CN109207508A - A method of improving crop yield - Google Patents

A method of improving crop yield Download PDF

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CN109207508A
CN109207508A CN201810876041.5A CN201810876041A CN109207508A CN 109207508 A CN109207508 A CN 109207508A CN 201810876041 A CN201810876041 A CN 201810876041A CN 109207508 A CN109207508 A CN 109207508A
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郑挺
沈志成
喻景权
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Zhejiang University ZJU
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    • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
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    • C12Y110/99—Oxidoreductases acting on diphenols and related substances as donors (1.10) with other acceptors (1.10.99)
    • C12Y110/99003—Violaxanthin de-epoxidase (1.10.99.3)
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/13—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen (1.14.13)
    • C12Y114/1309—Zeaxanthin epoxidase (1.14.13.90)

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Abstract

The present invention relates to a kind of methods for improving farming yield, and the expression by increasing PsbS, VDE, ZEP gene increases the photosynthetic efficiency of crop, and the expression by increasing TEL CYP78A gene increases the seed of crop.The screening that yield increases crops can screen respectively the crops of the increased crops of photosynthetic efficiency and seed increase by transgenosis, then pass through hybridization polymerization.The expression cassette for increasing photosynthetic PsbS, VDE, ZEP and TEL or CYP78A can also be placed in the same overexpression T-DNA carrier, screen the increased crops of yield.Crop yield can be significantly improved using method of the invention.

Description

A method of improving crop yield
Technical field
The present invention relates to a kind of methods for improving crop yield using gene technology.
Background technique
Improving crop yield is an important goal for improveing crops.There are many ways to providing crop yield is more Sample.A kind of method is to improve photosynthetic efficiency.Such as the photosynthetic work after increasing PsbS, VDE, ZEP expression such as Kromdjik It is improved with efficiency, the biomass of crop significantly improves, but the raising of biomass can not effectively improve the production of crops Amount.Another method for improving crop yield is to improve regulation crop growthing development and realize.For example, overexpression The size of the seed of crops can be improved in TEL gene (U.S. Patent application 20160010100) gene；It is overexpressed CYP78A The albumen of family can increase the size of the seed of crop, such as United States Patent (USP) US9,708,625B2.But improve farming species Sub- size may not be able to effectively improve yield.The problems such as big seed can bring grouting insufficient.Such as Zhao etc. is in mistake After expressing CYP78A72 gene, the soybean of seed increase is obtained, but not can increase soybean yields.
Summary of the invention
It is an object of the present invention to provide a kind of method for improving crop yield, this method is to improve the photosynthesis effect of crop Increase crop seed volume while rate, the crops of acquisition are than only improving photosynthetic efficiency or only regulating and controlling farming Object growth and development more efficiently improves crop yield.The photosynthetic method of raising is that importing is non-photochemical with crop Be quenched (Non photochemincal quenching, NPQ) relevant gene PsbS, VDE, ZEP, to improve these three bases The expression of cause；The method for increasing crop seed is to enhance the expression realization of CYP78A TEL gene by transgenosis.
The technical solution adopted by the present invention are as follows:
The present invention provides a kind of method for improving crop yield, and the method is by light protected protein PsbS gene, purple Flavine Violaxanthin De VDE gene and zeaxanthin epoxidase ZEP gene import receptor crop jointly, import simultaneously CYP78A gene or TEL gene and realize.The PsbS gene, VDE gene and ZEP gene pass through pAtRbcs1A promoter (SEQ ID NO:1), ZEP gene, pAtFBA2 promoter (SEQ ID NO:3), VDE gene, pGAPA-1 promoter (SEQ ID NO:2), PsbS gene, HSP18.2 terminator (SEQ ID NO:4) successively import receptor work jointly after functional connection Object.The CYP78A gene is by specific promoter shown in nucleotide sequence SEQ ID NO:10 and CYP78A gene and NOS Terminator (shown in SEQ ID NO:11) functional splice imports receptor crop together；The TEL gene is by nucleotides sequence Arrange specific promoter shown in SEQ ID NO:10 and TEL gene and NOS terminator (shown in SEQ ID NO:11) functional splice Receptor crop is imported together.
Further, the method that the PsbS gene, VDE gene and ZEP gene import receptor crop jointly are as follows: (1) PsbS gene, VDE gene and ZEP gene terminate sub-functionality with arabidopsis HSP18.2 respectively and connect, and obtain PsbS base respectively Because of segment, VDE genetic fragment, ZEP genetic fragment；The arabidopsis HSP18.2 terminator nucleotides sequence is SEQ ID NO:4 It is shown；(2) the hygromycin gene hpt II in pCAMBIA1300 carrier is replaced with into bar shown in SEQ ID NO:8 Gene is denoted as carrier 1300-B；(3) by pAtRbcs1A promoter and step (1) shown in carrier 1300-B and SEQ ID NO:1 The connection of ZEP genetic fragment, is denoted as plasmid 1300-Z-B；(4) pAtFBA2 shown in plasmid 1300-Z-B and SEQ ID NO:3 is opened Mover, the connection of step (1) VDE genetic fragment, are denoted as plasmid 1300-V-Z-B；(5) by plasmid 1300-Z-B and SEQ ID NO: PGAPA-1 promoter shown in 2, the connection of step (1) PsbS genetic fragment, are denoted as plasmid VPZB, and plasmid VPZB is imported receptor and is made Object.
Further, the enhancing receptor crop CYP78A gene or the method for TEL gene expression are as follows: (1) by nucleotides sequence CP4 gene cloning shown in SEQ ID NO:9 is classified as to carrier pUC57, is denoted as carrier pUC57-CP4；(2) by nucleotide sequence NOS terminator function shown in specific promoter shown in SEQ ID NO:10 and rice CYP78A11 gene and SEQ ID NO:11 Being stitched together for property, is denoted as plasmid pUC57-P；(3) by specific promoter shown in nucleotide sequence SEQ ID NO:10 and jade NOS terminator is functional shown in rice TEL gene ZmTE1 and SEQ ID NO:11 is stitched together, and is denoted as plasmid pUC57-T； (4) plasmid pUC57-P or plasmid pUC57-T is imported to the receptor crop of the VPZB containing plasmid.
Further, the crops include soybean, corn, rice or rape.
The present invention is by importing PsbS, VDE, ZEP gene, the transformant for selecting photosynthetic efficiency to improve；Pass through raising The expression of TEL or CYP78A promotes to be used as growth and development, the transformant for selecting seed to increase；PsbS, VDE, ZEP expression are added Strong transformant polymerize with the transformant that TEL or CYP78A expression is reinforced, the genetic engineering farming that yield potentiality significantly improves Object.
The method that the present invention improves crop photosynthesis efficiency is by accelerating the non-Photochemical quenching relaxation (NPQ of crop Relaxation velocity interpolation).Non- Photochemical quenching is the mechanism that plant is used to protect photosynthesizer under strong light. When plant is in the overgenerous environment of illumination, the energy of absorption can not be converted to chemical energy in time by photochemical reaction, just Protection mechanism be will start to protect the optical antenna complex excessively excited.In plant photosystem II (PSII), in excessive Excessive energy is converted to harmless thermal energy using NPQ by the antenna complex of excited state.Several different mechanism can lure PSII optical antenna complex is led into non-Photochemical quenching state, (energy is quenched including dependent on energy Dependent quenching, qE) and lutein (Violaxanthin) in Zeaxanthin cycle go epoxidation to generate Xanthin (anthoxanthin) and luteole (Zeaxanthin dependent quenching, qZ).When environment is from strong When light is transformed into dim light, the luminous energy of plant absorption declines, and optical antenna complex is no longer on the state excessively excited, but right and wrong Photochemical quenching relaxation lags behind environmental change so that part originally can be used for photochemically reactive energy by NPQ lose at Thermal energy, to affect photosynthetic efficiency.It is in tobacco studies have shown that enhance simultaneously relevant to qE PsbS gene, The expression of VDE, ZEP gene relevant to qZ, can accelerate NPQ relaxation, plant is dropped faster when illumination dies down Low NPQ is horizontal, to increase the energy for carbon solidification effect, improves photosynthetic efficiency.The present invention improves photosynthetic work It is by the expression of raising crop PsbS, VDE, ZEP gene, to accelerate what non-Photochemical quenching relaxation was realized with efficiency.
PsbS is the light protected protein in plant thylakoids film, is worked in the qE of NPQ, it is anticipated that different plants PsbS has similar function, and those skilled in the art can measure the function of PsbS by the prior art.Of the invention PsbS can be from any plant, and preferred PsbS comes from arabidopsis (amino acid sequence NP_001319163.1, nucleotide Sequence NM_001333231.1), corn (amino acid sequence NP_001105228.2, nucleotide sequence NM_001111758.2), Rice (amino acid sequence XM_015765683.1, nucleotide sequence XP_015621169.1), soybean (amino acid sequence NM_ 001289308.1, nucleotide sequence NP_001276237.1), rape (amino acid sequence XP_013667449.1, nucleotides sequence Arrange XM_013811995.2).
Violaxanthin de epoxidase VDE (violaxanthin de-epoxidase) is catalyzed viomellein (Violaxanthin) and the redox reaction of ascorbic acid (ascobate), generate luteole (Zeaxanthin) and Hydroascorbic acid, those skilled in the art can measure the activity of VDE by the prior art.VDE of the invention can be From any plant, preferred VDE comes from arabidopsis (amino acid sequence NP_001321301.1, nucleotide sequence NM_ 001331777.1), corn (amino acid sequence NP_001147756.1, nucleotide sequence NM_001154284.2), rice (ammonia Base acid sequence XP_015636342.1, nucleotide sequence XM_015780856.1), soybean (amino acid sequence NP_ 001241404.1, nucleotide sequence NM_001254475.1), rape (amino acid sequence XP_013641072.1, nucleotides sequence Arrange XM_013785618.2).
Zeaxanthin epoxidase ZEP (zeaxanthin epoxidase) is catalyzed the epoxidation of luteole, this field Technical staff can pass through the prior art measure VDE activity.ZEP of the invention can be from any plant, preferably VDE comes from arabidopsis (amino acid sequence NP_851285.1, nucleotide sequence NM_180954.3), corn (amino acid sequence NP_001151443.1, nucleotide sequence NM_001157971.1), rice (amino acid sequence XP_015636352.1, nucleosides Acid sequence XM_015780866.1), soybean (amino acid sequence NP_001276261.1, nucleotide sequence NM_ 001289332.1), rape (amino acid sequence NP_001302817.1, nucleotide sequence NM_001315888.1).
CYP78A gene is a kind of P450 gene, have a large number of studies show that, the table of the P450 gene of plant CYP78A family It reaches and spends and the size of seed is related, the expression for improving CYP78A gene can increase the seed of plant.The present invention is for increasing The gene of big vegetable seeds includes but is not limited to CYP78A1 (amino acid sequence NP_001106069.2, nucleotide sequence NM_ 001112599.2), CYP78A5 (amino acid sequence NP_172827.1, nucleotide sequence NM_101240.4), CYP78A6 (ammonia Base acid sequence NP_182189.1, nucleotide sequence NM_130231.4), CYP78A9 (amino acid sequence NP_191747.1, core Nucleotide sequence NM_116053.3), CYP78A11 (amino acid sequence XP_015613477.1, nucleotide sequence XM_ 015757991.1), CYP78A13 (amino acid sequence XP_015646235.1, nucleotide sequence XM_015790749.1), CYP78A72 (amino acid sequence XP_003554680.1, nucleotide sequence XM_003554632.3).
Have in U.S. Patent application 20160010100 in detail by improving the method that TEL gene expression increases vegetable seeds Thin description.The present invention includes but is not limited to rice Os TEL gene (amino acid sequence for increasing the TEL gene of crop seed XP_015646802.1, nucleotide sequence XM_015791316.1), corn ZmTE1 gene (amino acid sequence NP_ 001104903.1, nucleotide sequence NM_001111433.1).
The polynucleotides of coding PsbS, VDE, ZEP, CYP78, TEL gene can there are many variations.The change of polynucleotides It is different to include but is not limited to: 1) since the codon that encodes same amino acid different obtain different polynucleotide sequence, this A little nucleic acid sequence encodings have mutually homotactic polypeptide: 2) from the genetic diversity of biology, the i.e. difference of same plant Diversity between body or group；3) variation of polynucleotides is imported by manual operation.The variation manually imported can be with Machine, it is also possible to targetedly directed variation.Those of ordinary skill in the art can pass through the side of general molecular biology Method generates point mutation, insertion or deletion mutation etc..The variation that polynucleotides are imported by manual operation also includes engineer Synthesize gene.
The polynucleotide sequence of PsbS, VDE, ZEP, CYP78, TEL gene of the present invention, those skilled in the art Member usually can use the methods of PCR, DNA hybridization and clone from plant.The polynucleotide sequence design provided according to the present invention Primer can obtain PsbS, VDE, ZEP, CYP78, TEL gene of the present invention by PCR method, in addition can also be direct The sequence of offer according to the present invention, these artificial synthesized genes.
The method of the present invention includes the method by molecular biology, building improves crop photosynthesis comprising the present invention PsbS, VDE, ZEP gene, increase vegetable seeds PLA1 TEL gene expression cassette；Expression cassette can by one or Polynucleotide sequence (i.e. expression original part, including promoter, enhancer, terminator etc.) and the aforementioned base of the multiple control expression of person Because functionality connection obtains.Improving photosynthetic gene expression frame can lead respectively with the expression cassette for increasing crop seed gene Enter crop, then by the method polymerization of hybridization, object can also be turned into the same T-DNA carrier transfer by constructing.
Promoter for enhancing PsbS, VDE, ZEP gene of the present invention can be constitutive promoter, be also possible to green The promoter of organizing specific.The promoter that the present invention is used to express PsbS, VDE, ZEP includes but is not limited to AtRbsc1A starting Sub (SEQ ID NO:1), AtGAPA-1 promoter (SEQ ID NO:2), AtFBA2 promoter (SEQ ID NO:3).
Promoter for enhancing CYP78A, TEL gene expression of the present invention can be constitutive promoter, be also possible to plant The special promoter of son, preferred seed specific promoter.Promoter for CYP78A, TEL gene expression of the present invention include but It is not limited to SEQ ID NO:10.
The terminator for controlling gene expression of the present invention can be the natural terminator of the gene, be also possible to kindred plant The terminator of other genes or other plant.Common terminator includes NOS terminator (the SEQ ID from Agrobacterium NO:11), the HSP18.2 terminator (SEQ ID NO:4) etc. of arabidopsis.
