CN103911386A - Artificial fusion gene for improving stress tolerance of plants - Google Patents

Abstract

The invention relates to an artificial fusion gene for improving stress tolerance of plants and an application of the artificial fusion gene. A polynucleotide sequence of the gene contains: (a) a polynucleotide sequence encoding an NAD(P) combined functional structure domain of rice GPDH protein; (b) a polynucleotide sequence encoding an NAD_Gly3P dehydrogenase functional structure domain of escherichia coli gpsA protein. The artificial fusion gene provided by the invention can be used in the preparation of transgenic plants such as rice so as to improve the stress tolerance of plants.

Description

Improve the artificial fusion gene of the stress tolerance of plant

Technical field

The invention belongs to gene engineering technology field, be specifically related to artificial fusion gene and the application thereof of the stress tolerance that improves plant.

Background technology

Plant can suffer from various abiotic stresses in process of growth, natural disasteies such as arid, the gentle stagnant waterlogging of height, often cause a large amount of underproduction of farm crop, in addition, in China, the scarce phosphorus of ploughing is also that (China has arable land more than half to lack phosphorus to a kind of common abiotic stress environment, in production, often use in a large number phosphate fertilizer to ensure grain yield (Li Yongfu etc., Chinese Journal of Applied Ecology, 2005,16 (1): 119-124)).Therefore, plant abiotic stress biology is one of important goal of agricultural cience and farming techniques research.For example, when previous important breeding technique be attempt various adversity genes are carried out farm crop genetic engineering modified, to obtain degeneration-resistant new crop varieties.

Scientific research discovery, for resisting coercing of bad external environment, plant soma is experienced the variation of external environment and is passed the signal along in cell, and meeting inducing cell is expressed various response genes and is jointly resisted the injury of poor environment to plant materials.Signal transmits molecule and brought into play keying action in the process of plant stress-resistance/stress tolerant.For example, glycerol-3-phosphate (claiming again glycerol 3-phosphate, Glycerol-3-phosphate, G-3-P) is not only the synthetic precursor of various glyceryl ester materials (such as film fat etc.), also be signal small-molecule substance in plant materials, in the degeneration-resistant process of plant, play key effect simultaneously.

Glycerol-3-phosphate is the important as precursors thing of phosphatide in cytolemma plasma membrane, and the formation of the variation confrontation membrane phospholipid of its content has a significant effect.Moreover, experimental study is verified, the height of the expression level of the glycerol-3-phosphate in arabidopsis cell has reflected its resistance against diseases, has proved that glycerol-3-phosphate is the strong inductor of Systemic Acquired Resistance In Plants, is member important in plant immunization system.

It is one of key enzyme of plant generation glycerol-3-phosphate due to glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase claims again GPDH albumen).It is made up of two structural domains, it is NAD (P) combined function structural domain in its N end structure territory, its major function is in conjunction with NAD+/NADP+, its C end is NAD_Gly3P desaturase hydrolysis function structural domain, and its major function is that catalysis utilizes NADH and Dihydroxyacetone Phosphate to produce glycerol-3-phosphate.

For improving the expression level of glycerol-3-phosphate in vegetable cell, general way is to improve the expression level of glycerol-3-phosphate dehydrogenase in vegetable cell.Scientists attempts the glycerol-3-phosphoric desaturase gene in vegetable cell without the bacterial origin of retroactive effect to carry out arabidopsis thaliana transformation, referring to document Shen W et al, The Journal of Biological Chemistry, 2010,285:22957 – 22965, overexpression gpsA in arabidopsis cell ^fRgene (through the glycerol-3-phosphoric desaturase gene of improved bacterial origin) has changed a large amount of phospholipid metabolism related gene expression amounts, has changed lipid acid in membrane phospholipid and has formed and film fat composition.Scientists is also attempting this gene to import in the genome of rape, finds that this gene can improve the growth of rape under low-phosphorous condition under laboratory condition, and resists certain osmotic stress, but be only can realize under laboratory condition.It is limited that directly the gene of use bacterial origin carries out its effect of plant improvement.Moreover, do not find temporarily that at present this gpsAFR gene is applied to paddy rice or other cash crop.

At present, need a kind of new resistant gene, can improve the stress tolerance of plant, and can be more widely applicable for paddy rice and other cash crop.

Summary of the invention

One aspect of the present invention provides a kind of artificial fusion gene of the stress tolerance that improves plant, wherein,

The polynucleotide sequence that this gene has comprises:

(a) polynucleotide sequence of NAD (P) the combined function structural domain of coding paddy rice GPDH albumen; (b) polynucleotide sequence of the NAD_Gly3P dehydrogenase function structural domain of coding intestinal bacteria gpsA albumen.

In a preferred embodiment of the invention, described (b) is for adopting the sequence of favorite plant codon synthetic.In a more preferred of the present invention, the polynucleotide sequence of this artificial fusion gene comprises sequence as shown in SEQ ID NO.1.In addition, contriver is by nucleotide sequence called after shown in SEQ ID NO.1 oEGDgene.Preferably, the aminoacid sequence of this artificial fusion gene coding comprises sequence as shown in SEQ ID NO.2.

The present invention provides the recombinant vectors that contains above-mentioned artificial fusion gene on the other hand.

The host cell that provides above-mentioned recombinant vectors to transform on the one hand more of the present invention.

The present invention provides a kind of method of transgenic plant that produce stress tolerance on the other hand, wherein:

The method comprises the following steps:

1) artificial fusion gene claimed in claim 1 is operably connected to expression of plants regulating and controlling sequence, forms plant expression vector;

2) plant expression vector of step 1) gained is proceeded to vegetable cell;

3) transformant obtaining through screening, is regenerated as plant and offspring thereof, and described plant comprises vegetable cell, plant tissue or plant seed.

In a preferred embodiment of the present invention, described plant is selected from paddy rice, corn, wheat, barley, broomcorn millet, Chinese sorghum, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, oranges and tangerines or cucumber any one.

The present invention also provides the purposes aspect above-mentioned artificial fusion gene has stress tolerance transgenic plant in preparation on the other hand.

Artificial fusion gene of the present invention, can be used in preparation transgenic plant, improves ability, especially drought resisting, the high temperature of the anti-stress tolerance of plant, the ability of low-phosphorous, resistance to osmotic stress.

Brief description of the drawings

Fig. 1 is each kind of plant GPDH albumen comparison chart:

Adopt ClustalW software that the wild-type GPDH protein sequence of each kind of plant is compared; In figure, Glycine represents soybean (Glycine max), and serial ID is XP_003553315; Medicago represents clover (Medicago truncatula), and serial ID is XP_003622291; Cucumis represents cucumber (Cucumis sativus), and serial ID is XP_004156696; Citrus represents oranges and tangerines (Citrus clementina), and serial ID is XP_006446670; Ricinus represents castor-oil plant (Ricinus communis), and serial ID is XP_002526956; Solanum represents potato (Solanum tuberosum), and serial ID is XP_006357169; Setaria represents broomcorn millet (Setaria italic), and serial ID is XP_004971422; Zea represents corn (Zea mays), and serial ID is NP_001150493; Sorghum represents Chinese sorghum (Sorghum bicolor), and serial ID is XP_002459175; Orazy represents paddy rice (Orazy sativa), and serial ID is Os01g0971600; Hordeum represents barley (Hordeum vulgare), and serial ID is BAK07844; Triticum represents wheat (Triticum aestivum), and serial ID is AGS79224; Underscore in figure, solid line represents NAD (P)-b structural domain, dotted line represents NAD_Gly3P_DH structural domain.

Fig. 2 is expression vector pCB4004- oEGDstructure schematic diagram.

Fig. 3 is in transgenic paddy rice blade oEGDgene expression dose figure.Adopt real-time RT-PCR method to detect oEGDgene is expression amount in transgenic paddy rice blade, and wherein, the XQ of transverse axis represents the fine contrast in kind Hunan (non-transgenic paddy rice), and numbering H1G072-094 represents that difference turns oEGDgene strain; The longitudinal axis represents: log ₂(transgenosis with contrast XQ expression amount multiple).

