CN102604988B - Transmethylase gene in betaine synthesis process and application thereof - Google Patents

Transmethylase gene in betaine synthesis process and application thereof Download PDF

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CN102604988B
CN102604988B CN 201210104875 CN201210104875A CN102604988B CN 102604988 B CN102604988 B CN 102604988B CN 201210104875 CN201210104875 CN 201210104875 CN 201210104875 A CN201210104875 A CN 201210104875A CN 102604988 B CN102604988 B CN 102604988B
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glycine
apgsmt2
apdmt2
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CN102604988A (en
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张举仁
何影
李坤朋
何春梅
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Shandong University
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Abstract

The invention discloses a transmethylase gene in a betaine synthesis process, and modification and application thereof. The method comprises the following steps: cloning glycine sarcosine N-transmethylase gene (ApGSMT2) and dimethyl glycine N-transmethylase gene (ApDMT2) from aphanothece halophytica, substituting codons with lower occurrence possibility in higher plants in the ApGSMT2 and the ApDMT2 with higher plant preferred codons to generate a gene ApGSMT2d and a gene ApDMT2d, and constructing the serial genes, of which the coded products are respectively positioned in the chloroplast, by protein locating targeting sequence and transmethylase gene fusion. Glycine and sarcosine are used as the substrates, the methylation of the glycine is catalyzed, the methylation of the dimethyl glycine is catalyzed by ApDMT, and the glycinebetaine is synthesized under the concerted catalysis. The ApGSMT2/ApDMT2 or modifier genes thereof are synergically transferred into corn, wheat, cotton, soybean, tobacco and other crops, so high-concentration glycinebetaine is accumulated in the cells, thereby obviously enhancing the stress tolerance of the plant.

Description

Methyl transferase gene and utilization thereof in a kind of trimethyl-glycine route of synthesis
The application is dividing an application of publication number CN 101805744A application, the applying date of original application: 2009.09.09, and application number: 200910018647.6, denomination of invention: the methyl transferase gene in a kind of trimethyl-glycine route of synthesis and modification and utilization.
Technical field
The present invention disclose in a kind of cyanobacteria glycinebetaine route of synthesis two kinds of methyl transferase gene sequences with and the sequence of modifying factor and their encoded protein matter sequence respectively, and these two kinds of genes are used for the crop breeding for stress tolerance, belong to the genetically engineered field.Relate in particular to the clone and modify glycine sarkosine N-methyl transferase gene and N-methylsarcosine N-methyl transferase gene and the utilization that participates in the glycinebetaine biosynthetic pathway.
Background technology
In the natural synthetic plant of trimethyl-glycine; glycinebetaine is as a kind of osmotic protection material; can improve the resistance of plant to multiple abiotic stress (as saline and alkaline, arid, low temperature etc.), and closely related to the accumulating level of a certain resistance level of coercing and trimethyl-glycine.Genetically engineered studies show that, accumulates glycinebetaine by transgenic technology in plant and can improve its resistance.
At occurring in nature, the biosynthetic pathway of known glycinebetaine mainly contains two kinds, i.e. choline oxidative pathway and glycine methyl approach.
The choline oxidative pathway is substrate with the choline, by a step or the synthetic glycinebetaine of two-step oxidation reaction.This approach is widely distributed, by the choline dehydrogenase (CDH) and the betaine-aldehyde dehydrogenase catalysis that are present in microorganism, plant and the Mammals.Be present in the E.C. 1.1.99.1 in the microorganism, two successive reactions of energy catalysis synthesizing betaine.The catalysis glycinebetaine is synthetic successively for choline single oxygenase and betaine-aldehyde dehydrogenase in the higher plant of synthetic glycinebetaine.The enzyme gene of the synthetic glycinebetaine of existing a plurality of participation choline oxidation is cloned in succession.
The glycine methyl approach is a glycine through the continuous three step N-generation trimethyl-glycine that methylates, be by glycine sarkosine methyltransgerase (the glycine sarcosine methyltransferase that relies on S-adenosylmethionine (SAM), GSMT) and rely on the sarkosine N-methylsarcosine methyltransgerase of SAM (sarcosine dimethylglycine methyltransferase, SDMT) catalysis is finished respectively.From the eighties of last century later stage eighties, in some archeobacterias, eubacterium and extreme blue bacterium, found this approach successively, but understood seldom, only from a few species, clone so far and obtain genes involved.
The route of synthesis that with the choline is substrate is studied in multiple biology.Difference according to the choline mode of oxidizing in this approach relates to one or both enzymes.In higher plant, the research of trimethyl-glycine biosynthetic pathway mainly concentrates on chenopod, and research (McNeil et al., 1999,2001) is also arranged in Amaranthaceae and grass.In Chenopodiaceae and amaranthaceous plant, trimethyl-glycine is synthetic through the two-step oxidation reaction by choline, generates betaine aldehyde chloride by choline single oxygenase (CMO) catalysis earlier, and the latter generates trimethyl-glycine (Weretilnyk et al., 1989 under betaine-aldehyde dehydrogenase (BADH) catalysis; Akamoto, 2000).At present, CMO only finds in Chenopodiaceae and amaranthaceous plant, infers that other section plant may have different E.C. 1.1.99.1s.And the gene of coding BADH is cloned from spinach various plants such as (Weretilnyk and Hanson, 1990).BADH is not the enzyme of the synthetic special usefulness of glycinebetaine, it is identical with omega-amino-aldehyde dehydrogenase (a kind of in the polyamines metabolism extensively the enzyme of existence) (
Figure BDA0000152347860000011
Et al., 1994; Rathinasabapathi et al., 1994).
In intestinal bacteria, with the synthesis related reason bet operon coding of trimethyl-glycine (Andresen et al., 1988; Lamark et al., 1991): the betA CDH that encodes, the betB BADH that encodes, betT coding choline transport albumen, the betI modulin of encoding, similar in transition pathway from the choline to the trimethyl-glycine and the plant, be enzymatic by two kinds, be choline dehydrogenase (choline dehydrogenase, CDH) and BADH.CDH be a kind of film that relies on oxygen in conjunction with flavoprotein, not only can be oxidized to betaine aldehyde chloride by the catalysis choline, and can be with the oxidation of much at one speed catalysis betaine aldehyde chloride.BADH is the soluble proteins of a kind of NAD of depending on, and betaine aldehyde chloride is had very high affinity (Landfald and Strom, 1986).
In microorganism Arthrobacter globiformis (Arthrobacter globiformis) and nourishing Arthrobacter (A.pascens), it is E.C. 1.1.99.1 (choline oxidase that glycinebetaine synthetic only needs a kind of enzyme, COD) catalysis (Ikuta et al., 1977) produces H simultaneously 2O 2
The approach that with the glycine is the substrate synthesizing betaine at first is to have a liking for (Nyyssola et al., 2000 of finding among the microorganism Actinopolyspora halophila of salt and the Ectothiorhodospira halochloris, 2001 at two kinds of utmost points; Waditee et al., 2003).In these microorganisms, glycine is through the continuous three step N-generation glycinebetaines that methylate, the first step of reaction is by relying on S-adenosylmethionine (S-adenosylmethionine, SAM) glycine sarkosine methyltransgerase (glycine sarcosine methyltransferase, GSMT) catalytic, second step of reaction is by sarkosine N-methylsarcosine methyltransgerase (the sarcosine dimethylglycine methyltransferase of GSMT or dependence SAM, SDMT) catalysis, final step is by SDMT catalytic (Nyyssola et al., 2001; Waditee et al., 2003).
The biosynthetic genetically engineered research of trimethyl-glycine
Biochemical analysis to natural accumulation trimethyl-glycine organism shows that trimethyl-glycine is stable in plant materials, and the rate-limiting step of trimethyl-glycine accumulation is its synthetic rather than its degraded (Rhodes and Hanson, 1993).Along with people understand in depth trimethyl-glycine biosynthetic pathway molecular mechanism in plant and the microorganism, a large amount of trimethyl-glycine synthetic genes involveds are also cloned in succession.This provides the molecule foundation for setting up the trimethyl-glycine biosynthetic pathway by engineered means in the organism that does not accumulate trimethyl-glycine.The participation a plurality of genes of trimethyl-glycine synthetic such as COD, CDH, CMO, BADH etc. have changed into merit in various plants at present, and these transfer-gen plants have accumulated the trimethyl-glycine of different levels, have improved its resistance to multiple adverse circumstance.
In the higher plant, choline is converted into trimethyl-glycine by choline mono-oxygenase (CMO) catalysis, successively from spinach (Rathinasabapathi et al., 1997), beet (Russell et al., 1998) and prunella asiatica (Atriplex hortensis) (Shen Yiguo etc., 2001) be cloned into CMO in and carried out transgenic research, these genes possess signal peptide sequence, are used to guide ripe peptide chain to be positioned chloroplast stroma.Nuccio etc. (1998) change the CMO gene of spinach over to tobacco, and transgene product is positioned in the chloroplast(id), yet CMO is active and all very low (≤50nmol g of beet alkali content -1FW) (Nuccio et al., 2000).Yet when CMO expresses in tenuigenin, rather than when expressing in chloroplast(id), beet alkali content improves (430nmol g greatly in the transgene tobacco -1FW).McNeil etc. (2000) use 14The metabolic kinetics of choline in the choline of C mark (trimethyl-glycine synthesizes substrate) the follow transgene tobacco finds that choline is trimethyl-glycine synthetic principal element (McNeil et al., 2000) in the restriction chloroplast(id) by kytoplasm to the transhipment of chloroplast(id).Phosphorylethanolamine-N-methyltransgerase (PEAMT) is the key enzyme in the choline route of synthesis, and its catalysis phosphorylethanolamine generates the methylation reaction of phosphorylcholine.McNeil etc. (2001) with the BADH gene cotransformation of the PEAMT of spinach and CMO gene and beet to tobacco chloroplast, compare with single CMO genetic tobacco that changes, the free content of choline of changeing the polygene tobacco has improved 50 times, and beet alkali content has improved 30 times, is up to 1.8 μ mol g -1FW (McNeil et al., 2001).
From 1981 were separated to BADH from higher plant after, the research of relevant trimethyl-glycine synthetic enzyme had caused extensive concern very soon.From spinach (Weretilnyk and Hanson, 1990), beet (McCue and Hanson, 1992), Chinese sorghum (Wood et al., 1996), paddy rice (Nakamura et al., 1997), barley (Ishitani et al., 1995; Nakamura et al., 2001), be cloned into the BADH gene in prunella asiatica (Xiao Gang etc., 1995) and the prince's-feather (Legaria et al., 1998).In plants such as Chenopodiaceae, Amaranthaceae, BADH mainly is present in chloroplast stroma; And BADH mainly is present in the peroxisome in monocotyledonss such as paddy rice, barley.The difference location of BADH in cell has the performance that is beneficial to its enzymic activity.Plant BADH gene expression amount is very low under the normal condition, some data (Nakamura et al., 1997; Xiao Gang etc., 1995; Weretilnyk et al., 1989) show that the BADH expression of gene is subjected to Salt Stress-induced, but this reaction does not have salt inductive specificity, but to the reaction (Ishitani et al., 1995) of osmotic stress.
The research of changeing the BADH gene is more.Rathinasabapathi etc. (1994) will change tobacco at the BADH of spinach and beet gene the earliest, and transgene product is positioned in the chloroplast(id).The murder by poisoning that expression of exogenous gene render transgenic tobacco can be resisted betaine aldehyde chloride.When adding the 5mM betaine aldehyde chloride in substratum, the content of trimethyl-glycine has reached 20 μ mol g in the transgene tobacco -1FW.Yet when not adding betaine aldehyde chloride in substratum, transgene tobacco can not synthesizing betaine.Guo Yan etc. (1997) utilize particle bombardment with prunella asiatica BADH transgenosis in paddy rice, the transgenic paddy rice that filters out can be grown under salt stress and be set seeds.Kumar etc. (2004) have obtained to change the Radix Dauci Sativae of BADH gene, and beet alkali content is respectively 93-101 μ mol g -1DW has improved the salt tolerance of transfer-gen plant greatly.Yang etc. (2005,2007) will derive from the BADH gene transformation tobacco of spinach (Spinacia oleracea), transfer-gen plant can synthesize glycinebetaine, and the synthetic trimethyl-glycine mainly accumulates in chloroplast(id), and transfer-gen plant has improved the resistance to high temperature stress.They think that under the moderate high temperature stress photosynthetic the keeping of transgene tobacco is owing to the provide protection of trimethyl-glycine to the Rubisco activating enzymes; Under serious high temperature stress, transgene tobacco has alleviated high temperature stress to the active oxygen injury that photosynthetic mechanism causes by improving the function of antioxidase system in the body, has kept the higher PS II activity (Yang et al., 2007) of transgene tobacco.Existingly focus mostly in the conversion of individual gene about CMO and the engineered research of BADH, though the resistance of transgenic plant has raising in various degree, beet alkali content is very low.