The present invention can include a selection gene expression frame for constructing the carrier of gene expression frame simultaneously.This selection The cell that marker gene can be used to select to have converted, common selectable marker gene such as neomycin phosphotransferase, hygromycin Phosphotransferase, phosphinothrycin transacetylase, CP4EPSPS, G10evo EPSPS etc..Other selected marker bases The selection gene converted because being also used as the present invention.
PsbS, VDE, ZEP provided by the invention obtain the increased crop of photosynthesis, the present invention by importing target crop The CYP78A or TEL of offer obtain the crop that seed increases by importing target crop.It can be photosynthetic increasing respectively What gene and the gene transferred crop for increasing crop seed, the transformant for selecting photosynthesis to be remarkably reinforced respectively and seed increased Then transformant polymerize two kinds of characters by hybridizing, obtains the crop of volume increase, can also will enhance photosynthesis and increase kind The gene constructed of son converts target crop in a T-DNA carrier, and screening photosynthesis enhances and seed increases, so that producing Measure increased transformant.The present invention will preferably enhance the label of the expression cassette and a kind of antiweed of photosynthetic three genes Assortment of genes conversion crop screens the crop of photosynthetic crop enhancing, and antiweed marker gene includes but is not limited to bar, cp4 epsps, g10evo epsps.The present invention will preferably increase the expression cassette of the gene C YP78 or TEL of crop seed with it is aforementioned The different antiweed marker gene combination of antiweed marker gene, the anti-herbicide gene packet combined with CYP78 or TEL Include but be not limited to bar, cp4 epsps, g10evo epsps.Such as enhance photosynthetic gene and the bar assortment of genes, it sieves The crop transformant that photosynthesis enhances and is resistant to glufosinate is selected, gene and the cp4 epsps assortment of genes of seed are increased, The crop transformant that seed increases simultaneously energy resistance glyphosate is filtered out, the two can be sprayed simultaneously by the offspring of hybridization polymerization acquisition Glyphosate and glufosinate are applied, to simplify the process of screening-gene polymerization individual.
Those of ordinary skill in the art can utilize existing technology by channel genes of the invention into plant at present. More commonly used method is the method for Agrobacterium-mediated Transformation method and particle gun, and method of gene introduction of the invention includes but is not limited to Both methods.
The method for transformation and step of different plants are different.Commonly used approach is will by Agrobacterium or particle gun T-DNA imports immature embryo, mature embryo, undifferentiated callus or the protoplast of plant, then by screening accordingly Culture medium carries out screening and culturing, then carries out differentiation and obtain transformation bud, obtains transgenic seedling by culture of rootage.The present invention relates to Crop include corn, rice, soybean, rape.
The identification method for the crop transformant that the crop for the photosynthesis enhancing that the present invention obtains, seed increase includes phenotype Observation, sprinkling herbicide, molecular biology method etc..In practical applications, a variety of methods can be used together.The present invention increases Strong photosynthetic crop transformant has apparent phenotypic difference compared to non-transgenic crop, can be recognized by naked eyes, It is characterized in that the transformant of photosynthesis enhancing compares non-transgenic crop on biomass and increases significantly.The present invention The crop transformant that seed increases has apparent difference compared to non-transgenic control crop, can be recognized by naked eyes, special Sign is that seed significantly increases.
Compared with prior art, the beneficial effects of the present invention are embodied in: by photosynthesis enhancing and seed increase two kinds of property Shape is combined, and is solved to harvest seed as in the crop of target, enhancing photosynthesis does not increase seed can not be by life The increase of object amount is sufficiently used for the increase of seed quality, and only increase seed, which does not enhance photosynthesis, can not effectively increase yield Problem.The technology can effectively improve the yield of crop, and effect of increasing production is up to 10% or more.
Detailed description of the invention
Fig. 1 is VPZB carrier schematic diagram
Fig. 2 is PG carrier schematic diagram.
Fig. 3 is TG carrier schematic diagram.
Specific embodiment
Embodiment 1 enhances the building of three expression vector VPZB of photosynthetic efficiency
1, the acquisition of arabidopsis Rbcs1A promoter
With arabidopsis gene group (Arabidopsis thaliana) for template, primer pR-F-EcoRI and pR-R- are used BglII, using the PRIMESTAR archaeal dna polymerase of Takara company, reaction condition is 94 DEG C of 5min, (98 DEG C of 10s, 56 DEG C 10s, 72 DEG C of 90s) 32 circulations, 72 DEG C sufficiently extend 5min.Amplification obtains pAtRbcs1A (the SEQ ID that length is 1319bp No:1), PCR product is cloned into Peasy-simple-blunt by topo, and selection sequence is sequenced and correctly clones, and orders Entitled peasy-pRbcs1A is used for subsequent operation.
pR-F-EcoRI:5’-AATTCAAATTTATTATGTGTTTTTTTTCCGTGG-3’
pR-R-BglII:5’-AGATCTATTTAGTTAATATATAATGGTATGATTC-3’。
2, the acquisition of arabidopsis GAPA-1 promoter
Using arabidopsis gene group as template, using primer pG-F-SacI and pG-R-BglII, using the identical side of step 1 Method and condition, amplification obtain the pAtGAPA-1 (SEQ ID No:2) that length is 1247bp, and PCR product is cloned by topo Peasy-simple-blunt, and selection sequence is sequenced and correctly clones, peasy-pAtGAPA-1 is named as subsequent behaviour Make.
pG-F-SacI:5’-GAGCTCTCTCGTGTTGAGCTTAAGTCTTGTCTTA-3’
pG-R-BglII:5’-AGATCTGAGAGAATATTGGCCAAAGGATTTAGTA-3’。
3, the acquisition of arabidopsis FBA2 promoter
Using arabidopsis gene group as template, using primer p-F-KpnI and pF-R-BglII, using the identical method of step 1 And condition, amplification obtain the pAtFBA2 (SEQ ID No:3) that length is 1289bp, PCR product is cloned by topo Peasy-simple-blunt, and selection sequence is sequenced and correctly clones, peasy-pAtFBA2 is named as in subsequent operation.
p-F-KpnI:5’-GGTACCTGGAGGTGTTGAAAGTATAGAAGAAGAT-3’
pF-R-BglII:5’-AGATCTAGATTCGTTCAAGATACTGTTTCTTTAA-3’。
4, the acquisition for the arabidopsis VDE gene being connect with termination sub-functionality
(it is NM_001331777.1 that nucleotides sequence is classified as gene accession number to arabidopsis VDE gene, and amino acid sequence gene is stepped on Record number be NP_001321301.1) cds by with arabidopsis HSP18.2 terminator (nucleotide SEQ ID NO:4) manually spell Genetic fragment shown in nucleotide sequence SEQ ID NO:5 is obtained after connecing, and is manually closed according to genetic fragment shown in SEQ ID NO:5 At AtVDE-ter and it is cloned into carrier pUC57, sequencing correctly clone is chosen, is named as pUC57-VDE-ter for subsequent Operation.
5, the acquisition for the arabidopsis ZEP gene being connect with termination sub-functionality
(it is NM_180954.3 that nucleotides sequence is classified as gene accession number to arabidopsis ZEP gene, and amino acid sequence gene logs in Number be NP_851285.1) cds by with arabidopsis HSP18.2 terminator (SEQ ID NO:4) manually splicing after obtain core Genetic fragment shown in nucleotide sequence SEQ ID NO:6 according to the artificial synthesized AtZEP-ter of SEQ ID NO:6 and is cloned into carrier PUC57 chooses sequencing correctly clone, is named as pUC57-ZEP-ter for subsequent operation.
6, the acquisition for the arabidopsis PsbS gene being connect with termination sub-functionality
(it is NM_001333231.1, amino acid sequence gene that nucleotides sequence is classified as gene accession number to arabidopsis PsbS gene Accession number is NP_001319163.1) cds by with arabidopsis HSP18.2 terminator (SEQ ID NO:4) manually splicing after Obtain nucleotide sequence SEQ ID NO:7 shown in genetic fragment, according to the artificial synthesized AtPsbS-ter of SEQ ID NO:7 and gram Grand to carrier pUC57, selection sequencing is correctly cloned, and is named as pUC57-PsbS-ter for subsequent operation.
7, it is overexpressed the acquisition of VPZB carrier
With pCAMBIA1300 carrier (nucleotide sequence gene accession number AF234296) for skeleton, by hygromycin therein Resistant gene is come out with Xho I digestion, is replaced with artificial synthesized bar gene (SEQ ID NO:8), is named as 1300-B.
1300-B is used into (EcoR I, Sac I) double digestion, the carrier 1300-B (E, S) that recycling size is 8426bp.It will Peasy-pAtRbcs1A (EcoR I, Bgl II) double digestion, the segment pAtRbcs1A (E, B2) that recycling length is 1313bp. By pUC57-AtZEP-ter (BamHI, Sac I) double digestion, the segment AtZEP-ter (B1, S) that length is 2268bp is recycled. By 1300-B (E, S), pAtRbcs1A (E, B2), three sections of AtZEP-ter (B1, S) connections, connection product is converted to Escherichia coli TG1, picked clones identify that digestion products are in two bands, size in agarose gel electrophoresis with (EcoRI, Sac I) digestion The clone of respectively 8426bp, 3573bp are correctly to clone, and are denoted as 1300-Z-B, are used for subsequent operation.
By 1300-Z-B use (BamHI, Kpn I) double digestion, recycling size be 11986bp carrier 1300-Z-B (B1, K).By peasy-pAtFBA2 (Kpn I, Bgl II) double digestion, recycle segment pAtFBA2 that length is 1279bp (K, B2).By pUC57-VDE-ter BamHI digestion, the segment VDE-ter (B1) that length is 1649bp is recycled.By 1300-Z-B (B1, K), pAtFBA2 (K, B2), three sections of AtVDE-ter (B1) connections, connection conversion to e. coli tg1, picked clones are used Bgl II digestion identification, digestion products are in two bands in agarose gel electrophoresis, and size is respectively 11984bp, 2938bp's Clone is correctly clone, is denoted as 1300-V-Z-B, is used for subsequent operation.
1300-V-Z-B is used into (Sac I, Kpn I) double digestion, the carrier 1300-V-Z-B that recycling size is 14916bp (S,K).By peasy-pGAPA-1 (Sac I, Bgl II) double digestion, the segment pAtGAPA-1 that length is 1237bp is recycled (S,B2).By pUC57-PsbS-ter (BamHI, Kpn I) double digestion, the segment PsbS-ter that length is 1062bp is recycled (B1,K).By 1300-V-Z-B (S, K), pAtGAPA-1 (S, B2), three sections of AtPsbS-ter (B1, S) connections, connection conversion is extremely Escherichia coli, picked clones identify that digestion products are in two bands, size point in agarose gel electrophoresis with Sac I digestion Not Wei 15525bp, the clone of 1690bp is correct clone, is denoted as VPZB (Fig. 1), VPZB plasmid is used for subsequent Agrobacterium Conversion.
The acquisition of the increase of 2 seed of embodiment expression vector PG, TG
Artificial synthesized CP4 gene (SEQ ID NO:9) is cloned into pUC57, selects the correct clonal marker of sequencing and is PUC57-CP4 is used for subsequent operation.By the Xho I digestion of pCAMBIA1300 carrier, the carrier that length is 7864bp, mark are recycled It is denoted as 1300 (X).By pUC57-CP4 XhoI digestion, the segment that size is 1727bp is recycled, is labeled as CP4 (X), by 1300 (X) it is connect with CP4 (X), product is converted to e. coli tg1, picked clones, is identified with (Nco I, Kpn I) digestion, digestion Product is in two bands on agarose gel electrophoresis, and size distinguishes 8406bp, and the clone of 1173bp is correctly to clone, and is denoted as 1300-G is used for subsequent operation.
By soybean CYP78A72 gene promoter (SEQ ID NO:10), with rice CYP78A11 gene (nucleotide sequence Gene accession number XM_015791316.1, amino acid sequence gene accession number XP_015646802.1) cds, NOS terminator (SEQ ID NO:11) is functional to be stitched together (nucleotide sequence SEQ ID NO:12), is named as P, artificial synthesized P, And it is cloned into pUC57, it is pUC57-P that correct clone designation, which is sequenced,.
By soybean CYP78A72 gene promoter (SEQ ID NO:10), with corn TEL gene ZmTE1 (nucleotide sequence Gene accession number NM_001111433.1, amino acid sequence gene accession number NP_001104903.1), NOS terminator (SEQ ID NO:11) functional to be stitched together (SEQ ID NO:13), it is named as T, artificial synthesized T, and be cloned into pUC57, is sequenced Correct clone designation is pUC57-T.
It is poly- using the PRIMESTAR DNA of Takara company using primer r-F and r-R using pUC57-P plasmid as template Synthase, reaction condition are 94 DEG C of 5min, and (98 DEG C of 10s, 56 DEG C of 10s, 72 DEG C of 4min) 32 circulations, 72 DEG C sufficiently extend 10min.Amplification obtains the product that length is 3888bp, and PCR product is recycled by agarose gel electrophoresis, is named as P-r and is used for Subsequent operation.
r-F:5’-TTGCATGCCTGCAGGTCGACAACTCATGGAGTCCACTATCATCACTTTC-3’
r-R:5’-TACGAATTCGAGCTCGGTACCTCAAGCTGGGGATATCCCCGATCTAGTA-3’
It is poly- using the PRIMESTAR DNA of Takara company using primer r-F and r-R using pUC57-T plasmid as template Synthase, reaction condition are 94 DEG C of 5min, and (98 DEG C of 10s, 56 DEG C of 10s, 72 DEG C of 4min) 32 circulations, 72 DEG C sufficiently extend 10min.Amplification obtains the product that length is 4149bp, and PCR product is recycled by agarose gel electrophoresis, is named as T-r and is used for Subsequent operation.
1300-G (Sal I, Kpn I) double digestion, the carrier segments that recycling size is 9551bp, is labeled as 1300-G (S,K)。
Using the Clonexpress II one step cloning kit of Vazyme company, it is separately added into 45 × ce of μ L The μ L recombinase of 1300-G (S, K), the P-r of 300ng, 2 (exnase) of buffer, 300 ng, with sterile ultrapure water polishing to 20 μ It is immediately placed in after L, 37 DEG C of reaction 30min and stands 5min on ice, product is then converted into e. coli tg1.Selected clone is surveyed Sequence, it is PG (Fig. 2) that correct clonal marker, which is sequenced, and PG plasmid is used for subsequent Agrobacterium-mediated Transformation.
Using the Clonexpress II one step cloning kit of Vazyme company, it is separately added into 45 × ce of μ L The μ L exnase of 1300-G (S, K), the P-r of 300ng, 2 of buffer, 300ng, with sterile ultrapure water polishing to 20 μ L, 37 DEG C It is immediately placed in after reaction 30min and stands 5min on ice, product is then converted into e. coli tg1.Selected clone sequencing, sequencing Correct clonal marker is TG (Fig. 3), and TG plasmid is used for subsequent Agrobacterium-mediated Transformation.