Fig. 4 is plant height figure after the low-phosphorous processing of transgenic paddy rice.(when paddy growth is during to 4 leaf phase, bring into use without phosphorus formula nutritional liquid pouring paddy rice, the height of plant is added up in without phosphorus pouring for 10 days afterwards; Wherein, the XQ of transverse axis represents the fine contrast in Hunan (non-transgenic paddy rice); H1G072-78 is for turning for numbering oEGDtrans-genetic hybrid rice; The longitudinal axis represents: rice seedling plant height (unit for centimetre); * represent and t-test significance analysis P≤0.05 contrasting, * * represents P≤0.01.

Fig. 5 A is Plant Height of Rice figure after osmotic stress is processed; Fig. 5 B is paddy rice mortalogram after osmotic stress processing rehydration; When paddy growth is during to 4 leaf phase, bring into use the nutritive medium pouring paddy rice containing 20% PEG6000, process and after 7 days, use the processing of normal nutritive medium rehydration, height and the mortality ratio of within 3 days, adding up afterwards plant; In the transverse axis of Fig. 5 A and Fig. 5 B, XQ represents the fine contrast in Hunan, is non-transgenic paddy rice; H1G072-78 is for turning for numbering oEGDtrans-genetic hybrid rice; The longitudinal axis of Fig. 5 A represents: rice seedling plant height (unit for centimetre); The longitudinal axis of Fig. 5 B represents that dead plant number accounts for the ratio of total plant number; * represent and t-test significance analysis P≤0.05 contrasting, * * represents P≤0.01.

Fig. 6 is Plant Height of Rice figure after high temperature stress is processed; (when paddy growth is during to 4 leaf phase, seedling is displaced in high temperature booth, under hot environment, grows after 12 days, be transplanted to room temperature and carry out plant height statistics, unit is centimetre; In the transverse axis of Fig. 6, XQ represents the fine contrast in Hunan (non-transgenic paddy rice); H1G072-78 is for turning for numbering oEGDtrans-genetic hybrid rice; The longitudinal axis of Fig. 6 represents rice seedling plant height (unit for centimetre); * represent and t-test significance analysis P≤0.05 contrasting, * * represents P≤0.01.

Fig. 7 is for turning oEGDin trans-genetic hybrid rice blade, SOD is containing spirogram; When paddy growth is during to 4 leaf phase, 20% PEG6000 processes after 3 days and gets seedling leaves, measures SOD content in blade; In the transverse axis of Fig. 7, XQ represents the fine contrast in Hunan (non-transgenic paddy rice), and H1G072-87 is for turning for numbering oEGDtrans-genetic hybrid rice; The longitudinal axis of Fig. 7 represents SOD units activity in every milligram of fresh weight blade; * represent and t-test significance analysis P≤0.05 contrasting, * * represents P≤0.01.

Fig. 8 is for turning oEGDtrans-genetic hybrid rice is individual plant mean yield figure under Hainan Datian drought stress; When paddy rice enters ear differentiation period, start to carry out the plantation of drought pipe.After maturation, copy kind of a statistics output; The fine contrast in Hunan is non-transgenic paddy rice; In the transverse axis of Fig. 7, Hunan is fine is contrast (non-transgenic paddy rice), and H1G070-87 is for turning for numbering oEGDtrans-genetic hybrid rice; The longitudinal axis represents average every strain output, unit gram.

Fig. 9 is for turning oEGDtrans-genetic hybrid rice is individual plant mean yield figure under the booth drought stress of Shanghai; Each strain single-strain planting, left and right plantation contrast; When paddy rice enters ear differentiation period, start to carry out the plantation of drought pipe; After maturation, copy kind of a statistics output; Transfer-gen plant output and adjacent adjoining tree carry out output comparison; In the transverse axis of Fig. 9, the fine contrast in Hunan (non-transgenic paddy rice), H1G070-87 is for turning for numbering oEGDtrans-genetic hybrid rice; The longitudinal axis represents every strain output, unit gram; * t-test significance analysis P≤0.05 that represents to be adjacent contrast, * * represents P≤0.01.

Embodiment

Herein, term " separation ", " purifying " DNA or gene refer to, this DNA or gene fragment have been arranged in the sequence of its both sides and have separated from native state, also refer to that this DNA or gene fragment with under native state follow the component of nucleic acid to separate, and separate with the protein of following it in cell.

In a specific embodiments of the present invention, a kind of artificial fusion gene of the stress tolerance that improves plant, the polynucleotide sequence that this gene has comprises:

(a) polynucleotide sequence of NAD (P) the combined function structural domain of coding paddy rice GPDH albumen; (b) polynucleotide sequence of the NAD_Gly3P dehydrogenase function structural domain of coding intestinal bacteria gpsA albumen.

Described (a) and (b) sequence comprise the variant form of the polynucleotide sequence of wild-type and the identical function albumen of can encoding, and this variant form is open reading frame part generation nucleotide deletion, the insertion of wild-type sequence and/or replaces rear acquisition.These variant forms comprise, but be not limited to: several (are generally 1-90, preferably 1-60, better 1-20, best 1-10) disappearance, insertion and/or the replacement of Nucleotide, and 5 ' and/or 3 ' end add that several (being generally in 60, is preferably in 30, better is in 10, and best is in 5) Nucleotide.

In a specific embodiments of the present invention, the artificial fusion gene of the stress tolerance of raising of the present invention plant by (a) and (b) sequence form, (a) and (b) sequence be directly formed by connecting.In another specific embodiments of the present invention, to be (a) be formed by connecting by one section of connexon sequence with (b) sequence the artificial fusion gene of the stress tolerance of raising of the present invention plant, this connexon sequence does not affect the coding of albumen, does not affect the function of the albumen of last formation.

One preferred embodiment in, described (b) is for adopting the sequence of favorite plant codon synthetic.

In protein, each amino acid is corresponding to different passwords (few one, many has 6), and different plant species is used the frequency of different passwords different.Plant is used secret code Preference to have common data that Codon Usage Database(http is provided: //www.kazusa.or.jp/codon/).Use preference therefore change password, cannot in existing species, find, just must carry out synthetic.Adopting favorite plant codon artificial synthesized sequence is technique means well-known to those skilled in the art.

The polynucleotide sequence of this artificial fusion gene comprises sequence as shown in SEQ ID NO.1.In a specific embodiments of the present invention, the sequence of this artificial fusion gene is sequence as shown in SEQ ID NO.1.Contriver is by nucleotide sequence called after shown in SEQ ID NO.1 oEGDgene.

The polynucleotide sequence of artificial fusion gene of the present invention also comprises the variant form of open reading frame sequence in the SEQ ID NO. 1 of the albumen with OEGD identical function of encoding.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and hold and add several (being generally in 60 5 ' and/or 3 ', being preferably in 30, is more preferably in 10, is in 5 best) Nucleotide.

At the polynucleotide sequence of artificial fusion gene of the present invention, also comprise and refer to that coding has the degenerate sequence of SEQ ID NO. 1 sequence of OEGD identical function albumen.This degenerate sequence refers to the sequence that has one or more codons be encoded in described sequence to produce after the degenerate codon of same amino acid replaces.Due to the degeneracy of codon, so be low to moderate with SEQ ID NO. 1 sequence homology the aminoacid sequence that 89% degenerate sequence also can be encoded out described in SEQ ID NO. 2.This term also comprises can be under the rigorous condition of moderate, better under highly rigorous condition with the nucleotide sequence of SEQ ID NO. 1 nucleotide sequence hybridization.This term also comprises 1 nucleotide sequence homology at least 89% with SEQ ID NO., preferably at least 90%, and at least 95% nucleotide sequence best.

The aminoacid sequence of this artificial fusion gene coding comprises sequence as shown in SEQ ID NO.2.In a specific embodiments of the present invention, aminoacid sequence sequence as shown in SEQ ID NO.2 of this artificial fusion gene coding, oEGDthe albumen of genes encoding, contriver is by this albumen called after OEGD albumen.

