CN105349559A - Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics - Google Patents

Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics Download PDF

Info

Publication number
CN105349559A
CN105349559A CN201510980701.0A CN201510980701A CN105349559A CN 105349559 A CN105349559 A CN 105349559A CN 201510980701 A CN201510980701 A CN 201510980701A CN 105349559 A CN105349559 A CN 105349559A
Authority
CN
China
Prior art keywords
corn
gene
starch
zmwx
grain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510980701.0A
Other languages
Chinese (zh)
Inventor
张举仁
赵丫杰
王慧
李朝霞
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shandong University
Original Assignee
Shandong University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shandong University filed Critical Shandong University
Priority to CN201510980701.0A priority Critical patent/CN105349559A/en
Publication of CN105349559A publication Critical patent/CN105349559A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01021—Starch synthase (2.4.1.21)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention discloses an application of a corn ZmWx gene in increase of the corn yield and improvement of grain characteristics. A ZmWx gene sequence is cloned in corn endosperm cDNA (complementary deoxyribonucleic acid), the ZmWx gene encodes GBSSI (granule-bound starch synthase I), the GBSSI and an endosperm specific promoter form a fusion gene, the fusion gene is recombined in a plant expression carrier with a transgenic technology to transform corn, and corn transgenetic plants with yield increased and grain characteristics improved are obtained; alternatively, GBSSI overexpressed corn and mutant AGPase overexpressed high-starch corn are hybridized, a transgenetic polymer is obtained through screening, and the corn transgenetic plants whose grain yield, starch content and hundred-grain weight are remarkably higher than those of recipient materials are produced. The application provides a basis and a means for development and preparation of high-starch and high-amylose starch corn and also develops a new approach for increase of the corn yield and improvement of the starch quality.