Colibacillary CDH has the function of choline-monooxygenase (cmo) and betaine-aldehyde dehydrogenase concurrently, and this characteristic makes the genetically engineered that only shifts the betA gene can give the ability of plant accumulation glycinebetaine.1996, Lilius etc. changed the betA gene over to tobacco, and the salt tolerance of transgenic plant is compared with adjoining tree and is significantly improved, but they do not report the accumulating level of trimethyl-glycine.The betA gene that will comprise the plastosome signal for locating after Takabe etc. (1998) will modify imports paddy rice, and transgenic paddy rice can accumulate 5 μ mol g -1The trimethyl-glycine of FW, and its salt tolerant and drought resistance contrast be significantly improved.Wang Shufang etc. (2001) transfer to betA in the tomato, have improved the salt resistance of tomato.Under condition of salt stress, chlorophyll content of the Caulis et Folium Brassicae capitatae (Brassica oleracea var.capitata) of commentaries on classics betA gene and (Bhattacharya et al., 2004) that biomass all is significantly higher than the wild-type plant.We have obtained commentaries on classics betA gene corn and cotton by agrobacterium-mediated transformation in the laboratory, the resistance Physiological Analysis shows the cold-resistant and drought-resistant ability of transgenic corns (the Quan et al. that is significantly improved, 2004a, b), the drought resistance of transgene cotton also got tangible improvement the (Lv et al., 2007).
Synthetic of the intravital E.C. 1.1.99.1 of Arthrobacter (COD) catalysis trimethyl-glycine needs single enzyme, and therefore, the research of changeing the COD gene has more work.The COD enzyme is by codA (Arthrobactor globiformis) or cox (A.pascens) genes encoding, and using maximum at present is the codA gene.The codA gene has been changed over to Arabidopis thaliana (Hayashi et al., 1997,1998; Alia et al., 1998; Sakamoto et al., 2000), paddy rice (Akamoto et al., 1998; Mohanty et al., 2002) and in the tomato various plants such as (Park et al., 2004), the salt tolerant of transfer-gen plant (Hayashi et al., 1997,1998; Akamoto, 1998), cold-resistant (Park, 2004; Hayashi et al., 1997; Alia et al., 1998; Akamoto et al., 1998), heat-resisting (Alia et al., 1998) and freeze proof (Sakamoto et al., 2000) ability all is significantly increased.Express the COD gene that has the chloroplast(id) signal for locating in Arabidopis thaliana, trimethyl-glycine concentration is respectively 1.2 μ mol g in blade and the seed -1FW and 18 μ mol g -1DW (Hayashi et al., 1997), transfer-gen plant shows very strong salt tolerance.Coerce down freezing, the accumulation of GB in the transgenic arabidopsis plant significantly improved the survival rate of plant, and a lot of cool tone control gene (as cor6.6, cor15a, cor47 and cor78) expression level compare no significant difference (Sakamoto et al., 2000) with wild-type.In the conversion of paddy rice, COD is positioned chloroplast(id), similar in trimethyl-glycine concentration and the transgenic arabidopsis in the transgenic paddy rice blade; If COD is positioned in the kytoplasm, synthetic trimethyl-glycine concentration ratio COD is positioned the high 3-5 of plant times (Akamoto et al., 1998) of chloroplast(id).This may be because choline concentration (choline synthesize the site) in tenuigenin compares in chloroplast(id) (Mudd and Datko, 1989 due to the height; Alia et al., 1999).Yet the accumulation of trimethyl-glycine in chloroplast(id) but can better protect PS II complex body to avoid the injury of high salt and low temperature stress (Akamoto et al., 1998).Change codA gene tomato and can accumulate 0.3-1.2 μ mol g -1The FW trimethyl-glycine, transfer-gen plant all is improved significantly in the resistance to cold from seed germination to blocky each stage.Under cold stress conditions, the rate ratio wild-type of transgenic Fructus Lycopersici esculenti has improved 10-30% (Park et al., 2004).Owing to be accompanied by H in the process of COD catalysis choline generation trimethyl-glycine 2O 2Generation, and the transfer-gen plant body in H 2O 2The suitable raising of level has activated the expression of some oxygen scavenging activity mechanism as Vitamin C oxidase and catalase etc., and these enzymes can be with H 2O 2Change into H 2O, thus make H 2O 2Content maintains a level that lower pair cell is harmless.Simultaneously, transfer-gen plant activity in vivo oxygen removes that enzyme is active to be increased, and ability (Park et al., 2004) is coerced in the degeneration-resistant border that also helps improving plant.
Nyyssola etc. (2000) are respectively from two kinds of halophiles that greatly differ from each other in phylogenetics (Ectothiorhodospira halochloris and Actinopolyspora halophila, promptly have a liking for the salt aphanothece) in be separated to two catalysis trimethyl-glycine synthetic methyltransgerase GSMT (glycine sarkosine methyltransgerase) and SDMT (sarkosine N-methylsarcosine methyltransgerase), methyltransgerase sequence similarity on amino acid levels in two species is very high, and they all are that substrate passes through the synthetic glycinebetaine of two ground beetle glycosylation reactions with the glycine.Nyyssola etc. (2001) clone this two methyl transferase genes from E.halochloris, respectively called after EcGSMT and EcSDMT.Glycine N-methyltransgerase (GMT) catalysis glycine methyl changes into sarkosine in Mammals, and further methylation reaction does not take place.Lack homology (Waditee et al., 2003) between GMT in the Mammals and EcGSMT, EcSDMT and ApGSMT, the ApSDMT.2005, Waditee etc. arrive the gene cotransformation of GSMT that encodes among the A.halophila (ApGSMT) and SDMT (ApSDMT) in blue or green bacterium (Synechococcus sp.PCC 7942) of algae and the Arabidopis thaliana, discovery is changeed the GB concentration of PCC 7942 cells of ApGSMT/ApDMT gene up to 200mM under salt stress, content than the blue or green bacterium of the algae that changes bet (betA, betB) gene has improved 5 times, and the former can grow under the 600mMNaCl condition He in the seawater.The Arabidopis thaliana plant beet alkali content that changes ApGSMT/ApDMT significantly increases than the plant that changes the CMO gene, and supposition is owing to the substrate difference causes.In addition, GB has accumulation in the root that changes ApGSMT/ApDMT gene Arabidopis thaliana, stem, leaf and flower etc., and the salt tolerant of transfer-gen plant, cold-resistant comparing all with the Arabidopis thaliana of commentaries on classics CMO gene with drought resistance are significantly improved.Recently, Waditee etc. (2007) clone phosphoglycerate dehydrogenase gene (ApPGDH) from A.hanothece, and it is changed in intestinal bacteria E.coli and the Arabidopis thaliana.The result shows, no matter be in the intestinal bacteria of natural synthetic GB or can not the Arabidopis thaliana of self synthetic GB in, the heterogenous expression of ApPGDH has all improved the accumulating level of glycinebetaine.They are also with ApPGDH, ApGSMT and three genes of ApSDMT coexpression in Arabidopis thaliana, under 100mM NaCl condition, the beet alkali content of transgenic arabidopsis root is 1.5 times (Waditee et al., 2007) of changeing ApGSMT/ApSDMT, and accumulating level reaches 1.6 μ molg -1FW.These results show, will be that the GB route of synthesis of substrate is introduced in the plant of not synthesizing GB with the glycine, are expected to make plant to accumulate a large amount of GB.
How improving cell glycinebetaine accumulating level, thereby further to improve transgenic plant resistance be one of problem of very paying close attention in the plant science field.Carry out the genetically engineered of glycine methyl approach introducing and can utilize glycine abundant in the cell to be substrate, be expected to the glycinebetaine of accumulation high density in transfer-gen plant than choline.
Term of the present invention and substratum
" stem apex " is meant ten to tens microns shoot apical meristem (shoot meristemetic) and greatly to tens millimeters bud-end (shoot tip), " shoot apical meristem " is meant apical growth awl and contiguous leaf primordium thereof.
In the present invention, the Transformation Program of agrobacterium tumefaciens (Agrobacterium tumefaciens) mediation is basically with (Ishida etc. such as Ishida, 1996) report that (acetosyringone As) waits phenolic compound and neutral monose to add Syringylethanone in transfection liquid.
In the present invention, the used Agrobacterium of transfection contains improved Ti-plasmid, latter's portability or do not carry herbicide resistance gene or other selective agent resistant gene.
In the present invention, the plant resistance is expressed and given to used herbicide resistance gene or other resistant gene as long as enliven in transformed plant cells, the not restriction of the kind of the source of restriction gene, size, used promotor and institute's antiweed or other selective agent is not subjected to the restriction of agrobacterium strains yet.
In the present invention, minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic isoreactivity composition filtration sterilization adds behind medium sterilization.
In the present invention, selective agent such as weedicide sprays with neutral tap water dissolving back.
In the present invention, the general employing of Agrobacterium cultivation YEP substratum (yeast extract 10g/l, peptone 10g/l, NaCl 5g/l, pH 7.0, solid medium adds 15g/l agar) cultivate.
The minimum medium that cereal crop seed germination and embryo culture are adopted is modified MS medium: KNO 31900mg/l, NH 4NO 31650mg/l, CaCl 22H 2O 440mg/l, MgSO 47H 2O 370mg/l, KH 2PO 4H 2O 170mg/l, FeSO 47H 2O 27.8mg/l, ZnSO 47H 2O 10mg/l, MnSO 4H 2O 22mg/l, H 3BO 310mg/l, Na 2MoO 40.5mg/l, CuSO 45H 2O 0.05mg/l, CoCl 22H 2O 0.05mg/l, vitamin 10mg/l, pyridoxine hydrochloride 1mg/l, nicotinic acid 1mg/l, glycine 2mg/l, inositol 100mg/l, vitamin H 0.05mg/l, casein hydrolysate 250mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8-6.0.The kind of plant growth regulating substance and concentration are adjusted because of the genotype of cultivated material.
The minimum medium that leguminous crop seed germination and embryo culture are adopted is modified MS medium (the same) or improvement B5 medium.Medium component is planted in the back: KNO 32500mg/l, (NH 4) 2SO 4134mg/l, CaCl 22H 2O 150mg/l, MgSO 47H 2O 250mg/l, NaH 2PO 4H 2O 150mg/l, FeSO 47H 2O 27.8mg/l, ZnSO 47H 2O 2mg/l, MnSO 4H 2O 10mg/l, H 3BO 310mg/l, Na 2MoO 40.5mg/l, CuSO 45H 2O 0.05mg/l, CoCl 22H 2O 0.05mg/l, vitamin 10mg/l, pyridoxine hydrochloride 1mg/l, nicotinic acid 1mg/l, glycine 2mg/l, inositol 100mg/l, vitamin H 0.05mg/l, casein hydrolysate 250mg/l, sucrose 20g/l, agar powder 7g/l, pH 5.8-6.0.The kind of plant growth regulating substance and concentration are adjusted because of the genotype of cultivated material.
In the present invention, transplanted seedling waters 1/2 modified MS medium inorganic salt solution or 1/2 improvement B5 medium inorganic salt solution, pH 6.0-7.0 every other day.
Summary of the invention
The purpose of this invention is to provide methyl transferase gene and modification and utilization in a kind of trimethyl-glycine route of synthesis at prior art.