In the present invention, also comprise and having and variant form OEGD identical function, SEQ ID NO.2 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several (being generally in 20 at C-terminal and/or N-terminal, being preferably in 10, is more preferably in 5) amino acid.For example, in the art, while replacement with the close or similar amino acid of performance, conventionally can not change the function of protein.Again such as, the function of adding or several amino acid and conventionally also can not change protein at C-terminal and/or N-terminal.

The percent homology of albumen is by GAP(Needleman and Wunsh, 1970) analyze (GCG program) determine, wherein parameter gap creation penalty=5, gap extension penalty=0.3.When analyzed sequence length is at least 15 amino acid, GAP just analyzes and tests in 15 the amino acid whose regions that are at least of two sequences that participate in test.More preferably, when analyzed sequence length is at least 50 amino acid, GAP just analyzes and tests in 50 the amino acid whose regions that are at least of two sequences that participate in test.More preferably, when analyzed sequence length is at least 100 amino acid, GAP just analyzes and tests in 100 the amino acid whose regions that are at least of two sequences that participate in test.More preferably, when analyzed sequence length is at least 250 amino acid, GAP just analyzes and tests in 250 the amino acid whose regions that are at least of two sequences that participate in test.Even more preferably, when analyzed sequence length is at least 500 amino acid, GAP just analyzes and tests in 500 the amino acid whose regions that are at least of two sequences that participate in test.

By methods known in the art can synthesize, separation and purifying polynucleotide (DNA or RNA), carrier, transformant and organism.

The present invention separates the polynucleotide of synthetic artificial fusion gene, includes but not limited to: the encode nucleotide sequence of OEGD gene of SEQ ID NO. 1; Or this nucleotide sequence can with SEQ ID NO. 1 in from the nucleotide sequence hybridization of Nucleotide 1-1062 position; Or its function is equivalent to the subfragment of sequence shown in SEQ ID NO. 1.

Recombinant vectors for artificial fusion gene of the present invention can be as phage, plasmid, clay, minichromosome, virus or retroviral vector.The carrier that can be used for clone and/or express polynucleotide of the present invention is the carrier that can copy and/or express polynucleotide in need copy and/or express the host cell of polynucleotide.In general; the recombinant expression vector that carries nucleotide sequence of the present invention can use Ti-plasmids, plant viral vector; the conventional biotechnological means such as directly delivered DNA, microinjection, electroporation imports vegetable cell (Weissbach; 1998; Method for Plant Molecular Biology VIII; Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology (2nd Edition).

Having developed several different methods makes polynucleotide operationally be connected with carrier for the sticky end via complementary or recombinase.For example, can be wanting DNA segment ends in insertion vector DNA and add the same aggressiveness sequence fragment of special design.Then by the hydrogen bond connection carrier between complementary homopolymeric tail and DNA section to form recombinant DNA molecule; Or utilize DNA restructuring principle, use specific recombinase to carry out recombining reaction, form recombinant DNA molecules.

Term " is operationally connected " and is expressed as follows situation: some part of linear DNA sequence can affect the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA is operably connected to polypeptid DNA exactly so; If transcribing of promotor control sequence, it is to be operably connected to encoding sequence so; If when ribosome bind site is placed in the position that can make its translation, it is to be operably connected to encoding sequence so.Generally, " being operably connected to " means adjacent, means in reading frame adjacent for secretion leader sequence.

Polynucleotide inset should be operably connected to in the compatible suitable promotor of the host cell of expressing polynucleotide, and promotor can be strong promoter and/or inducible promoter.The example of some promotors of enumerating comprises phageλ PL promotor, intestinal bacteria lac, trP, phoA, tac promotor, SV40 in early days and late promoter and retrovirus LTR promotor; Other suitable promotor is well known by persons skilled in the art.Express recombinant vectors and further contain transcription initiation, termination site, and contain at transcriptional domain the ribosome bind site that is useful on translation.The encoding part of the transcript that recombinant vectors is expressed can comprise the terminator codon (UAA, UGA or UAG) that is positioned at the translation initiation codon at starting point place and is suitably positioned at the end that is translated polypeptide.

As mentioned above, expression vector can comprise at least one selective marker.Described mark comprises the antibiotic resistant gene of coding, for example: neomycin phosphotransferase (Neomycin phosphotransferase) gene npt II, hygromix phosphotransferase (Hygromycin phosphotransferase) gene hpt and Tetrahydrofolate dehydrogenase (Dihydrofolate reductase) gene dhfr; Another kind of is coding herbicide resistance gene, for example, careless fourth phosphinothricin acetyl transferring enzyme (Phosphinothricin acetyltransferase) Bar gene, 5-enol pyruvoyl oxalic acid-3-phosphate synthase (5-Enoylpyruvate shikimatr-3-phosphate) gene epsps.Suitably host's representative example includes but not limited to: protoplasm somatocyte and vegetable cell.The appropriate culture medium of above-mentioned host cell and culture condition are known in the art.

The method for transformation of goal gene or polynucleotide of interest a: class is carrier mediated method for transformation, be inserted on the carrier molecule such as the plasmid of Agrobacterium or the DNA of virus by goal gene, along with the transfer of carrier DNA, goal gene imported in Plant Genome; Agriculture bacillus mediated and virus-mediated method just belongs to this method.Equations of The Second Kind is gene direct guiding method, refers to by the method for physics or chemistry and directly external source goal gene is imported in the genome of plant.Physical method comprises via Particle Bombardment Transformation method, Electroporation method, supersonic method, microinjection and laser microbeam method etc.; Chemical process has PEG mediated transformation method and liposome method etc.The 3rd class is germplasm systems approach, and this comprises pollen tube passage method, sexual cell dip method, blastular and Ovary injection etc.In the present invention, the term of use " transformant " (transformant), with host cell or the organism of allogeneic dna sequence DNA molecule.

The present invention also comprises the host cell that contains nucleotide sequence of the present invention, and described nucleotide sequence can operate and be connected with one or more allos control region (as promotor and/or enhanser) through technology known in the art.Can select to regulate the expression of the gene order of inserting, or can be according to required particular form modification and the host strain of processed gene product.Under the existence of some inductor, the expression that some promotor starts can raise.

Can identify the cell successfully being transformed by well-known technology, contain cell or the organism of the recombinant vectors of nucleotide sequence of the present invention.

In a specific embodiments of the present invention, a kind of method of transgenic plant that produce stress tolerance, the method comprises the following steps:

1) artificial fusion gene claimed in claim 1 is operably connected to expression of plants regulating and controlling sequence, forms plant expression vector;

2) plant expression vector of step 1) gained is proceeded to vegetable cell;

3) transformant obtaining through screening, is regenerated as plant and offspring thereof, and described plant comprises vegetable cell, plant tissue or plant seed.

Above-mentioned " artificial fusion gene is operably connected to expression of plants regulating and controlling sequence ", refers to artificial fusion gene sequence is effectively connected to plant constructive expression or stress-inducing expression promotor.

Described plant is selected from paddy rice, corn, wheat, barley, broomcorn millet, Chinese sorghum, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, oranges and tangerines or cucumber any one.In a specific embodiment of the present invention, the carrier of OEGD gene is proceeded in rice cell and obtains and turn OEGD trans-genetic hybrid rice.

Due to the homology of the height of the GPDH protein sequence of paddy rice, corn, wheat, barley, broomcorn millet, Chinese sorghum, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, oranges and tangerines and cucumber, referring to following embodiment 1, therefore, can be applicable to the artificial fusion gene of rice cell, also can be applicable to corn, wheat, barley, broomcorn millet, Chinese sorghum, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, oranges and tangerines and cucumber, this is that persons skilled in the art can be inferred according to general knowledge known in this field.

Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.

embodiment 1: plant GPDH protein sequence comparison

Download from NCBI gene database platform (http://www.ncbi.nlm.nih.gov/) species such as paddy rice, corn, wheat, barley, broomcorn millet, Chinese sorghum, soybean, cucumber, clover, potato, oranges and tangerines, castor-oil plant GPDH albumen aminoacid sequence (wherein, the aminoacid sequence of the GPDH albumen of paddy rice is SEQ ID NO.7), adopt ClustalW software that plant GPDH protein sequence is compared, concrete outcome is shown in Fig. 1.As can be seen from Figure 1, these derive from dicotyledonous and GPDH albumen unifacial leaf species is very conservative, no matter be NAD (P) combined function structural domain or NAD_Gly3P dehydrogenase structure domain, its sequence similarity is very high, and function between these albumen is also extremely similar.