Description

Corn ZmWx gene is improving the application in corn yield and improvement grain characters
Technical field
The invention belongs to crop bioengineering breeding field, specifically, relate to a kind of corn ZmWx gene and improving the application in corn yield and improvement grain characters.
Background technology
Starch is the indispensable moiety of human foods, is also the important raw and processed materials of foodstuffs industry and chemical industry, in human lives, plays key player.Along with the fast development of starch processing industry, the purposes of starch is constantly widened, particularly the production of alcohol fuel, causes the demand of starch sharply to rise.Corn is as the large crop that beats the world, and its output is higher than wheat and paddy rice, and in various crop starch, W-Gum has best chemical constitution, and output is the highest.Global starch yield about 6,880 ten thousand tons in 2013, wherein W-Gum about 6,100 ten thousand tons, accounts for 89% of total amount.China's starch processing industry more than 90% is starting material with corn.In conventional corn seed, starch content is generally about 65%.Starch content has good industrial use higher than the high starch maize of 74%.The high starch maize that current China mainly promotes based on mixed type high starch maize, its grain is heavy, endosperm weight average higher than conventional corn, and endosperm proportion is comparatively large, and not quite, but crude fat content is lower than conventional corn for grain protein content and conventional corn difference.
Starch is the macromolecular carbohydrate that glucose molecule is polymerized by α-Isosorbide-5-Nitrae glycosidic link or α-1,6 glycosidic link, according to the difference of structure, can be divided into amylose starch and amylopectin.In conventional corn seed starch, amylose starch generally accounts for 25-30%, and amylopectin accounts for 70-75%.Amylose molecule amount is less, is generally the linear molecule be made up of with α-Isosorbide-5-Nitrae glycosidic link 200-5000 glucose unit, only containing a small amount of branch (being on average less than in every 1000 glucose units).The polymerization degree of conventional corn amylose starch is generally 230-2370, similar with amylose in rice, and the polymerization degree of potato amylose is comparatively large, is generally 970-9770 (HanashiroandTakeda1998).The most molecular weight of amylopectin is large, is generally made up of more than 5000 glucose units, and containing comparatively multiple-limb, tapping point is sentenced α-1,6 glycosidic link and connected, and the length of branched chain is not from 6-100DP (polymerization degree, degreeofpolymerization) etc.The number-average degree of polymerization (DPn) of corn amylopectin is 15900, and its DPn that is large-scale, medium-sized and small molecule is respectively 26500,8400,1100, and molar fraction is respectively 54%, 16%, 30%; Massfraction is respectively 90%, 8%, 2% (Takedaetal.2003).The constructional feature of amylose starch and amylopectin determines the two and has larger physicochemical property difference.
The molecular structure of starch determines cooking properties and the industrial use of starch, and structural parameter comprise amylose starch/amylopectin ratios (a straight/ratio), the amylose starch polymerization degree and amylopectin branch chain length and distribution.Wherein a straight/comparison starch property and purposes have the greatest impact, and highly purified amylopectin or amylose starch more can meet the different demands of food and industrial processes.Amylopectin has larger viscosity and the coefficient of expansion, is applied in food-processing the volume that can increase sticky glutinous mouthfeel and puffed food, and uses as tackiness agent industrial circle is many.Amylose starch is compared to amylopectin, purposes is more extensive, with it, there is water insoluble characteristic, low viscosity, higher gelatinization point, good film-forming properties, anti-swelling, high stability, become the starting material that candy, papermaking, weaving, pharmacy and plastics-production field are desirable.Especially amylose starch can be applied to production photodissociation type agricultural plastic film, can replace traditional non-degradable plastics, effectively reduces " white pollution ", is the focus product that the whole world is all paid close attention to.In recent years, take amylose starch as the new focus that the Resistant starch of raw material production becomes healthy diet aspect.Resistant starch has the structural performance of similar food fibre, digestedly can not be hydrolyzed to glucose, not easily enter the recycle system and blood sugar is increased in small intestine.Therefore, Resistant starch is the ultimate food of diabetics, also can be developed to healthy effective diet food simultaneously.Thus, increasing researchist is devoted to the purification of amylose starch and the breeding work of high amylose starch crop.
In Cereal endosperm, the synthesis of starch is enzymatic complex process more than, relates generally to four large fermentoids: ADP-glucose pyrophosphorylase (AGPase), amylosynthease (SSs), Q-enzyme (SBEs), starch debranching enzyme (DBEs).This four large fermentoid all comprises many family members, and each member has again the meticulous division of labor, in Starch synthesis process, play different effects.In recent years, along with the discovery of much starch synthesis relevant enzymes mutant and the fast development of genetic engineering technique, in conjunction with forward genetics and reverse genetic Epidemiological Analysis, the research of scholars to the expression pattern of enzyme classes and family member, structure, Subcellular Localization, catalysis characteristics and expression regulation etc. makes significant progress.
Amylose starch mainly by Granule-Bound Starch Synthase GBSSI be responsible for synthesize.Amylosynthease adds the glucosyl group in ADPG (ADP-glucose) to the branched chain of amylopectin or the non reducing end of amylose starch.Amylosynthease can be divided into 5 families: SSI, SSII, SSIII, SSIV and GBSS.They extend the side chain of different polymerization degree in amylopectin respectively, or the amylose chain of elongated linear.There are two member: GBSSI and GBSSII in GBSS family.GBSSI by Wx genes encoding, the synthesis of amylose starch in primary responsibility endosperm and pollen granule.GBSSII mainly expresses in blade, is responsible for the synthesis of the amylose starch in non-storage organ.In the GBSSI deletion mutant of corn, wheat, paddy rice, potato, only containing amylopectin fraction in storage organ's starch small grain, hardly containing amylose component.In these mutant, the disappearance of amylose starch does not affect the phenotype of starch small grain, and this shows to only have amylopectin to be the indispensable integral part (Zeemanetal.2010) of starch granule structure.The two large features that GBSSI is different from other family's amylosyntheases are that it is only limitted to the strict Subcellular Localization of starch small grain and its pattern of playing a role.Different from soluble starch synthase, GBSSI is the amylosynthease that uniquely can extend amylose starch continuously, generates long oligonucleotide chain.In contrast, other amylosynthease all has location in starch small grain He in amyloplast matrix, and only the synthesis of responsible amylopectin branched chain, can not synthesize amylose starch.
Increasing evidence shows, in higher plant, the amylose starch synthesis that GBSSI is responsible for originates in introduction MOS (Denyeretal.1996).The ability of GBSSI synthesis amylose starch shows as and Oligomeric maltose (MOS) can either be utilized for primer synthesis amylose starch, also can act on the side chain of amylopectin, be responsible for the synthesis of overlength chain.When iodine combined techniques measures amylose content, the overlength chain in amylopectin is also by a part of can be regarded as apparent amylose content.By in normal Wx channel genes paddy rice wx mutant, endosperm apparent amylose content is increased to about 22%, and wherein real amylose content is about 16%, other parts are overlength chain, show that the importing of Wx gene is the increase in overlength chain content (Hanashiroetal.2008) to the impact that amylopectin is maximum.Also similar report (Kitaharaetal.2007) is had in wheat and potato.
In higher plant starch storage allelotaxis, the increase of amylose content starts from growing the middle and later periods.In corn, the content of endosperm amylose starch is increased to 26.5% by 18% in pollination in latter 14 days to 28 days.There are 4 class factors to affect GBSSI and synthesize the ability of amylose starch, namely have the structural performance of amylopectin in the Wx gene copy number of function and transcript thereof, avidity, substrate A DPG content and starch small grain to primer MOS.In Maize mutant endosperm, the Wx gene copy number of function is had to become positive correlation with GBSSI enzymic activity and amylose content (AC), wherein Wx gene copy number and enzymic activity linear (Tsai1974).Although other amylosynthease also can add a glucosyl group on MOS, but amylosynthease (SS) and Oligomeric maltose dissociate after this, can not continue again to extend, and glucosyl group can be connected into MOS until synthesis amylose starch by GBSSI continuously one by one.The avidity of GBSSI to MOS is more than 20 times of other amylosynthease, therefore, when MOS concentration is very low in vivo GBSSI still can effectively in conjunction with MOS and make it extend.Denyer etc. also find that in vitro soluble g BSSI is very low and no longer can extend MOS constantly to the avidity of MOS, but when adding a small amount of amylopectin in system, GBSSI improves the avidity of MOS and can continue to extend.This shows that GBSSI is to the important of MOS and the characteristic of uniqueness may depend on the interaction with amylopectin grid in starch small grain.
ADPG is another important factor affecting GBSSI synthesis amylose starch ability as its concentration of substrate.Thermomechanical analysis and experimental evidence show, the synthesis of amylose starch in the supply status restriction body of ADPG.Compared to other amylosyntheases that responsible amylopectin synthesizes, the avidity of GBSSI to ADPG is low, such as in pea GBSSI to the K of ADPG mfor 1.4mmol/L, and other SS is to the K of ADPG mat 0.06-0.6mmol/L.In amyloplast, the concentration of ADPG is close to the saturation value of other SS catalyzed reaction, and lower than the suitable substrate concentration of GBSSI.In ADPG biosynthesis block mutant, amylose content reduces the amplitude that amplitude is greater than amylopection content reduction.Can say that ADPG content is an important factor of restriction amylose starch synthesis.But the research of this aspect is reported in shortage corn embryosperm.
GBSSI is fixed on along with the growth of starch small grain wherein, can not move as other SS in amyloplast matrix.In starch small grain, the structure matrix of amylopectin can affect GBSSI activity and amylose content in several ways.The infiltration of dense matrix to ADPG and MOS of amylopectin has inhibition, and along with the increase with starch small grain top layer distance, the concentration of ADPG and MOS reduces thereupon, causes the substrate of GBSSI under-supply.But the lower MOS of this matrix closely polymerization degree that also makes GBSSI newly synthesize is trapped in around GBSSI and not easily spreads out, be conducive to the lasting extension of the poly sugar chain that GBSSI catalysis is formed.Therefore, the existence of amylopectin structure not give only GBSSI and can effectively combine and the dynamics extending MOS, have impact on the perviousness of ADPG and MOS yet, affects the enzyme activity of GBSSI.This explains why in common starch grain amylose content only no longer improve at about 25-30%.
Show the deletion mutantion of GBSSI and the research of suppression Wx genetic expression, GBSSI enzymic activity becomes positive correlation with amylose content.Lacking completely of Wx gene function will cause the serious reduction (0-5%) of amylose content, even cause in starch only containing 100% amylopectin.Although the synthesis of GBSSI to amylose starch has so important effect, whether Grain Amylose content report is affected seldom to the process LAN of Wx gene.Wheat Wx-B1 gene proceeds in durum wheat by particle bombardment by the report such as Sestili, the amylose content of transgenic wheat does not significantly improve (Sestilietal.2012), infer that 2 kinds of different GBSSI hypotypes (Wx-A1, Wx-B1) can provide enough enzymic activity catalysis amylose starch synthesis in durum wheat, in wheat, the impact of process LAN Wx-B1 gene pairs amylose content is very limited.Because GBSSI albumen is fixed on wherein along with the growth of starch small grain, can not move in amyloplast matrix as other SS, the substrate limited amount that can be utilized around it, therefore, increase the abundance of GBSSI albumen in endosperm and be still to improve amylose starch synthesis capability the approach merited attention.