The clone of ApGSMT2 gene and ApDMT2 gene
The present invention extracts and has a liking for salt aphanothece (Aphanothece halophytica) genomic dna, conserved sequence design PCR primer according to mammals glycine N-methyltransgerase (GMT), clone ApGSMT2, ApDMT2 gene, make up plant expression vector, change over to and carry out the gene function checking in the tobacco.By detecting the glycinebetaine content of transgene tobacco, in conjunction with gene sequencing relatively, determined that ApGSMT2 and ApDMT2 gene have function, be respectively the homologous gene of ApGSMT and ApSDMT.Concrete steps are:
30ml bacterium liquid is packed in the 50ml centrifuge tube, and 4 ℃ of centrifugal 10min of 5000g collect thalline.Resuspended 2 times of STE (0.1M NaCl, 10mM Tris-HCl (pH 8.0), 1mM EDTA (pH 8.0)).In the 50ml centrifuge tube that fills thalline, add 20ml 2 * CTAB (100mmol/L Tris-HCl pH 8.0,1.4mol/L NaCl, 20mmol/L EDTA pH8.0,2%CTAB) damping fluid, mixing, incubation 90min in 65 ℃ of water-baths (adding 1% beta-mercaptoethanol before the preheating), the centre shakes up 2-3 time.Then centrifuge tube is taken out from water-bath, thing to be mixed adds equal-volume chloroform/primary isoamyl alcohol (24: 1) after being chilled to room temperature, puts upside down centrifuge tube gently several times, the centrifugal 10min of 10000rpm under the room temperature.Again supernatant is changed in the new 50ml centrifuge tube over to 0.8 times of volume isopropanol precipitating.Flocks changes over to and fills in the 70% alcoholic acid 7ml centrifuge tube washed twice.Discard ethanol, dry, 2ml TER (2ml TE+2 μ l 10mg/ml RNaseA) dissolving, 37 ℃ of water-bath hydrotropies.Add isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), put upside down gently, make its abundant mixing extracting 10min, 4 ℃ of centrifugal 10min of 12000rpm.Get supernatant, add isopyknic chloroform/primary isoamyl alcohol (24: 1), put upside down gently, make its abundant mixing extracting 10min, 4 ℃ of centrifugal 10min of 12000rpm.Get supernatant liquor, add 1/10 volume 3M sodium-acetate (PH 5.2), behind the mixing, add 2 times of dehydrated alcohols that volume is cold.After mixing leaves standstill a little while gently, tick DNA with the rifle head, behind 70% alcohol flushing 2 times, vacuum-drying.TE dissolving DNA according to the DNA that carries of institute amount adding 100-500ul.
Retrieval GenBank database is collected relevant methyl transferase gene, the corresponding protein sequence is analyzed design degenerated primer GSMT-F 5 ' ATGGCNAARA ARGTNGA 3 ' and GSMT-R 5 ' YTARTCYTTY TTNGC3 '; DMT-F 5 ' ATGACNAARG CNGAYGC 3 ' and DMT-R 5 ' YTANGGYTTR TGRAA 3 '.
Note: R:A/G; W:A/T; K:G/T; B:C/G/T; M:A/C; D:A/G/T; V:A/C/G;
H：A/C/T；N：A/C/G/T；Y：C/T；S：G/C.
To have a liking for salt aphanothece genomic dna is template, uses degenerated primer GSMT-F and GSMT-R respectively, DMT-F and DMT-R, and amplification obtains dna fragmentation.Response procedures is 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 45 ℃ of annealing 1min, 72 ℃ are extended 30s, circulate 35 times; 72 ℃ are extended 7min.Adopt Takara to reclaim Kit and reclaim the dna fragmentation that pcr amplification obtains, select for use
Figure BDA0000152347860000051
(Promega USA) clones the PCR fragment Easy Vector Systems Kit, send order-checking company to check order.
Sequencing result shows, has obtained two long nucleotide sequences for 798bp and 834bp.The sequence that obtains is analyzed, determined their two ORF that encode respectively.The protein sequence of two ORF sequence encodings is carried out the blast analysis, find them and report that the GSMT and the DMT sequence that derive from other kinds biology have very high similarity.They all contain feature motif (the Motif I of methyltransgerase further analysis revealed, Post I, Motif II, and MotifIII), tentatively inferring them is the newcomers that have a liking for salt aphanothece methyl transferase gene, respectively called after ApGSMT2, ApDMT2 gene.
The present invention clone's the nucleotide sequence of having a liking for salt aphanothece glycine sarkosine N-methyl transferase gene (ApGSMT2) is shown in sequence table SEQ ID NO.5.
The ApGSMT2 amino acid sequence coded is shown in sequence table SEQ ID NO.6.
The present invention clone's the nucleotide sequence of having a liking for salt aphanothece N-methylsarcosine N-methyl transferase gene (ApDMT2) is shown in sequence table SEQ ID NO.7.
The ApDMT2 amino acid sequence coded is shown in sequence table SEQ ID NO.8.
The modification of ApGSMT2 gene and ApDMT2 gene
The present invention can efficiently express in cereal crop in order to make the gene of having a liking for salt aphanothece (a kind of blue-green algae), we select for use the preference codon of cereal plants and corn to replace the codon of part A pGSMT2 and ApDMT2, ApGSMT2 called after ApGSMT2d after the modification, the ApDMT2 unnamed gene after the modification is ApDMT2d.ApGSMT2d and ApDMT2d gene synthetic, and through sequence verification.
The nucleotide sequence of ApGSMT2d is shown in sequence table SEQ ID NO.9.
The nucleotide sequence of ApDMT2d is shown in sequence table SEQ ID NO.10.
Methylase and chloroplast protein are led peptide sequence and are merged
The present invention can effectively improve the resistance of plant in order to make ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d gene in higher plant cell, from realizing that glycinebetaine is in intracellular compartmentation accumulation angle, ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d are merged with peptide (transit peptide) coding region of leading of location chloroplast protein respectively, ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d albumen are positioned in the chloroplast(id) after synthetic, promote a large amount of glycinebetaine of accumulation in the chloroplast(id).Clone Arabidopis thaliana the RUBISCO small ylidene gene lead peptide-coding region, respectively with ApGSMT2 and ApGSMT2d and ApDMT2 and ApDMT2d gene fusion, produce ApTpGSMT2 and ApTpGSMT2d and ApTpDMT2 and ApTpDMT2d gene.
The RUBISCO small ylidene gene of Arabidopis thaliana lead peptide-coding region shown in sequence table SEQ ID NO.11.
Its coding lead peptide shown in sequence table SEQ ID NO.12.
Fusion rotein ApTpGSMT2 that the present invention produces and ApTpGSMT2d aminoacid sequence are shown in sequence table SEQ ID NO.13.
Fusion rotein ApTpDMT2 that the present invention produces and the aminoacid sequence of ApTpDMT2d are shown in sequence table SEQ ID NO.14.
Clone's methylase gene and mitochondrial protein are led peptide sequence and are merged
Gene fusion construct of the present invention accumulates the purpose of the resistance that reaches further raising transgenic plant to realize glycinebetaine in plant mitochondria.Plastosome is the main place in cellular energy source as ATP synthetic place in the cell, realizes that trimethyl-glycine helps improving the resistance of transgenic plant in Intramitochondrial accumulation.As with corn citrate synthase gene lead peptide-coding sequence and ApGSMT2 and ApDMT2 gene fusion, produce ApTpGSMT2m and ApTpDMT2m gene.
The corn mitochondrial protein is led peptide sequence shown in sequence table SEQ ID NO.15.
The fusion rotein ApTpGSMT2m sequence that the present invention produces is shown in sequence table SEQ ID NO.16.
The fusion rotein ApTpDMT2m sequence that the present invention produces is shown in sequence table SEQ ID NO.17.
The structure of plant expression vector
The present invention is with ApGSMT2/ApDMT, ApTpGSMT2d/ApTpDMT2d, ApTpGSMT2m/ApTpDMT2m etc. respectively with higher plant promotor such as cauliflower mosaic virus (CAMV) 35S rna gene promotor, corn Ubiquitin gene promoter, Arabidopis thaliana RD29 promotor links to each other with gene 3 ' tail district, the fusion gene that formation efficiently expresses, and then construct and carry ApGSMT2 and ApDMT respectively, the plant expression vector of ApTpGSMT2d and ApTpDMT2d and ApTpGSMT2m and ApTpDMT2m (target gene is started by different promotors respectively, and various combination can be arranged).As utilize mini-Ti plasmid generation T-DNA district to be different plant expression vectors shown in Figure 1.Conventional Protocols in Molecular Biology is adopted in gene recombination.
Gene function is determined and the assessment of using value
The present invention is in order to determine whether ApGSMT2 and ApDMT2 have the catalytic methylation reactive activity, can the two coordinate expression improve the content of cell glycinebetaine, ApGSMT2 and ApDMT2 are recombinated on the prokaryotic expression carrier, change intestinal bacteria over to, measure the glycinebetaine content of transgenosis intestinal bacteria and wild-type e. coli.Glycinebetaine extracts Xing and Rajashekar (2001) method of adopting, and content is used 1H NMR measures (Bruker AM500 NMR spectrometer).The content of glycinebetaine is than (in the LB nutrient solution) about the transgenosis control cells does not double in e. coli bl21 (DE3) plysS of ApGSMT2 and ApDMT2 coexpression cell, in nutrient solution, increase 0.3-0.5mol/LNaCl, the glycinebetaine content ratio of transgenic cell not transgenosis contrast increases 30%-60%, transgenic cell shows the comparison illumination and shows the salt tolerance that improves, and growth velocity is significantly higher than contrast.Be that ApGSMT2 and ApDMT2 coexpression in intestinal bacteria can synthesize glycinebetaine in a large number, improve the salt tolerance of cell.
The present invention is an acceptor with the plant expression vector pCPE that contains CaMV 35S promoter, multiple clone site and plant anti-herbicide gene EPSPs (5-enolpyruvyl-shikimate-3-phosphate synthase gene), insert ApGSMT2 and ApDMT2 gene (starting), produce plasmid pCPE-ApGSMT2, pCPE-ApDMT2 and pCPE-ApGSMT2-ApDMT2 by the CaMV 35S promoter.These plasmids are changed over to respectively in the agrobacterium tumefaciens lba4404, transform the tobacco cell that can not synthesize glycinebetaine then, produce transfer-gen plant.In the blade of different transfer-gen plants, measure glycinebetaine content, determine whether transgenosis produces the product function.Genetic transformation and transfer-gen plant analytical procedure are: getting the tobacco aseptic seedling is material, adopts Ye Panfa to transform.(Monsanto screens on inducing culture USA) the transgenosis budlet, takes root on root media containing selective agent 4mg/L glyphosate.Adopt PCR and Southern hybridization analysis method to determine transfer-gen plant.Detect the expression level of transgenosis in tobacco leaf by RT-PCR (Reverse Transcription PCR) method, utilize 1Glycinebetaine content in the quantitative blade of H NMR assay method.The result shows, the tobacco of coexpression ApGSMT2/ApDMT2 not only has the transgenosis transcript than high abundance in the cell, and (strain is that 9 concentration are 0.1832 μ molg to have accumulated the glycinebetaine of higher concentration -1FW), be higher than the glycinebetaine content that changes the betA gene plant significantly.And transfer-gen plant and only contain ApGSMT2 or the transfer-gen plant of ApDMT2 gene does not detect glycinebetaine accumulation not.
ApGSMT2/ApDMT2 gene pairs tobacco plant grows and the influence of resistance in order detect to change, with the transfer-gen plant seed and not transgenosis adjoining tree planting seed in flowerpot, the variation that observation is grown.Transgenic tobacco plant does not have phenotypic difference under the normal growth condition.Yet, by detecting survival rate and the growth velocity of transgene tobacco leaf dish under high salt and osmotic stress condition, draw change the ApGSMT2/ApDMT2 gene tobacco compared with the control drought-and salt-tolerance increase substantially, the change of transgene tobacco drought-and salt-tolerance is because ApGSMT2/ApDMT2 genetic expression and glycinebetaine accumulation cause.