Therefore,, in genetic manipulation, the domain gene of identical function is exchanged gene function impact very little in these above-mentioned plant species.

Certainly, the gpsA albumen homology in the GPDH albumen of plant origin and intestinal bacteria source is very low, for example, the consistence of paddy rice GPDH albumen and intestinal bacteria gpsA albumen (ID accession:P37606) is only 21.4%, consider congeniality amino acid factor, its similarity is only also 35.1%.The NAD_Gly3P_DH structural domain of the NAD of plant origin (P) combined function structural domain and bacterial origin is manually merged, no matter use which kind of plant sequence, it will be very little to artificial fusion gene function effect.Be the sequence that NAD in the present invention (P) combined function structural domain can use different plant origins, and NAD_Gly3P_DH structural domain carry out source sequence with intestinal bacteria, does not affect the performance of fusion gene function.

The present invention is using paddy rice as the plant origin of embodiment below.

embodiment 2: synthetic separating clone oEGDgene

Download rice Os 01g0971600 and bacterium P37606 sequence from ncbi database platform (http://www.ncbi.nlm.nih.gov/).First separating clone paddy rice GPDH gene.Adopt the total RNA of TRIzol reagent (GIBCO BRL, USA) extracting rice leaf.Utilize ThermoScript II MLV(Tiangen, China) its reverse transcription is become to cDNA.With primer GPDF (5 '-atggagaacggacacgccaagaatc-3 ') and primer GGR(5 '-gctggactccaatgaagtcctctacaacagaaaccagg-3 '), amplification is containing the PCR product of gene NAD (P) combined function structural domain cDNA.PCR reaction conditions is: 94 DEG C of denaturation 3min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 90sec totally 35 circulations; 72 DEG C are extended 5min.The PCR product that amplification is obtained is connected into pGEM-T carrier (Promega, USA), and screening positive clone order-checking, obtain osGPDHthe Partial cDNA Sequence (SEQ ID NO. 3) of gene.Then with reference to bacterium gpsA protein sequence (SEQ ID NO. 5), according to the NAD_Gly3P dehydrogenase structure domain of paddy rice codon preference synthetic bacterium gpsA on DNA synthesizer (SEQ ID NO. 4).Taking composition sequence as template, with primer GGF (5 '-cctggtttctgttgtagaggacttcattggagtccagc-3 ') and primer gpsAR (5 '-cagtgactggagcgctcgtcc-3 '), amplification is containing the PCR product of synthetic DNA fragment.PCR reaction conditions is the same.Subsequently two PCR products are mixed, carry out 94 DEG C of sex change 3min, then make temperature be reduced to gradually 60 DEG C with 1 DEG C of reduction per second, add Taq enzyme and extend 5min at 72 DEG C.Finally add primer GPDF and gpsAR, the synthetic gene that amplification comprises paddy rice NAD (P) combined function structural domain and intestinal bacteria NAD_Gly3P dehydrogenase structure domain.PCR reaction conditions is the same.Pack PCR product into pGEM-T carrier (Promega, USA), screening positive clone order-checking, obtain oEGDthe cDNA sequence (SEQ ID NO. 1) of gene.

embodiment 3: oEGDthe structure of gene overexpression vector and genetic transformation

3.1 structure containing destination gene expression carrier:

According to oEGDthe full length sequence (SEQ ID NO. 1) of gene, design amplifies the primer of complete coding reading frame, and on upstream primer and downstream primer, introduce respectively BP Clonase enzyme recombination sequence site, so that by GATEWAY technique construction expression vector.Taking in embodiment 1 obtain amplified production as template, carry out after pcr amplification through high-fidelity Taq enzyme pfu enzyme (Tiangen, China), will oEGDgene cDNA clone is to intermediate carrier (as pDONR207), further transform bacillus coli DH 5 alpha, ensureing to identify intermediate carrier under the correct prerequisite of reading frame, then extracting plasmid, use pCB4004 plant expression plasmid to carry out the restructuring of LR Clonase enzyme, so just gene two is added respectively to CAMV35S transcripting promoter and terminator, formed one and comprised oEGDgene complete is expressed in the plant expression vector pCB4004::OEGD of unit, transforms Agrobacterium EHA105, finally carries out Rice Callus transformation experiment.

3.2 rice transformation

3.2.1 seed disinfection

After shelling, the ripe fine rice paddy seed in Hunan puts into aseptic triangular flask, with 75% alcohol-pickled 1-2 min, aseptic water washing 2 times; With 30% NaClO, 30 min that sterilize, need frequent shake therebetween again, then with aseptic washing 3-4 time, blot unnecessary moisture with aseptic filter paper, seed is inoculated on callus inducing medium (MS+2,4-D 2.0 mg/L), approximately 30, every ware, in 28 DEG C of dark cultivations.

3.2.2 succeeding transfer culture

Through the induction in nearly January, paddy rice grows the callus that yellow is expanded, and removes its scultellum, and callus is gone on fresh callus inducing medium (MS+2,4-D 2.0 mg/L) and carries out subculture.Once, general subculture can obtain for 2-4 time and be applicable to genetically modified bright yellow, granular embryo callus every 2 weeks subcultures.After 2 weeks, select embryo particle for genetic transformation at succeeding transfer culture.

3.2.3 the cultivation of Agrobacterium

On conversion flat board, picking list bacterium colony is cultivated in 1ml Agrobacterium substratum.In 50ml Agrobacterium substratum (containing corresponding microbiotic), add the above-mentioned culture of 1ml, 200rpm, 28 DEG C are cultivated 5-6hr to OD600 is 0.6-1.0, cultivation finishes front 2hr and adds Syringylethanone (AS, final concentration 100uM).Get above-mentioned bacterium liquid at room temperature, 4000rpm, 10min, abandons supernatant, adds the resuspended thalline of MS liquid nutrient medium (containing AS 100uM), with on cultivate 2hr under identical condition, make the OD600=0.5-1 of bacterium liquid, now can be used to transformed calli.AS=acetosringone Syringylethanone.

3.2.4 cultivate altogether

EMBRYO IN RICE callus is immersed to Agrobacterium bacterium liquid 20-30min, then use aseptic thieving paper suck dry moisture, the callus infecting is placed in to common culture medium (MS+2,4-D 2.0 mg/L+AS 100 uM) upper, 28 DEG C of dark cultivations three days.

3.2.5 wash bacterium

The callus of cultivating altogether is first used aseptic water washing 3 times, then is immersed in containing after 20-30min in the MS liquid nutrient medium of Cef/CN 400 mg/L, and callus is proceeded on aseptic filter paper and blotted.

3.2.6 select to cultivate

The callus of suck dry moisture is inoculated in and is selected on substratum (MS+2,4-D 2.0 mg/L+Hyg 30 mg/L+Cef 400 mg/L).After 3 weeks, select the callus newly growing and be inoculated in selection substratum (MS+2,4-D 2.0 mg/L+Hyg 50 mg/L+Cef 250 mg/L) above, then select 2 weeks.

3.2.7 differentiation culture

The resistant calli obtaining through 2 selections is transferred to upper dark the cultivation about 10 days of pre-division culture medium (N6+KT 2.0 mg/L+NAA 0.2 mg/L+6-BA 2.0 mg/L+Hyg 30 mg/L+Cef 200 mg/L+agar 9g/L+sucrose 45g/L), then forwards the upper illumination cultivation of division culture medium (N6+KT 2.0 mg/L+NAA 0.2 mg/L+6-BA 2.0 mg/L+Hyg 30 mg/L+agar 4.5g/L+sucrose 30 g/L) to.