Summary of the invention
For current present Research, the problem to be solved in the present invention is to provide corn ZmWx gene and is improving the application in corn yield and improvement grain characters.
In the present invention, from ncbi database and Maize genome order-checking data, inquiry obtains the cDNA sequence of corn ZmWx gene (encodes granule bound starch synthetic enzyme GBSSI), finds to only have a kind of GBSSI albumen in corn.Carry out GC% to the encoding sequence of the latter and analyze discovery, its GC% is up to 66.36%.
With corn embryosperm cDNA for template, according to Maize genome order-checking Data Design primer amplification ZmWx full length gene sequence.Because GC content is high, fail to increase out object fragment with common high-fidelity enzyme; Using LATaq enzyme instead coordinates GCbufferII successful clone to go out ZmWx full length gene sequence.Order-checking is delivered after described full length sequence is connected into pEASY-Blunt cloning vector.The ZmWx gene amplified contains complete encoding sequence, but has sequence in 3 bases and database inconsistent, and the amino acid of its coding does not change.These differences may be that the sequence of its ZmWx gene and self-mating system B73 exists single nucleotide polymorphism and caused because the donor of cDNA template is inbred Zheng 58.
Corn ZmWx gene of the present invention is improving the application in corn yield and improvement grain characters.
Wherein: the cDNA sequence of described corn ZmWx gene is as shown in SEQIDNo.1, and the aminoacid sequence of its coding is as shown in SEQIDNo.2; Described corn yield and grain characters refer to the single plant yield of corn, 100-grain weight and amylose content.
The application method of corn ZmWx gene of the present invention in raising corn yield and improvement grain characters is: from corn embryosperm cDNA, clone ZmWx gene order, described ZmWx genes encoding corn particle mating type amylosynthease GBSSI, itself and endosperm specificity promoter are formed fusion gene, fusion gene to be recombinated to maize transformation in plant expression vector by transgenic technology, obtain the corn gene plant of output increased and grain characters improvement; Or the high starch maize of GBSSI process LAN corn and saltant type AGPase process LAN is hybridized, screening obtains transgene pyramiding body, produces the corn gene strain that grain yield, starch content and 100-grain weight are significantly higher than acceptor material.
Wherein, described endosperm specificity promoter is preferably zein spirit-soluble gene promotor P27kD or P22kD.
In above-mentioned application, described corn ZmWx gene is improving the application method in corn yield and improvement grain characters preferably: adopt RT-PCR method from corn embryosperm cDNA, clone corn ZmWx gene, then merge with P27kD, and with herbicide resistance gene als for selective marker, construct plant conversion carrier; Adopt agriculture bacillus mediated maize bud point genetic transforming method transgenic corn plant and offspring thereof, select the transgenic line that starch content and 100-grain weight are significantly higher than acceptor material.
In the present invention, be inserted in plant expression vector by the ZmWx gene of clone and produce conversion carrier pCAMBIA1300-P27kD::ZmWx-als, use zein spirit-soluble gene promotor P27kD or P22kD etc., endosperm-specific ground starts ZmWx genetic expression.Als is weedicide chlorsulfuron resistant gene, encoding Arabidopsis mutant acetolactate synthase.Adopt freeze-thaw method that the conversion carrier built is proceeded to agrobacterium tumefaciens bacterial strain LBA4404, identify that correct transformant transforms for follow-up maize genetic by colony polymerase chain reaction (PCR) method.
In the present invention, with the prosperous 7-2 of maize elite inbred line and Zheng 58 for acceptor (respectively referred to as Wx-C and Wx-Z) carries out genetic transformation.Herbicid resistant screening and PCR detection are carried out to transformed plant and offspring thereof, judges whether to reach transgenic homozygous according to Herbicid resistant segregation ratio.In the plant of each transgenic corns strain and acceptor self-mating system, ZmWx gene is specificity process LAN in corn embryosperm, and the abundance of ZmWx gene transcripts improves along with the growth of grouting time, reaches climax, then reduce after pollination when the 30th day.Compared with acceptor self-mating system plant, in selected transgenic line, the expression intensity of ZmWx gene is significantly higher than acceptor self-mating system, but increasing degree is variant between each transgenic line.Under prosperous 7-2 background, the ZmWx Gene expression intensities of transgenic line Wx-C-5 is the highest; Under Zheng 58 background, the ZmWx Gene expression intensities of transgenic line Wx-Z-20 is the highest.
In the present invention, transgenic line GBSSI activity significantly improves, and this means that there is more zymoprotein in albuminous cell can play a role.Genetically modified process LAN increases corresponding with GBSSI enzymic activity.In order to confirm to proceed to ZmWx gene and realize process LAN whether have impact to GBSSI activity in corn embryosperm, to difference grouting period (10,15,20,25,30,35DAP) T3 carry out GBSSI determination of activity for the endosperm of transfer-gen plant and acceptor self-mating system plant.Show that in each transgenic line and acceptor self-mating system, GBSSI activity increases along with the propelling of filling process, peaks in pollination for latter 35 days.Compared with acceptor self-mating system, transgenic line GBSSI activity is significantly increased, and increasing degree is variant between different strain.
It should be noted that all strains were when latter 10 days of pollination, GBSS enzymic activity is very low, is only 0.007-0.0165nmolmin -1mgprotein -1, and to pollination latter 35 days time be increased to 0.232-0.297nmolmin -1mgprotein -1.The Changing Pattern of this enzymic activity is different from AGPase.Infer that GBSSI is active in relevant with Gene expression intensities, also relevant with the hypocrystalline texture of the starch residing for it, namely at Early filling stage, the order degree of starch hypocrystalline texture is low, and GBSSI activity is restricted.
In the present invention, analyze the change of process LAN ZmWx gene corn seed starch composition.By T3 for transgenic corn plant and acceptor self-mating system plant kind in experimental field, bagging selfing, gathers in the crops after Grain Ripening, for measuring total starch content and amylose content.Result shows, the process LAN of ZmWx gene adds the amylose content of corn embryosperm.The amylose content of each transfer-gen plant comparatively non-transgenic reference all significantly increases, but between different transgenic line, amplification has difference.Under prosperous 7-2 background, transgenic line amylose content is increased to 35.8-41.1% by 25.3% of acceptor self-mating system, and wherein transgenic line Wx-C-5 amplification is maximum, is increased to 41.1%.Under Zheng 58 background, transgenic line amylose content is increased to 35.3-41.9% by 26.4% of acceptor self-mating system, and wherein transgenic line Wx-Z-20 amylose content is the highest, reaches 41.9%.
In storage Starch synthesis process, the increase of amylose starch synthesis often causes the reduction of total starch content, therefore, measures in the present invention to the Grain starch content of each transgenic line and acceptor self-mating system.Under two kinds of self-mating system backgrounds, compared with acceptor self-mating system, ZmWx transgenic line Grain starch content does not reduce, and slightly increases, and the raising of partial transgenic strain starch content reaches the significance level of difference.Wherein the starch content of transgenic line Wx-C-1 (being increased to 71.18% by contrast 64.18%) and Wx-Z-6 (being increased to 71.24% by contrast 66.21%) is the highest.But, contrast the variation tendency of amylose content and total starch content in each ZmWx transgenic line, still show that the transgenic line total starch content that amylose content is the highest is minimum, close with acceptor self-mating system.
In the present invention, by ZmWx gene overexpression transgenic corns and acceptor self-mating system kind in field, growth conditions is identical with control measures, bagging selfing, and harvest-seed extraction after Grain Ripening, takes 100-grain weight after seed drying.The Grain Development of each transgenic line is normal, and 100-grain weight slightly increases, and its increasing degree is different in the strain of different independent transformants.Under prosperous 7-2 background, the process LAN of ZmWx gene makes 100-grain weight improve more than 13% than acceptor self-mating system, reaches difference pole conspicuous level.Under Zheng 58 background, the 100-grain weight of ZmWx transgenic line increases about 17% than acceptor self-mating system, and difference reaches conspicuous level.Show that, under identical self-mating system background, transgenic corns Grain starch content and 100-grain weight increasing degree have dependency, its seed 100-grain weight of the strain that namely Grain starch content is higher is also higher.Meanwhile, the transgenic line 100-grain weight that amylose content is high is low.Namely in endosperm, process LAN GBSSI can promote Starch synthesis, improves Grain Amylose content, and not to sacrifice total starch content for cost, so that partial transgenic strain shows and significantly improves starch content and 100-grain weight.
In the present invention, in order to determine the impact of process LAN GBSSI because growing on transgenic corn plant in endosperm, getting transgenic line and acceptor self-mating system is seeded in large Tanaka, carrying out community comparison test.Open pollination, system carries out Observation on Agronomic Characters and determination of yield.Observe plant type at Early filling stage and add up plant height and Ear height.Measure spike length and row grain number after mature fruit cluster dries, measure 100-grain weight and grain number per spike, calculate single plant yield in conjunction with community strain number.In whole breeding time, there is not obvious change in the Plant Type in Maize of process LAN ZmWx gene, grows and blossom and have seeds normal.Drawn by the field statistic data of 8 transgenic lines and acceptor self-mating system, plant height and Ear height change between different transgenic line, but luffing is less, wherein only the plant height of 1 transgenic line of Zheng 58 background and Ear height significantly reduce than the plant of acceptor self-mating system.But fruit ear and grain characters change obviously between transgenic line and acceptor self-mating system.Compared with acceptor self-mating system, transgenic line output and 100-grain weight under open pollination conditions obviously increase.Wherein the single plant yield of transgenic line Wx-C-31 increases to 77.84 ± 5.34 by the 66.46 ± 5.72g contrasting prosperous 7-2, and transgenic line Wx-Z-6 is increased to 80.11 ± 7.55g by the 67.68 ± 10.67g contrasting Zheng 58.Namely the process LAN of GBSSI can improve maize grain yield and 100-grain weight.
The present invention by agriculture bacillus mediated stem apex genetic transformation by clone the prosperous 7-2 of corn Wx channel genes maize elite inbred line, in Zheng 58, achieve process LAN, obtain the transgenic line that 100-grain weight, single plant yield and endosperm amylose content significantly improve.
In the present invention, also the strain from same acceptor self-mating system of Wx gene overexpression material and transposon mutant type AGPase gene is hybridized, create the transgene pyramiding strain that single plant yield, 100-grain weight, endosperm starch content and amylose starch ratio significantly improve, obtain starch content about 78% high starch maize breeding material.
The invention has the beneficial effects as follows: after corn Wx gene and corn embryosperm specificity promoter P27kd being merged, import maize elite inbred line, produce transfer-gen plant; The transgenic line that 100-grain weight, single plant yield, endosperm starch content and amylose starch ratio significantly improve is obtained from stable transgenic line, and other proterties of transfer-gen plant and acceptor self-mating system are without marked difference, for the material having important value has been formulated in the exploitation of high starch maize and amylomaize.This invention is that corn yield improves and a new way is explored in starch quality improvement.