VPZB, PG, TG vector introduction Agrobacterium LBA4404 are obtained the Agrobacterium for being used to convert plant by embodiment 3
LB culture medium composition: 5g/L yeast extract, 10g/L peptone, 10g/L NaCl, solvent are pure water, pH value It is natural.
YEP solid medium composition: 10g/L yeast extract, 10g/L peptone, 5g/L NaCl, 15g/L agar are molten Agent is pure water, and pH value is natural.
The plasmid of VPZB, PG, TG are imported into Agrobacterium LBA4404 bacterial strain with the method for Agrobacterium-mediated Transformation respectively.Specifically Method is to be shocked by electricity after mixing the plasmid of 200ng with 100 μ L Agrobacterium LBA4404 competence using 1800V, 5ms, after electric shock Mixed liquor is transferred to sterile 1.5mL centrifuge tube, and the LB culture medium of the non-added with antibiotic of 1ml is added, 28 DEG C of stationary culture 1h Afterwards, 100 μ L bacterium solutions are taken to be applied on the YEP solid medium containing 50mg/L kanamycins+15mg/L tetracycline, 28 DEG C of cultures After 48h, picked clones Liquid Culture identifies correctly clone, obtains the Agrobacterium containing VPZB, the agriculture bar containing PG respectively Bacterium and Agrobacterium containing TG are used for Plant Transformation.
Embodiment 4 obtains the genetically engineered soybean for importing VPZ gene
B5 medium: a great number of elements (KNO32500mg/L、MgSO4·7H2O 250mg/L、CaCl2·2H2O 150mg/L、 (NH4)2SO4134mg/L、NaH2PO4·H2O150mg/L), microelement (KI 0.75mg/L, H3BO33.0mg/L、 MnSO4· 4H2O10mg/L、ZnSO4·7H2O2.0mg/L、Na2MoO4·2H2O 0.25mg/L、CoCl2·6H2O 0.025 mg/L、 CuSO4·5H2O 0.025mg/L), molysite (Na2-EDTA 37.3mg/L、FeSO4·7H2O 27.8mg/L), organic principle (inositol 100mg/L, niacin 1.0mg/L, puridoxine hydrochloride 1.0mg/L, thiamine hydrochloride 1.0mg/L), solvent is deionized water.
Infect culture medium: 1/10 × B5 a great number of elements, 1/10 × B5 microelement, 1/10 × B5 organic matter, 2.8mg/L FeSO4·7H2O, 3.8mg/L Na2Then the gibberellic acid (GA3) of filtration sterilization is added in-EDTA, 120 DEG C of high pressure sterilization 15min 0.25mg/L, 6- benzylaminopurine (BAP) 1.67mg/L, cysteine (Cysteine) 400mg/L, dithiothreitol (DTT) (Dithiothrietol) (storing liquid is 40mg/ml acetosyringone to 154.2mg/L and 40mg/L acetosyringone, molten Agent is dimethyl sulfoxide), solvent is deionized water, pH 5.4.
SIM culture medium: 1 × B5 a great number of elements, 1 × B5 microelement, 1 × B5 organic matter, 28mg/L FeSO4·7H2O, 38mg/L Na2- EDTA, 30g/L sucrose, 0.59g/L MES (2-morpholine ethane sulfonic acid), 2.9g/L coagulator (gelrite) are high BAP (1.11mg/L) is added after warm high pressure sterilization, Ticarcillin/Clavulanate Acid (Timentin) 50mg/L, pH 5.6.
MS culture medium: a great number of elements (KNO3 1900mg/L、NH4NO3 1650mg/L、MgSO4·7H2O 370mg/L、 CaCl2·2H2O 440mg/L、KH2PO4170mg/L, niacin 0.5mg/L, Vt-B6 (Pyridoxin-HCl) 0.5mg/L, Vt-B1 (Thiamine-HCl) 0.1mg/L, inositol 100mg/L, glycine 2.1mg/L), microelement (MnSO4·4H2O 22.3mg/L、KI0.83mg/L、H3BO3 6.2mg/L、ZnSO4·7H2O 8.6mg/L、CuSO4·5H2O 0.025mg/L、 Na2MO4·2H2O 0.25mg/L、CoCl2·6H2O 0.025mg/L、FeSO4·7H2O 27.8mg/L、Na2EDTA 37.3 Mg/L), solvent is deionized water.
SEM culture medium: 1 × MS a great number of elements, 1 × MS microelement, 1 × B5 organic matter, 28mg/L FeSO4·7H2O, 38mg/L Na2EDTA, 30g/L sucrose, 0.59g/L 2-morpholine ethane sulfonic acid (MES) and 2.9g/L gelrite, high temperature are high After pressure sterilizing, asparagine acid 50mg/L, the Pidolidone 100mg/L of filtration sterilization, heteroauxin (IAA) 0.1mg/ is added L, GA3 (0.5mg/L), zeatin (Zeatin) 1mg/L, Timentin (50mg/L), glufosinate-ammonium (Glufosinate) 8mg/ L, solvent are deionized water, pH 5.7.
RM culture medium: 1/2 × MS a great number of elements, 1/2 × MS microelement, 1/2 × B5 organic matter, 28mg/L FeSO4· 7H2O, 38mg/L Na2EDTA, 20g/L sucrose, 0.59g/L MES, 2.9g/L gelrite after autoclave sterilization, are added Indolebutyric acid (IBA) 1mg/L, glufosinate (3mg/L) of filtration sterilization, pH 5.6.
The technology of transformation of soybean current comparative maturity, such as Paz et al. report " Improved in 2004 cotyledonary node method using an alternative explant derived from mature Seed for efficient Agrobacterium-mediated soybean transformation. ", and it is international special It is discussed in detail in benefit application PCT/US2003/019212, here is the detailed description to transformation of soybean method.
The grand No.1 soya seeds in mature day of full health are selected as receptor soybean, are put into containing chlorine (mass concentration 36% concentrated hydrochloric acid 5ml+ sodium hypochlorite 20ml+ water 80ml reaction generate) container in sterilize 16h.Soybean after disinfection is placed in Super-clean bench blows 30min or so, removes the chlorine on surface, is then seeded in GM culture medium (i.e. B5 medium), in 25 DEG C, 140 umol/s/m2Under illumination, culture 18h or so, until cotyledon greening, and rough leaf not yet grows completely, it is square in parallel along hilum To cutting, remove kind of a skin, and two panels cotyledon is separated, obtains two panels and respectively contain a piece of cotyledon, half of hypocotyl and half of epicotyl Explant.
The Agrobacterium containing VPZB carrier that embodiment 3 obtains is in+15mg/L the tetracycline of kanamycins containing 50mg/L Plate culture is drawn for 24 hours on YEP solid medium.It scrapes cultured Agrobacterium and is in infecting to be resuspended in culture medium to OD600 0.4-0.6, as the Agrobacterium infected liquid containing VPZB carrier.
Explant and the Agrobacterium infected liquid containing VPZB carrier are mixed, then 22 DEG C of dark training 2h outwell infected liquid, Explant is in the infected liquid for removing surface on the dry filter paper of sterilizing.Explant is gone in sky culture dish, sealed membrane is used (parafilm) it seals, after 24 DEG C are secretly trained 3 days, explant is gone into SIM culture medium, in 24 DEG C, illumination/8 hour are black within 16 hours Dark period, intensity of illumination 140umol/s/m2, after a week, explant is transferred to containing 8mg/L glufosinate bud inducement cultivation SIM culture medium, in 24 DEG C, 16 hours illumination/8 hour dark cycles, intensity of illumination 140umol/s/m2, screened Culture, replaces a subculture every 2 weeks, until screening obtains the bud of 2-3cm.The tissue for removing screening necrosis, by what is filtered out Bud goes to SEM culture medium, and in 24 DEG C, 16 hours illumination/8 hour dark cycles, intensity of illumination is 140 umol/s/m2, elongation Culture 2-3 weeks, obtains the bud of 8-10cm.The bud of elongation is cut, is transferred to RM culture medium, 24 DEG C, 16 hours illumination/8 hour Dark photoperiod, intensity of illumination 140umol/s/m2, carry out culture of rootage.Healthy and strong root is born after 1-2 weeks, by what is taken root Seedling is transferred to greenhouse, peat culture medium (- 1P) in potting culture, until harvest VPZB transformant T0 generation kind Son.
Potting is in greenhouse together for the seed of VPZB transformant T0 generation and non-transgenic receptor soybean, and transgenosis T1 is for plant 1:100 times of volume ratio diluted protect is sprayed in 3 leaf phases to try to plant up to the isolated non-transgenic of (18% glufosinate-ammonium soluble concentrate) removing Strain.The transformant of photosynthesis enhancing is presented as that the biomass of plant increases in phenotype, highly gets higher.Biomass is chosen to increase Add 5% or more single copy transformant GmVPZB.
Embodiment 5 obtains the transgenic corns for importing VPZ gene
The Technical comparing of corn transformation is mature, such as the report of Ishida in 2007 et al. " Agrobacterium- Mediated transformation of maize. ", here are the specific descriptions to corn transformation method.
1, corn transformation culture medium mother liquor is prepared:
10 × LS a great number of elements: 19.0g KNO3, 16.5g NH4NO3, 4.4g CaCl2·2H2O, 3.7gMgSO4· 7H2O, 1.7g KH2PO4, deionized water is settled to 1L.
100 × FeEDTA (molysite): 2.78g FeSO4·7H2O, 3.73g Na2EDTA, deionized water are settled to 1L.
100 × LS microelement: 2.23gMnSO4·5H2O, 1.06gZnSO4·7H2O, 620mg H3BO3, 83mg KI, 25.0mg Na2MoO4·2H2O, 2.5mg CuSO4·5H2O, 2.5mgCoCl2·6H2O, deionized water are settled to 1L.
100 × LS organic matter: weighing 10g inositol (myo-inositol), 100mg thiamine hydrochloride (thiamine Hydrochloride), 50mg puridoxine hydrochloride (pyridoxine hydrochloride), 50mg niacin (nicotinic Acid), deionized water is settled to 1L.
100mg/L 2,4 dichlorophenoxyacetic acid (2,4-D): with 1N NaOH be slowly added dropwise in 100mg 2,4-D until 2,4-D powder are completely dissolved, and sterile deionized water is settled to 1L.
100mg/L zeatin (zeatin): the zeatin in 100mg is slowly added dropwise until zeatin powder with 1N NaOH It is completely dissolved, sterile deionized water is settled to 1L.
100mg/L indolebutyric acid (IBA): the IBA in 100mg is slowly added dropwise with 1N NaOH until IBA powder is completely molten Solution, sterile deionized water are settled to 1L.
100mg/L 6- benzylaminopurine (6-BA): the 6-BA in 100mg is slowly added dropwise until 6BA powder with 1N NaOH End is completely dissolved, and sterile deionized water is settled to 1L.
100mM acetosyringone (acetosyringone): 392.4mg acetosyringone is weighed, 10ml is used DMSO dissolution, is then added 10ml sterile deionized water, filtration sterilization after mixing.
LS-inf culture medium: into 700ml distilled water be added 100ml 10 × LS a great number of elements, 10ml 100 × 100 × LS microelement of FeEDTA, 10ml, 100 × LS organic matter of 10ml, the 2,4-D of the 100mg/L of 15ml, it mixes molten 68.46g sucrose, 36.04g glucose and 1.0g methionine (Casamino acids) are added into mixture by Xie Hou, mix It is settled to 1L, adjust pH to 5.2 and is filtered with 0.22 μm of filter.
LS-inf-As culture medium: 1 μ L 100mM acetosyringone is added into 1ml LS-inf culture medium.
LS-AS solid medium: by 100ml 10 × LS a great number of elements, 100 × LS of 10ml 100 × FeEDTA, 10ml Microelement, 10ml 100 × LS organic matter, 15ml 100mg/L 2,4-D, 0.05ml 100mM CuSO4It is dissolved in 700ml steaming Then distilled water weighs and 20g sucrose is added, 10g glucose, 0.7g proline, and 0.5g MES is settled to 1L, is adjusted to pH 5.8 And 3g gelrite is added, 1ml 100mM is added when temperature is down to 50 DEG C or so in autoclave sterilization Acetosyringone, 0.05ml 100mM AgNO3。
LSD1.5A culture medium: by 100ml 10 × LS a great number of elements, 100 × LS of 10ml 100 × FeEDTA, 10ml is micro- Secondary element, 10ml 100 × LS organic matter, 15ml 100mg/L 2,4-D are dissolved in 700ml distilled water, and 20g sucrose is then added, 0.7g proline, 0.5g MES are settled to 1L after mixing.PH to 5.8 is adjusted, 3g gelrite, autoclave sterilization is added. 50 DEG C or so are down to culture medium temperature after sterilizing, 1ml 0.1ml of 100mM AgNO is added3, 20 g/L grass ammonium of 0.25ml Phosphine (phosphinothricin), 1ml 200mg/L timentin.
LSD1.5B culture medium: by 100ml 10 × LS a great number of elements, 100 × LS of 10ml 100 × FeEDTA, 10ml is micro- Secondary element, 10ml 100 × LS organic matter, 15ml 100mg/L 2,4-D are mixed in 700ml distilled water, then weigh addition 20g sucrose, 0.7g proline and 0.5g MES are settled to 1L, adjust pH to 5.8 and 3g gelrite, high temperature and pressure is added Sterilizing is down to 50 DEG C or so to temperature, 0.1ml of 100mM AgNO is added3, 0.25ml 20g/L Phosphinothricin, 1ml 200mg/ml timentin.
LSZ culture medium: by 100ml 10 × LS a great number of elements, the micro member of 10ml 100 × LS of 100 × FeEDTA, 10ml Element, 10ml 100 × LS organic matter, 50ml 100mg/L zeatin, 0.1m 100mM CuSO4700 distilled water are mixed in, then 20g sucrose is added and 0.5g MES is settled to 1L.It adjusts pH to 5.8 and 3g gelrite, autoclave sterilization, wait train is added It supports base temperature and is down to 50 DEG C or so, 1ml 200mg/ml timentin, 0.25ml 20g/L phosphinothricin is added.
LSF culture medium: by 100ml 10 × LS a great number of elements, the micro member of 100 × FeEDTA of 10ml, 10ml100 × LS Element, 100 × LS of 10ml is organic, and the 100mg/L indolebutyric acid of 2ml is dissolved in 700ml pure water, and 15g sucrose is then added, 0.5g MES is settled to 1L, and pH is adjusted to be 5.8 and 3g gelrite, autoclave sterilization is added.