3.2.8 root culture

About 1-2 month, seedling high 2cm left and right is forwarded to the generation of the upper inducing adventitious root of root media (1/2MS+Hyg 15 mg/L+agar 4.5g/L+sucrose 20g/L).

3.2.9 the transplanting of transgenic seedling

When seedling grows to 10cm when high, seedling is taken out, clean the solid medium adhering to sterilized water, move in earth, just start, with lens cover several days, after plant to be planted stalwartness, to take off again lens, in greenhouse, cultivate.

3.3 the genetic transformation of tobacco

3.3.1 tobacco aseptic seedling numerous soon

First by 75% alcohol immersion 0.5min for tobacco seed, then soak 10min with 2%NaClO, aseptic water washing 3-4 time, with aseptic thieving paper suck dry moisture, is inoculated on MS substratum, and illumination cultivation at 25 DEG C, can obtain tobacco in vitro cuttings.Cut the blade of tobacco in vitro cuttings, remove its main lobe arteries and veins and leaf margin, be cut into about 1cm ²square vanelets, is inoculated in bud division culture medium MS1, after treating that bud grows, cuts single bud and is inoculated in MS substratum, about 20 days, can grow up to plant.

3.3.2 the cultivation of Agrobacterium

On conversion flat board, picking list bacterium colony is cultivated in 1ml Agrobacterium substratum.In 50ml Agrobacterium substratum (containing corresponding microbiotic), add the above-mentioned culture of 1ml, 200rpm, 28 DEG C of shaking culture are spent the night; 4000rpm under room temperature, 10min, abandons supernatant, thalline suspends with 1/2MS liquid nutrient medium, is diluted to the 5-20 of original volume doubly, with on cultivate 2hr under identical condition, make the OD600=0.5 left and right of bacterium liquid.

3.3.3 agrobacterium is infected

The aseptic blade of getting the tobacco of growth about two weeks, removes its main lobe arteries and veins and leaf margin, is cut into about 1cm ²square vanelets.Blade is put into the bacterium liquid preparing, soaked 2-5 min 190rpm, on aseptic filter paper, blot unnecessary bacterium liquid.

3.3.4 cultivate altogether

The leaf explant blotting is placed on to callus induction or division culture medium MS1(MS+6-BA 1.0 mg/L+NAA 0.1 mg/L), 25 DEG C of dark 48hr that cultivate.

3.3.5 select to cultivate

Be added with by proceeding to through the leaf explant of cultivating altogether the de-bacterium differentiation or the callus inducing medium MS29 MS+6-BA 1.0 mg/L+NAA 0.1 mg/L+Hpt 50 mg/L+Cb 250mg/L that select pressure) on, vacuum side of blade is downward, edge is pressed in substratum, under 25 DEG C of illumination, cultivates.Cultivate the formation of 7-15 days visible callus, approximately 20 days afterwards visible Bud Differentiation grow.

3.3.6 root culture

After bud is grown up, cut, be placed in to contain on the root media MS3 (1/2MS+NAA 0.5mg/L+Hpt 25mg/L) that selects to press and carry out root culture, within about 2-7 days, grow adventive root.

3.3.7 the transplanting of transgene tobacco

After well developed root system, plant is taken out, clean with sterilized water the solid medium that adheres to, move in soil, just start, with lens cover several days, after plant to be planted stalwartness, to take off again lens, in greenhouse, cultivate.

embodiment 4: oEGDgene overexpression transfer-gen plant expression analysis

4.1 material is prepared

The transgenic rice plant T2 that embodiment 3.2 is obtained for the fine seed germination of seed and check variety Hunan after, transplant in liquid nutrient medium (international paddy rice institute water-culturing rice nutritive medium, formula is shown in http://irri.org/).After growth of seedling 15 d, clip blade drops into fast liquid nitrogen and preserves, for the extracting of RNA.

4.2 without total RNA preparation of DNA

The leaf RNA providing by Shanghai Hua Shun Bioisystech Co., Ltd is the extracting of extraction agent box working instructions in a small amount.Use Beckman Coulter DU 640 ultraviolet spectrophotometers to measure RNA concentration.For removing the DNA remaining in RNA, each total RNA sample is got 5 μ g, adds 1 μ L DNAase (American I nvitrogen company) and 1 μ L10 × reaction buffer, supply volume to 10 μ L, normal-temperature reaction 30 min, and then every pipe adds 1 μ L 2 mmol L-1 EDTA termination reactions, finally makes DNAase at 70 DEG C of heating 10 min inactivation.

4.3 the first chain cDNA's is synthetic

Above-mentioned RNA sample is respectively got to 2 μ L, the reagent providing by Promega company of U.S. reverse transcription test kit adds 4 μ L 25 mmol L-1 MgCl2 successively, 2 μ L10 × RT damping fluids, the mixed liquid of 2 μ L dNTP and 1 μ L oligo (dT) 15, add water and supply volume to 18.5 μ L, at 70 DEG C of heat denatured 10 min, fast in cooled on ice.Then add 0.5 μ L RNase inhibitor and 1 μ L AMVRTase, at 42 DEG C of water-bath 60 min, heat 10 min termination reactions at 70 DEG C.

4.4 quantitative PCR

According to gene oEGDsequences Design Auele Specific Primer RF:5 '-agcttgcccaccgcttcggag-3 ', RR:5 '-cagtgactggagcgctcgtcc-3 ' is for quantitative fluorescent PCR, according to Actin(GenBank accession No. AY212324) cDNA sequences Design Auele Specific Primer the AF:5 '-cttcctcatgccatcctgc-3 ' of gene, ar:5 '-gcaagcttctccttgat gtcc-3 ' is for the quantitative fluorescent PCR of reference gene.PCR uses American AB I PRISM 7000 quantitative PCR instrument, and each PCR arranges once and repeats.Reaction system comprises SYBR Premix Ex Taq (2 ×) 10 μ L, the each 0.5 μ L of forward and reverse primer, and the cDNA template 1 μ L of various processing, adds water and supplies volume to 25 μ L.Response procedures is: 95 DEG C of 30 s, then at 95 DEG C of 10 s, circulates 40 times under 61 DEG C of 34 s, while being set in each circulation 60 DEG C of 34 s, read fluorescent value, carry out ROX value simultaneously and proofread and correct, finally add the analysis of fluorescence PCR products melt curve analysis, other operations refer to instrument working instructions.In order to detect the pollution that whether has DNA in RNA sample, choose at random 3 samples, respectively to get 1 μ L RNA and carry out PCR as template, method is the same.

4.5 analytical procedure

Ct is defined as 0.2 rear generation in the fluorescence thresholding of PCR by craft by 7000 system SDS Version1.2.3 softwares, enters data into EXCEL and carries out computational analysis.Data analysis employing method is 2 ^{-Δ Δ CT}, then utilize EXCEL table to make differential expression histogram (Δ Δ CT).

4.6 analytical results

As can be seen from Figure 3, oEGDgene all obtains high expression level in transgenic paddy rice strain H1G072-094, and its expression amount compared with the control, all exceedes 100 times, and high expression level amount even exceedes 500(2 ⁹) doubly, show in these transfer-gen plants oEGDgene high expression level normally in paddy rice, can further carry out next step and detect test.

embodiment 5: oEGDgene overexpression transfer-gen plant is resistance to contrary test under laboratory condition

5.1 material is prepared

The transgenic paddy rice T2 that embodiment 3.2 is obtained for the fine seed germination of seed and check variety Hunan after, transplant in liquid nutrient medium (international paddy rice institute water-culturing rice nutritive medium, formula is shown in http://irri.org/).Growth of seedling during to 4 leaf, starts to carry out resistance to osmotic stress, resistance to low-phosphorous and high temperature test.

5.2 stress treatment

For low-phosphorous processing, by the NaH in former nutrient solution prescription ₂pO ₄2H ₂o did not add in the time again preparing nutritive medium, then changed one time of nutrition liquid every 3 days, observed growth of seedling height at the 10th day.

For osmotic stress processing, will in nutritive medium, add every liter of 200 grams of PEG6000, PEG6000 processes after 10 days and changes normal nutritive medium, observes growth of seedling and death condition.