Can influence the reaction of transgenic tobacco plant in order to understand ApGSMT2/ApDMT2 genetic expression to drought stress, place the MS inorganic salt solution that contains 10% (W/V) PEG 6000 to carry out osmotic stress the transgene tobacco seedling and handle, variations such as the variation of detection blade relative water content and cell membrane damage.Drawing tobacco plant blade relative water content in the osmotic stress process reduces gradually, cell membrane damage constantly increases the weight of, and the comparison of transfer-gen plant relative water content is low according to the plant changing down, cell membrane damage is obviously lighter, growth is very fast, shows that the tobacco plant drought tolerance of ApGSMT2/ApDMT2 genetic expression improves a lot.This gene has good using value in the crop resistance breeding.
Crop genetic transforms and transgenic line obtains
Select for use different transgenic methods to obtain transgenic plant according to transgenic acceptor.
Method for transformation commonly used has: 1) agriculture bacillus mediated genetic transforming method as by inflorescence dip method arabidopsis thaliana transformation, obtains positive transformed plant by dull and stereotyped antibiotic-screening and PCR checking and carries out subsequent analysis.2) direct conversion method promptly extracts recombinant plasmid dna, adopts particle gun blast technique, polyoxyethylene glycol revulsion, electro fusion method, silicon-carbon fibre method etc. directly with the plasmid DNA transfered cell.3) planting is conversion method, comprises pollen tube passage method, ovary injection etc.In addition, also has the Transformation Program that the inhomogeneity method is used in combination, as particle gun blast technique and Agrobacterium are used in combination to improve transformation efficiency.The present invention is to be that material carries out genetic transformation with crops such as corn, wheat, jowar, soybean, cottons, and the method for transformation of employing is mainly agrobacterium-mediated transformation and particle gun blast technique.
It is example summary testing sequence and method that the present invention obtains resistant strain with the agrobacterium-mediated transformation transformed wheat:
Wheat seed soaks 7-10min with 0.1% mercuric chloride solution again with 70% alcohol immersion 1-3min, then with sterilized water washing 5 times.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.The sterilization seed is placed on to fill with sterilized water and soaks in the Cans of filter paper, is placed under the dark condition (24 ± 2 ℃) 1-2 days after sealing.Treat seed sprout grow radicle and plumule after, place it on the M1 substratum in dark condition (24 ± 2 ℃) and continue down to cultivate.When treating that plumule is stretched to 2-5cm, peel off coleoptile and 1-2 sheet spire, expose the point that sprouts with scalper, again with scalper from the middle part vertical cut wound shoot tip meristem, cut primary root.The aseptic seedling of cut wound shoot tip meristem is used for Agrobacterium-mediated Transformation.
To have agrobacterium tumefaciens lba4404 28 ℃ of concussion cultivations down in additional antibiotic YEP substratum of purpose plasmid, concussion speed is 180rpm, makes bacterium be in logarithmic phase.Under 3000rpm centrifugal 5 minutes then, abandon supernatant.(acetosyringone, MS liquid nutrient medium AS) is resuspended with adding the 100mg/l Syringylethanone with thalline again.Then bacterium liquid is poured in the culture dish of diameter 15cm, the inclination culture dish makes to be immersed in the bacterium liquid by the aseptic seedling stem end of cut wound, vacuumizes (0.05MPa) and infects 7～10min.
Stem apex after the dip-dye blots with aseptic filter paper, seedling is inserted wheat germination substratum (MS substratum inorganic salt composition, N vitamin (Li Shirun etc., 1990), sucrose 30g/L, agar 7g/L, pH 5.8-6.0) upward cultivated altogether 3 days in the dark.Culture temperature is 22-24 ℃.Aseptic seedling after will cultivating altogether after 3 days is transplanted in the flowerpot of upper strata vermiculite lower soil, and covers the plant top with loose moistening vermiculite.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, temperature 15-21 ℃ of night.Water the inorganic salt solution of 1/2MS substratum every other day.
When being transplanted to transformed plant length in the vermiculite to tri-leaf period, the weedicide aqueous solution that evenly sprays suitable concn screens.Sprinkling is fallen drop with plant leaf and is advisable.Spray the back plant and be placed on the greenhouse growth, day temperature 15-20 ℃, about 10 ℃ of nocturnal temperatures about day illumination 12h, watered 1 time in per 3 days.Period of seedling establishment is got leaf DNA and is carried out the PCR detection.
With the epsps gene is the transformed plant sprinkling 0.12%Roundup aqueous solution of selection markers gene, promptly adds 1.2ml " Roundup " (41% glyphosate isopropyl amine salt) in every liter of tap water.Spray back about 15 days not the transgenosis adjoining tree begin death, the adjoining tree mortality ratio is about 90% after 25 days.Transformed plant is behind the spraying herbicide aqueous solution, and some individual variations are similar with adjoining tree, and other individualities then continue growth, change not obvious.The survival plant is long is transplanted to the field to the 4-5 leaf during phase.
Transformed plant (T0) selfing of antiweed screening produces T1 for seed, and the latter sprouts in flowerpot, and seedling is long to 3-4 leaf spraying herbicide aqueous solution during the phase, and weedicide concentration and plant strain growth condition are with T0 generation.The survival plant grows to draw materials behind 3 young leaves and carries out Molecular Detection.PCR detects positive plant and partly is transplanted into flowerpot, every strain one basin; Part is transplanted to the land for growing field crops, manages according to the Routine Management method, survives the winter naturally, gets leaf DNA after turning green and carries out Molecular Detection.Individual plant is isolated pollination self and is got T2 for seed.At field seeding, survive the winter naturally by seedling for seed for part T2, gets young leaves after turning green and carry out PCR detection, Southern hybridization and sxemiquantitative RT-PCR checking.The seed of transgenosis homozygous lines is used for salt tolerant, drought-enduring physiological is measured.
The resistance of transfer-gen plant detects
The present invention detects and is utilized as the resistance detection of example summary transfer-gen plant and general procedure and the method for utilizing with the salt tolerant of transgenic wheat.
With the non-transgenic plant is contrast, selects to carry out resistance from the transgenosis homozygous lines of the independent transformant of difference and measures.In seed germination test, plant in sand basin of the same size after seed dried, pouring contains the aqueous solution of different concns NaCl (0,100mM, 200mM, 300mM) respectively, and each strain is 300 seeds of every concentration kind.Be consistent between each basin of irrigation amount, observe the reguarity of seed sprouting situation and seedling every day, and avoid drenching with rain.The sand that statistics seed final seedling rate after 20 days, coleoptile expose covering is promptly thought and is sprouted.
According to the mensuration of seed bud ratio, select the salt concn during suitable concn NaCl solution is handled as salt stress.Choose respectively from the transgenosis homozygous lines and non-transgenic contrast the carrying out salt tolerant physiology of the independent transformant of difference and measure.With each strain is planting seed (diameter 10cm, high 15cm) in sand basin of the same size, 20 of every basin kinds, and each strain system kind of 8 basin waters the Hoagland nutritive medium every day, and plant is long to the final singling during phase of 3 leaves, stays seedling 15 strains of big or small growing way unanimity in each basin.Plant is long during the phase, to be divided into two groups with husky basin by each strain system to 5 leaves at random.Wherein one group begins to water the Hoagland nutritive medium that contains NaCl, continuous pouring 10 days, and another group continues pouring Hoagland nutritive medium as the untreated control group.In the salt stress treating processes, measure glycinebetaine content and other physical signs of handling the plant and the plant of being untreated and comprise photosynthesis index, ion content, RWC, chlorophyll content, cytosol gesture, soluble sugar and proline content, cytolemma ion seepage, mda content and biomass.
The wheat strain system of isozygotying in saltings plantation transgenosis carries out the salt tolerance analysis, and can the research transgenosis improve the mechanism of wheat salt tolerance in the time of infertility and salt tolerance raising.Winter wheat to the critical porion of salt stress be respectively emerge and the seedling stage and early spring period of seedling establishment, the salt tolerance power finally shows the economic yield in the saltings.
Measurement result shows that the seed of transgenic line germination rate under condition of salt stress is significantly higher than wild-type, and the seedling growth better.Wherein the part strain ties up to 200mM NaCl concentration and handles down that germination rate is about 95%, is about 1.5 times of wild-type, and seedling injury is lighter.5 leaf phase seedlings are carried out salt stress handle, NaCl concentration with every day 50mM be incremented to final concentration 200mM, then pouring every day once, salt concn is constant.After salt stress was handled, the transfer-gen plant growing way obviously was better than adjoining tree, and the over-ground part biomass comparison of handling first quarter moon rear section transfer-gen plant is high more than 30% according to plant, and the root biomass is also compared according to plant high by about 30%.
Measure under the condition of salt stress not that homophyletic is the variation of the physiological parameter of seedling, no matter draw transfer-gen plant or adjoining tree, the Na in blade and the root +Content all obviously raises, K +Content slightly raises earlier and then reduces.Compare transfer-gen plant Na with adjoining tree +Increasing amount is few, K +The reduction amplitude is little, the Na of part strain system +, K +All difference is remarkable compared with the control for content, and K +/ Na +Ratio also is significantly higher than adjoining tree.Under condition of salt stress, the chlorophyll content of all strains system all is to have earlier to raise by a small margin to descend with different rates then, but the chlorophyll content of transfer-gen plant is significantly higher than adjoining tree handling the after date phase.Photosynthetic rate, stomatal conductance, intercellular CO 2The measurement result of concentration and PS II photochemistry efficient shows that under long period NaCl coerced processing, photosynthetic rate reduced significantly because but stomatal conductance reduction and PS II complex body activity are subjected to cause jointly.But in salt stress was handled, transfer-gen plant showed obviously higher photosynthetic rate, stomatal conductance and maximum photochemistry efficient (Fv/Fm), promptly can synthesize more carbohydrate in salt stress is handled.
Under the condition of salt stress, the leaf cell solute potential reduces gradually along with the increase of the time of coercing, but the solute potential of part transgenic line significantly is lower than adjoining tree, promptly has the ability of stronger water conservation and absorption moisture.Compare with adjoining tree, rotaring gene plant blade contains more soluble sugar and proline(Pro).These results show, accumulated more soluble substance in the transgenic line cell and (comprise Na under condition of salt stress +With proline(Pro) and soluble sugar etc.), make cell keep lower solute potential, help the growth of cell eubolism and photosynthesis and plant.Under condition of salt stress, transfer-gen plant shows lower MDA level and ion percolation ratio, show that the damaged membrane degree is less than adjoining tree, the higher glycinebetaine level of accumulation is remarkable negative correlation in this and the transfer-gen plant, has higher PS II activity consistent with transfer-gen plant.The stability and the biomacromolecule function that show the excess accumulation cell membrane of glycinebetaine in transfer-gen plant have produced useful provide protection.
Show at the transgenic line of beach saline land plantation and the test-results of check variety, the survival and grow of some transgenic lines in the saltings obviously is better than check variety, economic characters significantly are better than check variety as individual plant tiller number, boot leaf area, single plant yield etc., show the salt tolerance that significantly improves.Promptly change the salt tolerance that glycine sarkosine N-methyl transferase gene (ApGSMT2) and N-methylsarcosine N-methyl transferase gene (ApDMT2) wheat are significantly increased.
Beneficial effect of the present invention
The invention discloses the glycine sarkosine N-methyl transferase gene (ApGSMT2) in a kind of cyanobacteria glycinebetaine route of synthesis and the coded protein sequence of N-methylsarcosine N-methyl transferase gene (ApDMT2) sequence and supposition.These two gene sources are in having a liking for salt aphanothece (Aphanothece halophytica), after merging with higher plant constitutive promoter (cauliflower mosaic virus (CAMV) 35SRNA gene promoter, corn Ubiquitin gene promoter), change over to jointly in the crops such as corn, wheat, cotton, tobacco, significantly improved degeneration-resistant (drought, salt, cold, high temperature) property of plant, shown that these two genes have good using value in the crop breeding for stress tolerance.