2, the preparation of immature embryo: 10 days or so after pollination A188 corncob are taken, with 20% sodium hypochlorite of mass concentration After aqueous solution surface sterilization, immature embryo (length is 1mm or so) is collected, is placed in equipped with 1.4mlLS-inf culture medium In 2ml centrifuge tube (every Guan Yueke places 100 embryos).By the embryo of suspension 2,700rpm vortex 5s, LS-inf is then absorbed, New LS-inf culture medium 1.8ml is added, again 2,700rpm vortex 5s.Embryo after vortex is then fast in 46 DEG C of water-bath 3min Speed is transferred on ice, ice bath 1min.It absorbs LS-inf and new LS-inf culture medium 1.8ml is added.In 4 DEG C, 20, 000g is centrifuged 10min.
3, the preparation of Agrobacterium infected liquid: the Agrobacterium containing VPZB carrier prepared by embodiment 3 draws plate in YEP culture medium On, 25 DEG C are cultivated 24 hours.Agrobacterium is scraped, being resuspended in LS-inf-As culture medium to OD600 is 1.0, and as Agrobacterium is invaded Dye liquor.
4, corn immature embryo is infected and is co-cultured: by the culture in centrifuge tube after 20,000g centrifugation in above-mentioned steps 2 Base suction discards, and the Agrobacterium infected liquid that the ready OD600 of 1ml above-mentioned steps 3 is 1.0 is added, by centrifuge tube in 2, 700 rpm vortex 30s, are then stored at room temperature 5min.The mixed liquor of embryo and bacterium is transferred to sky culture dish, is sucked out therein 0.7ml liquid, discards.Embryo is transferred to LS-AS solid medium, scultellum to be made is paid attention to when putting upward, in 25 DEG C of dark trainings 7 It.
5, kanamycin-resistant callus tissue: after dark training co-cultures 7 days, embryo is transferred to LSD1.5A culture and is cultivated 10 days based on 25 DEG C Carry out primary screening.After one sieve, embryo is transferred to LSD1.5B culture and carries out two sieves within culture 21 days based on 25 DEG C.By two sieves The callus of generation is transferred to new LSD1.5B culture medium, 25 DEG C dark culture 21 days, screening obtains kanamycin-resistant callus tissue three times.
6, the regeneration of seedling: being transferred to LSZ culture medium for step 5 kanamycin-resistant callus tissue, and in 25 DEG C, 5,000lx light are trained 14 days, obtains Obtain regeneration bud.Regeneration bud is transferred to LSF culture medium, trains 14 days in 25 DEG C, 5,000lx light, obtains the plant to take root.It takes root Plant be transferred to greenhouse peat culture medium (- 1P) plantation, it is expanded to female fringe, when filigree is not yet revealed, by female fringe bagging, It when filigree is more, is pollinated using non-transgenic A188 plant pollen, obtains the T0 of VPZB corn transformation body for seed.
The T0 of VPZB corn transformation body for seed and non-transgenic A188 together potting in glasshouse (28-32 DEG C, it is natural Light), transgenosis transformant tries the nontransgenic plants isolated up to removing in diluted protect of 4 leaf phases spray volume ratio 1:100.It is photosynthetic The corn transformation body of effect enhancing is presented as that the biomass of plant increases in phenotype, highly gets higher.To the height of different transformant Degree is analyzed, and single copy transformant ZmVPZB that biomass increases by 5% or more is chosen.
Embodiment 6 obtains the transgenic paddy rice for importing VPZ gene
The technology of rice conversion is highly developed at present, usually infects mature embryo-derived callus then with Agrobacterium It is screened by selective agent and obtains kanamycin-resistant callus tissue, finally by kanamycin-resistant callus tissue seedling differentiation and induce root system, such as Sahoo et al. Report in 2011 " An improved protocol for efficient transformation and Regeneration of diverse indica rice cultivars " is specifically retouching to rice conversion method below It states.
1, the preparation of rice conversion culture medium mother liquor:
10 × N6 a great number of elements: 28.3g/L KNO3, 1.66g/L CaCl2·2H2O, 1.85g/L MgSO4·7H2O, 4g/ L KH2PO4, 4.63g/L (NH4)2SO4, solvent is deionized water.
100 × B5 microelement: 75mg/L KI, 300mg/L H3BO3, 200mg/L MnSO4·7H2O, 25mg/L Na2Mn·2H2O, 2.5mg/L CoCl2·6H2O, 2.5mg/L CuSO4·5H2O, solvent are deionized water.
100 × B5 organic matter: 10g/L inositol, 100mg/L niacin, 100mg/L vitamin B6,1g/L thiamine hydrochloride (thiamine HCl), solvent is deionized water.
100 × Fe:3.73g/L Na2EDTA, 2.78g/L FeSO4·7H2O, solvent are deionized water.
1mg/ml 2,4-D: with 1N NaOH be slowly added dropwise in 1mg 2,4-D until 2,4-D powder be completely dissolved, sterilize Deionized water is settled to 1mL.
10 × AA a great number of elements: 29.5g/L KCl, 2.5g/L MgSO4·H2O, 1.5g/L NaH2PO4·H2O, 1.5g/L CaCl2·2H2O, solvent are deionized water.
100 × AA amino acid: 87.6g/L Pidolidone, 26.6g/L L-Aspartic acid, 17.4g/L L-arginine, 0.75g/L glycine, solvent are deionized water.
100 × MS organic matter: 10g/L inositol, 50mg/L niacin, 50mg/L vitamin B6,40mg/L vitamin B1 are molten Agent is deionized water.
10 × MS a great number of elements: 16.5g/L NH4NO3, 19g/L KNO3, 4.4g/L CaCl2·2H2O, 3.7g/L MgSO4·7H2O, 1.7g/L KH2PO4, solvent is deionized water.
100 × MS microelement: 80mg/L KI, 620mg/L H3BO3, 2.23g/L MnSO4·4H2O, 25mg/L Na2MoO4·2H2O, 2.5mg/L CoCl2·6H2O, 2.5mg/L CuSO4·5H2O, solvent are deionized water.
NBD culture medium: 100ml 100 × N6 a great number of elements, 10ml 100 × B5 microelement, 100 × B5 of 10ml have Machine object, 10ml 100 × Fe, 2ml 1mg/ml 2, after 4-D is mixed with 700ml distilled water, weighs 500mg L-PROLINE, 500mg Pidolidone, 300mg methionine (Bacto casamino acids), 30g sucrose are settled to 1L after mixing, adjust pH To 5.8,2.4g gelrite, autoclave sterilization is added.
AAM culture medium: 100ml 10 × AA a great number of elements, 10ml 100 × AA amino acid, 1ml 100 × MS organic matter, It is mixed with 700ml distilled water, weighs and 500mg bacto casamino acids, 68.5g sucrose is added, 36g glucose mixes It is settled to 1L after even, adjusts pH to 5.2,100 μM of acetosyringone of 1ml are added after cooling in autoclave sterilization.
NBDC culture medium: 100ml 10 × N6 a great number of elements, 10ml N6 microelement, 1ml 1000 × N6 organic matter, 700ml distilled water is added in 2ml 1mg/ml 2,4-D, weighs after mixing and 1g Bacto casamino acids, 30g sugarcane is added Sugar, 10g glucose are settled to 1L after mixing, adjust pH to 5.2, and 2.5g gelrite is added, and autoclave sterilization is dropped to temperature 1ml 100uM acetosyringone is added when to 50 DEG C.
NBDS culture medium: 100ml 100 × N6 a great number of elements, 10ml 100 × B5 microelement, 100 × B5 of 10ml have Machine object, 10ml 100 × Fe, 2ml 1mg/ml 2, after 4-D is mixed with 700ml distilled water, weighs 500mg L-PROLINE, 500mg Pidolidone, 300mg Bacto casamino acids, 30g sucrose are settled to 1L after mixing, adjust pH to 5.8, 2.4g gelrite is added, 0.25ml 20mg/L is added when culture medium temperature is down to 50 DEG C in autoclave sterilization phosphinothrithin。
PRE culture medium: 100ml 10 × MS a great number of elements, 10ml 100 × MS organic matter, 5ml 1mg/ml ABA, 1ml 1mg/ml methyl α-naphthyl acetate (NAA), 5ml 1mg/ml 6-BA after mixing with 700ml distilled water, are weighed and 30g sucrose, 20g L- are added D-sorbite is settled to 1L after mixing, adjust pH to 5.8, and 3g gelrite, autoclave sterilization is added.
MSD culture medium: 100ml 10 × MS a great number of elements, 10ml 100 × MS microelement, 10ml MS organic matter, 3ml 1mg/ml 6-BA, 200 μ L 1mg/ml NAA are mixed with 700ml distilled water, weigh and 30g sucrose is added, and are mixed, constant volume To 1L, pH to 5.8 is adjusted, 3g gelrite, autoclave sterilization is then added.
RM culture medium: 50ml 10 × MS a great number of elements, 5ml 100 × MS microelement, 10ml 100 × MS organic matter, 200 μ L 1mg/ml NAA are mixed with 700ml distilled water, weigh and 20g sucrose is added, and mixing is settled to 1L, adjust pH to 5.8, so 2.8g gelrite, autoclave sterilization are added afterwards.
2, the mature seed decladding of rice varieties show water 134,75% alcohol surface sterilization 2min, sterile washing 3 times, then use Just face sterilizes 25min to 20% aqueous sodium hypochlorite solution of mass concentration, and after drying on filter paper, seed is put for sterile washing 4 times It puts to NBD culture medium, secretly trains induction in 7 days for 28 DEG C and obtain callus.The callus that induction obtains is cut, and changes to new NBD Culture medium squamous subculture is spare to diameter 5mm or more.The Agrobacterium containing VPZB plasmid that embodiment 3 obtains is drawn to be cultivated in YEP Basal growth for 24 hours after, scrape and be resuspended in AAM culture medium, make OD600 0.4 or so.The Agrobacterium being resuspended is cured with ready Injured tissue mixing, then 28 DEG C, 80rpm dark culturing 30min outwell liquid, callus are placed in the dry filter paper of sterilizing On dry, be then placed into NBDC culture medium, 28 DEG C after dark training 2 days, are washed callus 4 times with sterile water, then in the dry of sterilizing It is dried on dry filter paper, callus is put to 28 DEG C of NBDS culture medium dark trainings, a NBDS culture medium is replaced every two weeks, until resistance Callus is grown.The good kanamycin-resistant callus tissue of vigor is transferred to 28 DEG C of PRE culture medium dark trainings to carry out within 7 days after breaking up in advance, callus is turned Shifting has in the culture dish of aseptic filter paper, and 28 DEG C of dark trainings are 3 days dry.Dry callus is transferred to MSD culture medium, 30 DEG C, 16 is small Shi Guangzhao/8 hour dark photoperiod, 350 uE/m of intensity of illumination2/ s, differentiation culture turn seedling until generating the seedling of 3cm or so Move to RM culture medium, 28 DEG C culture of rootage 2 weeks or so.Seedling after taking root is transferred to the T0 of greenhouse harvest VPZB rice conversion body Generation seed.
The T0 of VPZB rice conversion body for seed and non-transgenic rice together potting in glasshouse (28-32 DEG C of temperature, Natural light), transgenosis transformant is tried to plant up to the isolated non-transgenic of removing in diluted protect of after planting 4 weeks spray volume ratio 1:100 Strain.The rice conversion body of photosynthesis enhancing is presented as that the biomass of plant increases in phenotype, highly gets higher.Choose biology Matter increases by 5% or more single copy transformant OsVPZB.
Embodiment 7 obtains the genetically engineered soybean for improving TEL expression
The soybean that TEL is overexpressed is obtained by Agrobacterium-medialed transformation, and step is similar to Example 4, the main distinction Be using carrier be TG that embodiment 2 constructs, the selection markers of TG carrier are Antiglyphosate gene CP4, and selective agent is Glyphosate, method particularly includes:
The mature grand No.1 soya seeds in day for selecting full health, are put into containing chlorine (36% concentrated hydrochloric acid of mass concentration 5ml+ sodium hypochlorite 20ml+ water 80ml reaction generate) container in sterilize 16h.Soybean after disinfection is placed in super-clean bench to blow 30min or so, removes the chlorine on surface, is then seeded in GM culture medium (i.e. B5 medium), in 25 DEG C, 140umol/s/m2 18h or so is cultivated under intensity of illumination, until cotyledon greening, and rough leaf not yet grows completely, cuts along hilum parallel direction, Remove kind of a skin, and two panels cotyledon is separated, obtains two panels and respectively contain the outer of a piece of cotyledon, half of hypocotyl and half epicotyl Implant.
The Agrobacterium containing TG carrier that embodiment 3 obtains is in the YEP with the+15mg/L tetracycline of kanamycins containing 50mg/L Plate culture is drawn for 24 hours for 28 DEG C on solid medium.It scrapes cultured Agrobacterium and is weighed in infecting in culture solution (with embodiment 4) Hanging to OD600 is 0.4-0.6.
Explant and the Agrobacterium infected liquid containing TG carrier are mixed, then 22 DEG C of dark training 2h outwell infected liquid, outside Implant is in the infected liquid for removing explant surface on the dry filter paper of sterilizing.Explant is gone in sky culture dish, is used Parafilm is sealed, and explant after dark training 3 days, is gone to SIM culture medium (with embodiment 4) by 24 DEG C, in 24 DEG C, 16 small time According to/8 hours dark cycles, intensity of illumination 140umol/s/m2, bud induce after a week, explant is transferred to containing 30mg/L The SIM culture medium of glyphosate (glyphosate), in 24 DEG C, 16 hours illumination/8 hour dark cycles, intensity of illumination 140umol/s/m2It is screened, replaces a subculture every 2 weeks, until screening obtains the bud of 2-3cm.Remove screening necrosis Tissue, goes to SEM culture medium (with embodiment 4, glufosinate-ammonium is replaced with 10mg/L glyphosate) for the bud filtered out, in 24 DEG C, 16 hours illumination/8 hour dark cycles, intensity of illumination 140umol/s/m2, culture 2-3 weeks is extended, the bud of 8-10cm is obtained. The bud of elongation is cut, is transferred to RM culture medium (with embodiment 4, removing glufosinate-ammonium), 24 DEG C, illumination/8 hour are black within 16 hours Dark photoperiod, intensity of illumination 140umol/s/m2, carry out culture of rootage.Healthy and strong root is born after 1-2 weeks, and the seedling taken root is turned Move on to greenhouse, peat culture medium (- 1P) in potting culture, until harvest TG transformant T0 generation seed.