For pyroprocessing, during summer high temperature, transplant seedlings in airtight glasshouse, its diurnal temperature scope is between 30-55 DEG C, and nutritive medium water temperature is the highest exceedes 52 DEG C, cultivates and observes seedling growth after 12 days.

Improve the Physiology and biochemistry reason of plant stress tolerance ability in order to understand these transfer-gen plants, the seedling that 20% PEG6000 is processed 3 days is got blade, measures SOD(superoxide-dismutase in blade) content.Liquid nitrogen grinding blade, gets 0.05g sample powder, adds 1ml physiological saline (0.86%), homogenate, and 3500-4000 rev/min of centrifugal 10min, gets supernatant and measures.Test kit adopts Nanjing to build up the total superoxide-dismutase (T-SOD) of biological study institute and measures test kit (article No.: A001-1 hydroxylamine assay).

5.3 analytical results

Use low-phosphorous nutritive medium pouring rice seedling after 10 days, in 4 transgenic lines, there are 3 transgenic line H1G072,073 and 074 its plant height highly significant or extremely remarkable height and adjoining tree, show that these 3 transgenic lines tolerate low-phosphorous ability apparently higher than contrast, see Fig. 4.Overexpression in paddy rice is described oEGDafter gene, obviously improve the ability that paddy rice tolerates low phosphorus nutrition, can promote plant normal growth on the barren soil of phosphorus nutrition.

Use PEG6000 to process seedling, find between transfer-gen plant and adjoining tree, to present notable difference after 3 days, when 7 days treatment times to the, adjoining tree starts death.Within the 10th day, after rehydration, add up surviving rate and the upgrowth situation of plant, the numbering H1G072-74 transfer-gen plant not only plant height utmost point is significantly higher than contrast, and its mortality ratio is lower than 5%, and contrast mortality ratio exceedes 30%(and sees Fig. 5).After this gene of overexpression is described, obviously improve the Osmotic Stress Tolerance ability of paddy rice, and after rehydration, recovered the ability of growth.

In between high-temperature cultivation, the growth of adjoining tree starts to be suppressed, along with the prolongation of time, adjoining tree poor growth even stops, but contrast transfer-gen plant still can be grown preferably, its plant height is significantly obviously better than contrasting (see figure 6), illustrates that transfer-gen plant has good repairing effect under hot conditions, can ensure that plant maintains the normal growth of plant under the condition that has certain day and night temperature.

After processing, PEG6000 checks SOD content in rice plant blade, find that in transfer-gen plant, content is all significantly higher than adjoining tree (see figure 7), illustrate that transfer-gen plant actively changes the biochemical reactions in plant body after coercing, and resists bad external environment.

embodiment 5: oEGDgene overexpression transgenic paddy rice is at field Drought resistant experiment

Chosen the gene overexpression transgenosis T2 obtaining in embodiment 3.2 and carried out the drought stress test of fringe phase for family plant (numbering H1G070,71,72,81,83,85 and 87), this experiment is carried out in Hainan and Shanghai respectively.First by transfer-gen plant T2 for the fine presprouting of seeds of seed and check variety Hunan after at field sowing.After 3 week by seedling replanting.It is as follows that Hainan Province's winter drying is coerced test: every part of material is planted 3 row, every row 7 strains, when most of material in colony enters ear differentiation period (paddy rice is to tricky time of moisture), start emptying field moisture, after about 2 week, field starts to occur part plant leaf roll, shows to start drought stress symptom.Solid and ripe when most of plant, results seed is weighed, statistics individual plant mean yield.Result shows, under drought stress, the fine single plant yield in check variety Hunan is only 7.27 grams/strain, the present invention clone's oEGDthe transfer-gen plant single plant yield major part of gene overexpression exceedes 9 grams/strain, the highest reaches 10.3 grams/strain, illustrates that its output performance will be better than contrasting (see figure 8) significantly after Drought Stress Stress treatment.

In order to improve the precision of test, in the booth of Shanghai, carry out drought stress test.By seedling replanting, in booth cement pit, each transgenic line is planted 5 strains, in field stochastic distribution, plants 2 strain adjoining trees in transfer-gen plant left and right simultaneously.When most of material in colony enters ear differentiation period, emptied of water is divided and is carried out arid and process.After about 3 week, field starts to occur part plant leaf roll, shows to start drought stress symptom.Along with increasing the weight of of the degree of coercing, part plant leaf starts withered and yellow dying, but there is part transfer-gen plant, still blade maintenance is green.Solid and ripe when most of plant, results seed is weighed, statistics individual plant mean yield.The mean number of transfer-gen plant and adjacent adjoining tree output is compared, find under drought stress, transfer-gen plant single plant yield is obviously better than the fine single plant yield (see figure 9) in check variety Hunan, and wherein contrasting the Hunan average per unit area yield of fine plant is 6.2-10.8 gram/strain, and the present invention clone oEGDthe transfer-gen plant H1G072,73,74 of gene overexpression, 87 average per unit area yields, between 17.6-22.6 gram/strain, are significantly better than contrast, but plant height statistics does not have difference.Illustrate after Drought Stress Stress treatment oEGDthe expression of gene can be alleviated paddy rice and is obstructed entering the plant strain growth that after reproductive growth, drought stress causes; can effectively protect paddy rice to avoid the damage of abiotic stress; improve the setting percentage of plant and seed grouting, strengthened the resistance of transgenic paddy rice to abiotic stress and reduce the loss of output.

embodiment 6:OEGD gene overexpression transgene tobacco is at field Drought resistant experiment

By the transgene tobacco extracting DNA obtaining, PCR is detected to positive tobacco plant and further breed acquisition T2 for seed.Positive transgene tobacco T2 is directly seeded in nutrition pot for seed and contrast non-transgenic tobacco seed.In the time that tobacco seedling grows into 5 leaves, stop moisture and irrigate.After 7 days, contrast the tobacco plant blade wrinkle that starts to wither, and transgenic tobacco plant blade is still extremely unfolded.After fortnight, contrast tobacco stops growing, and has a half vane withered, and adjoining tree blade just starts the wrinkle of withering.After one month, adjoining tree is completely withered, and transgene tobacco still has 2 leaves to keep green.After rehydration three days, contrast tobacco cannot survive, and transgene tobacco starts to bring back to life, and blade is unfolded and come, and grows young leaves.As can be seen from the above results, overexpression OEGD gene in tobacco, can significantly improve the tolerance of tobacco to drought stress, improves the surviving rate of tobacco.Illustrating that this modifying gene can be applied to improves the resistibility of plant to environment stress in various plants.

Scope of the present invention is not subject to the restriction of described specific embodiments, and described embodiment, only as the single example of illustrating all respects of the present invention, also comprises method and the component of functional equivalent in the scope of the invention.In fact,, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to description and accompanying drawing above.Within described improvement also falls into the scope of appended claims.Every section of reference mentioned above is listed in herein as a reference all in full.