The present invention is based on clone's ApGSMT2 and ApDMT2 gene, needs according to the crop breeding for stress tolerance, gene order is modified, (1) replaces the low codon of frequency of utilization in higher plant with the codon of cereal plants and corn preference, synthesized ApGSMT2d and ApDMT2d gene; (2) protein is merged respectively at the coding region of chloroplast(id) positioning sequence and ApGSMT2 and ApDMT2 gene, ApGSMT2d and ApDMT2d gene, produce ApTpGSMT2 and ApTpDMT2 gene, ApTpGSMT2d and ApTpDMT2d gene, realized the location of transgene expression product in chloroplast(id); (3) protein is merged respectively at the coding region of mitochondrial matrix positioning sequence and ApGSMT2 and ApDMT2 gene, ApGSMT2d and ApDMT2d gene, produce ApTpGSMT2m and ApTpDMT2m gene, ApTpGSMT2dm and ApTpDMT2dm gene, realized that the transgene expression product is in Intramitochondrial location; (4), the location and the high local concentrations accumulation of glycinebetaine under the stress-inducing condition of transgene expression product different zones in cell have been obtained by the application of polygene conversion carrier and the application of transgenosis polymerization process.Like this, transgenosis breeding for stress tolerance novel material or the new variety that can recombinate out various as required.
Description of drawings
The T-DNA district collection of illustrative plates of Fig. 1, plasmid pCUA-APGSMT2-ApDMT2-epsp
P35S promoter, the cauliflower mosaic virus 35S promoter; PUbi, corn ubiquitin ubiquintin gene promoter; Epsp, the selection markers gene; RB and LB, the left and right border of the T-DNA of Ti-plasmids; Tnos, the no terminator.
Embodiment
The creation of embodiment 1:ApGSMT2/ApDMT2 gene clone and the degeneration-resistant material of corn gene
1) ApGSMT2/ApDMT2 gene clone
Cultivation is had a liking for salt aphanothece (Aphanothece halophytica) to OD600=1.2, gets 30ml bacterium liquid and packs in the 50ml centrifuge tube centrifugal collection thalline into.With STE (0.1MNaCl, 10mMTris-HCl (pH 8.0), 1mMEDTA (pH8.0)) the resuspended thalline of liquid, centrifugal collection repeats 1 time.Add again 20ml 2 * CTAB (20mmol/LEDTApH 8.0 for 100mmol/L Tris-HCl pH8.0,1.4mol/LNaCl, 2%CTAB) damping fluid, mixing, incubation 90min in 65 ℃ of water-baths (preheating before add 1% beta-mercaptoethanol), the centre shakes up 2-3 time.Then centrifuge tube is taken out from water-bath, be cooled to and add equal-volume chloroform/primary isoamyl alcohol (24: 1) after the room temperature, put upside down centrifuge tube gently several times, the centrifugal 10min of 10000rpm under the room temperature.Again supernatant is changed in the new 50ml centrifuge tube, with 0.8 times of volume isopropanol precipitating.Precipitation changes in 70% ethanol and washs 2 times, dries then, uses 2ml TER (2ml TE+2 μ l RNaseA (10mg/ml)) 37 ℃ of dissolvings.Add 2 times of dehydrated alcohols that volume is cold then.Mixing is ticked DNA with the rifle head after leaving standstill a little while gently, behind 70% alcohol flushing 2 times, and vacuum-drying.TE dissolving DNA according to the DNA that carries of institute amount adding 100-500ul.
Design degenerated primer GSMT-F 5 ' ATGGC (T/C/A/G) AA (A/G) AA (A/G) GT (T/C/A/G) GA 3 ' and GSMT-R 5 ' (C/T) TA (G/A) TC (C/T) TT (C/T) TT (C/T/G/A) GC 3 '; DMT-F 5 ' ATGAC (T/C/A/G) AA (A/G) GC (T/C/A/G) GA (T/C) GC 3 ' and DMT-R 5 ' (C/T) TA (T/C/A/G) GG (C/T) TT (G/A) TG (G/A) AA 3 '.With carried DNA is template, uses degenerated primer GSMT-F and GSMT-R respectively, DMT-F and DMT-R, and amplification obtains dna fragmentation.Response procedures is 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 1min, 45 ℃ of annealing 1min, 72 ℃ are extended 30s, circulate 35 times; 72 ℃ are extended 7min.Adopt Takara to reclaim Kit and reclaim the dna fragmentation that pcr amplification goes out, select for use
Figure BDA0000152347860000101
(Promega USA) clones the PCR fragment Easy Vector Systems Kit, the order-checking of order-checking company.Obtained the nucleotide sequence that length is respectively 798bp and 834bp, each own coding ORF.Based on the GSMT and the DMT sequence of other kinds biology of having reported, name institute clone gene is ApGSMT2, ApDMT2 gene.
2) structure of plant expression vector
Adopt conventional DNA recombinant technology, with the plant expression vector pCPE that contains CaMV 35S promoter, multiple clone site and plant anti-herbicide gene EPSPs (5-enolpyruvyl-shikimate-3-phosphate synthase gene) is acceptor, insert ApGSMT2 and ApDMT2 gene (starting) in the T-DNA district, produce plasmid pCPE-ApGSMT2-ApDMT2 by the CaMV 35S promoter.Adopt freeze-thaw method to change agrobacterium tumefaciens lba4404 or AGL0 etc. over to this plasmid, be used for corn gene.
3) foundation of transgene receptor system
With key selfing used in China's agriculture production is material, as Zheng 58, tuck in 478, tuck in 515 etc. self-mating system seed.Isolated culture induces stem apex to produce the sprout tuber that grows thickly, and is that acceptor carries out genetic transformation with the sprout tuber that grows thickly.Used substratum has:
Seed germination substratum: KNO 31900mg/l, NH 4NO 31650mg/l, CaCl 22H 2O 440mg/l, MgSO 47H 2O 370mg/l, KH 2PO 4H 2O 170mg/l, FeSO 47H 2O 27.8mg/l, ZnSO 47H 2O 10mg/l, MnSO 44H 2O 22.3mg/l, H 3BO 310mg/l, KI 0.83mg/l, Na 2MoO 42H 2O 0.5mg/l, CuSO 45H 2O 0.025mg/l, CoCl 26H 2O 0.025mg/l, vitamin 10.0mg/l, pyridoxine hydrochloride 1.0mg/l, nicotinic acid 1.0mg/l, glycine 2.0mg/l, inositol 100.0mg/l, vitamin H 0.05mg/l, casein hydrolysate 500mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8-6.0.Be used for seed germination.Liquid nutrient medium then removes agar powder.
The A substratum: the seed germination substratum adds 6-BA 4.5-9.0 μ mol/l and 2, and 4-D 1.0-3.0 μ mol/l is used to induce isolated culture bud point to produce grow thickly the bud tissue block and the bud tissue block succeeding transfer culture of growing thickly.
B substratum: additional 6-BA 4.5 μ mol/l of seed germination substratum and IBA (indolebutyric acid) 1.8 μ mol/l, the bud tissue block that is used to grow thickly differentiation seedling.
Become the seedling substratum: additional 6-BA 2.25 μ mol/l of seed germination substratum and IBA 3.6 μ mol/l, the budlet that is used to grow thickly develops into seedling.
Root media: the seed germination substratum adds IBA 2.8～3.6 μ mol/l, is used for the little seedling rooting of unrooted.
Minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic and weedicide isoreactivity composition filtration sterilization add behind medium sterilization.
Seed sterilization and sprouting: corn seed with 0.1% mercury chloride immersion 10-15 minute, washs 3-5 time with sterilized water more then with 70% alcohol immersion 10 minutes.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of (30-40 milliliter/250 milliliter triangular flask) sterilized water in the bottle, is placed under the dark condition (23-30 ℃) 1--2 days after sealing.(showing money or valuables one carries unintentionally) back seed of sprouting is placed on the minimum medium and continues to sprout down in dark condition.
Stem tip culture and grow thickly that the bud tissue block is induced, subculture, differentiation: when the plumule of germinating seed is stretched to 3-5 centimetre, peel off coleoptile and spire, cut the epicotyl and the stem apex that are about 5 millimeter, be inoculated into (24-27 ℃) cultivation under dark on the A substratum, and in time cut the plumular axis of elongation and peel off spire.Cultivate after 6-10 days, stem apex begins the irregular growth of expanding, and occurs several wartys or digitation on the meristematic tissue that expands.After 20 days, begin to form indefinite bud and embryoid on the surface of warty or digitation.General per 4 all succeeding transfer culture once.In succeeding transfer culture, budlet is on the high side 2 if the bud tissue block of growing thickly is grown thickly, and 4-D concentration is got 3.0 μ mol/l; Bud tissue block callusization is heavier if grow thickly, though a large amount of meristematic cell groups is arranged, indefinite bud seldom appears in the surface, can be with 2, and 4-D concentration is reduced to 1.0 μ mol/l, continues to cultivate then to produce a large amount of wartys or digitation again.The bud tissue block of growing thickly on the A substratum, minority has the generation of adventive root.Exist equally with spire, adventive root also influences the generation of expanding growth and embryoid and the budlet that grows thickly of tissue block, need remove early.The bud tissue block of growing thickly was transferred on the B substratum after 2-3 days, and color and luster is flavescence gradually, and quality is more pliable and tougher, and microspike appearred in the rear surface in 5-6 days.Observe visible each phase embryoid and indefinite bud under the scanning electron microscope.Embryoid and indefinite bud are grown rapidly, form the budlet that grows thickly on the tissue block surface.
4) with the bud tissue block of growing thickly be the conversion and the plant regeneration of acceptor
(every liter of substratum contains: tryptone 10g at additional antibiotic LB substratum will to have the agrobacterium tumefaciens (as AGL0 and LBA4404) of goal gene, yeast extract 5g, NaCl 10g, pH 7.0, autoclaving) 28 ℃ of concussion cultivations down in, concussion speed be 110rpm (rev/min), make bacterium be in logarithmic phase.Under 3000rpm centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal again collection with the liquid seeds germination medium (be that the seed germination medium component reduces by half, remove agar powder) of 1/2 concentration.(acetosyringone, As) the liquid inducing clumping bud substratum of 1/2 concentration of 100 μ mol/l suspends, and dilutes 5-10 and doubly is used for transforming with adding Syringylethanone with thalline again.
Getting the bud tissue block of growing thickly of cultivating 13--20 days behind the subculture is transgene receptor.Transform with agrobacterium-mediated transformation, transform the back material and recover darkling to cultivate.Grow thickly budlet or the bud tissue block of growing thickly of agroinfection were cultivated 7-12 days bacteria growing inhibiting in dark on the substratum that is added with cephamycin (Cefotaxime) 250mg/l or Pyocianil (Carb) 500mg/L.Grow thickly budlet or the bud tissue block of growing thickly of recover cultivating after back or the antibacterial cultivation are being added with step sizing 3-4 generation on the substratum of selective agent, obtain transgenic cell and budlet.The exhausted big bud tissue block of growing thickly of counting is dead gradually in screening and culturing.Remove 2 with what the tissue block of survival was transferred to no selective agent, recover to cultivate generation budlet in back on the A substratum of 4-D.
Budlet is placed on into irradiation growth down on the seedling substratum, light intensity 2000-3000lx, illumination 14-15 hour/day.Seedling length changes in the root media during to 3-4 sheet leaf takes root.Cultivated about 15 days, about 40% seedling produces new root.For the seedling of not taking root, its base portion of cut wound is transferred on the new root media and is cultivated, and most of plant produce root system after 10 days.Take root behind the seedling flush away substratum, being transplanted to the vermiculite is to grow in the flowerpot of cultivation medium.Plant grows under natural lighting, day warm 22-28 ℃, at temperature 15-21 ℃ of night, waters the inorganic salt composition of the seed germination substratum of 1/2 concentration every other day.Grow about 2 weeks, nursery transplant produces flourishing root system, is colonizated in the field then.
5) resistance of transfer-gen plant detects and selects and utilizes
The blade of getting the transplant survival plant carries out molecular Biological Detection and determines transfer-gen plant.With transfer-gen plant bagging self-fertility.Get planting seed in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and glycinebetaine assay, and the bagging selfing is reserved seed for planting according to qualifications.The transgenosis homozygous lines is carried out salt tolerant, drought tolerance respectively detect, and numerous kind of bagging selfing, obtain the transgenosis self-mating system that resistance significantly improves than transgenosis self-mating system not.The latter can be used for preparing the drought-enduring cross-fertilize seed of corn salt tolerant.