Potting is in greenhouse together for the seed of the T0 generation of TG transformant and non-transgenic receptor soybean, and transgenosis T1 is for plant 1:200 times of volume ratio diluted agriculture is sprayed in 3 leaf phases, and isolated non-transgenic plant is removed up to (41% glyphosate isopropylamine salt water solution) Strain.The transformant of TG carrier is presented as that seed increases in phenotype, and 100-grain weight increases.Hundred are counted to the seed of different transformant Grain weight, the transformant list that selected seed 100-grain weight increases by 5% or more copy GmTG.
Embodiment 8 obtains the transgenic corns for improving TEL expression
The corn that TEL is overexpressed is obtained by Agrobacterium-medialed transformation, and step is similar to Example 5, the main distinction Be using carrier be TG that embodiment 2 constructs, the selection markers of TG carrier are Antiglyphosate gene CP4, and selective agent is Glyphosate.Detailed process is as follows.
The A188 corncob for taking 10 days or so after pollinating, after 20% aqueous sodium hypochlorite solution surface sterilization of mass concentration, Immature embryo (length is 1mm or so) is collected, the 2ml equipped with 1.4mlLS-inf culture medium (composition is with embodiment 5) is placed in In centrifuge tube (every Guan Yueke places 100 embryos).By the embryo of suspension 2,700rpm vortex 5s, LS-inf is then absorbed, is added New LS-inf 1.8ml, again 2,700rpm vortex 5s.Embryo after vortex is in 46 DEG C of water-bath 3min, then rapidly transfer It arrives on ice, ice bath 1min.It absorbs LS-inf and new LS-inf 1.8ml is added.In 4 DEG C, 20,000g are centrifuged 10min.
The preparation of Agrobacterium infected liquid: the Agrobacterium containing TG carrier that embodiment 3 obtains draws plate and trains on YEP culture medium It supports 24 hours.Agrobacterium is scraped, being resuspended in LS-inf-As culture medium (with embodiment 5) to OD600 is 1.0.
Corn immature embryo is infected and is co-cultured: the culture medium in centrifuge tube after above-mentioned 20,000g centrifugation being sucked out and is abandoned It goes, and the Agrobacterium infected liquid that the above-mentioned ready OD600 of 1ml is 1.0 is added, by centrifuge tube and 2,700rpm vortex 30s, Then it is stored at room temperature 5min.The mixed liquor of embryo and bacterium is transferred to sky culture dish, 0.7ml liquid therein is sucked out, discards.It will Embryo is transferred to LS-AS solid medium (with embodiment 5), and scultellum to be made is paid attention to when putting upward, secretly trains 7 days in 25 DEG C.
It screens kanamycin-resistant callus tissue: after dark training 7 days, embryo being transferred to LSD1.5A culture medium (with embodiment 5) in 25 DEG C Cultivate 10 days progress primary screenings.After one sieve, embryo is transferred to LSD1.5B culture medium (with embodiment 5, glufosinate-ammonium replacement For 1ml 100mg/ml glyphosate) two sieve of progress in 21 days is cultivated in 25 DEG C.The callus that two sieves generate is transferred to new LSD1.5B culture medium, 25 DEG C are screened acquisition kanamycin-resistant callus tissue in dark culture 21 days three times.
The regeneration of transformation seedlings: kanamycin-resistant callus tissue is transferred to LSZ culture medium, and (with embodiment 5, glufosinate-ammonium replaces with 1ml 100mg/ml glyphosate), in 25 DEG C, 5,000lx light train 14 days to obtain regenerated bud.The bud that regeneration obtains is transferred to LSF training It supports base (with embodiment 5), is trained 14 days in 25 DEG C, 5,000lx light, obtain the plant to take root.The plant to take root is transferred to greenhouse mud The plantation of charcoal culture medium, expands to female fringe, when filigree is not yet revealed, female fringe bagging is used non-transgenic when filigree is more A188 plant pollen pollination, obtain TG transformant T0 for seed.
The seed and non-transgenic receptor A188 corn of the T0 generation of TG transformant together potting in glasshouse (28-32 DEG C, natural light), transgenosis T1 sprays 1:200 times of diluted agriculture in 3 leaf phases for plant and reaches (41% glyphosate isopropylamine salt water solution) Remove isolated nontransgenic plants.The transformant of TG carrier is presented as that seed increases in phenotype, and 100-grain weight increases.To not Seed with transformant counts 100-grain weight, and selected seed 100-grain weight increases by 5% or more single copy transformant ZmTG.
Embodiment 9 obtains the transgenic paddy rice for improving TEL expression
The rice that TEL is overexpressed is obtained by Agrobacterium-medialed transformation, and step is same as Example 5, the main distinction Be using carrier be TG that embodiment 2 constructs, the selection markers of TG carrier are Antiglyphosate gene CP4, and selective agent is Glyphosate is detailed process below.
The 134 mature seed decladding of rice varieties show water, 75% alcohol surface sterilization 2min, sterile washing 3 times, then use quality After drying on filter paper, seed is placed into for 20% aqueous sodium hypochlorite solution surface sterilization 25min of concentration, sterile washing 4 times NBD culture medium (with embodiment 5), 28 DEG C are secretly trained 7 days evoked callus.The callus that induction obtains is cut, and is changed to new NBD culture medium (with embodiment 5) squamous subculture is spare to diameter 5mm or more.The agriculture bar containing TG plasmid that embodiment 3 obtains Bacterium is drawn after 28 DEG C of YEP culture medium growths for 24 hours, is scraped and is resuspended in AAM culture medium (with embodiment 5), keeps OD600 0.4 left It is right.The Agrobacterium being resuspended mixes with ready callus, then 28 DEG C, 80rpm dark culturing 30min outwell liquid Body, callus is placed on the dry filter paper of sterilizing and is dried, and is then placed into NBDC culture medium (with embodiment 5), and 28 DEG C dark After training 2 days, callus is washed 4 times with sterile water, is then dried on the dry filter paper of sterilizing, callus is put to NBDS culture medium (with embodiment 5) 28 DEG C of dark training screenings, replace a subculture, until kanamycin-resistant callus tissue is grown every two weeks.Vigor is good anti- Property callus be transferred to PRE culture medium (with embodiment 5), 28 DEG C of dark trainings carry out after breaking up in advance for 7 days, and callus transfer is had sterile filter In the culture dish of paper, 28 DEG C of dark trainings are 3 days dry.Dry callus is transferred to MSD culture medium (with embodiment 5), 30 DEG C, 16 Hour illumination/8 hour dark photoperiod, intensity of illumination 350uE/m2/ s, differentiation culture, until the seedling of 3cm or so is generated, by seedling It is transferred to RM culture medium, 16 hours illumination/8 hour dark photoperiods, intensity of illumination 350uE/m2/s, 28 DEG C of culture of rootage.It is raw Seedling after root is transferred to the T0 generation seed of greenhouse harvest TG transformant.
The seed and non-transgenic of the T0 generation of TG transformant by rice together potting in glasshouse (28-32 DEG C, it is natural Light), transgenosis T1 sprays 1:200 times of volume ratio diluted agriculture in 3 leaf phases for plant and removes up to (41% glyphosate isopropylamine salt water solution) Remove isolated nontransgenic plants.The transformant of TG carrier is presented as that seed increases in phenotype, and 100-grain weight increases.To difference The seed of transformant counts 100-grain weight, and selected seed 100-grain weight increases by 5% or more single copy transformant OsTG.
Embodiment 10 obtains the genetically engineered soybean for improving CYP78A11 expression
The soybean that CYP78A11 is overexpressed is obtained by Agrobacterium-medialed transformation, and step is same as Example 7, mainly Difference be using carrier for 2 buildings PG, selected seed weight increases by 5% or more single copy transformant GmPG.
Embodiment 11 obtains the transgenic corns for improving CYP78A11 expression
The soybean that CYP78A11 is overexpressed is obtained by Agrobacterium-medialed transformation, and step is same as Example 8, mainly Difference be using carrier be PG that embodiment 2 constructs, selected seed weight increases by 5% or more single copy transformant ZmPG。
Embodiment 12 obtains the transgenic paddy rice for improving CYP78A11 expression
The rice that CYP78A11 is overexpressed is obtained by Agrobacterium-medialed transformation, and step is same as Example 9, mainly Difference be using carrier be PG that embodiment 2 constructs, selected seed mass of 1000 kernel increases by 5% or more single copy transformant OsPG。
Embodiment 13 obtains genetically engineered soybean, corn and the rice that PsbS, VDE, ZEP are superimposed with TEL
Embodiment 4 is obtained the GmTG that the seed that the increased GmVPZB transformant of biomass and embodiment 7 obtain increases to turn Changing body hybridization, obtained F1 is reached using 200 times of diluted agricultures and 100 times of diluted guarantors try to spray screening after up to isometric mixing, The hybrid soybean of photosynthesis enhancing and seed increase is obtained, F1 generation selfing obtains F2, continues to be reached with 200 times of diluted agricultures Screening is sprayed up to after mixing in equal volume with 100 times of diluted guarantor's examinations, F2 selfing obtains F3, and F3 generation continues with 200 times of diluted agricultures Screening is sprayed up to after mixing in equal volume up to 100 times of diluted guarantor's examinations, it is complete up to all plant of mixing screening that F3 middle peasant reaches and protect examination The strain all survived is homozygous lines, is compared with the yield of non-transgenic receptor, GmVPZB, GmTG.Yield is selected to increase 10% or more GmVPZB × GmTG F3 strain.
The ZmVPZB transformant that embodiment 5 obtains hybridizes with the ZmTG transformant that embodiment 8 obtains, and obtained F1 is used 200 times of diluted agricultures reach and 100 times of diluted guarantor examinations are up to spraying screening after isometric mixing, acquisition photosynthesis enhance and The hybrid maize that seed increases, F1 generation selfing obtain F2, continue to be reached with 200 times of diluted agricultures and 100 times of diluted guarantor try to reach etc. Screening is sprayed after volume mixture, F2 selfing obtains F3, and F3 generation continues to be reached with 200 times of diluted agricultures and 100 times of diluted guarantor's examinations reach Screening is sprayed after isometric mixing, it is homozygous lines that F3 middle peasant, which reaches and protects examination and screens all plant up to mixing and all survive, with Non-transgenic receptor, ZmVPZB, ZmTG yield be compared.Yield is selected to increase by 10% or more ZmVPZB × ZmTG strain System.
The OsVPZB transformant that embodiment 6 obtains hybridizes with the OsTG transformant that embodiment 9 obtains, and obtained F1 is used 200 times of diluted agricultures reach and 100 times of diluted guarantors try to spray screening after up to isometric mixing, obtain photosynthesis enhancing and plant The hybrid maize that son increases, F1 generation, which is selfed, obtains F2, continues to be reached with 200 times of diluted agricultures and 100 times of diluted guarantor's examinations reach equal bodies Screening is sprayed after product mixing, F2 selfing obtains F3, and F3 generation continues to be reached with 200 times of diluted agricultures and 100 times of diluted guarantors try to reach etc. Spray screening after volume mixture, it is homozygous lines that F3 middle peasant, which reaches and protects examination and screens all plant up to mixing and all survive, and non- Transgene receptor, ZmVPZB, ZmTG yield be compared.Select F3 plants of OsVPZB × OsTG of 10% or more yield increase System.
(number represents different transformant numbers, similarly hereinafter) is compared in 1. different lines soybean yields of table
Strain Yield/kilogram mu-1
Its grand No.1 (non-transgenic receptor) 162
GmVPZB-9 173
GmTG-19 178
GmVZPB-9 X GmTG-19 198
2 different lines corn yield of table compares
Strain Yield/kilogram mu-1
A188 (non-transgenic receptor self-mating system) 321
ZmVPZB-6 350
ZmTG-12 355
ZmVZPB-6 X ZmTG-12 388
3. different lines rice yield of table compares
Strain Yield/kilogram mu-1
Elegant water 134 (non-transgenic receptor) 546
OsVPZB-33 578
GmTG-26 606
OsVPZB-33 X OsTG-26 660
Embodiment 14 obtains transgenic paddy rice, corn and the soybean that PsbS, VDE, ZEP are superimposed with PLA1
The GmVPZB transformant that embodiment 4 obtains is hybridized with the GmPG transformant that embodiment 10 obtains, obtained F1 makes It is reached with 200 times of diluted agricultures and 100 times of diluted guarantors tries to spray screening after up to isometric mixing, obtaining photosynthesis enhances simultaneously And the hybrid soybean that seed increases, F1 generation selfing obtain F2, continue to be reached with 200 times of diluted agricultures and 100 times of diluted guarantor's examinations reach Screening is sprayed after isometric mixing, F2, which is selfed, obtains F3, and F3 generation continues to be reached with 200 times of diluted agricultures and 100 times of diluted guarantors try Screening is sprayed after up to isometric mixing, F3 middle peasant reaches and protects examination and screens the strain that all plant all survive up to mixing as homozygosis Strain is compared with the yield of non-transgenic receptor, GmVPZB, GmPG.Yield is selected to increase by 10% or more GmVPZB X GmPG F3 strain.
The ZmVPZB transformant that embodiment 5 obtains hybridizes with the ZmPG transformant that embodiment 8 obtains, and obtained F1 is used 200 times of diluted agricultures reach and 100 times of diluted guarantor examinations are up to spraying screening after isometric mixing, acquisition photosynthesis enhance and The hybrid maize that seed increases, F1 generation selfing obtain F2, continue to be reached with 200 times of diluted agricultures and 100 times of diluted guarantor try to reach etc. Screening is sprayed after volume mixture, F2 selfing obtains F3, and F3 generation continues to be reached with 200 times of diluted agricultures and 100 times of diluted guarantor's examinations reach Screening is sprayed after isometric mixing, it is homozygous lines that F3 middle peasant, which reaches and protects examination and screens all plant up to mixing and all survive, with Non-transgenic receptor, ZmVPZB, ZmPG yield be compared.Yield is selected to increase by 10% or more ZmVPZB X ZmPG F3 strain.
The OsVPZB that embodiment 6 obtains hybridizes with the OsPG that embodiment 9 obtains, and obtained F1 uses 200 times of diluted agricultures Screening, the hybridization that the enhancing of acquisition photosynthesis and seed increase are sprayed up to after mixing in equal volume up to 100 times of diluted guarantor's examinations Rice, F1 generation selfing obtain F2, continue to be reached with 200 times of diluted agricultures and 100 times of diluted guarantors try to spray after up to isometric mixing Screening, F2, which is selfed, obtains F3, and F3 generation continues to be reached with 200 times of diluted agricultures and 100 times of diluted guarantors try to spray after up to isometric mixing Apply screening, it is homozygous lines that F3 middle peasant, which reaches and protects examination and screens all plant up to mixing and all survive, with non-transgenic receptor, OsVPZB、
The yield of OsPG is compared.Yield is selected to increase by 10% or more OsVPZB X OsPG F3 strain.
