drops to critical wilting point, or the sugarcane leaf is in wilted condition, early morning when not having guttation all day, and two of test sites repeat to continue drought stress, and the irrigation that repeats to water is to keep normal growth.

● the detection of mda content

Adopt artificial water stress test.Test divides water stress treatment group and normal water supply control group, and every group every part strain is planted 20 strains, and quantitative water supply is to keep the consistence of soil moisture.The sugarcane elongation is during the initial stage, except that the normal water supply control group continues to supply water, the water stress treatment group begins to stop to supply water and handles, after cutting off the water when the unified contrast in the whole nation kind of (ROC10) sugarcane leaf relative water content descend 10～20% or the sugarcane leaf be in wilted condition all day, when not having " guttation " early morning, every 2 days determination and analysis mda contents, totally four times.

Get+5 on 1 leaf from the sugarcane strain, take back the laboratory rapidly, to remove the surface contamination thing, more once, blot the moisture on surface gently with clean gauze with deionized water rinsing with tap water rinsing blade.Take by weighing sugarcane blade 0.5g, add 5mL 10% TCA (Tricholroacetic Acid) solution and grind to form homogenate, the centrifugal 10min of 3000rpm, get supernatant 2mL, add equal-volume 0.67% TBA (thiobarbituricacid), react 20min in the boiling water bath, the cooling back is centrifugal, and supernatant liquor is measured light absorption value at 532nm and 600nm place respectively.

In the formula: ε=155L/mmoLcm; B is the cuvette light path; V1 is for adding the volume of extracting solution; V2 is for drawing the volume of supernatant.

● the detection of membrane permeability relatively

Get+5 on 1 leaf from the sugarcane strain of above-mentioned drought stress, take back the laboratory rapidly, to remove the surface contamination thing, more once, blot the moisture on surface gently with clean gauze with deionized water rinsing with tap water rinsing blade.With the aperture is that the cork drill of 1cm is beaten and got disk (avoiding middle arteries and veins); Each handles two groups of divide A and Bs, and every group is repeated every part of 1.0g 3 times; Put into the glass small beaker of numbering, material is pushed down gently, add the 20mL deionized water immediately, sample is immersed in the water fully with the little glass stick of band+prefix.Be positioned in the vacuum drier in the first group sample sets, bleed repeatedly with vacuum pump, vacuum pressure is 0.04～0.05MPa, exits 3～4 times, remove anhydrate and blade surface between air, make blade contact tight with water and ionogen is easy to ooze out.Can recover normal pressure behind the decompression infiltration 30min, between 26～30 ℃, be incubated 2h; After covering glass sphere, second group sample in the beaker directly puts the 15min that heats on the boiling water bath, kill tissues.After first group sample insulation back and second group sample kill cooling, in the small beaker of the outer sepage difference of tissue impouring cleaning, measure specific conductivity (μ Ω/cm) with conductivitimeter.

● the drought resistance evaluation:

Drought-resistant variety requires mda content to be lower than 7.0 μ molg under water stress 30 days or potted plant moderate water stress ^-1FW, membrane permeability is lower than 30%, and plant height injury rate is lower than 30%

● the result: the drought resistance of transgenic sugarcane is apparently higher than the acceptor kind, and it is under the moderate water stress, and blade mda (MDA) content is at 3.5～5.5 μ molg ^-1FW, membrane permeability are 5～24%, and plant height injury rate is all less than 30%.

(2) growth and workers and peasants' character screening of planting:

Field sugarcane seedling rate reaches after 50%, institutes an inquiry seedling rate, investigates once weekly, until stable; Sugarcane production speed investigation is tillered and is begun after finishing, and investigation was once up to November in per 30 days.At the sugarcane technical maturity, measure its hammer degree, plant height, stem footpath and calculate the effective diameter number; Sugar part analysis graded every 15 days sampling analysis sucrose parts, sugarcane juice purity, hammer degree, fibers in mid-November.

Claims (4)

1, a kind of method of utilizing the DREB2B gene to cultivate the drought resisting sugar cane breed, comprise the structure that contains DREB2B gene plant expression vector, the cultivation of drought resisting commentaries on classics DREB2B genetic material and the Screening and Identification of drought resisting transgenic sugarcane novel material, it is characterized in that may further comprise the steps:

(1) make up plant expression vector, described plant expression vector is pRD29A-DREB-Hyg and pRD29A-DREB-pmi;

(2) method by the particle gun transduction changes pRD29A-DREB-Hyg and pRD29A-DREB-pmi in the sugarcane explant over to;

(3) through in containing Totomycin or seminose substratum subculture, the acquisition resistant plant breaks up, takes root;

(4) detect the sugarcane plant that obtains to change the DREB2B gene by PCR, southern dot-blotting.

2, a kind of method of utilizing the DREB2B gene to cultivate the drought resisting sugar cane breed according to claim 1, it is characterized in that described pRD29A-DREB-Hyg is is goal gene with DREB2B, RD29A is a promotor, Nos-PolyA is a terminator, and Hyg is the plant expression vector of selection markers gene; Described pRD29A-DREB-pmi is to be goal gene with DREB2B, and RD29A is a promotor, and Nos-PolyA is a terminator, and pmi is the plant expression vector of selection markers gene.

3, a kind of method of utilizing the DREB2B gene to cultivate the drought resisting sugar cane breed according to claim 1 and 2 is characterized in that the described Totomycin substratum that contains is to add 10～50gL respectively in subculture medium, division culture medium and root media ^-1Totomycin.

4, a kind of method of utilizing the DREB2B gene to cultivate the drought resisting sugar cane breed according to claim 1 and 2 is characterized in that the described seminose substratum that contains is to add 20～70% the alternative sucrose of seminose respectively in subculture medium, division culture medium and root media.