Embodiment
Embodiment 1: process LAN GBSSI improves maize grain yield and amylose content Wx gene clone and restructuring
Get the corn kernel be in the milk mid-term and be about 0.1g, liquid nitrogen grinding becomes powder, transfers to and fills 800 μ L extracting solution (100mMTris, pH9.0; 200mMNaCl; 1% sodium laurate; 20mMEDTA; In 1.5mL centrifuge tube 5mMDTT), vortex mixes 1 minute.Centrifugal 10 minutes of 12000rpm at 4 DEG C, then gets supernatant 600 μ L, adds the NaAc (2M, pH4.0) of 1/10 volume, mixing, then adds 300 μ L chloroform isoamyl alcohols (24:1) and 300 μ L water-saturated phenols, vortex 2 minutes.Centrifugal 10 minutes of 12000rpm at 4 DEG C, gets supernatant 500 μ L, adds 500 μ L chloroform isoamyl alcohols (24:1), vortex extracting 2 minutes again.Then centrifugal 10 minutes of 12000rpm at 4 DEG C, gets supernatant 360 μ L, adds 120 μ L8MLiCl, 4 DEG C of precipitations 3 hours, centrifugal 15 minutes of 12000rpm under low temperature.Wash 2 times with 75% ethanol again, drying at room temperature is to transparence.Add 20 μ LDEPC water dissolution, obtain qualified Total RNAs extraction thing.
Endosperm total serum IgE is carried out quantitatively, then shift in the centrifuge tube of about 300ng to RNasefree, add RNasefreeH 2o mends to 500 μ L.Add OBBbuffer (20mMTris-Cl, the pH7.5 of equal-volume (500 μ L) again; 1MNaCl; 2mMEDTA; 0.2%SDS) He 30 μ L10% (W/V) OligotexSuppressoionparticles (suppressionoligotexparticlessuspendedinbufferof10mMTric-Cl, pH7.5; 500mMNaCl; 1mMEDTA; 0.1%SDS; 0.1%NaN 3), then fully mix, be put in 70 DEG C of incubations 3 minutes.Sample is taken out, is chilled to room temperature, oligotexparticles and mRNA is fully combined.16000g centrifugal force 2 minutes, oligotex-mRNA complex precipitate, carefully sucks supernatant liquor, in precipitation, add OW2Buffer (10mMTris-Cl, pH7.5; 150mMNaCl; 1mMNaCl; 1mMEDTA) resuspended Oligotex-mRNA precipitation (mode with vortex or piping and druming), with 16000g centrifugal 2 minutes, abandons supernatant, then repeats this step once.In precipitation, add the OEBbuffer (5mMTric-Cl, pH7.5) of 20-100 μ L70 DEG C of preheating again, resuspended precipitation, centrifugal 2 minutes, is transferred to supernatant in new centrifuge tube for subsequent use, is the mRNA of purifying.
Reverse transcription is carried out with OligodTprimer.Reaction system is RNA500ng, 2 μ L5 × PrimeScriptBuffer, 0.5 μ LOligodTprimer, 0.5 μ LPrimeScriptRTEnzymeMixI, RNaseFreedH 2o complements to 10 μ L.In 37 DEG C of reactions 30 minutes, then in 85 DEG C of deactivations in 5 seconds.The strand cDNA obtained is stand-by-20 DEG C of preservations.
According to the nucleotide sequence (Accessionnumber:NM_001111531) of the corn particle mating type amylosynthease GBSSI encoding gene ZmWx submitted in ncbi database, design synthetic primer, to increase its coding region full length sequence by PCR method from corn embryosperm cDNA storehouse.With corn embryosperm cDNA for template, GCbufferII is coordinated to carry out PCR reaction with LATaq enzyme.The primer sequence of amplification Wx gene: Primer-1:5 '-G cTCTAGAaTGGCGGTCTTGGCCACG-3 ' (underscore part is the XbaI enzyme cutting site of introducing, and G is protection base) Primer-2:5 '-CGG gGTACCtCAGGGCGCGGC-3 ' (underscore part is the KpnI restriction enzyme site introduced, and CGG is protection base).Comprise in 50 μ LPCR reaction systems: 5 μ L10 × LAPCRBufferII (Mg 2+plus), 8 μ LdNTPMixture (2.5mMeach), 2.5ng template cDNA, 1 μ Lprimer-1,1 μ Lprimer-2,32 μ LddH 2o, 0.5 μ LLATaq (5U/uL).PCR response procedures is: 95 DEG C of denaturations 5 minutes, 38 circulations: 95 DEG C 1 minute, 62 DEG C 1 minute, 72 DEG C 2 minutes, 72 DEG C excessively extend 7 minutes.
The PCR primer agarose gel electrophoresis of 1% is separated, and cuts 1.8kb object fragment under ultraviolet lamp, reclaims test kit reclaim with AxyProDNA gel.Object fragment is connected in pEASY-Blunt carrier, the super competent cell of transformation of E. coli DH5 α.LB solid medium flat board containing 50mg/L kantlex screens transformant, adopts blue hickie method to screen, i.e. picking white colony on Selective agar medium.The latter to contain in the LB liquid nutrient medium of 50mg/L kantlex 37 DEG C of concussion cultivations 12 hours at 3mL.Carry out enzyme by alkaline lysis method of extracting plasmid DNA to cut and identify and carry out sequence verification.
The ZmWx gene of sequence verification is used for building plant expression vector pCAMBIA1300-P27kD::ZmWx-als.Carrier used is plasmid pCAMBIA1300-P35S-Tnos-als and pCAMBIA3300-p27kD::Bt2-bar.
Use HindIII and XbaI double digestion plasmid pCAMBIA3300-p27kD::Bt2-bar, after agarose gel electrophoresis is separated, reclaim the P27kD promoter fragment of 1368bp.Use HindIII and XbaI double digestion plasmid pCAMBIA1300-P35S-Tnos-als simultaneously, reclaim carrier segments after electrophoretic separation, be connected into P27kD promoter fragment.Cut after qualification through enzyme, obtain the correct carrier pCAMBIA1300-P27kD-als inserted.Use XbaI and KpnI double digestion carrier pEASY-Blunt-ZmWx, after agarose gel electrophoresis is separated, reclaim the ZmWx fragment of 1859bp; Use XbaI and KpnI double digestion carrier pCAMBIA1300-P27kD-als, after electrophoretic separation, reclaim carrier segments.By ligation, ZmWx full length fragment is inserted in pCAMBIA1300-P27kD-als, produce conversion carrier pCAMBIA1300-P27kD::ZmWx-als.
The plant expression vector pCAMBIA1300-P27kD::ZmWx-als enzyme of structure is cut qualification, determine that the correct rear freeze-thaw method that adopts is proceeded in agrobacterium strains LBA4404, and identify that correct transformant is for follow-up maize genetic transformation experiment with colony polymerase chain reaction (PCR) method.
Corn inbred line genetic transformation
With Inbred Lines Zheng 58 used in China's agriculture production, prosperous 7-2, DH4866 for acceptor self-mating system.Seed 70% alcohol immersion 10 minutes, then soak 10-15 minute with 0.1% mercury chloride, then with sterilized water washing 3-5 time.After sterilizing, seed is placed in aseptic triangular flask and sprouts, and puts into a small amount of sterilized water in bottle, is placed on 1-2 days under dark condition (24 DEG C) after sealing.(showing money or valuables one carries unintentionally) seed of sprouting proceeds on minimum medium and continues to sprout under dark condition.When coleoptile elongation stops 3-4 centimetre, peel off coleoptile and 2-3 sheet spire, expose stem apex growing tip for agroinfection.
By the agrobacterium tumefaciens LBA4404 with binary vector (Mini--Ti plasmid is pCAMBIA1300-P27kD::ZmWx-als), at additional antibiotic LB substratum, (often liter of substratum contains: tryptone 10g, yeast extract 5g, NaCl10g, pH7.0, pressure sterilizing) at 28 DEG C concussion cultivate, concussion speed is 110rpm (rev/min), makes bacterium be in logarithmic phase.Then at 3,000 rpm centrifugal 10 minutes, supernatant liquor is abandoned.The thalline MS liquid nutrient medium of 1/2 concentration washs, then collected by centrifugation.Again the MS liquid nutrient medium of thalline by 1/2 concentration of adding Syringylethanone (acetosyringone, As) 100 μm of ol/l is suspended, dilution OD 6000.25-0.65 for transforming.During conversion, first bacterium liquid is poured in the culture dish of 4.5 cm diameters, inclination culture dish, makes aseptic seedling stem apex be immersed in bacterium liquid, 0.5 × 10 58-12 minute is processed under Pa normal atmosphere.Then blotted by the stem apex aseptic filter paper after dip-dye, be put back into by seedling on solidified MS media and cultivate 2-3 days in dark, culture temperature is 22-24 DEG C.Thereafter seedling is put and cultivate 2 days under diffuse light, be transplanted in the flowerpot of upper strata vermiculite lower floor loam, and cover plant top with vermiculite.After plant grows vermiculite, the 3 leaf phases that grew under natural lighting spray selective agent chlorsulfuron (Shenyang Agricultural Chemicals Factory produces, 15% effective constituent) 25ppM solution, day temperature 22-28 DEG C, and night, temperature 15-21 DEG C, watered 1/2MS substratum inorganic salt every other day.
After 3 leaf phase unconverted plant spraying herbicide chlorsulfurons, within about 7 days, stop growing, after 15 days, start death.After spraying, some individual changes are with unconverted adjoining tree, and other individual continued propagation, change not obvious for transformed plant.When the plant that survives grows to 5 leaf, by its field planting to field, bagging selfing is set seeds.T1 seed from different T0 plant is broadcast in greenhouse or the field with safeguards, 3 the leaf phase plant spray chlorsulfuron 25ppM or 30ppM the solution susceptibility of the factor receptor self-mating system (and variant).After first quarter moon, non-transfer-gen plant is dead, the survival of transfer-gen plant majority, continued growth.The blade getting transplant survival plant carries out Molecular Detection determination transfer-gen plant.Then by transfer-gen plant (T1) bagging self-fertility.By continuous 2-3 for selfing, obtain transgenic homozygous strain.Through detecting transgene expression level, GBSSI enzymic activity and the Analysis and Identification to tree characteristics, filtering out transfer-gen plant and offspring thereof that grain characters obviously changes, createing in breeding the new germ plasm with application prospect.
In PCR detects, with herbicide resistance gene als and goal gene ZmWx for detected object.Comprise in 25 μ LPCR reaction systems: 2.5 μ L10 × PCRBufferII (Mg 2+free), 1.5 μ LMg 2+, 0.5 μ LdNTPMixture (10mMeach), 2.5ng template DNA, 1 μ L10uMprimer-1,1 μ L10uMprimer-2,18.375 μ LddH 2o, 0.125 μ LrTaq.The PCR reaction conditions detecting als gene is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 1 minute, 56 DEG C of annealing 1 minute, 72 DEG C extend 1 minute, circulate 40 times; 72 DEG C excessively extend 7min, amplified production electrophoresis on the sepharose of 1% (w/v).Upstream primer als-F:5 '-GAGGACACGCTGAAATCACC-3 ', downstream primer als-R:5 '-CATCGGCTAATCCGCTAA-3 '.Because ZmWx gene intron sequence is shorter, if the primer that PCR detects is taken in ZmWx genes encoding frame, then being difficult to differentiation is native gene or genetically modified amplified production, and therefore the design of the detection primer one of transgenosis ZmWx held in P27kD promotor 3 ', another is positioned at ZmWx coding region.Upstream primer P27kD:5 '-TCTCCATCACCACCACTGGG-3 ', downstream primer Wx-R:5 '-GAAATCGAAGGACGACTTGAATC-3 '.PCR reaction conditions is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 1 minute, 60 DEG C of annealing 1 minute, 72 DEG C extend 1 minute, circulate 40 times; 72 DEG C excessively extend 7min, and amplified production 1% (w/v) agarose gel electrophoresis is separated.
Be 26 independent transformants of acceptor and Zheng 58 at prosperous 7-2 be that in 19 independent transformants of acceptor, T2 and T3 meets Mendelism for the segregation ratio of weedicide chlorsulfuron resistance, most of transfer-gen plant is integrated into Maize genome in the mode of single copy.
Transgene expression level detects and adopts real-timeRTPCR method.Transfer-gen plant seed RNA extracts and reverse transcription system and the homogenic clone of method.Reference gene is actin1.Reaction system: premixExTaq tM(2 ×) 5 μ L, PCRsensePrimer (10 μm of ol/L) 0.2 μ L, PCRantisensePrimer (10 μm of ol/L) 0.2 μ L, cDNA template 1 μ L, SterileddH 2o3.6 μ L.PCR reaction conditions is: 95 DEG C of denaturations 5 minutes; 95 DEG C of sex change 15 seconds, 60 DEG C of annealing 15 seconds, 72 DEG C extend 37s, circulate 40 times; 72 DEG C excessively extend 5min, amplified production electrophoresis on the sepharose of 2% (w/v).The primer sequence of amplifying target genes ZmWx: GBSSI-F:5 '-GCCTGTCGCTGGAACGGACT-3 ', GBSSI-R:5 '-CTTTGCGTCCCTGTAGATGC-3 '.Detected result is with 2 -Δ Δ Ct(LivakandSchmittgen, 2001) method calculates, and be 1 unit with the expression amount of the non-transgenic reference actin1 gene of 10DAP, the expression amount of transgenic line represents with the multiple of actin1 expression amount.From difference grouting period (10,15,20,25,30,35DAP) transgenic line plant and the fruit ear of its acceptor self-mating system plant (WT) strip seed, extracting RNA, is template after reverse transcription.In different milpa, ZmWx gene transcripts abundance increases along with the growth of grouting time, reaches climax, then reduce after pollination when the 30th day.Compared with acceptor self-mating system plant, in transgenic line, the expression intensity of ZmWx gene significantly improves, but increasing degree is variant between each transgenic line, to be generally all significantly higher than acceptor self-mating system.Under prosperous 7-2 background, the ZmWx Gene expression intensities of transgenic line Wx-C-5 is the highest, is the 1.5-3.2 of acceptor self-mating system doubly (variant because of Grain Development difference in period); Under Zheng 58 background, the ZmWx Gene expression intensities of transgenic line Wx-Z-20 is the highest, is 1.5-3.9 times of acceptor self-mating system.