4. different lines soybean yields of table is compared
Strain Yield/kilogram mu-1
Its grand No.1 (non-transgenic receptor) 162
GmVPZB-9 173
GmPG-11 181
GmVZPB-9 X GmPG-11 203
5. different lines corn yield of table compares
Strain Yield/kilogram mu-1
A188 (non-transgenic receptor) 321
ZmVPZB-6 350
ZmPG-16 362
ZmVpzB-6 X ZmPG-16 397
6. different lines rice yield of table compares
Strain Yield/kilogram mu-1
Elegant water 134 (non-transgenic receptor) 556
OsVPZB-33 578
OsPG-4 615
OsVPZB-33 X OsPG-4 671
Sequence table
<110>Zhejiang University
<120>a kind of method for improving crop yield
<160> 13
<170> SIPOSequenceListing 1.0
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<213>unknown (Unknown)
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aaatttatta tgtgtttttt ttccgtggtc gagattgtgt attattcttt agttattaca 60
agacttttag ctaaaatttg aaagaattta ctttaagaaa atcttaacat ctgagataat 120
ttcagcaata gattatattt ttcattactc tagcagtatt tttgcagatc aatcgcaaca 180
tatatggttg ttagaaaaaa tgcactatat atatatatat tattttttca attaaaagtg 240
catgatatat aatatatata tatatatata tatgtgtgtg tgtatatggt caaagaaatt 300
cttatacaaa tatacacgaa cacatatatt tgacaaaatc aaagtattac actaaacaat 360
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ctgatcgaat ttttaaagtt gaatatctga cgtaggattt ttttaatgtc ttacctgacc 540
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atttcccatt tttattcatg aatcatacca ttatatatta actaaat 1307
<210> 2
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tctcgtgttg agcttaagtc ttgtcttagt agtgataaga atagtgagtt tagtggtcca 60
atcacttcat ttgattggaa tgaagctgag cctagacgaa ttggtacttc tagtattgat 120
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agtggtgagc ctagtactcc tttggtgcga cttagttgga acaagcagga tccgaggtat 360
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ggatgtaaaa actcgttgct attgttgatt aatgatcttt tgatctctga ctctaagcct 780
cattgagttt cctttcatag tatggtctgt gtaggaacta tgtaccttca aaattgtgaa 840
taactagctt tagcattgga tttgattaga cgtatggaag ttagttttga acagagagca 900
tgtgacataa cggggaactt gaagtagatt atgactaaca ccagcattta tgtttctctg 960
ccctcgagct gatggagttg caggattttg tctgcctcac catcgtatgg aatgtgtagc 1020
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gatttgtgaa attaggcatt tgtaacatcg tacagatgat gtattcttca ttacttcaat 1140
acaggcatat agactctctg tccaacttaa aaggcataca aggtctgtgg tttaccaaaa 1200
tcagctttac taaatccttt ggccaatatt ctctc 1235
<210> 3
<211> 1277
<212> DNA
<213>unknown (Unknown)
<400> 3
tggaggtgtt gaaagtatag aagaagatgg gtcagtgagc aatggagagg ctaaagaaga 60
aaaaccagcg tcagtatcgt ctcaagtatt caaggatgat ttcatgagca catacgttga 120
agacaatgct gaggattgta atgttacaga ggatcctgtc aaagtaactt cacaagagat 180
tttcaaagtg tgggatctag aggttgtggg ggacaatgat gaggaagatg ggttggtgct 240
gcagctgaag aaggccctcg atgagtcttc cactgtccag ccccttccac agccactgaa 300
tgatgatcaa gttgtatcag agaagagcaa tatcgatgat ctgatatctg gaatatcgga 360
tctgtctctt gcagaaactt ttaagtgaac tatgtgtatg tgttttattt tgacatcaca 420
aactctcttt tgctttacta ctccttaacc gacacaaacc gggtttgaga tttcaaaatc 480
tgctactaga ccagaccgaa aggtttactg ggtcaatttg gtgcctacac atccacttgg 540
ccacctgttg tgcaatgatg acgatgagat tgcagtgtct ctcgactgat gatgtagact 600
tcacaatctc ttaaaaatcc cagtacttaa cctcagcctt aagaaaacgc agggaactac 660
gtcctcacaa aatctttctc tttgagtgta actttcagac gcatctttgg ctctaaattc 720
taaaaaggaa aattttaata ggttttcata atcatgggtt cactggatta gcatataagt 780
ctatggttga gaaacttgag acccagacta acgaaacctc tttccggatc caaaggtcct 840
cttgtgtagt tgacgtggta aactctctac cgtcaaattt agacattagc taatctgatc 900
aataatctcg gcagctctta aaaattaaaa attagaaatg atacgaacct cataattttt 960
ctttctctta tcaaaacacc atctttgtat ctttataagc ctgttgccac tatttttaat 1020
tgaaaatgat gcgtttgtct tatgttttct gtcctggagt tcaacattat gacaatatgt 1080
atagtaaatt agtgatatac aagacgtttg caattcaaga aaaaaactta taaactaatt 1140
aatattatgg tccacggtgc tacatattaa ctcttgatgg ttttatacat cttttctaca 1200
tgctaatatg cttttaatat tgtagcctaa cgttataatt tgttttttct taaagaaaca 1260
gtatcttgaa cgaatct 1277
<210> 4
<211> 250
<212> DNA
<213>unknown (Unknown)
<400> 4
atatgaagat tgaaaatgca gatttggtgt gtgcaataaa aagcttgtgt gcttaagttt 60
gtgtttcttt tttcttttgg gtttgttgtg ttgtgaaatt gtcgctttct ttctaaaatc 120
atatgaatgt aagatctcat cataatgaat aaacgaatgt ttctataatc catcgtgaat 180
gttatgttga atctcttctg caaattataa cggatacgct ttggaatatg atttaaagat 240
aagatgggct 250
<210> 5
<211> 1655
<212> DNA
<213>unknown (Unknown)
<400> 5
ggatccaaca atggcagtag ctacacattg tttcacttca ccttgtcatg accgtattcg 60
atttttctca agtgatgatg gtattggtag gcttggcatt acaagaaaga ggatcaatgg 120
cactttcttg ctcaagattt tacctccaat ccaaagtgct gatctcagaa caactggtgg 180
gagatcctca cgtcctttat ctgcattcag gtcaggattc tctaagggga tatttgacat 240
tgtgccatta ccatcaaaga atgagctgaa agagctgacc gctccgctgt tgctaaaact 300
cgtgggtgtt ttagcttgcg cgttccttat tgttccatct gcagatgcag ttgatgcact 360
taaaacttgt gcatgcttat tgaagggatg caggatagaa ctcgcaaagt gcattgccaa 420
ccctgcctgt gcagccaatg tcgcgtgcct tcagacctgc aataaccgtc cagatgaaac 480
cgagtgccag attaaatgtg gggatctgtt tgagaacagt gttgttgatg agttcaacga 540
gtgtgctgtg tcgagaaaaa agtgtgttcc tagaaaatct gatctcggag aatttcctgc 600
cccagaccct tctgttcttg tacagaactt caacatctcg gactttaacg ggaagtggta 660
cattacaagt ggcttgaatc caacctttga tgccttcgac tgccagctgc atgagttcca 720
cacagaaggt gacaacaagc ttgttggaaa catctcttgg agaataaaga ccctagacag 780
tggattcttt actaggtcag ccgtacaaaa attcgtgcaa gatcctaacc aacctggtgt 840
tctctacaat catgacaacg agtaccttca ctatcaagat gactggtata tcctgtcatc 900
aaagatagag aataaacctg aagactatat atttgtatac taccgtgggc gaaacgatgc 960
ttgggatgga tatggtggtg cagttgtata cacgagaagt tctgtattac ccaatagcat 1020
tataccagaa ctcgaaaaag cagcaaaaag cataggcaga gacttcagca cattcattag 1080
aacggataac acatgtggtc ctgaacctgc gctcgtggag agaattgaga agacagtgga 1140
agaaggtgaa aggataatcg taaaagaggt tgaagagata gaagaagagg tagagaagga 1200
agtggagaag gtcggtagga ctgagatgac cttgttccag agattggctg aaggatttaa 1260
tgaactgaag caagacgagg agaatttcgt gagagagtta agtaaagaag agatggagtt 1320
tttggatgag atcaaaatgg aagcaagtga ggttgaaaaa ttgtttggga aagctttgcc 1380
aatcaggaag gtcaggtaga tatgaagatt gaaaatgcag atttggtgtg tgcaataaaa 1440
agcttgtgtg cttaagtttg tgtttctttt ttcttttggg tttgttgtgt tgtgaaattg 1500
tcgctttctt tctaaaatca tatgaatgta agatctcatc ataatgaata aacgaatgtt 1560
tctataatcc atcgtgaatg ttatgttgaa tctcttctgc aaattataac ggatacgctt 1620
tggaatatga tttaaagata agatgggctg gatcc 1655
<210> 6
<211> 2270
<212> DNA
<213>unknown (Unknown)
<400> 6
ggatccaaca atgggttcaa ctccgttttg ctactctatc aatccatctc catcaaagct 60
tgatttcacg aggacccatg tgtttagtcc tgtttctaaa cagttttact tagatttatc 120
atcgttttcc ggaaaacccg gaggagtatc tgggtttagg agccgtcgag ctttgctcgg 180
agtaaaggcg gcgacggcgt tagttgagaa ggaggagaag agagaggcgg tgacggagaa 240
gaagaagaaa tcgagggttt tagttgccgg aggtggaatc ggaggattgg tgtttgcttt 300
agcggctaag aagaaaggat tcgatgtgtt agtgtttgag aaagatttga gtgctataag 360
aggagaagga aaatacagag gcccgattca aatacagagc aacgctttag ctgctttgga 420
agctattgat attgaagttg ctgaacaagt tatggaagct gggtgtatca ctggtgatcg 480
gattaacggt ctcgttgatg gtatctctgg tacttggtat gtaaagtttg atactttcac 540
tcctgcggcg tcacggggac ttcctgtgac tagagtaatt agtagaatga ctctgcagca 600
gattctagca cgtgcggttg gagaagatgt gattagaaac gagagtaatg ttgttgattt 660
tgaagattct ggagataagg ttactgtggt actcgagaat ggtcaacgct atgaaggtga 720
tctgcttgtg ggtgcagatg gcatttggtc taaggtgaga aataatttgt ttggccgtag 780
tgaagctact tattcaggct acacttgtta cacggggatt gcagatttta taccagcgga 840
tatcgagtct gttggctacc gggttttctt gggacacaaa cagtactttg tttcttcgga 900
tgttggtggt ggaaaaatgc aatggtatgc atttcacgag gaaccagctg gtggggctga 960
tgctccaaat ggtatgaaga aaaggttgtt tgaaatattt gacggttggt gcgacaatgt 1020
actcgacttg ttgcatgcga ctgaggagga agccattctg agaagagata tttatgatag 1080
aagtcctggt tttacttggg gtaaagggcg tgttacgctg ctcggggatt ctatccatgc 1140
gatgcagcca aatatgggtc aaggtggatg catggccatt gaggatagtt ttcaactagc 1200
attggagctt gatgaagcat ggaaacagag tgttgaaacg actacacctg ttgatgttgt 1260
ttcctctttg aaaagatatg aggaatctag aagactgaga gtcgctatta tccatgcaat 1320
ggcgaggatg gctgcaatta tggcttccac ttacaaagca tacttaggtg ttgggcttgg 1380
tcctctgtct ttcttgacaa agtttagagt accacatcca ggaagagttg gtggtagatt 1440
cttcgttgac attgctatgc catcgatgct tgactgggtc cttggaggta acagtgaaaa 1500
actccaagga aggccaccta gttgcagact cactgacaaa gccgatgacc ggcttcgaga 1560
gtggtttgaa gatgacgatg ctcttgaacg tactataaag ggagaatggt atctaattcc 1620
acacggcgac gattgttgcg tttcggaaac attatgtcta accaaagatg aagatcaacc 1680
ttgcatcgtc ggaagcgaac cagatcaaga ttttcctgga atgcgcattg tgatcccttc 1740
gtctcaggtt tcgaagatgc atgctcgtgt gatttacaaa gacggagctt tcttcttgat 1800
ggatcttcga agcgaacacg gaacctatgt gaccgataac gaaggaagaa gatatagagc 1860
aacaccgaat tttcccgcgc ggtttagatc gtccgacatc atcgagtttg gttcagataa 1920
gaaggcggcg tttagggtga aagtaatcag gaaaactccg aaatcgacga ggaagaatga 1980
gagtaacaac gataaattac ttcagacagc ttgaatatga agattgaaaa tgcagatttg 2040
gtgtgtgcaa taaaaagctt gtgtgcttaa gtttgtgttt cttttttctt ttgggtttgt 2100
tgtgttgtga aattgtcgct ttctttctaa aatcatatga atgtaagatc tcatcataat 2160
gaataaacga atgtttctat aatccatcgt gaatgttatg ttgaatctct tctgcaaatt 2220
ataacggata cgctttggaa tatgatttaa agataagatg ggctgagctc 2270
<210> 7
<211> 2270
<212> DNA
<213>unknown (Unknown)
<400> 7
ggatccaaca atgggttcaa ctccgttttg ctactctatc aatccatctc catcaaagct 60
tgatttcacg aggacccatg tgtttagtcc tgtttctaaa cagttttact tagatttatc 120
atcgttttcc ggaaaacccg gaggagtatc tgggtttagg agccgtcgag ctttgctcgg 180
agtaaaggcg gcgacggcgt tagttgagaa ggaggagaag agagaggcgg tgacggagaa 240
gaagaagaaa tcgagggttt tagttgccgg aggtggaatc ggaggattgg tgtttgcttt 300
agcggctaag aagaaaggat tcgatgtgtt agtgtttgag aaagatttga gtgctataag 360
aggagaagga aaatacagag gcccgattca aatacagagc aacgctttag ctgctttgga 420
agctattgat attgaagttg ctgaacaagt tatggaagct gggtgtatca ctggtgatcg 480
gattaacggt ctcgttgatg gtatctctgg tacttggtat gtaaagtttg atactttcac 540
tcctgcggcg tcacggggac ttcctgtgac tagagtaatt agtagaatga ctctgcagca 600
gattctagca cgtgcggttg gagaagatgt gattagaaac gagagtaatg ttgttgattt 660
tgaagattct ggagataagg ttactgtggt actcgagaat ggtcaacgct atgaaggtga 720
tctgcttgtg ggtgcagatg gcatttggtc taaggtgaga aataatttgt ttggccgtag 780
tgaagctact tattcaggct acacttgtta cacggggatt gcagatttta taccagcgga 840