6) salt tolerant of transgenic corns, drought tolerance detect and utilize
To change the milpa bagging selfing of isozygotying of ApGSMT2d/ApDMT2d gene, planting seed in the sand basin, the pouring 0.8%NaCl aqueous solution, and be contrast with transgenosis self-mating system seed not.The not homophyletic of transfer-gen plant ties up to the salt solution pouring and shows different salt tolerances down, most genetically modified strains are to show the comparison illumination to show the salt tolerance that improves, the 5 leaf phase surviving rates that strains more than half are are more than 70%, and contrast self-mating system seed is seldom emerged, also dead gradually after emerging, the surviving rate of 5 leaf phases is less than 10%.Real-time quantitative RT-PCR detects and shows do not having detection signal in the transfer-gen plant by transgene expression intensity height.The transfer-gen plant bagging selfing of picking out the salt tolerance excellence is isozygotied, and carries out combining ability test, selects combining ability identical with the donor self-mating system or to have the transgenosis self-mating system of raising to be used for haloduric corn breeding hybridized.
With the seed that changes the seed of the milpa of isozygotying of ApGSMT2/ApDMT2 gene and not genetically modified contrast self-mating system broadcast flowerpot in, carry out drought stress in seedling stage (3-7 leaf phase), female tassel growth period (9-13 leaf phase), the pollination phase of blooming respectively and handle, detect arid and handle the difference of influence, damaged membrane degree, single plant yield and other economic characters and the physical signs of corn growth etc.Comprehensive many-sided test result, genetically modified corn comparison shows the drought tolerance of obvious raising according to plant.The transfer-gen plant bagging selfing of selecting the drought tolerance excellence is isozygotied, and carries out combining ability test, and selection combining ability is identical with the donor self-mating system or have the transgenosis self-mating system of raising to be used for the seed selection of the corn seed of single cross.
The creation of the synthetic and degeneration-resistant material of corn gene of embodiment 2:ApGSMT2d, ApDMT2d gene
1) structure of the synthetic and expression of plants structure of ApGSMT2d, ApDMT2d gene
Select the preference codon replacement part A pGSMT2 of cereal plants and corn and the codon of ApDMT2 for use, synthetic ApGSMT2d and ApDMT2d gene.Be connected gene fusion construct with the RD29 promotor then, the latter is recombinated among the plant expression vector pCPE, produce pCPE-ApGSMT2d-ApDMT2d.Recombinant plasmid imported be used for genetic transformation in the agrobacterium tumefaciens.
2) acquisition of corn aseptic seedling
Bagging obtains seeds such as the key self-mating system of corn such as Zheng 58, neat 319, prosperous 7-2, with 70% alcohol immersion 10 minutes, again with 0.1% mercury chloride immersion 10-12 minute, washs 3-5 time with sterilized water then.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium, under dark condition, continue to sprout.When treating that plumule is stretched to 3-5 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.
3) Agrobacterium is cultivated and activation
(the Mini-Ti plasmid is pCPE-ApGSMT2d-ApDMT2d will to have binary vector, this plasmid has herbicide glyphosate resistant gene epsps) agrobacterium tumefaciens (AGL1 or LBA4404) 28 ℃ of concussions in additional antibiotic YEP substratum cultivate, concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal collection with 1/2 improvement MS liquid nutrient medium.Thalline is suspended with the 1/2MS improvement liquid nutrient medium that adds the 100mg/L Syringylethanone, dilution 5-10 doubly is used for transforming again.
4) the corn aseptic seedling transforms.
A. bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish, the aseptic seedling top that will expose the stem apex growing tip is immersed in 2-5 minute (AGL15 minute, LBA44049 minute) in the bacterium liquid.
B. the bud point after contaminating blots with aseptic filter paper, root is inserted in the modified MS medium cultivated 2-3 days in dark, and culture temperature is 22-24 ℃, then aseptic seedling is placed under the scattered light to cultivate 2 days.
C. the aseptic seedling after irradiation being cultivated is transplanted in the flowerpot that is covered with upper strata vermiculite and lower floor's loam, and vermiculite covers the plant top.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
5) transformed plant screening and field planting
After transformed plant grows 3 leaves, spraying the glyphosate aqueous solution, is contrast with unconverted plant.The sprinkling amount is not fallen drop with plant and is advisable.Adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 15 days.Plant after the conversion processing, the variation of some individualities is similar to adjoining tree, and other individualities then continue growth, no considerable change.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.
6) evaluation of transfer-gen plant, management and offspring analysis
The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene, and the PCR positive plant is carried out Southern blotting checking and RT-PCR detection.Bagging selfing or sisters handed over solid after transfer-gen plant was bloomed.Planting seed is in the land for growing field crops or the greenhouse, and plant is long gets blade to the 4-6 leaf and extract DNA during the phase, adopt round pcr to detect and whether have foreign gene, the PCR positive plant carried out Southern blotting detects and the RT-PCR detection, therefrom selects transfer-gen plant.Simultaneously transfer-gen plant is carried out the bagging selfing and reserve seed for planting, analyze the ratio of transgenosis individuality among the next generation, select transgenosis and isozygoty and be.
7) resistance of transfer-gen plant is measured
Selecting the transgenosis homozygous lines to be used for measuring, is contrast with the non-transgenic selfing.Transfer-gen plant seed and contrast sowing seed same period are seeded in respectively in the flowerpot that size is consistent, the soil amount equates, make plant be in suitable growth season.The plant strain growth environment is consistent with management process.Kind grow to 10 leaf after dates in the milpa of flowerpot, every day is watered the water yield (about 600ml) in control, makes that relative water content remains on about 15-16% in the soil, 9-16 on daytime plant blade wilting between period, night, blade recovered to stretch, and continued to handle for 2 weeks, recovered then normally to water.The all backs plant that recovers normally to water is taken out hero.Again that flowerpot is placed apart, so that the plant self-fertility.Results mature fruit cluster and cauline leaf, biomass and grain yield are measured in the oven dry back.With plant control water the 2nd day (blade began to wilt about at 9 o'clock in the morning) be designated as drought stress and handled the 0th day, drew materials in the 7th day the 0th, 1,5,10,14 day that handles and the back of recovering normally to water respectively, adopt conventional plant physiology method to measure and degeneration-resistant relevant every physical signs.And female tassel developmental state, plant height, seed weight, pollen vigor etc. are observed.With the 10 leaf phase of part plant stop to water, observe continuously arid to the influence of vine growth and development and survival rate.In entire test, plant is under natural lighting and the temperature and grows, and avoids plant to be drenched with rain during the control water treatment.
Under the normal growth condition, grow and the morphological specificity of transfer-gen plant and adjoining tree do not have significant difference.After drought stress was handled, transfer-gen plant and adjoining tree notable difference all occurred in proterties such as plant height, tassel branch amount, the loose powder of blooming time, the loose powder-pitch time of weaving silk, pollen vigor, fruit ear length, 100-grain weights.Transfer-gen plant tassel branch amount is not few, stamen development is bad, pollen abortion, loose powder-pitch time of weaving silk is long, the individual plant grain yield is low, and transfer-gen plant shows the drought tolerance of obvious raising, pollen development is normal, the individual plant grain yield apparently higher than the contrast.It is more normal to be that transfer-gen plant grows in arid treating processes, has obvious enhanced drought tolerance.Measure the blade relative water content, the solubility total sugar content, the ion percolation ratio, mda content, physical signs such as proline content show that slow that plant reduces shone in the comparison of rotaring gene plant blade relative water content under drought stress, water retention capacity is stronger; Ion percolation ratio and mda content show that the rotaring gene plant blade cell membrane damage is less; soluble sugar and proline content illustrate that transfer-gen plant may be to keep eubolism by accumulating more osmotic protection material apparently higher than adjoining tree under drought stress.
To the salt solution of corn pouring in seedling stage 0.8%, the salt tolerance of transfer-gen plant is apparently higher than transgenosis adjoining tree not.Measure the discoveries such as over-ground part and underground part fresh weight, dry weight under the salt treatment condition of plant in seedling stage, transfer-gen plant grows normally, situations such as growth retardation, plant dwarfing do not occur.
Present embodiment has createed the transgenic corns material that drought-enduring salt tolerance significantly improves.
The creation of the structure of embodiment 3, ApTpGSMT2d, ApTpDMTd2 gene and the degeneration-resistant material of corn gene
1) structure of the synthetic and expression of plants structure of ApTpGSMT2d, ApTpDMTd2 gene
For ApGSMT2 and ApDMT2 are efficiently expressed in maize cell, and the accumulation of realization glycinebetaine in chloroplast(id) (plastid), ApGSMT2d and ApDMT2d gene are merged with peptide (transit peptide) coding region of leading of location chloroplast protein respectively, ApGSMT2 and ApDMT2 albumen are positioned in the chloroplast(id) (plastid) after synthetic, and catalysis is synthetic a large amount of glycinebetaine in chloroplast(id).According to the Arabidopis thaliana RUBISCO small ylidene gene sequences Design PCR primer of having reported, from Arabidopis thaliana, clone the peptide-coding region of leading of RUBISCO small subunit, be connected respectively to the 5` end of ApGSMT2d gene and ApDMT2d gene opening code-reading frame after the sequence verification, produce fusion gene, i.e. ApTpGSMT2d and ApTpDMT2d gene.Replace ApGSMT2d and ApDMT2d among the plasmid pCUA-ApGSMT2d-ApDMT2d-epsps respectively with the fusion gene that makes up then, produce plant genetic transferring structure pCUA-ApTpGSMT2d-ApTpDMT2d-epsps.Adopt freeze-thaw method that recombinant plasmid is imported and be used for the maize genetic conversion in the agrobacterium tumefaciens.
2) resistance of generation of corn gene plant and transgenosis homozygous lines is measured
With embodiment 2, obtained the transgenic corns material that salt tolerance of drought significantly improves.
The creation of the structure of embodiment 4, ApTpGSMT2dm, ApTpDMT2dm gene and the degeneration-resistant material of corn gene
1) structure of the synthetic and expression of plants structure of ApTpGSMT2dm, ApTpDMTd2m gene
This laboratory work finds that when corn suffered severe drought or salt stress, mitochondrial function reduced significantly, improves the working efficiency of plastosome when cell is coerced, and helps improving the resistance of corn.For ApGSMT2 and ApDMT2 are efficiently expressed in maize cell, and realize that glycinebetaine accumulates in Intramitochondrial compartmentation, ApGSMT2d and ApDMT2d gene are merged with proteic peptide (transit peptide) coding region of leading of position line plastochondria respectively, be positioned in the plastosome after making ApGSMT2 and ApDMT2 albumen synthetic, at the tired more glycinebetaine of plastosome inner product.According to reporting sequences Design PCR primer, from the corn spire, clone the peptide-coding sequence of leading of citrate synthase gene, be connected respectively to 5 ' end of ApGSMT2d gene and ApDMT2d gene opening code-reading frame after the sequence verification, produce fusion gene, i.e. ApTpGSMT2dm and ApTpDMT2dm gene.Then the fusion gene that makes up is recombinated in the DATEWAY entry vector that has stress induced promoter RD29, start its expression respectively by RD29.By fixed point reorganization fusion gene is recombinated in the DATEWAY plant expression vector again, produce plasmid pZH-ApTpGSMT2dm-ApTpDMT2dm-epsps.Adopt freeze-thaw method that this plasmid is imported and be used for the maize genetic conversion in the agrobacterium tumefaciens.
2) resistance of generation of corn gene plant and transgenosis homozygous lines is measured
With embodiment 2, obtained the transgenic corns material that salt tolerance of drought significantly improves, but material resistance increase rate is less than the transgenosis homozygous lines of embodiment 2 and embodiment 3 generations.
Embodiment 5, ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 import jointly and create corn breeding for stress tolerance novel material
For ApGSMT2 and ApDMT2 are efficiently expressed in maize cell, realize the not only accumulation in chloroplast(id) (plastid) but also in tenuigenin of glycinebetaine, the probability of avoiding transgene silencing to take place simultaneously, and the accumulation of excessive concentrations causes that plant strain growth is slack-off in the prevention glycinebetaine tenuigenin, therefore, on the one hand ApGSMT2 and ApDMT2 gene are merged with peptide (transit peptide) coding region of leading of location chloroplast protein respectively, impel synthetic ApGSMT2 and ApDMT2 albumen to be positioned in the chloroplast(id) (plastid), a large amount of glycinebetaines of accumulation in chloroplast(id); On the other hand ApGSMT2d and ApDMT2d gene are merged with stress induced promoter respectively, be implemented in transgene product accumulation in a large number in kytoplasm under the stress conditions.