In GBSS Enzyme assay, first carry out zymoprotein extraction.Namely get the pollination each 0.1g of endosperm of latter 10,15,20,25,30,35 days, add 2ml Extraction buffer (50mmol/LHepes-NaOH, pH7.5,5mmol/LMgCl respectively 2, 1mmol/LDTT, and1mg/mLBSA), ice bath grinds, and is transferred in 7mL centrifuge tube, then in mortar, add the flushing of 2ml Extraction buffer, is incorporated in the lump in centrifuge tube.At 4 DEG C, centrifugal 20 minutes of 20000g, abandons supernatant.Adding 2mL Extraction buffer will precipitate resuspended, and at 4 DEG C, centrifugal 20 minutes of 20000g, abandons supernatant; Repeat again once.Finally add 1mL Extraction buffer, resuspended precipitation.In GBSS determination of activity, containing 100mmol/LBicine (pH8.3) in 100 μ L reaction systems, 4.5mmol/LEDTA, 25mmol/LKCl, 10mmol/LGSH, 7.5mmol/L [ 14c] ADPG (59.2dpm/nmol), and 25 μ L enzyme liquid.In 25 DEG C of reactions 60 minutes, add 50 μ L0.25NNaOH termination reactions.The a identical system of another preparation adds 50 μ L0.25NNaOH termination reactions immediately, as blank after adding enzyme liquid.The methyl alcohol adding 0.5mL precooling makes starch sedimentation, and mixing is placed on-20 DEG C of precipitations 10 minutes.At 4 DEG C, centrifugal 5 minutes of 12400g, abandons supernatant, and precipitation is redissolved in 300 μ L0.1NNaOH.Add the alcohol settling of 1mL precooling again, mixing is placed on-20 DEG C of precipitations 10 minutes, at 4 DEG C with 12400g centrifugal 5 minutes, and abandon supernatant, precipitation is redissolved in 300 μ L0.1NNaOH; Repeat again once, to remove the radiolabeled substrate not mixing reaction.Abandon supernatant after last pelleting centrifugation, in starch sedimentation, add 100 μ L1NHCl, boiling water bath 10 minutes, draw 90 μ L after cooling in scintillation vial, add 1mL scintillation solution, measure radioactive intensity.Quantification of protein (Bradford, 1976) is carried out with Coomassie Brilliant Blue.Every milligram of total protein in 1 minute is mixed 1nmolADPG and is defined as 1 unit of enzyme activity.In each transgenic line and acceptor self-mating system, GBSSI activity increases along with Filling velocity, within latter 35 days, peaks in pollination.Compared with acceptor self-mating system, transgenic line GBSSI activity is significantly increased, and increasing degree is variant between different strain, and majority is 1.2-1.5 times of acceptor self-mating system.It should be noted that all strains were when latter 10 days of pollination, GBSS enzymic activity is very low, is only 0.007-0.0165nmolmin -1mgprotein -1, and to pollination latter 35 days time be increased to 0.232-0.297nmolmin -1mgprotein -1.The Changing Pattern of this enzymic activity is different from AGPase.Infer that GBSSI is active in relevant with Gene expression intensities, also relevant with the hypocrystalline texture of the starch residing for it, namely at Early filling stage, the order degree of starch hypocrystalline texture is low, and GBSSI activity is restricted.
The Agronomic characteristic of transgenic corns strain and determination of yield
By by for selfing and detection, obtain the corn strain of the transgenic homozygous of the ZmWx gene overexpression of Absorbable organic halogens heredity.By them and acceptor self-mating system together field planting, adopt same field management, observe and record and gather in the crops fruit ear, get seed and measure 100-grain weight, total starch content and amylose content.In order to whether the different strains understood from same independent transformants also exist difference, from each independent transformants offspring, select 3 strains carry out parallel test.
The synthesis of Granule-Bound Starch Synthase GBSSI primary responsibility amylose starch.By transgenic corn plant and the selfing of acceptor self-mating system plant bagging, results seed is for measuring total starch content and amylose content.Result shows, the process LAN of ZmWx gene adds corn embryosperm total starch content and amylose content, and the total starch content of each transgenic line and amylose content comparatively acceptor self-mating system significantly increase, but between different transgenic line, amplification has difference.Compared with acceptor self-mating system, part turns ZmWx gene strain Grain starch content obviously to be increased, and difference reaches conspicuous level.The Grain starch content that wherein Grain starch content of transgenic line Wx-C-1 is increased to 71.18%, Wx-Z-6 by 64.18% of acceptor self-mating system is increased to 71.24% by 66.21% of acceptor self-mating system.Under prosperous 7-2 background, the amylose content of transgenic line also brings up to 35.8-41.1% by 25.3% of acceptor self-mating system, and wherein transgenic line Wx-C-5 amplification is maximum, is increased to 41.1%.Under Zheng 58 background, the amylose content of transgenic line is also increased to 35.3-41.9% by 26.4% of acceptor self-mating system, and wherein transgenic line Wx-Z-20 amylose content is the highest, reaches 41.9%.If amylose content in each ZmWx transgenic line and starch content variation tendency correspondence analysis are drawn, the transgenic line Grain starch content that amylose content is the highest is low, but still higher than acceptor self-mating system.
By ZmWx gene overexpression corn and acceptor self-mating system kind in land for growing field crops, community is planted, repeat for 4 times, growth conditions is identical with control measures, and system carries out Observation on Agronomic Characters, adds up plant height and Ear height in Early filling stage observation plant type.Harvest drying after fruit ear maturation, mix threshing, weigh, take dry rear hundred grain weight.Field data statistical study draws, plant height and Ear height change between different transgenic line, but luffing is less, little with acceptor self-mating system difference.But fruit ear and grain characters have considerable change between transgenic line and acceptor self-mating system, the Grain Development of each transgenic line is normal, and 100-grain weight has and increases in various degree.。Compared with acceptor self-mating system, transgenic line its 100-grain weight and output under open pollination conditions obviously increase.Wherein the single plant yield of transgenic line Wx-C-31 increases to 77.84 ± 5.34 by the 66.46 ± 5.72g contrasting prosperous 7-2, and 100-grain weight brings up to 24.96 ± 0.64g from 22.06 ± 0.4g of acceptor self-mating system; Transgenic line Wx-Z-6 is increased to 80.11 ± 7.55g by the 67.68 ± 10.67g contrasting Zheng 58, and 100-grain weight is increased to 34.17 ± 1.14g by 29.22 ± 1.23g of acceptor self-mating system, improves 16.94%.Draw from these data, the process LAN of GBSSI can improve corn 100-grain weight and grain yield, difference reaches conspicuous level compared with acceptor self-mating system, and transgenic corns Grain starch content becomes dependency with the increasing degree of 100-grain weight, and its 100-grain weight of strain that namely Grain starch content is higher is also higher.In endosperm, process LAN GBSSI can not only improve Grain Amylose content, and also promoting 100-grain weight increases and total starch increase.
This work also finds the obvious increase compared with acceptor self-mating system of the spike length of transgenic line, under prosperous 7-2 background, rises to 11.5 ± 0.8cm, reach the significance level of difference by 8.8 ± 1.0cm of WT fruit ear.Under Zheng 58 background, the process LAN of ZmWx gene also makes the spike length of each transgenic line be increased to 16.9 ± 0.6cm by 13.8 ± 1.3cm of acceptor self-mating system.Draw the further analysis of Ear Characters, compared with acceptor self-mating system, the tassel row number of each strain fruit ear of process LAN ZmWx gene pairs has no significant effect, but the row grain number of transgenic line fruit ear increases.Draw thus, it is caused by the increase acting in conjunction of row grain number and 100-grain weight that the transgenic line grain yield that ZmWx gene overexpression causes improves.
Embodiment 2: polymerization transgenosis ZmWx and saltant type AGPase gene improve maize grain yield and starch content
Because GBSSI controls the ability of amylose starch synthesis, except by except the impact of GBSSI protein content, be also subject to the restriction of the factors such as substrate A DPG.In the well-off situation of substrate A DPG, the amylose content that GBSSI is responsible for synthesizing likely improves further.In Starch synthesis process, AGPase catalysis G1P and ATP generates ADPG and inorganic phosphate PPi, and the synthesis for starch provides precursor substance ADPG.It is generally acknowledged that the synthesis of ADPG is the important regulating and controlling point of Starch biosynthesis, AGPase is rate-limiting enzyme important in Starch synthesis approach.Therefore, the transgenic corns of the saltant type AGPase process LAN of acquisition is carried out hybridization with the transgenic corns of GBSSI process LAN and is polymerized, detect grain characters and starch content and composition.
Embodiment 1 is shown in the acquisition of GBSSI process LAN transgenic corns.
Step and the method for the generation of saltant type AGPase process LAN transgenic corns are as follows:
Corn AGPase is the allos tetramer be made up of two large subunits and two small subunits, and the AGPase of 85% is plasmotype, and regulates and controls by the activation of 3-PGA and the restraining effect of Pi.The large and small subunit of corn AGPase is encoded by Sh2 and Bt2 respectively.The large subunit of the saltant type Sh2r6hs coding of Sh2 has two site mutations, namely the Histidine mutagenesis of its coded amino acid the 333rd is tyrosine and inserts tyrosine and serine residue after the 476th amino acids, sudden change causes with the interaction of small subunit stronger, and AGPase is reduced the restraining effect susceptibility of Pi.In the present invention, overlapping primers extension method is utilized to obtain the large subunit coding gene Sh2r6hs of saltant type AGPase, corn embryosperm specificity promoter P22kD and P27kD is selected to drive the expression of Sh2r6hs gene and Bt2 gene respectively, construct the process LAN plant vector pCAMBIA3300-P27kD::Bt2-P22kD::Sh2r6hs-bar of Sh2r6hs gene and Bt2 gene tandem, adopt agriculture bacillus mediated maize bud point method for transformation goal gene to be proceeded to the prosperous 7-2 of Elite inbred and Zheng 58.
The seed (T1 generation) produced for transformed plant by T0 is broadcast in vinyl disc, and 3 leaf phases, with the screening of weedicide grass fourth phosphine, then carried out PCR detection.The seedling of Herbicid resistant and the PCR positive is transplanted to large field, bagging selfing, obtains T2 for seed.T2 is continued herbicide spraying screening for seedling, and resistant plant is used for detection GMOs.Therefrom select the transgenic line of inheritance stability to carry out grain characters to observe and starch content mensuration.
For measuring the material of starch content and grain weight for non-non-transgenic control and T3 are for transfer-gen plant, being numbered C72-WT, Z58-WT, Sh2r6hsBt2 representative pCAMBIA3300-P27kD::Bt2-P22kD::Sh2r6hs-bar respectively and transforming the transgenic line obtained.C represents self-mating system prosperous 7-2, Z and represents inbred Zheng 58.
The PCR of transgenic corns detects with CTAB method Trace bio-element maize leaf DNA.Detect the primer of riddled basins bar: P1:5 '-TGACGCACAATCCCACTATCC-3 ', P2:5 '-AACCCACGTCATGCCAGTTCC-3 ', amplified production length: 600bp.The primer of testing goal gene Sh2r6hs: upstream primer 706F:5 '-GGCGATCAGCTTTATCGG-3 ', downstream primer 1499R:5 '-TTCCCAATCCTAGCATTCA-3 '.Contain in 25 μ L reaction systems: 10 × PCR reaction buffer 2.5 μ L, Mg 2+(25mM) 1.5 μ L, primer 1 (10 μMs) 1 μ L, primer 2 (10 μMs) 1 μ L, dNTP (10mMeach) 0.5 μ L, Taq DNA polymerase (5U/ μ L) 0.15 μ L, DNA profiling 50-100ng, aseptic double-distilled water supplies 25 μ L.Response procedures: denaturation 95 DEG C 7 minutes; Sex change 95 DEG C 1 minute, anneal 56 DEG C 1 minute, extend 72 DEG C 1 minute, circulate 38 times; Excessive extension 7 minutes.Amplification is detected by 1% agarose gel electrophoresis.