tatcgagtct gttggctacc gggttttctt gggacacaaa cagtactttg tttcttcgga 900
tgttggtggt ggaaaaatgc aatggtatgc atttcacgag gaaccagctg gtggggctga 960
tgctccaaat ggtatgaaga aaaggttgtt tgaaatattt gacggttggt gcgacaatgt 1020
actcgacttg ttgcatgcga ctgaggagga agccattctg agaagagata tttatgatag 1080
aagtcctggt tttacttggg gtaaagggcg tgttacgctg ctcggggatt ctatccatgc 1140
gatgcagcca aatatgggtc aaggtggatg catggccatt gaggatagtt ttcaactagc 1200
attggagctt gatgaagcat ggaaacagag tgttgaaacg actacacctg ttgatgttgt 1260
ttcctctttg aaaagatatg aggaatctag aagactgaga gtcgctatta tccatgcaat 1320
ggcgaggatg gctgcaatta tggcttccac ttacaaagca tacttaggtg ttgggcttgg 1380
tcctctgtct ttcttgacaa agtttagagt accacatcca ggaagagttg gtggtagatt 1440
cttcgttgac attgctatgc catcgatgct tgactgggtc cttggaggta acagtgaaaa 1500
actccaagga aggccaccta gttgcagact cactgacaaa gccgatgacc ggcttcgaga 1560
gtggtttgaa gatgacgatg ctcttgaacg tactataaag ggagaatggt atctaattcc 1620
acacggcgac gattgttgcg tttcggaaac attatgtcta accaaagatg aagatcaacc 1680
ttgcatcgtc ggaagcgaac cagatcaaga ttttcctgga atgcgcattg tgatcccttc 1740
gtctcaggtt tcgaagatgc atgctcgtgt gatttacaaa gacggagctt tcttcttgat 1800
ggatcttcga agcgaacacg gaacctatgt gaccgataac gaaggaagaa gatatagagc 1860
aacaccgaat tttcccgcgc ggtttagatc gtccgacatc atcgagtttg gttcagataa 1920
gaaggcggcg tttagggtga aagtaatcag gaaaactccg aaatcgacga ggaagaatga 1980
gagtaacaac gataaattac ttcagacagc ttgaatatga agattgaaaa tgcagatttg 2040
gtgtgtgcaa taaaaagctt gtgtgcttaa gtttgtgttt cttttttctt ttgggtttgt 2100
tgtgttgtga aattgtcgct ttctttctaa aatcatatga atgtaagatc tcatcataat 2160
gaataaacga atgtttctat aatccatcgt gaatgttatg ttgaatctct tctgcaaatt 2220
ataacggata cgctttggaa tatgatttaa agataagatg ggctgagctc 2270
<210> 8
<211> 570
<212> DNA
<213>unknown (Unknown)
<400> 8
ctcgagtcta ccatgagccc agaacgacgc ccggccgaca tccgccgtgc caccgaggcg 60
gacatgccgg cggtctgcac catcgtcaac cactacatcg agacaagcac ggtcaacttc 120
cgtaccgagc cgcaggaacc gcaggagtgg acggacgacc tcgtccgtct gcgggagcgc 180
tatccctggc tcgtcgccga ggtggacggc gaggtcgccg gcatcgccta cgcgggcccc 240
tggaaggcac gcaacgccta cgactggacg gccgagtcga ccgtgtacgt ctccccccgc 300
caccagcgga cgggactggg ctccacgctc tacacccacc tgctgaagtc cctggaggca 360
cagggcttca agagcgtggt cgctgtcatc gggctgccca acgacccgag cgtgcgcatg 420
cacgaggcgc tcggatatgc cccccgcggc atgctgcggg cggccggctt caagcacggg 480
aactggcatg acgtgggttt ctggcagctg gacttcagcc tgcctgtacc gccccgtccg 540
gtcctgcccg tcaccgagat ttgactcgag 570
<210> 9
<211> 1733
<212> DNA
<213>unknown (Unknown)
<400> 9
ctcgagtcaa cacaacatat acaaaacaaa cgaatctcaa gcaatcaagc attctacttc 60
tattgcagca atttaaatca tttcttttaa agcaaaagca attttctgaa aattttcacc 120
atttacgaac gatagccatg gcggcgacca tggcgtccaa cgctgcggct gcggctgcgg 180
tgtccctgga ccaggccgtg gctgcgtcgg cagcgttctc gtcgcggaag cagctgcggc 240
tgcctgccgc agcgcgcgga gggatgcggg tgcgggtgcg ggcgcggggt cggcgggagg 300
cggtggtggt ggcgtccgcg tcgtcgtcgt cggtggcagc gccggcggcg aaggctgaga 360
tgctacacgg tgcaagcagc cggccggcaa ccgctcgcaa atcttccggc ctttcgggaa 420
cggtcaggat tccgggcgat aagtccatat cccaccggtc gttcatgttc ggcggtcttg 480
ccagcggtga gacgcgcatc acgggcctgc ttgaaggtga ggacgtgatc aataccggga 540
aggccatgca ggctatggga gcgcgtatcc gcaaggaagg tgacacatgg atcattgacg 600
gcgttgggaa tggcggtctg ctcgcccctg aggcccctct cgacttcggc aatgcggcga 660
cgggctgcag gctcactatg ggactggtcg gggtgtacga cttcgatagc acgttcatcg 720
gagacgcctc gctcacaaag cgcccaatgg gccgcgttct gaacccgttg cgcgagatgg 780
gcgtacaggt caaatccgag gatggtgacc gtttgcccgt tacgctgcgc gggccgaaga 840
cgcctacccc gattacctac cgcgtgccaa tggcatccgc ccaggtcaag tcagccgtgc 900
tcctcgccgg actgaacact ccgggcatca ccacggtgat cgagcccatc atgaccaggg 960
atcataccga aaagatgctt caggggtttg gcgccaacct gacggtcgag acggacgctg 1020
acggcgtcag gaccatccgc cttgagggca ggggtaaact gactggccaa gtcatcgatg 1080
ttccgggaga cccgtcgtcc acggccttcc cgttggttgc ggcgctgctc gtgccgggga 1140
gtgacgtgac catcctgaac gtcctcatga acccgaccag gaccggcctg atcctcacgc 1200
ttcaggagat gggagccgac atcgaggtga tcaacccgcg cctggcaggc ggtgaagacg 1260
ttgcggatct gcgcgtgcgc tcctctaccc tgaagggcgt gacggtcccg gaagatcgcg 1320
cgccgtccat gatagacgag tatcctattc tggccgtcgc cgctgcgttc gccgaagggg 1380
ccacggtcat gaacggtctt gaggaactcc gcgtgaagga atcggatcgc ctgtcggcgg 1440
tggccaatgg cctgaagctc aacggtgttg actgcgacga gggtgagacc tcactcgtgg 1500
tccgtggccg gcctgatggc aagggcctcg gcaacgccag tggagcggcc gtcgccacgc 1560
acctcgatca tcgcatcgcg atgtccttct tggtgatggg tctcgtctca gagaacccgg 1620
tgaccgtcga tgacgccacg atgatagcga cgagcttccc agagttcatg gatctgatgg 1680
cgggcctcgg ggccaagatc gaactgtctg acacgaaggc cgcttgactc gag 1733
<210> 10
<211> 1889
<212> DNA
<213>unknown (Unknown)
<400> 10
aactcatgga gtccactatc atcactttcc aagtcaccct ctgcgtcctt ctctttaccc 60
tcttgttcac gatgttcttc actctcggtg ggcttacttg ggcctggccc agcccagaat 120
cattgtccct ggcctagtaa cggccttgct cggggtcttc atgggctcca cacctcactg 180
cactttatcc aaattggccc gcacttacca cgcggaaaag ctgatggctt tctccattgg 240
tttaacctag ttcgttatct cgagcgaaca agaaaccgcg aaggagattc tcaacagccc 300
cgatttcgcc gacaggccgg tgaaggaatc tgcttatgag tttctcttcc accggtatta 360
cgtagttcat ttggccatag gcgaatcata tgcctaagta atatcttagt gttagttagt 420
tatttggaag ttagtttagt tggttgcatg ttgcaactaa tgataattag ttagttagtt 480
ggaagttagt ttagttggtt ggaagttagt tgctatatat aaccatttgt gcaaacgtgt 540
gcagatttag aggaacaact ttgagaatta attctttcaa tacatagaac tccattcact 600
gcttcccttt ttcttttagc ttttcttgca agcttacttg gttagatatg cattcaacaa 660
tggcaggcac aaggtttatg gtaataagca tacgttgaat tttttttttc ttttctttcc 720
aataaacata attttctttt tgctatatat tatgcatatg ttgaagttga aaggacaatg 780
aaaatcacca accaaacgac caatgaaaaa cagaaaaaca atgaatgaaa attataatca 840
tttgcgtaag gtgagtctga acccgattca ttataaaaat taaaaagggt atttatatgg 900
atgcagctgc gactccattt ccgaaaccct aaattgattc tcccaaagta ccattatagg 960
taaactttgt cttaattgtc attaaattat ggtgttgttt tccctaataa tgttagtgta 1020
aactcttcgg ctaaatattc attttcaaat ttcaattgac ataaacataa gctaattttt 1080
ttttaaaaaa ataacataaa ctactatact ttaatctttt tcaaccctaa ccccaaggag 1140
ttcgctaagt ggaacaagac acaaaaactt ttcctatttt gtattagggt gtcactatgt 1200
tcttcttcta aatttaactt ataaaaaata tttttcaacc ctaacttcaa attgcaaatt 1260
gttattgttt gtgttcttac ttactcatat tttttagccg agttaaaaat aaatataaat 1320
ttctttatta aaaaaatgat aggaagttag ggacaatttc ttgggtatac tctttttttt 1380
tatcactttt tactaattaa atttattcca attaacaatt ttatgtatta tattttagta 1440
ttatttaata aactttattt cttttttctt tccaataaaa aaattattta taattttata 1500
gttttaaata aattaaataa aatatccttt tttattgacc tcttttcaat atatcgttaa 1560
ttatatttaa aattcatttc actctcttga ttttacaaat ttttatataa ttttttttat 1620
gcaagcagta atgttttttt aaaagcaaac attttcttaa taatttaatt atttttttct 1680
tgatttttat tttttttatt tgcatatgca tttcctttac tccgtgtaac catggttaac 1740
caaactttaa ttttaaccac acccctcacc aactttctat ataagcagta gctaactaac 1800
tcttactcac accatccagt actcttcttt tcagcacaac ctcttcatct ctcccaggct 1860
ttcttttctt tgcaacaaca ctaccaaaa 1889
<210> 11
<211> 274
<212> DNA
<213>unknown (Unknown)
<400> 11
ccgatcgttc aaacatttgg caataaagtt tcttaagatt gaatcctgtt gccggtcttg 60
cgatgattat catataattt ctgttgaatt acgttaagca tgtaataatt aacatgtaat 120
gcatgacgtt atttatgaga tgggttttta tgattagagt cccgcaatta tacatttaat 180
acgcgataga aaacaaaata tagcgcgcaa actaggataa attatcgcgc gcggtgtcat 240
ctatgttact agatcgggga tatccccagc ttga 274
<210> 12
<211> 3847
<212> DNA
<213>unknown (Unknown)
<400> 12
aactcatgga gtccactatc atcactttcc aagtcaccct ctgcgtcctt ctctttaccc 60
tcttgttcac gatgttcttc actctcggtg ggcttacttg ggcctggccc agcccagaat 120
cattgtccct ggcctagtaa cggccttgct cggggtcttc atgggctcca cacctcactg 180
cactttatcc aaattggccc gcacttacca cgcggaaaag ctgatggctt tctccattgg 240
tttaacctag ttcgttatct cgagcgaaca agaaaccgcg aaggagattc tcaacagccc 300
cgatttcgcc gacaggccgg tgaaggaatc tgcttatgag tttctcttcc accggtatta 360
cgtagttcat ttggccatag gcgaatcata tgcctaagta atatcttagt gttagttagt 420
tatttggaag ttagtttagt tggttgcatg ttgcaactaa tgataattag ttagttagtt 480
ggaagttagt ttagttggtt ggaagttagt tgctatatat aaccatttgt gcaaacgtgt 540
gcagatttag aggaacaact ttgagaatta attctttcaa tacatagaac tccattcact 600
gcttcccttt ttcttttagc ttttcttgca agcttacttg gttagatatg cattcaacaa 660
tggcaggcac aaggtttatg gtaataagca tacgttgaat tttttttttc ttttctttcc 720
aataaacata attttctttt tgctatatat tatgcatatg ttgaagttga aaggacaatg 780
aaaatcacca accaaacgac caatgaaaaa cagaaaaaca atgaatgaaa attataatca 840
tttgcgtaag gtgagtctga acccgattca ttataaaaat taaaaagggt atttatatgg 900
atgcagctgc gactccattt ccgaaaccct aaattgattc tcccaaagta ccattatagg 960
taaactttgt cttaattgtc attaaattat ggtgttgttt tccctaataa tgttagtgta 1020
aactcttcgg ctaaatattc attttcaaat ttcaattgac ataaacataa gctaattttt 1080
ttttaaaaaa ataacataaa ctactatact ttaatctttt tcaaccctaa ccccaaggag 1140
ttcgctaagt ggaacaagac acaaaaactt ttcctatttt gtattagggt gtcactatgt 1200
tcttcttcta aatttaactt ataaaaaata tttttcaacc ctaacttcaa attgcaaatt 1260
gttattgttt gtgttcttac ttactcatat tttttagccg agttaaaaat aaatataaat 1320
ttctttatta aaaaaatgat aggaagttag ggacaatttc ttgggtatac tctttttttt 1380
tatcactttt tactaattaa atttattcca attaacaatt ttatgtatta tattttagta 1440
ttatttaata aactttattt cttttttctt tccaataaaa aaattattta taattttata 1500
gttttaaata aattaaataa aatatccttt tttattgacc tcttttcaat atatcgttaa 1560
ttatatttaa aattcatttc actctcttga ttttacaaat ttttatataa ttttttttat 1620
gcaagcagta atgttttttt aaaagcaaac attttcttaa taatttaatt atttttttct 1680
tgatttttat tttttttatt tgcatatgca tttcctttac tccgtgtaac catggttaac 1740
caaactttaa ttttaaccac acccctcacc aactttctat ataagcagta gctaactaac 1800
tcttactcac accatccagt actcttcttt tcagcacaac ctcttcatct ctcccaggct 1860
ttcttttctt tgcaacaaca ctaccaaaag gatccaacaa tggcaatggc caccgccacc 1920
gcctcctcct gcgtcgacgc cacgtggtgg gcgtacgccc tcccggcgct cctcggcgcc 1980
gacaccctct gcgcccaccc ggcgctgctc gccggcgccg tcctcctggc cttcgccacc 2040
gccgcggtgc tcgcctgggc cgcgtccccc ggcgggccgg cgtgggcgca cggccgcggc 2100
cgcctcggcg cgacgcccat cgaggggccc cgggggctcc ccgtgttcgg cagcatcttc 2160
gcgctctccc ggggcctccc gcaccgcgcg ctcgacgcga tgtcgcgcga cgcggcggcg 2220
ccacgggcga gggagctcat ggcgttctcc gtcggggaga cgccggcggt ggtgtcgtcg 2280
tgcccggcga cggcgaggga ggtgctcgcg cacccgtcgt tcgccgaccg cccgctgaag 2340
cgctcggcgc gggagctgct gttcgcgcgc gccatcgggt tcgcccccag cggcgagtac 2400