<110> Shanghai City Agricultural biological Gene Center

<120> improves the artificial fusion gene of the stress tolerance of plant

<160> 7

<170> PatentIn version 3.3

<210> 1

<211> 1062

<212> DNA

<213> synthetic

<220>

<221> CDS

<222> (1)..(1062)

<400> 1

atggagaacg gacacgccaa gaatcttgtg gccgtcatcg gcagcggcaa ctggggcagc 60

gtcgcctccc gcctcatcgc ttctaacacc gctaagttgc cctcctttca tgatgaagta 120

aggatgtggg tgtttgaaga aatattacca acaggcaaga agctctctga gtccattaac 180

caagcaaatg agaattgcaa atacttacct ggtataaagc tcggagctaa tgtaattgct 240

gatcctgatt tggagaatgc agtgaaagat gcaaatatgc ttgtttttgt gactccccat 300

caatttgtgg agggtatatg taagaagctt gtgggtaaac taaggccagg aactgagggt 360

atctccctca tcaagggcat ggaggtcaag atggaaggac catgcatgat atctaaatta 420

attacaaaca tacttggaat caattgctgt gtccttatgg gtgctaacat tgcaaatgag 480

attgctgttg agaaattcag tgaagcgaca attggatata agaaagacaa ggaagtggca 540

acccgatggg ctaaactttt tacaacacct tacttcctgg tttctgttgt agaggacttc 600

attggagtcc agctcggagg agccgtcaag aacgtcattg ccattggagc cggaatgagt 660

gacggaattg gattcggagc caacgcccgc acagccctca ttacacgcgg actcgccgag 720

atgtcccgcc tcggagccgc cctcggagcc gacccggcca cattcatggg aatggccgga 780

ctcggagacc tcgtcctcac atgcacagag aaccagagtc gcaaccgccg cttcggaatg 840

atgctcggac agggaatgga cgtccagtcc gcccaggaga agattggaca ggtcgtcgag 900

ggatatcgca acacaaagga ggtccgcgag cttgcccacc gcttcggagt cgagatgccg 960

attacagagg agatttatca ggtcctctat tgcggaaaga acgcccgcga ggccgccctc 1020

acactcctcg gacgcgcccg caaggacgag cgctccagtc ac 1062

<210> 2

<211> 354

<212> PRT

<213> synthetic

<400> 2

Met Glu Asn Gly His Ala Lys Asn Leu Val Ala Val Ile Gly Ser Gly

1 5 10 15

Asn Trp Gly Ser Val Ala Ser Arg Leu Ile Ala Ser Asn Thr Ala Lys

20 25 30

Leu Pro Ser Phe His Asp Glu Val Arg Met Trp Val Phe Glu Glu Ile

35 40 45

Leu Pro Thr Gly Lys Lys Leu Ser Glu Ser Ile Asn Gln Ala Asn Glu

50 55 60

Asn Cys Lys Tyr Leu Pro Gly Ile Lys Leu Gly Ala Asn Val Ile Ala

65 70 75 80

Asp Pro Asp Leu Glu Asn Ala Val Lys Asp Ala Asn Met Leu Val Phe

85 90 95

Val Thr Pro His Gln Phe Val Glu Gly Ile Cys Lys Lys Leu Val Gly

100 105 110

Lys Leu Arg Pro Gly Thr Glu Gly Ile Ser Leu Ile Lys Gly Met Glu

115 120 125

Val Lys Met Glu Gly Pro Cys Met Ile Ser Lys Leu Ile Thr Asn Ile

130 135 140

Leu Gly Ile Asn Cys Cys Val Leu Met Gly Ala Asn Ile Ala Asn Glu

145 150 155 160

Ile Ala Val Glu Lys Phe Ser Glu Ala Thr Ile Gly Tyr Lys Lys Asp

165 170 175

Lys Glu Val Ala Thr Arg Trp Ala Lys Leu Phe Thr Thr Pro Tyr Phe

180 185 190

Leu Val Ser Val Val Glu Asp Phe Ile Gly Val Gln Leu Gly Gly Ala

195 200 205

Val Lys Asn Val Ile Ala Ile Gly Ala Gly Met Ser Asp Gly Ile Gly

210 215 220

Phe Gly Ala Asn Ala Arg Thr Ala Leu Ile Thr Arg Gly Leu Ala Glu

225 230 235 240

Met Ser Arg Leu Gly Ala Ala Leu Gly Ala Asp Pro Ala Thr Phe Met

245 250 255

Gly Met Ala Gly Leu Gly Asp Leu Val Leu Thr Cys Thr Glu Asn Gln

260 265 270

Ser Arg Asn Arg Arg Phe Gly Met Met Leu Gly Gln Gly Met Asp Val

275 280 285

Gln Ser Ala Gln Glu Lys Ile Gly Gln Val Val Glu Gly Tyr Arg Asn

290 295 300

Thr Lys Glu Val Arg Glu Leu Ala His Arg Phe Gly Val Glu Met Pro

305 310 315 320

Ile Thr Glu Glu Ile Tyr Gln Val Leu Tyr Cys Gly Lys Asn Ala Arg

325 330 335

Glu Ala Ala Leu Thr Leu Leu Gly Arg Ala Arg Lys Asp Glu Arg Ser

340 345 350

Ser His

354

<210> 3

<211> 614

<212> DNA

<213> Oryza sativa

<220>

<221> misc_feature

<222> (1)..(614)

<400> 3

atggagaacg gacacgccaa gaatcttgtg gccgtcatcg gcagcggcaa ctggggcagc 60

gtcgcctccc gcctcatcgc ttctaacacc gctaagttgc cctcctttca tgatgaagta 120

aggatgtggg tgtttgaaga aatattacca acaggcaaga agctctctga gtccattaac 180

caagcaaatg agaattgcaa atacttacct ggtataaagc tcggagctaa tgtaattgct 240

gatcctgatt tggagaatgc agtgaaagat gcaaatatgc ttgtttttgt gactccccat 300

caatttgtgg agggtatatg taagaagctt gtgggtaaac taaggccagg aactgagggt 360

atctccctca tcaagggcat ggaggtcaag atggaaggac catgcatgat atctaaatta 420

attacaaaca tacttggaat caattgctgt gtccttatgg gtgctaacat tgcaaatgag 480

attgctgttg agaaattcag tgaagcgaca attggatata agaaagacaa ggaagtggca 540

acccgatggg ctaaactttt tacaacacct tacttcctgg tttctgttgt agaggatatt 600

gaaggagttg aatt 614

<210> 4

<211> 447

<212> DNA

<213> synthetic

<220>

<221> misc_feature

<222> (1)..(447)

<400> 4

ggagccgtca agaacgtcat tgccattgga gccggaatga gtgacggaat tggattcgga 60

gccaacgccc gcacagccct cattacacgc ggactcgccg agatgtcccg cctcggagcc 120

gccctcggag ccgacccggc cacattcatg ggaatggccg gactcggaga cctcgtcctc 180

acatgcacag agaaccagag tcgcaaccgc cgcttcggaa tgatgctcgg acagggaatg 240

gacgtccagt ccgcccagga gaagattgga caggtcgtcg agggatatcg caacacaaag 300

gaggtccgcg agcttgccca ccgcttcgga gtcgagatgc cgattacaga ggagatttat 360

caggtcctct attgcggaaa gaacgcccgc gaggccgccc tcacactcct cggacgcgcc 420

cgcaaggacg agcgctccag tcactga 447

<210> 5

<211> 339

<212> PRT

<213> E.coli

<220>

<221> PEPTIDE

<222> (1)..(339)