Real-timeRTPCR detects and adopts LiCl method to extract seed RNA, carries out reverse transcription quantitatively with random primer.Reverse transcription system is: RNA500ng, 5 × PrimerScriptBuffer2 μ L, Random6mers0.5 μ L, OligodTPrimer0.5 μ L, PrimerScriptRTEnzymeMixI0.5, SterileddH 2o complements to 10 μ L.In 37 DEG C of reactions 30 minutes, then in 85 DEG C of deactivations in 5 seconds.The strand cDNA obtained is stand-by-20 DEG C of preservations.
Reverse transcription gained cDNA is diluted 10 times, as the template of pcr amplification.The reagent that real-timeRTPCR adopts is SYBRGreenRT-PCRKit (Takara, Dalian, China).Rna transcription change multiple is originally with 2 -Δ Δ Ctmethod calculates, and with corn native gene actin for internal reference, calculation formula is Δ Δ C t=(C t, Targetgene-C t, actin) transgenic-(C t, Target gene-C t, actin) wT.Each sample repeats 3 times.Reaction system: premixExTaq tM(2 ×) 5 μ L, PCRsensePrimer (10 μMs) 0.2 μ L, PCRantisensePrimer (10 μMs) 0.2 μ L, cDNA template 1 μ L, SterileddH 2o3.6 μ L.PCR reaction conditions is: 95 DEG C of denaturations 5 minutes; 95 DEG C of sex change 15 seconds, 60 DEG C of annealing 15 seconds, 72 DEG C extend 37s, circulate 40 times; 72 DEG C excessively extend 5min, amplified production electrophoresis on the sepharose of 2% (w/v).The primer sequence: corn native gene actin gene primer P1:5 '-GCTACGAGATGCCTGATGGTC-3 '; P2:5 '-CCCCCACTGAGGACAACG-3 '; Upstream primer the P3:5 '-GCCGCTGCAAATGATTCAACATACC-3 ' of goal gene Bt2, downstream primer P4:5 '-GCAGGCTTGGCACGCTTCTTTG-3 '.Upstream primer the P5:5 '-GGGAGCGGACACCTATGAA-3 ' of goal gene Sh2, downstream primer P6:5 '-AGCCTCTTGGATGCCCTTAC-3 '.
In AGPase Enzyme assay, get the pollination seed 2-3 grain of latter 10,15,20,25 days respectively, be wrapped in gauze, in liquid nitrogen after quick-frozen 30s, be stored in-70 DEG C.Take the material that 0.5g is freezing, add 2ml Extraction buffer (50mMHEPES-NaOH, pH7.5; 5mMEDTA; 1mMDTT; 2mMKCl; 1%PVP) ice bath grinding.Again homogenate is proceeded to 10ml centrifuge tube, rinse mortar at twice with 6ml extracting solution, washing fluid is poured in centrifuge tube in the lump, and centrifugal 15 minutes of 15000g at 4 DEG C, getting its supernatant liquor is zyme extract.Reaction system is: add in 1.5mLEppendorf pipe: 100 μ L5mmol/LADPG, 50 μ L50mmol/LMgCl 2, 100 μ L50mmol/LHEPES-NaOH (pH7.5), the thick zyme extract of 50 μ L or the thick zyme extract through comparatively high temps process.By reaction mixture 30 DEG C of incubations 10 minutes; The same reaction system of preparation is put into boiling water bath 1 minute immediately simultaneously, contrast as unreacted.Add 100 μ L20mmol/LPPi again and start reaction, in 30 DEG C of reactions 15 minutes, boiling water bath 1 minute termination reaction.After being cooled to room temperature, add 100 μ L20mmol/LNADP +, 1.5IUPGM, 5IUG6PDH, 50 μ L50mmol/LMgCl2,200 μ L50mmol/LHEPES-NaOH (pH7.5), react 10 minutes in 30v, under 340nm wavelength, measure absorbance value.Typical curve is done with G-1-P.Quantification of protein (Bradford, 1976) is carried out with Coomassie Brilliant Blue.
In starch content measures, get the ripe dry seed 4-5 grain of each strain, be put in 40 DEG C of baking ovens and be dried to constant weight, record seed gross weight.Then in boiling water, boil 1-2 minute, peel off seed coat and embryo, be put in 40 DEG C of baking ovens and be dried to constant weight, record endosperm fraction gross weight.Use beveller (MM400, Retsch, Germany) with 25 hertz of grindings 1 minute, to wear into fine powder, cross 80 mesh sieves, namely obtain the sample measured for starch content.Slough the soluble sugar in Crude starch, then with perchloric acid hydrolysis-anthrone-dense H 2sO 4method measures total reducing sugar amount, calculates seed total starch content.
Transgenosis T1 is for strain after herbicide screening, and antagonism plant carries out PCR detection.Have 37 independent transformants to be detected as the PCR positive, wherein 21 independent transformants are from Zheng 58.Measured and hereditary segregation ratio by continuous selfing and Herbicid resistant, obtain transgenic homozygous strain.
The mensuration of 6 transgenic homozygous strains for Analysis of agronomic characters, grain yield and starch content is gone out according to starch content measurement result and seed phenotypic screen.Being sowed by their late March, is contrast with acceptor selfing.Zone leader 3m, wide 1.8m, point 3 row plantations, line-spacing 0.60m, spacing in the rows 0.25m, often row stays seedling 10 strain.If 3 repetitions, every transgenic line is kept a full stand of seedings 90 strains altogether.Result shows, the process LAN of saltant type AGPase does not make significant difference to plant forms feature, but adds the starch content of corn embryosperm.Increasing degree has difference because of transgenic line difference, and wherein the 100-grain weight of 5 strains, seed starch difference compared with acceptor self-mating system reach pole conspicuous level.The increase of starch content causes DW/FW to increase, and shows as the increase of seed dry weight and seed volume.Infer that the process LAN of saltant type AGPase improves endosperm AGPase active, and the raising of AGPase activity facilitates carbohydrate from soluble sugar to the conversion of starch, thus cause Starch synthesis and accumulation to increase.Under identical self-mating system background, transgenic corns Grain starch content and 100-grain weight increasing degree have dependency, and its 100-grain weight of strain that namely Grain starch content is high is also high.
In the present invention, transposon mutant type AGPase gene strain Sh2r6hsBt2-Z-54, Sh2r6hsBt2-Z-80, Sh2r6hsBt2-C-20, Sh2r6hsBt2-C-46, Sh2r6hsBt2-C-144 and GBSSI process LAN transgenic line Wx-Z-3, Wx-Z-20, Wx-C-7, Wx-C-8, Wx-C-12 and non-non-transgenic control (acceptor self-mating system) are seeded in land for growing field crops, bagging hybridization (comprising reciprocal cross) is carried out in plant pollination period, polymerization transgenosis, obtains F1 seed.The sister of F1 seed, corresponding parent handed over the seed of generation and acceptor self-mating system plant sister to hand over the planting seed of generation in land for growing field crops, carry out economical character observation and Yield compari@test.Community random alignment, if 4 times are repeated, open pollination, observes plant type at Early filling stage and adds up plant height and Ear height.After Grain Ripening, gather in the crops fruit ear, dry rear statistics spike length and row grain number, measure 100-grain weight and grain number per spike, calculate single plant yield in conjunction with community strain number.
F1 plant strain growth and blossom and have seeds normal, plant type is without obvious change, although plant height and Ear height change between different strain, luffing is less, does not reach significant difference conspicuous level.But, fruit ear and grain characters hands over plant in different F1 strain and sister parent, acceptor self-mating system sister hands between plant considerable change.Compared to contrast self-mating system, sisters hand over plant, and the sisters of each transgenic line hand over the single plant yield of plant and F1 plant all to increase, and wherein the output amplification of transgene pyramiding strain reaches difference and conspicuous level.The single plant yield of one of them polymerized strain increases to 116.74 ± 11.97g by contrast 68.77 ± 5.43.The spike length of transgenic line all increases, and the 13.5 ± 0.8cm of plant can be handed over to rise to 16.7 ± 0.7cm by acceptor self-mating system sisters, reach the significance level of difference.Strain its 100-grain weight under open pollination conditions that grain yield is high also obviously increases.In the polymerized strain that ZmWx process LAN transgenic line and they are maternal, row grain number hands over plant apparently higher than acceptor self-mating system sisters, reaches the significance level of difference.Data analysis draws, the polymerized strain being female parent with ZmWx process LAN strain, and its output increase is jointly caused by the increase of 100-grain weight and row grain number.The economical character of transgene pyramiding strain is all more similar to its female parent, shows that the impact of the transgenosis of these 3 endosperm specificity expressions on fruit ear and seed phenotype has obvious maternal effect.
To corn different grouting period (10,15,20,25,30,35DAP) the endosperm real-timeRTPCR that carries out large small ylidene gene Sh2, Bt2 and Wx gene of AGPase detects, the expression intensity that draws Sh2, Bt2 and Wx gene in different biopsy method point transgenosis parent's strain and polymerized strain all hands over plant higher than acceptor self-mating system and its sisters.In the transgene pyramiding strain being female parent with saltant type AGPase process LAN corn, the expression intensity of Sh2, Bt2 higher than the polymerized strain being female parent with GBSSI process LAN strain, but lower than saltant type AGPase process LAN parent's.Similar with it, in the transgene pyramiding strain being female parent with GBSSI process LAN strain, the gene expression abundance of Wx gene is higher than the transgene pyramiding strain being female parent with saltant type AGPase process LAN plant, but lower than GBSSI process LAN parent's.Infer that transgenosis becomes positive correlation at the copy number of albuminous cell with transgene expression intensity, in stock plant albuminous cell, transgenosis Sh2 and Bt2 or Wx is 3 copies, and in transgene pyramiding strain albuminous cell transgene copy number or be 2 (from female parents) or be 1 (from male parent).
In order to confirm transgenosis polymerization plant in really played effect, strip grouting different times (10,15,20,25,30,35DAP) corn embryosperm, measure itself AGPase and GBSSI enzymic activity.Result shows, the AGPase activity of transgene pyramiding strain comparatively nontransgenic plants is significantly increased, but lower than AGPase process LAN parent's; The GBSSI enzymic activity of transgene pyramiding strain comparatively nontransgenic plants is significantly increased, but lower than GBSSI process LAN parent's.These results show, in the transgene pyramiding plant of process LAN AGPase gene and Wx gene, transgenosis can play one's part to the full, thus AGPase enzyme and Wx enzymic activity are increased substantially.This prompting, further improves endosperm AGPase and GBSSI enzymic activity if wish, seed selection 3 transgenosiss can all realize the transgenic line that isozygotys.
The starch content of each transgene pyramiding plant comparatively acceptor self-mating system sisters is handed over plant to have significantly to increase.Consistent with transgene expression abundance and enzyme activity assay result, the starch content of polymerization plant changes not quite compared with the parent of process LAN AGPase gene.The starch content about 66.1% of the prosperous 7-2 of acceptor self-mating system, amylose content is lower than 30%; The prosperous 7-2 strain starch content of 3 process LAN AGPase genes is respectively 74.9%, 75.3% and 77.3%, with its be the starch content of maternal polymerized strain for 73.3%-78.0%, and with the starch content of its polymerized strain being male parent for 73.5%-76.6%.The amylose content of transgene pyramiding plant comparatively acceptor self-mating system is all significantly increased, parent's amylose content of process LAN GBSSI is 39.7%, 40.7% and 41.8%, with its be the amylose content of maternal polymerized strain for 37.7%-42.8%, the amylose content of the polymerized strain being male parent with it is 37.5%-41.2%.The starch content of acceptor inbred Zheng 58 is 68.8%, and amylose content is also lower than 30%; Zheng 58 strain starch content of 2 process LAN AGPase genes is respectively 75.2%% and 76.3%, with its be the starch content of maternal polymerized strain for 76.0%-78.2%, and with the starch content of its polymerized strain being male parent for 77.7%-78.6%.And parent's amylose content of process LAN GBSSI is 36.7% and 39.7%, with its be the amylose content of maternal polymerized strain for 35.3%-41.2%, the amylose content of the polymerized strain being male parent with it is 34.6%-39.6%.These results show, transgene pyramiding plant, compared with acceptor self-mating system plant, had both improve Grain Amylose content, the starch content be significantly increased again; Compare with the strain of process LAN GBSSI gene with the strain of process LAN AGPase gene, both the high starch property that process LAN AGPase gene produces had been maintained, maintain again the characteristic compared with high amylose content that process LAN GBSSI gene produces, not only promote that grain yield increases considerably, and improve corn kernel quality.This invention is that available kind of matter has been formulated in the breeding of high starch amylomaize.