tggcgcctcc tccgccgcat cgcctccacc cacctcttct cccctcgccg cgtcgccgcg 2460
cacgagccgg ggcgccaggc cgacgccacg gcgatgctgt ccgccatggc cgccgagcag 2520
tccgccaccg gcgccgtcgt gctccgcccc cacctccagg ccgccgcgct caacaacatc 2580
atgggcagcg tgttcggccg gcgctacgac gtctcctcct cctccggcgc cgccgccgac 2640
gaggccgagc agctcaagag catggtgcgc gaggggttcg agctcctcgg cgcgttcaac 2700
tggtccgacc acctcccatg gctcgcccac ctctacgacc ccaaccacgt cgcccgccgc 2760
tgcgccgcgc tcgtcccccg cgtccaggcg ttcgtccgcg gcgtcatccg cgaccaccgc 2820
ctccgccgcg actcctcctc caccgccgcc gacaatgccg acttcgtcga cgtcctcctc 2880
tccctcgagg cccacgagaa cctcgccgag gacgacatgg tcgccgtcct ctgggagatg 2940
atatttcgtg ggacggacac gacggcgttg gtgacggagt ggtgcatggc ggaggtggtg 3000
aggaacccgg cggtgcaggc gaggctgagg gcggaggtgg acgcggcggt gggcggcgac 3060
gggtgtccca gcgacggcga cgtggcgcgg atgccgtacc tgcaggcggt ggtgaaggag 3120
acgctgaggg cgcacccgcc ggggccgctg ctgagctggg cgcggctggc caccgccgac 3180
gtggggctcg ccaacggcat ggtggtgccg gcgggcacga cggcgatggt gaacatgtgg 3240
gccatcaccc acgacggcga ggtgtgggcc gacccggagg cgttcgcgcc ggagcggttc 3300
atcccgtcgg agggcggcgc cgacgtcgac gtccgcggcg gcgacctccg cctggcgccg 3360
ttcggcgccg ggcgccgcgt ctgccccggc aagaacctcg gcctcgccac cgtcaccctc 3420
tgggtcgccc gcctcgtcca cgccttcgac tggttcctcc ccgacggctc gccgccggtg 3480
tccctcgacg aggtcctcaa gctctccctc gagatgaaga cccctctcgc cgccgccgcc 3540
accccccgcc gccgccgcgc cgcctgagaa ttcccgatcg ttcaaacatt tggcaataaa 3600
gtttcttaag attgaatcct gttgccggtc ttgcgatgat tatcatataa tttctgttga 3660
attacgttaa gcatgtaata attaacatgt aatgcatgac gttatttatg agatgggttt 3720
ttatgattag agtcccgcaa ttatacattt aatacgcgat agaaaacaaa atatagcgcg 3780
caaactagga taaattatcg cgcgcggtgt catctatgtt actagatcgg ggatatcccc 3840
agcttga 3847
<210> 13
<211> 4150
<212> DNA
<213>unknown (Unknown)
<400> 13
aactcatgga gtccactatc atcactttcc aagtcaccct ctgcgtcctt ctctttaccc 60
tcttgttcac gatgttcttc actctcggtg ggcttacttg ggcctggccc agcccagaat 120
cattgtccct ggcctagtaa cggccttgct cggggtcttc atgggctcca cacctcactg 180
cactttatcc aaattggccc gcacttacca cgcggaaaag ctgatggctt tctccattgg 240
tttaacctag ttcgttatct cgagcgaaca agaaaccgcg aaggagattc tcaacagccc 300
cgatttcgcc gacaggccgg tgaaggaatc tgcttatgag tttctcttcc accggtatta 360
cgtagttcat ttggccatag gcgaatcata tgcctaagta atatcttagt gttagttagt 420
tatttggaag ttagtttagt tggttgcatg ttgcaactaa tgataattag ttagttagtt 480
ggaagttagt ttagttggtt ggaagttagt tgctatatat aaccatttgt gcaaacgtgt 540
gcagatttag aggaacaact ttgagaatta attctttcaa tacatagaac tccattcact 600
gcttcccttt ttcttttagc ttttcttgca agcttacttg gttagatatg cattcaacaa 660
tggcaggcac aaggtttatg gtaataagca tacgttgaat tttttttttc ttttctttcc 720
aataaacata attttctttt tgctatatat tatgcatatg ttgaagttga aaggacaatg 780
aaaatcacca accaaacgac caatgaaaaa cagaaaaaca atgaatgaaa attataatca 840
tttgcgtaag gtgagtctga acccgattca ttataaaaat taaaaagggt atttatatgg 900
atgcagctgc gactccattt ccgaaaccct aaattgattc tcccaaagta ccattatagg 960
taaactttgt cttaattgtc attaaattat ggtgttgttt tccctaataa tgttagtgta 1020
aactcttcgg ctaaatattc attttcaaat ttcaattgac ataaacataa gctaattttt 1080
ttttaaaaaa ataacataaa ctactatact ttaatctttt tcaaccctaa ccccaaggag 1140
ttcgctaagt ggaacaagac acaaaaactt ttcctatttt gtattagggt gtcactatgt 1200
tcttcttcta aatttaactt ataaaaaata tttttcaacc ctaacttcaa attgcaaatt 1260
gttattgttt gtgttcttac ttactcatat tttttagccg agttaaaaat aaatataaat 1320
ttctttatta aaaaaatgat aggaagttag ggacaatttc ttgggtatac tctttttttt 1380
tatcactttt tactaattaa atttattcca attaacaatt ttatgtatta tattttagta 1440
ttatttaata aactttattt cttttttctt tccaataaaa aaattattta taattttata 1500
gttttaaata aattaaataa aatatccttt tttattgacc tcttttcaat atatcgttaa 1560
ttatatttaa aattcatttc actctcttga ttttacaaat ttttatataa ttttttttat 1620
gcaagcagta atgttttttt aaaagcaaac attttcttaa taatttaatt atttttttct 1680
tgatttttat tttttttatt tgcatatgca tttcctttac tccgtgtaac catggttaac 1740
caaactttaa ttttaaccac acccctcacc aactttctat ataagcagta gctaactaac 1800
tcttactcac accatccagt actcttcttt tcagcacaac ctcttcatct ctcccaggct 1860
ttcttttctt tgcaacaaca ctaccaaaag gatccaacaa tgggtgggtt cccggaagcc 1920
acgggtaacc ttctcgatgc cgcagctcag gagttccacc ctacggtctg tgccccctat 1980
cctctacagc cgcttccgca acagctatac tgcccccacc catatccagc catgccggtg 2040
cctccgccgc cgcaaatagc catgttacag ccagtgcctc cgatggcgat ggccatggcg 2100
ccgcagccgg ggtacacctt gccaacgacg acgccggtgg tcaatggccc gtcgagccgc 2160
gtcgtggtgc tgggccttgt cccgccgcac gcgcaggagg ccgacgtggc gcaggcgatg 2220
gcgccattcg gcgcgatccg ctcggtcgac gcgtgcgcgg tggcgtccga gggcgtggcc 2280
accgtccatt tcttcgacat ccgcgccgcc gagctcgcct tgacctgtgt ccgcgagcag 2340
cacatgcgcc agcagagccg cctcgggcag ctctacgcgg cggccgccgt agccccggcg 2400
tgggctcctg caccgacgcc ccaggcctgg gactggcccc accccaacga cgacggccgc 2460
ggcctcgtcc tcgggcacgc cgtgtgggcc cacttcgcca ccggcgccga cgacggcgac 2520
aaccgcggct ccctggtggt cctgagcccc ctgcccggcg tctcggtcgc tgacctccgc 2580
caagtcttcc aggccttcgg ggacttgaag gatgtgaggg agtcggcgca gcggcccagc 2640
cacaagttcg tggacttctt cgacacgcgc gacgccgcgc gcgcgctcgc cgagctcaac 2700
ggccaggagc ttttcggccg ccgcctcgtc gtcgagttca cgcgcccttc cggccccggg 2760
ccccgcaggc gcgggtacgc accccaccag caccggccca ccgcgccgac tccgccgagg 2820
cttcaagcga cgtggcgacc gtcccaaccg acgtcgtctc agccgccggc atcctcgtcg 2880
tcgtccggtt ccgtaagggc gagggaagga gtggtgcttc tgaggaggag ctcctgtaag 2940
tctagcgcgg gcagcgacca gtcgtccaag ggaggcaatg ccggaacgag ccatgagcgc 3000
aagaccaagg gcggcaagat cgtggtggcg gcggcggcgg catcctcgtc gaccccgaca 3060
gcgtccggga agcaaaccca gaaaggcgtc gggagcagcg gcggcgggag ctggaaagga 3120
cgaaagagcg ggtgggaggc gcgcttcctg ttcaaggagc ccgaggccgg cggcggcgcc 3180
gacacgcaag caacgccggc ttcggagatg gatacgagga ccaccgtcat gatcaggaac 3240
ataccgaaca agtacagcca gaagctgctg ctcaacatgc tggacaacca ctgcatccaa 3300
tccaacgagt ggatcgtggc gagcggcgag gagcagccct tctccgccta cgatttcgtc 3360
tacctcccca tagatttcaa caacaagtgt aatgtgggct acggcttcgt caacctgaca 3420
tcgccggagg ctcgcgtgcg gctgtacaag gcgttccaca agcagccatg ggaggtgtac 3480
aactcgcgca agatctgcca agtgacatac gcgcgcgtac aaggcctgga agcgctgaag 3540
gagcacttca agaactccaa gttcccgtgc gacagcgacg agtacctgcc cgtggcgttc 3600
tcgccggcgc gcgacggcaa ggagcttacg gatccagtgc ccatcgtggg ccgctcgccc 3660
gcggcgtcgt ccgcgtcgtc gcctcccaag agccgggcgg ctagcgtgga ccggcttggg 3720
caggagctga tgccggcgcc gtcgtcatcc gcggacggcg cgtcgtcgac cactacgtcc 3780
acccacgcgc cgtccgaaca cgacgaggag gaggaggagg gagacatcag gctcgcaggc 3840
gagctgcggc ggcttggcta cgacgactag gaattcccga tcgttcaaac atttggcaat 3900
aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt 3960
tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg 4020
tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc 4080
gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat cggggatatc 4140
cccagcttga 4150

Claims (7)

1. a kind of method for improving crop yield, it is characterised in that the method is by light protected protein PsbS gene, purple Huang Plain Violaxanthin De VDE gene and zeaxanthin epoxidase ZEP gene import receptor crop jointly, while importing CYP78A base Because or TEL gene due to realize.
2. improving the method for crop yield as described in claim 1, it is characterised in that the PsbS gene, VDE gene and ZEP Gene by pAtRbcs1A promoter, ZEP gene, pAtFBA2 promoter, VDE gene, pGAPA-1 promoter, PsbS gene, HSP18.2 terminator successively imports receptor crop after functionality connects jointly.
3. improving the method for crop yield as described in claim 1, it is characterised in that the CYP78A gene is to pass through nucleosides Specific promoter shown in acid sequence SEQ ID NO:10 imported together with CYP78A gene and NOS terminator functional splice by Body crop.
4. improving the method for crop yield as described in claim 1, it is characterised in that the TEL gene is by nucleotide sequence Specific promoter shown in SEQ ID NO:10 imports receptor crop together with TEL gene and NOS terminator functional splice.
5. improving the method for crop yield as described in claim 1, it is characterised in that the PsbS gene, VDE gene and ZEP The method that gene imports receptor crop jointly are as follows: (1) PsbS gene, VDE gene and ZEP gene respectively with arabidopsis HSP18.2 Sub-functionality connection is terminated, obtains PsbS genetic fragment, VDE genetic fragment, ZEP genetic fragment respectively；The arabidopsis HSP18.2 terminator nucleotides sequence is shown in SEQ ID NO:4；(2) by the hygromycin resistance base in pCAMBIA1300 carrier Because hpt II replaces with bar gene shown in SEQ ID NO:8, it is denoted as carrier 1300-B；(3) by carrier 1300-B and SEQ ID PAtRbcs1A promoter shown in NO:1 is connected with step (1) ZEP genetic fragment, is denoted as plasmid 1300-Z-B；(4) by plasmid 1300-Z-B is connect with pAtFBA2 promoter shown in SEQ ID NO:3, step (1) VDE genetic fragment, is denoted as plasmid 1300-V- Z-B；(5) plasmid 1300-Z-B is connect with pGAPA-1 promoter shown in SEQ ID NO:2, step (1) PsbS genetic fragment, It is denoted as plasmid VPZB, plasmid VPZB is imported into receptor crop.
6. improving the method for crop yield as described in claim 1, it is characterised in that the enhancing receptor crop CYP78A base Cause or the method for TEL gene expression are as follows: (1) nucleotides sequence is classified as CP4 gene cloning shown in SEQ ID NO:9 to carrier PUC57 is denoted as carrier pUC57-CP4；(2) by specific promoter and rice shown in nucleotide sequence SEQ ID NO:10 NOS terminator is functional shown in CYP78A11 gene and SEQ ID NO:11 is stitched together, and is denoted as plasmid pUC57-P；(3) By NOS shown in specific promoter shown in nucleotide sequence SEQ ID NO:10 and corn TEL gene ZmTE1 and SEQ ID NO:11 Terminator is functional to be stitched together, and is denoted as plasmid pUC57-T；(4) plasmid pUC57-P or plasmid pUC57-T importing is contained The receptor crop of plasmid VPZB.
7. improving the method for crop yield as described in claim 1, it is characterised in that the receptor crop includes soybean, jade Rice, rice or rape.
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CN109880832A (en) * 2019-04-09 2019-06-14 西南大学 Rice leaf Leaf angle mutated gene PLA1 and its application
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