1) structure of the synthetic and expression of plants structure of ApTpGSMT2, ApTpDMT2 gene
Utilize the peptide-coding region of leading of RUBISCO small ylidene gene in the Arabidopis thaliana cloned, be connected respectively to ApGSMT2 gene and ApDMT2 gene (without ApGSMT2d and ApDMT2d sequence, the generation of minimizing transgene silencing incident) 5 ' end of opening code-reading frame, produce fusion gene, i.e. ApTpGSMT2 and ApTpDMT2 gene.
Adopt conventional Protocols in Molecular Biology, ApTpGSMT2d and ApTpDMT2d gene, ApGSMT2d and ApDMT2d are recombinated respectively in the GATEWAY entry vector, merge with different plant promoter and gene 3 ' tail district respectively, the fusion gene that formation is adapted at expressing in the cereal crop, wherein ApGSMT2d is connected with the promotor RD29 of stress-inducing respectively with the ApDMT2d gene, and ApTpGSMT2 links to each other with constitutive promoter Ubiquitin respectively with ApTpDMT2.By in the polygene conversion carrier of fixed point reorganization importing based on the GATEWAY plant expression vector, produce plasmid pZH-ApGSMT2d-ApDMT2d-ApTpGSMT2-ApTpDMT2-epsps then.Adopt freeze-thaw method that the polygene conversion carrier is imported in the agrobacterium tumefaciens and be used for the seeding corn and other crops genetic transformation.
2) resistance of generation of corn gene plant and transgenosis homozygous lines is measured
With embodiment 2, obtained the transgenic corns material that salt tolerance of drought significantly improves, the latter's resistance significantly is better than the material of embodiment 2 and embodiment 3 generations.
Embodiment 6, commentaries on classics ApTpGSMT2, ApTpDMT2 gene are created cotton breeding for stress tolerance novel material
1) expression of plants structure and Agrobacterium are cultivated
The structure of plant expression vector and Agrobacterium are cultivated and activate with embodiment 1.Agrobacterium strains is LBA4404.
2) the cotton aseptic seedling obtains
Get the cotton improved seeds or recover the seed of system, slough surperficial fine hair with the vitriol oil, 70% alcohol immersion 1 minute was soaked 10-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium that ammonium salt reduces by half and sprout down in dark condition.When treating that plumular axis is stretched to 3-5 centimetre, peel off a slice cotyledon, emergent stem end growing tip.
3) the cotton aseptic seedling transforms
(1) dip-coating of Agrobacterium bacterium liquid is being separated on the stem apex growing tip that digs pricking wound, and covering thereon with agrobacterium liquid wetted cotton balls, cultivating darkling then 2-3 days, culture temperature is 24-26 ℃.Again aseptic seedling is placed under the scattered light and cultivated 2 days.
(2) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot of upper strata vermiculite lower floor loam, allows plant under natural lighting, grow, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
4) transformed plant screening and field planting
After transformed plant grew 3 leaves, spraying herbicide glyphosate (concentration is different because of the kind) aqueous solution did not fall drop with plant and is advisable.Unconverted adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 12 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and the very strong resistance of other individual performances continues growth.The survival plant is long during the phase, to arrive field with its field planting to 5 leaves.
5) molecular biology identification of antiweed plant
When antiweed plant length arrives the 7-8 leaf, take a sample, mix the back and extract DNA, employing round pcr detection foreign gene from 3-4 sheet leaf.Gather in the crops the seed (selfing) of PCR positive plant, plant the back in the spraying herbicide glyphosate screening during phase of 3-4 leaf, the survival plant is got blade extraction DNA and carries out the PCR detection, and positive plant carries out Southern blotting checking and RT-PCR detects.Transfer-gen plant selfing knot peach, the results seed continues plantation.T2 for plant still at 3-4 leaf spraying herbicide glyphosate during the phase, statistics survival plant and dead plant ratio, in conjunction with previous generation Southern blotting result, determine that can transgenosis genetic stability, and therefrom select the high transgenosis homozygous lines of target gene expression intensity.Further adopt transgene expression intensity and glycinebetaine content in real-time quantitative RT-PCR technology and the different transgenosiss of glycinebetaine assay technical Analysis then, select suitable strain system to carry out the resistance analytical test.
6) resistance of transfer-gen plant is identified and is utilized
Get the seed of the transfer-gen plant that isozygotys, sowing is in flowerpot, and plant 2 leaves carry out subzero treatment during the phase, and by survival rate statistics and physiological index determining, filtering out cold-resistant strain is to be used for producing.
The seed of transgenic line is broadcast in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and salt tolerance detect, and the bagging selfing isozygotys, obtained the salt tolerant kind.
The plant of transgenic line is carried out the drought stress test in seedling stage (5 leaf phase), flower bud phase and flowering period respectively, measure relevant physical signs analysis, determine the plant drought tolerance.5 leaf phase cotton seedlings are handled in containing the Hogland nutritive medium of 12%PEG-6000, the blade dehydration of wild-type plant is serious after 24 hours, plant is seriously wilted, and rotaring gene plant blade stretches, plant do not take place to wilt or degree lighter, the blade relative water content of transfer-gen plant is significantly higher than wild-type, and Net Photosynthetic Rate, stomatal conductance and transpiration rate all are significantly higher than wild-type.Osmotic stress has reduced the solute potential of cotton seedling cell, wherein the solute potential of transfer-gen plant cell reduces more, Physiologic Studies shows, under the drought stress condition, accumulated more solute in the cell, strengthened that cell absorbs water and water-holding power, kept normal morphology and cell turgor thereby help plant.Cotton grows to flower bud during the phase in flowerpot, disposablely stop to water after watering sufficient water, carries out drought stress and handles.At flower bud phase drought stress after 8 days, the blade beet alkali content of transfer-gen plant is significantly higher than the wild-type plant, the blade dehydrating speed is obviously compared according to slow, blade is wilted evening, blade cell film ion percolation ratio and mda content significantly are lower than the wild-type plant, illustrate that cell membrane damage and film fat peroxidation degree are all lighter.In addition, the chlorophyll content of transfer-gen plant and superoxide dismutase (SOD) are active also higher in drought stress is handled.The branches of transfer-gen plant contrasts than wild-type and manys 15%-26% behind the drought stress, and the individual plant number of blooming contrasts than wild-type and manys 10%-20%, and individual plant seed cotton yield fall is obviously little to be contrasted than wild-type.Correlation analysis draws, and transgene cotton individual plant seed cotton yield and trimethyl-glycine level are remarkable positive correlation.These results show; the increase of trimethyl-glycine level can improve the drought tolerance of transgene cotton greatly; the raising of transgene cotton drought resistance is not only because the provide protection of trimethyl-glycine cell membrane stability and integrity under the stress conditions, and the accumulation that will give the credit to trimethyl-glycine has improved the osmotic adjustment ability of transfer-gen plant.
Embodiment 7, commentaries on classics ApTpGSMT2d, ApTpDMT2d gene are created wheat breeding for stress tolerance novel material
1) expression of plants structure and Agrobacterium are cultivated
The expression of plants structure is with embodiment 3.Agrobacterium is cultivated and activates with embodiment 1.Agrobacterium strains is LBA4404.
2) the wheat aseptic seedling obtains and transforms
The seed of wheat improved seeds Jimai 20, Jimai 22 etc. is used 70% alcohol immersion 1-3 minute respectively, soaks 8-12 minute with 0.1% mercury chloride, then with sterilized water washing 3-5 time again.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water in the bottle in dark condition (20-25 ℃) 1-2 days down.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium and sprout down in dark condition.When treating that plumule elongation ends 3-4 centimetre, peel off coleoptile and 1-2 sheet spire, expose the point that sprouts with scalper, again with scalper from the middle part vertical cut wound shoot tip meristem.The aseptic seedling of cut wound shoot tip meristem (germinating seed) is used for Agrobacterium-mediated Transformation.Bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish is immersed in the bacterium liquid, 0.5 * 10 the aseptic seedling of exposing the stem apex growing tip 5The Pa normal atmosphere was handled 6-10 minute down.Bud point after the dip-dye blots with aseptic filter paper, and germinating seed is placed on the modified MS medium of adding the 200mg/l glutamine and cultivated 2-3 days in dark, and culture temperature is 22-24 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.Aseptic seedling after the irradiation cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, and covers the plant top with vermiculite.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
3) transformed plant screening, field planting and Molecular Detection
After transformed plant grows 3 leaves, the spraying herbicide glyphosate aqueous solution, concentration is 0.12%Roundup (41% glyphosate isopropyl amine salt), falls drop with plant and is advisable.Unconverted adjoining tree stops growing after back 6 days in sprinkling, and beginning is dead about 15 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.When the plant of waiting to survive length arrives the 7-8 leaf, the field is arrived in its field planting, and promoted to tiller.
The antiweed plant is got blade in the jointing stage of standing up and extracts DNA after vernalization treatment, adopts round pcr to detect transformed plant.In the antiweed plant that obtains, about about 20% is the PCR positive individuals.Latter's self-fertility divides the fringe results.
4) the transfer-gen plant offspring identifies and analyzes
Plant by vernalization stage (the subzero treatment condition is different because of kind) was stood up under the long day, jointing, blossom and bear fruit.The seed that transfer-gen plant produces is put after immersion is sprouted in the refrigerator (0-2 ℃) and was carried out vernalization treatment 30-65 days.Seed after the part vernalization treatment is handled with the 0.12%Roundup aqueous solution, observes the individual ratio of statistics resistance and susceptibility, and the survival seedling is broadcast into flowerpot; Planting seed after another part vernalization treatment carries out drought stress processing and high-salt stress respectively and handles at land for growing field crops and sand basin, screens drought-enduring and/or salt tolerant transfer-gen plant.Extract the leaf DNA of antiweed plant and salt tolerant and drought resisting plant, adopt round pcr to detect foreign gene.Get upper blade extraction DNA behind the PCR positive plant jointing and carry out Southern blotting checking.The transfer-gen plant selfing is set seeds, and following generation will be continued plantation, continues at plant 3-4 leaf spraying herbicide glyphosate during the phase, the ratio of statistics survival plant and dead plant, in conjunction with previous generation Southern blotting result, determine that can transgenosis genetic stability, and therefrom select transgenosis to isozygoty to be.In conjunction with the result of real-time quantitative RT-PCR and the acquisition of glycinebetaine assay, selecting suitable strain is to carry out resistance to measure.Obtain to be directly used in wheat breeding or to enter security interim test and environmental release test after the transgenosis pure lines.
5) the drought resistance analysis of transgenic wheat
Emerge and be wheat to one of tricky time of lack of water seedling stage, this stage drought impact wheat seedling rate and tiller number can make a big impact to output.The seed of transgenic line and adjoining tree is sprouted in the MS inorganic salt solution that contains 0,10%, 15%, 20%, 25% (W/V) PEG-6000 respectively, statistics germination rate, root length and bud are long, discovery is under 25% (W/V) PEG-6000 osmotic stress condition, the seed germination comparison of transgenic line is according to early, the length of bud and root is not also respectively greater than genetically modified, and difference reaches significance level.
Potted plant wheat seedling is carried out continuous drought handle, the biomass of observation plant forms difference and mensuration ground and underground part.Transfer-gen plant in arid is handled growth better, well developed root system, biomass are significantly higher than adjoining tree.The drought tolerance of different transgenic lines has certain difference, and the best transgenic line of drought tolerance is being coerced when handling 25 days on the ground and the biomass of underground part is compared photograph plant high 60% and about 40% respectively.Under the drought stress condition, higher, the stretching, extension of the blade relative water content of transgenic line, the wilting degree is low, and chlorophyll content, Net Photosynthetic Rate, stomatal conductance and transpiration rate are compared respectively according to significantly high.