<400> 5

Met Asn Gln Arg Asn Ala Ser Met Thr Val Ile Gly Ala Gly Ser Tyr

1 5 10 15

Gly Thr Ala Leu Ala Ile Thr Leu Ala Arg Asn Gly His Glu Val Val

20 25 30

Leu Trp Gly His Asp Pro Glu His Ile Ala Thr Leu Glu Arg Asp Arg

35 40 45

Cys Asn Ala Ala Phe Leu Pro Asp Val Pro Phe Pro Asp Thr Leu His

50 55 60

Leu Glu Ser Asp Leu Ala Thr Ala Leu Ala Ala Ser Arg Asn Ile Leu

65 70 75 80

Val Val Val Pro Ser His Val Phe Gly Glu Val Leu Arg Gln Ile Lys

85 90 95

Pro Leu Met Arg Pro Asp Ala Arg Leu Val Trp Ala Thr Lys Gly Leu

100 105 110

Glu Ala Glu Thr Gly Arg Leu Leu Gln Asp Val Ala Arg Glu Ala Leu

115 120 125

Gly Asp Gln Ile Pro Leu Ala Val Ile Ser Gly Pro Thr Phe Ala Lys

130 135 140

Glu Leu Ala Ala Gly Leu Pro Thr Ala Ile Ser Leu Ala Ser Thr Asp

145 150 155 160

Gln Thr Phe Ala Asp Asp Leu Gln Gln Leu Leu His Cys Gly Lys Ser

165 170 175

Phe Arg Val Tyr Ser Asn Pro Asp Phe Ile Gly Val Gln Leu Gly Gly

180 185 190

Ala Val Lys Asn Val Ile Ala Ile Gly Ala Gly Met Ser Asp Gly Ile

195 200 205

Gly Phe Gly Ala Asn Ala Arg Thr Ala Leu Ile Thr Arg Gly Leu Ala

210 215 220

Glu Met Ser Arg Leu Gly Ala Ala Leu Gly Ala Asp Pro Ala Thr Phe

225 230 235 240

Met Gly Met Ala Gly Leu Gly Asp Leu Val Leu Thr Cys Thr Glu Asn

245 250 255

Gln Ser Arg Asn Arg Arg Phe Gly Met Met Leu Gly Gln Gly Met Asp

260 265 270

Val Gln Ser Ala Gln Glu Lys Ile Gly Gln Val Val Glu Gly Tyr Arg

275 280 285

Asn Thr Lys Glu Val Arg Glu Leu Ala His Arg Phe Gly Val Glu Met

290 295 300

Pro Ile Thr Glu Glu Ile Tyr Gln Val Leu Tyr Cys Gly Lys Asn Ala

305 310 315 320

Arg Glu Ala Ala Leu Thr Leu Leu Gly Arg Ala Arg Lys Asp Glu Arg

325 330 335

Ser Ser His

339

<210> 6

<211> 1056

<212> DNA

<213> Oryza sativa

<220>

<221> CDS

<222> (1)..(1053)

<400> 6

atggagaacg gacacgccaa gaatcttgtg gccgtcatcg gcagcggcaa ctggggcagc 60

gtcgcctccc gcctcatcgc ttctaacacc gctaagttgc cctcctttca tgatgaagta 120

aggatgtggg tgtttgaaga aatattacca acaggcaaga agctctctga gtccattaac 180

caagcaaatg agaattgcaa atacttacct ggtataaagc tcggagctaa tgtaattgct 240

gatcctgatt tggagaatgc agtgaaagat gcaaatatgc ttgtttttgt gactccccat 300

caatttgtgg agggtatatg taagaagctt gtgggtaaac taaggccagg aactgagggt 360

atctccctca tcaagggcat ggaggtcaag atggaaggac catgcatgat atctaaatta 420

attacaaaca tacttggaat caattgctgt gtccttatgg gtgctaacat tgcaaatgag 480

attgctgttg agaaattcag tgaagcgaca attggatata agaaagacaa ggaagtggca 540

acccgatggg ctaaactttt tacaacacct tacttcctgg tttctgttgt agaggatatt 600

gaaggagttg aattatgtgg aacactgaaa aatgtcgtgg ccattgcagc aggtcttgtt 660

gatggtttgg atatggggaa caataccaag gctgcaataa tgaggattgg tttgcgggaa 720

atgcgtgctt tctctaagct tctatcccct acagtcaggg acaacacttt ctttgagagc 780

tgtggtgtgg ctgacctaat aactacatgc cttggtggga gaaacagaag agtcgctgag 840

gcctttgcac gaaatggtgg caaaaggtct tttgatgagt tggaggccga gatgctacat 900

ggccaaaaac ttcagggagt gtccacagca aaagaagttt atgaagtctt gacttatcga 960

gggtggcaag aattgtttcc tcttctgtcc acagtgcatg agatttgtat tggtcagcta 1020

cctcctacat caatagttga atacagaatc cactga 1056

<210> 7

<211> 351

<212> PRT

<213> Oryza sativa

<400> 7

Met Glu Asn Gly His Ala Lys Asn Leu Val Ala Val Ile Gly Ser Gly

1 5 10 15

Asn Trp Gly Ser Val Ala Ser Arg Leu Ile Ala Ser Asn Thr Ala Lys

20 25 30

Leu Pro Ser Phe His Asp Glu Val Arg Met Trp Val Phe Glu Glu Ile

35 40 45

Leu Pro Thr Gly Lys Lys Leu Ser Glu Ser Ile Asn Gln Ala Asn Glu

50 55 60

Asn Cys Lys Tyr Leu Pro Gly Ile Lys Leu Gly Ala Asn Val Ile Ala

65 70 75 80

Asp Pro Asp Leu Glu Asn Ala Val Lys Asp Ala Asn Met Leu Val Phe

85 90 95

Val Thr Pro His Gln Phe Val Glu Gly Ile Cys Lys Lys Leu Val Gly

100 105 110

Lys Leu Arg Pro Gly Thr Glu Gly Ile Ser Leu Ile Lys Gly Met Glu

115 120 125

Val Lys Met Glu Gly Pro Cys Met Ile Ser Lys Leu Ile Thr Asn Ile

130 135 140

Leu Gly Ile Asn Cys Cys Val Leu Met Gly Ala Asn Ile Ala Asn Glu

145 150 155 160

Ile Ala Val Glu Lys Phe Ser Glu Ala Thr Ile Gly Tyr Lys Lys Asp

165 170 175

Lys Glu Val Ala Thr Arg Trp Ala Lys Leu Phe Thr Thr Pro Tyr Phe

180 185 190

Leu Val Ser Val Val Glu Asp Ile Glu Gly Val Glu Leu Cys Gly Thr

195 200 205

Leu Lys Asn Val Val Ala Ile Ala Ala Gly Leu Val Asp Gly Leu Asp

210 215 220

Met Gly Asn Asn Thr Lys Ala Ala Ile Met Arg Ile Gly Leu Arg Glu

225 230 235 240

Met Arg Ala Phe Ser Lys Leu Leu Ser Pro Thr Val Arg Asp Asn Thr

245 250 255

Phe Phe Glu Ser Cys Gly Val Ala Asp Leu Ile Thr Thr Cys Leu Gly

260 265 270

Gly Arg Asn Arg Arg Val Ala Glu Ala Phe Ala Arg Asn Gly Gly Lys

275 280 285

Arg Ser Phe Asp Glu Leu Glu Ala Glu Met Leu His Gly Gln Lys Leu

290 295 300

Gln Gly Val Ser Thr Ala Lys Glu Val Tyr Glu Val Leu Thr Tyr Arg

305 310 315 320

Gly Trp Gln Glu Leu Phe Pro Leu Leu Ser Thr Val His Glu Ile Cys

325 330 335

Ile Gly Gln Leu Pro Pro Thr Ser Ile Val Glu Tyr Arg Ile His

340 345 350

Claims (9)

1. an artificial fusion gene that improves the stress tolerance of plant, is characterized in that:

The polynucleotide sequence that this gene has comprises:

(a) polynucleotide sequence of the NAD of coded plant GPDH albumen (P) combined function structural domain; With

(b) polynucleotide sequence of the NAD_Gly3P dehydrogenase function structural domain of coding intestinal bacteria gpsA albumen.

2. artificial fusion gene as claimed in claim 1, is characterized in that:

Described (b) is for adopting the sequence of favorite plant codon synthetic.

3. artificial fusion gene as claimed in claim 2, is characterized in that: the polynucleotide sequence of this artificial fusion gene comprises sequence as shown in SEQ ID NO.1.

4. the albumen of artificial fusion gene coding claimed in claim 3, is characterized in that: the aminoacid sequence of this artificial fusion gene coding comprises sequence as shown in SEQ ID NO.2.

5. contain the recombinant vectors of artificial fusion gene claimed in claim 1.

6. the host cell that adopts recombinant vectors claimed in claim 5 to transform.

7. a method of producing the transgenic plant of stress tolerance, is characterized in that:

The method comprises the following steps:

1) artificial fusion gene claimed in claim 1 is operably connected to expression of plants regulating and controlling sequence, forms plant expression vector;

2) plant expression vector of step 1) gained is proceeded to vegetable cell;

3) transformant obtaining through screening, is regenerated as plant and offspring thereof, and described plant comprises vegetable cell, plant tissue or plant seed.

8. method claimed in claim 7, is characterized in that: described plant is selected from paddy rice, corn, wheat, barley, broomcorn millet, Chinese sorghum, soybean, cucumber, clover, potato, castor-oil plant, peanut, cotton, tobacco, oranges and tangerines or cucumber any one.

9. artificial fusion gene claimed in claim 1 has the purposes aspect the transgenic plant of stress tolerance in preparation.