Claims (5)

1. corn ZmWx gene is improving the application in corn yield and improvement grain characters.
2. apply as claimed in claim 1, it is characterized in that: the cDNA sequence of described corn ZmWx gene is as shown in SEQIDNo.1, and the aminoacid sequence of its coding is as shown in SEQIDNo.2; Described corn yield and grain characters refer to the single plant yield of corn, 100-grain weight and amylose content.
3. apply as claimed in claim 1, it is characterized in that: the application method of described corn ZmWx gene in raising corn yield and improvement grain characters is: from corn embryosperm cDNA, clone ZmWx gene order, described ZmWx genes encoding corn particle mating type amylosynthease GBSSI, itself and endosperm specificity promoter are formed fusion gene, fusion gene to be recombinated to maize transformation in plant expression vector by transgenic technology, obtain the corn gene plant of output increased and grain characters improvement; Or the high starch maize of GBSSI process LAN corn and saltant type AGPase process LAN is hybridized, screening obtains transgene pyramiding body, produces the corn gene strain that grain yield, starch content and 100-grain weight are significantly higher than acceptor material.
4. apply as claimed in claim 3, it is characterized in that, described endosperm specificity promoter is zein spirit-soluble gene promotor P27kD or P22kD.
5. apply as claimed in claim 3, it is characterized in that, the application method of described corn ZmWx gene in raising corn yield and improvement grain characters is: adopt RT-PCR method from corn embryosperm cDNA, clone corn ZmWx gene, then merge with P27kD, and with herbicide resistance gene als for selective marker, construct plant conversion carrier; Adopt agriculture bacillus mediated maize bud point genetic transforming method transgenic corn plant and offspring thereof, select the transgenic line that starch content and 100-grain weight are significantly higher than acceptor material.
CN201510980701.0A 2015-12-23 2015-12-23 Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics Pending CN105349559A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510980701.0A CN105349559A (en) 2015-12-23 2015-12-23 Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510980701.0A CN105349559A (en) 2015-12-23 2015-12-23 Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics

Publications (1)

Publication Number Publication Date
CN105349559A true CN105349559A (en) 2016-02-24

Family

ID=55325621

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510980701.0A Pending CN105349559A (en) 2015-12-23 2015-12-23 Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics

Country Status (1)

Country Link
CN (1) CN105349559A (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107177601A (en) * 2017-07-03 2017-09-19 安徽省农业科学院烟草研究所 A kind of cornstarch synthesis regulation gene ZmMIKC2a and its application
CN107266595A (en) * 2016-04-05 2017-10-20 玉米产品开发公司 Composition and method for producing the starch with New function
CN110563827A (en) * 2019-09-27 2019-12-13 中国农业科学院作物科学研究所 Protein related to corn kernel yield and coding gene thereof
CN110982826A (en) * 2019-11-22 2020-04-10 中国农业大学 Cloning, function research and marker excavation of corn kernel development control gene ZmDek605
CN111518940A (en) * 2020-05-09 2020-08-11 华南师范大学 Method for detecting Wx1 molecular marker and applying Wx1 molecular marker to sweet waxy corn breeding
CN114410663A (en) * 2020-10-28 2022-04-29 中国农业科学院作物科学研究所 Application of corn ZmDek701 gene in regulation and control of plant grain quality and mutant thereof
CN114885829A (en) * 2022-04-06 2022-08-12 甘肃省河西玉米生物育种及制种工程研究中心 High-starch corn variety breeding method
CN116042653A (en) * 2022-12-21 2023-05-02 安徽农业大学 Corn ZmMYB155 gene and application thereof

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107266595B (en) * 2016-04-05 2021-02-05 玉米产品开发公司 Composition and method for producing starch having novel function
CN107266595A (en) * 2016-04-05 2017-10-20 玉米产品开发公司 Composition and method for producing the starch with New function
CN107177601B (en) * 2017-07-03 2019-11-05 安徽省农业科学院烟草研究所 A kind of cornstarch synthesis regulation gene ZmMIKC2a and its application
CN107177601A (en) * 2017-07-03 2017-09-19 安徽省农业科学院烟草研究所 A kind of cornstarch synthesis regulation gene ZmMIKC2a and its application
CN110563827A (en) * 2019-09-27 2019-12-13 中国农业科学院作物科学研究所 Protein related to corn kernel yield and coding gene thereof
CN110563827B (en) * 2019-09-27 2021-07-06 中国农业科学院作物科学研究所 Protein related to corn kernel yield and coding gene thereof
CN110982826A (en) * 2019-11-22 2020-04-10 中国农业大学 Cloning, function research and marker excavation of corn kernel development control gene ZmDek605
CN110982826B (en) * 2019-11-22 2021-08-13 中国农业大学 Cloning, function research and marker excavation of corn kernel development control gene ZmDek605
CN111518940A (en) * 2020-05-09 2020-08-11 华南师范大学 Method for detecting Wx1 molecular marker and applying Wx1 molecular marker to sweet waxy corn breeding
CN114410663A (en) * 2020-10-28 2022-04-29 中国农业科学院作物科学研究所 Application of corn ZmDek701 gene in regulation and control of plant grain quality and mutant thereof
CN114885829A (en) * 2022-04-06 2022-08-12 甘肃省河西玉米生物育种及制种工程研究中心 High-starch corn variety breeding method
CN116042653A (en) * 2022-12-21 2023-05-02 安徽农业大学 Corn ZmMYB155 gene and application thereof
CN116042653B (en) * 2022-12-21 2024-02-13 安徽农业大学 Application of corn ZmMYB155 gene in regulation and control of corn grain morphology and starch synthesis process

Similar Documents

Publication Publication Date Title
CN105349559A (en) Application of corn ZmWx gene in increase of corn yield and improvement of grain characteristics
CN101970668B (en) Plants with modified starch metabolism
Keeling et al. Effect of temperature on enzymes in the pathway of starch biosynthesis in developing wheat and maize grain
Kang et al. Increasing the starch content and grain weight of common wheat by overexpression of the cytosolic AGPase large subunit gene
RU2377303C2 (en) Wheat with modified activity of brahching enzyme, and also starch and starch-containing products produced from it
CN1886507B (en) Rice and products thereof having starch with an increased proportion of amylose
Umemoto et al. Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties
RU2619636C2 (en) High amylose wheat
EP1012250B1 (en) Regulation of gene expression in plants
CN102933072B (en) Fructus Hordei Vulgaris and application thereof
Schaffer et al. ADPglucose pyrophosphorylase activity and starch accumulation in immature tomato fruit: the effect of a Lycopersicon hirsutum-derived introgression encoding for the large subunit
US20080201807A1 (en) Gelatinization Temperature Manipulation
CN110358772B (en) OsEBP89 gene for improving abiotic stress resistance of rice, and preparation method and application thereof
CN101768602A (en) Scheme and application for increasing amylose content of corn seeds through overexpression of Sh2 and Bt2 genes and RNAi suppression expression of SBEIIa and SBEIIb genes
Diedhiou et al. Carbohydrate profiling in seeds and seedlings of transgenic triticale modified in the expression of sucrose: sucrose-1-fructosyltransferase (1-SST) and sucrose: fructan-6-fructosyltransferase (6-SFT)
CN113862265A (en) Method for improving rice grain shape and appearance quality
CN107245490B (en) method for adjusting starch content of rice grains based on ZmMIKC2a gene
CN103146746A (en) Transgenic rice with excellent eating quality and cultivation method
CN100532553C (en) Amylose content control gene DU1 of rice endosperm and application thereof
CN101519660B (en) Method for improving content of amylose in rice by using RNA interference
CN101792773A (en) Application of sh2 and bt2 genes in enhancing starch content in corn seeds and increasing weight of seeds
JP4711762B2 (en) Function elucidation of starch synthase type IIIa and new starch production method
WO2022266081A2 (en) Agronomic practices and sustainability methods involving yield traits
Sun et al. Overexpression of starch branching enzyme 1 gene improves eating quality in japonica rice
CN111197106A (en) PCR detection primer for coix seed wax gene mutation and application thereof

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
WD01 Invention patent application deemed withdrawn after publication
WD01 Invention patent application deemed withdrawn after publication

Application publication date: 20160224