Drought stress has reduced the solute potential of wheat seedling cell, has improved cell soluble sugar and proline content.Wherein to reduce amplitude bigger for the solute potential of transfer-gen plant cell, and the accumulation volume of soluble sugar and proline(Pro) is significantly higher than wild-type.Under the drought stress condition, more soluble substance accumulation helps the cell suction and keeps normal turgescence in the transgenic cell.Transgenic line blade cell ion percolation ratio and MDA content all significantly are lower than adjoining tree, illustrate that the cell membrane damage of transgenic line and film fat peroxidation degree are lighter.Superoxide dismutase of transfer-gen plant (SOD) and catalase (POD) activity also are higher than adjoining tree in drought stress is handled, show that transfer-gen plant has stronger anti-peroxidation ability under the drought stress condition.These results suggest glycinebetaines have the membrane stability of raising and the active effect of stabilized enzyme.The excess accumulation that these results show glycinebetaine is in the osmoregulation of transgenic wheat cell and alleviate and brought into play vital role in the free radical injury, has improved the transfer-gen plant drought-resistant ability.
Embodiment 8, cotransformation ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 gene are created soybean breeding for stress tolerance novel material
1) expression of plants structure and Agrobacterium are cultivated
Plant expression vector, Agrobacterium are cultivated and activate with embodiment 5.Agrobacterium strains is LBA4404.
2) the soybean aseptic seedling obtains and Agrobacterium cultivation and activation
Get the seed of improved seeds,, soaked 10-12 minute, then with sterilized water washing 3-5 time with 0.1% mercury chloride with 70% alcohol immersion 2-3 minute.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (50-60 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 2-3 days after sealing.After treating that seed is sprouted, place it on the improvement B5 medium and sprout down in dark condition.When treating that plumular axis grows to 3-5 centimetre, peel off cotyledon and emergent stem end growing tip.
To have the destination gene expression structure agrobacterium tumefaciens (AGL1 or LBA4404) in additional antibiotic YEP substratum 28 ℃ down concussion cultivate, concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under the 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal again collection with 1/2 improvement B5 liquid nutrient medium (promptly improveing B5 medium basal component reduces by half).Thalline is suspended with the 1/2 improvement B5 liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-10 doubly is used for transforming again.
3) the soybean aseptic seedling transforms
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish makes the aseptic seedling top of exposing the stem apex growing tip be immersed in the bacterium liquid 3-6 minute.
(2) the bud point after the dip-dye blots with aseptic filter paper, the seedling butt is inserted on the improvement B5 medium cultivated 3-4 days in dark, and culture temperature is 20-23 ℃.
(3) aseptic seedling is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, the top covers vermiculite, and the long day (16 hours/day) is growth down, warm 23-28 ℃ of day, at temperature 20-25 ℃ of night, waters 1/2 improvement B5 medium inorganic salt every other day.
4) transformed plant screening and field planting
After transformed plant grows 3 leaves, spray the 0.12%Roundup aqueous solution and handle, do not fall drop with plant and be advisable, unconverted adjoining tree stopped growing in sprinkling in back 6 days, and beginning is dead about 15 days.Transformed plant is after sprinkling, and the variation of some individualities is similar to adjoining tree, and other individual continuing grow, and change not obvious.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.
The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene.The PCR positive plant keeps solid.
5) transfer-gen plant offspring's molecular biology identification
The seed that transfer-gen plant produces is sowed in the land for growing field crops or the greenhouse.Plant 3-4 leaf is got blade extraction DNA and is carried out PCR detection transgenosis ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 during the phase.Get PCR positive plant blade and carry out Southern blotting checking, transfer-gen plant self-fertility.Second filial seedling (5 leaf phase) is added up survival plant and dead plant ratio after spraying the 0.12%Roundup aqueous solution, the survival plant is carried out PCR again detect, and determines the segregation ratio of foreign gene in progeny plant.The transgenosis homozygous lines is carried out real-time quantitative RT-PCR detect and the glycinebetaine assay, select the high strain system of transgene expression intensity to be used to detect resistance.
6) transfer-gen plant offspring's resistance is measured
Physiology method routinely and thremmatology method are carried out.
Embodiment 9, realize that by transgenosis polymeric method glycinebetaine creates the high resistance to cold and diseases material in the distribution of cell different zones
1) the transgenosis polymerization produces corn high resistance to cold and diseases self-mating system
To produce plant, the plant that has ApGSMT2d/ApDMT2d and ApTpGSMT2dm/ApTpDMT2dm gene that has ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2 gene, the plant that has ApTpGSMT2d/ApTpDMTd2 and ApTpGSMT2dm/ApTpDMT2dm gene respectively from the milpa hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same genotype (self-mating system).Then transgenosis polymerization plant is carried out continuous selfing, selection, the acquisition transgenosis is isozygotied and is.Also different transfer-gen plants and transgenosis polymerization plant can be hybridized once more, obtain to carry different genetically modified self-mating systems by selfing with selection, as carry the corn inbred line of ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene, realize that different critical functions district in the cell all accumulates the target of the glycinebetaine of higher level under the environment stress condition.The hybridization of transgenic corns, selfing and system of selection be with the corn conventional breeding, but need carry out in the transgenic corns isolated area.Whether and in the self-generation of transgenosis polymerization plant, need to adopt PCR method to detect all target genes and exist, only selecting and remain, those have the individuality of whole target genes.Coding region corresponding to gene of the PCR primer of each target gene, one corresponding to promoter region, has good specificity.The resistance measuring method of transgenosis polymeric material is with example 2.
2) the degeneration-resistant novel material of transgenosis polymerization wheat produces
To produce plant, transgenosis ApTpGSMT2d/ApTpDMTd2 and the ApTpGSMT2dm of the plant, transgenosis ApGSMT2d/ApDMT2d and the ApTpGSMT2dm/ApTpDMT2dm that carry transgenosis ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2, the plant of ApTpDMT2dm respectively from the wheat plant hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same kind.Then transgenosis polymerization plant is carried out continuous selfing, selection, the acquisition transgenosis is isozygotied and is.Also different transfer-gen plants and transgenosis polymerization plant can be hybridized once more, selfing and selection, obtain to carry the transgenosis polymerized strain of different target genes.The hybridization of transgenic wheat, selfing and system of selection be with the wheat conventional breeding, but need carry out in the transgenic wheat isolated area.And adopt PCR method to detect whether target gene all exists among the transgenosis polymerization plant self progeny, those individual selfings that have whole target genes of only selecting and remain are isozygotied.The resistance measuring method of transgenosis polymeric material is with embodiment 7.
3) the transgenosis polymerization produces the degeneration-resistant novel material of cotton
To produce the plant of plant, transgenosis ApTpGSMT2d/ApTpDMTd2 and the ApTpGSMT2dm/ApTpDMT2dm of the plant, transgenosis ApGSMT2d/ApDMT2d and the ApTpGSMT2dm/ApTpDMT2dm that carry transgenosis ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2 respectively from the cotton plants hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same kind or recovery system.Then transgenosis polymerization plant is carried out continuous selfing, selection, the acquisition transgenosis is isozygotied and is.Also different transfer-gen plants and transgenosis polymerization plant can be hybridized once more, selfing and selection, obtain to carry the transgenosis polymerized strain of different target genes.The hybridization of transgene cotton, selfing and system of selection are with the cotton conventional breeding, but need carry out in the transgene cotton isolated area, and adopt PCR method to detect whether target gene exists among the transgenosis polymerization plant self progeny, only selecting and remain, those have individual selfing, the selection of whole target genes.The resistance measuring method of transgenosis polymeric material is with example 6.
4) the degeneration-resistant novel material of transgenosis polymerization soybean produces
To produce the plant of plant, transgenosis ApTpGSMT2d/ApTpDMTd2 and the ApTpGSMT2dm/ApTpDMT2dm of the plant, transgenosis ApGSMT2d/ApDMT2d and the ApTpGSMT2dm/ApTpDMT2dm that carry transgenosis ApGSMT2d/ApDMT2d and ApTpGSMT2d/ApTpDMTd2 respectively from the soybean plant strain hybridization of changeing ApGSMT2d/ApDMT2d, ApTpGSMT2d/ApTpDMTd2, ApTpGSMT2dm/ApTpDMT2dm gene respectively of same kind.Then transgenosis polymerization plant is carried out continuous selfing, selection, the acquisition transgenosis is isozygotied and is.Also different transfer-gen plants and transgenosis polymerization plant can be hybridized once more, selfing and selection, obtain to carry the transgenosis polymerized strain of different target genes.The hybridization of genetically engineered soybean, selfing and system of selection are with the soybean conventional breeding, but need carry out in the genetically engineered soybean isolated area, and adopt PCR method to detect whether target gene exists among the transgenosis polymerization plant self progeny, only selecting and remain, those have individual selfing, the selection of whole target genes.The resistance measuring method of transgenosis polymeric material is seen conventional breeding for stress tolerance method.
Embodiment 10, utilization are changeed ApGAMT2 and are prepared degeneration-resistant cross-fertilize seed with the different corn inbred lines of ApDMT2 and derivation gene thereof
To have the excellent heterotic different corn inbred line hybridization that change ApGSMT2d/ApDMT2d or ApTpGSMT2d/ApTpDMTd2 or ApTpGSMT2dm/ApTpDMT2dm gene respectively over to, and produce and carry this genetically modified cenospecies of father and mother respectively.The latter can be directly used in production, promptly is the degeneration-resistant cross-fertilize seed of transgenosis that has competitive edge on producing.The selfing that also can change ApGSMT2d/ApDMT2d and ApTpGSMT2/ApTpDMT2 gene is male parent or the female parent of cross-fertilize seed, another selfing with commentaries on classics ApTpGSMT2dm/ApTpDMT2dm gene is female parent or the male parent of cross-fertilize seed, thereby realizes that the interior different critical functions district of hybrid cell all can accumulate the target of higher concentration glycinebetaine under the environment stress condition.The hybrid of transgenic corns produces with the corn conventional breeding, but need carry out in the transgenic corns isolated area.The resistance measuring method of transgenosis cross-fertilize seed is with reference to example 2.
Figure IDA0000152347950000011
Figure IDA0000152347950000021
Figure IDA0000152347950000031
Figure IDA0000152347950000041
Figure IDA0000152347950000051
Figure IDA0000152347950000061
Figure IDA0000152347950000071
Figure IDA0000152347950000091
Figure IDA0000152347950000101
Figure IDA0000152347950000111

Claims (3)

1. the methyl transferase gene in the cyanobacteria trimethyl-glycine route of synthesis, it is characterized in that: described gene is ApTpGSMT2d and ApTpDMT2d, be that ApGSMT2d and ApDMT2d are merged with the peptide-coding region of leading of locating chloroplast protein respectively, the transgene expression product that obtains is positioned the fusion gene of chloroplast(id), wherein the fusion rotein aminoacid sequence of ApTpGSMT2d generation is shown in SEQ ID NO.13, and the fusion rotein aminoacid sequence that ApTpDMT2d produces is shown in SEQ ID NO.14; Described ApGSMT2d and ApDMT2d gene are to obtain with the codon replacement ApGSMT2 of cereal plants preference and low codon, the synthetic of ApDMT2 frequency of occurrences in higher plant, the nucleotide sequence of ApGSMT2d is shown in SEQ ID NO.9, and the nucleotide sequence of described ApDMT2d is shown in SEQ ID NO.10; Described ApGSMT2 and ApDMT2 are meant the glycine sarkosine N-methyl transferase gene ApGSMT2 in the glycinebetaine route of synthesis that clones and the gene A pDMT2 of N-methylsarcosine N-methyltransgerase from have a liking for salt aphanothece (Aphanothece halophytica), wherein the nucleotide sequence of ApGSMT2 is shown in SEQ ID NO.5, and the nucleotide sequence of ApDMT2 is shown in SEQ ID NO.7.
2. the application of methyl transferase gene in the anti-salt of cereal plants is drought-enduring in the cyanobacteria trimethyl-glycine route of synthesis, be to adopt transgenic technology that ApTpGSMT2d and ApTpDMT2d gene are imported the drought-enduring transfer-gen plant of the screening anti-salt of generation in the cereal plants cell, it is characterized in that: the fusion rotein aminoacid sequence that ApTpGSMT2d produces is shown in SEQ ID NO.13, and the fusion rotein aminoacid sequence that ApTpDMT2d produces is shown in SEQ ID NO.14.
3. application as claimed in claim 2 is characterized in that: described cereal plants is a corn.
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