CN103805616A - C4 type pyruvate orthophosphate dikinase (PPDK) gene of corn and application thereof in wheat - Google Patents
C4 type pyruvate orthophosphate dikinase (PPDK) gene of corn and application thereof in wheat Download PDFInfo
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- CN103805616A CN103805616A CN201410064052.5A CN201410064052A CN103805616A CN 103805616 A CN103805616 A CN 103805616A CN 201410064052 A CN201410064052 A CN 201410064052A CN 103805616 A CN103805616 A CN 103805616A
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Abstract
The invention discloses a C4 type pyruvate orthophosphate dikinase (PPDK) gene of corn and an application thereof in wheat. The novel PPDK gene provided by the invention can be stably expressed and inherited in the wheat, and the photosynthetic efficiency is obviously improved in comparison with that of an acceptor material, thus an important gene source and a parent material support are provided for improving the photosynthetic efficiency of C3 type crops by utilizing a C4 type high-photosynthetic efficiency gene and breeding a high-photosynthetic efficiency transgenic wheat variety of which the yield level is greatly increased.
Description
Technical field
The present invention relates to biology field, be specifically related to the gene of the two kinases (PPDK) of a kind of corn C 4 type phosphopyruvates and the application in wheat thereof.
Background technology
Wheat is the main food crop of China, and its throughput and state between supply and demand are related to the Vital Strategic Problems such as Chinese national economy development and grain security all the time.From world wide, world food production status is closely connected with various countries' politics, Economic Development Status all the time, and grain-production amount and margin one have decline, and international grain price is just unprecedented soaring, panic mood spreads with regard to the general trend of events, and the political economy situation of various countries is caused to great effect; From domestic, along with the growth of China's population, the fast development of industrialization and urbanization, will continue to strengthen to the demand of grain.Simultaneously, because China's plant of grain crops area can not significantly recover to increase, therefore will ensure China's grain security, the only way out is to continue to improve per unit area yield, and this just requires to obtain the progress of leap property aspect the Inheritance of Yield Traits improvement of the staple food crop such as wheat, paddy rice.
Photosynthesis is the basis of all green plants material productions, and according to estimates, 90% of plant shoot dry-matter comes from photosynthesis.Therefore, the key that improves crop yield is to improve optical energy utilization efficiency, thereby improves the output of unit surface.And will increase substantially the efficiency of light energy utilization and realize super high-yielding, the technological line that must take reasonable plant type (external photosynthetic efficiency) and high light efficiency (inherent photosynthetic efficiency) to combine.The raising of the crop yield prediction levels such as current wheat, paddy rice, it is mainly the result due to variety yield potentiality genetic improvement and working condition improvement, and the genetic improvement of yield potential is mainly to realize by the approach such as improvement and hybrid vigour of morphological characters, relatively high at variety of crops yield levels such as existing wheat, paddy rice, and all there is good morphological structure, leaf area index and coefficient of harvest have all approached in the situation of the limit, the more simple great-leap-forward raising that relies on above-mentioned approach to be difficult to realize yield potential.Under this background, domestic and international many scholars invest sight the genetic improvement research of optical energy utilization efficiency one after another, wish to realize increasing substantially of biological yield by improving photosynthetic efficiency, and then obtain the breakthrough of yield potential improvement.
According to the difference of photosynthesis approach, plant can be divided into C
3type, C
4type and CAM(crassulacean acid) type.C
4to tame through long-term under adverse circumstance with CAM plants, from C
3plant evolution and come.With C such as wheat, paddy rice, soybean
3plant is compared, the C such as corn, Chinese sorghum, sugarcane
4plant has CO
2concentrating mechanism, light compensation point is high, CO
2compensation point is low, a little less than photorespiration, has especially obvious Photosynthetic Advantages under the adverse environmental factor such as high temperature, arid.Due to C
4plant has high photosynthetic, high-moisture, high nitrogen utilization efficiency and high biological yield, therefore by C
4approach is introduced C
3plant, to improve its photosynthetic efficiency and grain yield, is the great science proposition in domestic and international biological study field always.Research main path was in this respect the methods such as the screening of same chamber, cytogamy and distant hybirdization in the past, but did not obtain breakthrough always.The transgenic technology reaching its maturity in recent years, due to the reproduction isolation that can break between species, realizes the interchange of gene in different plant species, has therefore become the Major Technology that solves farm crop important character genetic improvement bottleneck problem.
At present by C
4there are reports for pathway key enzyme gene, but C
4the high light efficiency key gene of approach imports C
3after crop, its mechanism of action is always not yet clear and definite.Therefore, to import C
4the C of the high light efficiency gene of type
3crop is research material, further investigation C
4the impact that the importing of high light efficiency gene produces acceptor material associated metabolic approach, discloses C
4pathway key gene is at C
3the mechanism of action in crop, can be and utilize C
4pathway key gene improvement C
3the Photosynthetic Efficiency of crop and yield potential provide solid theoretical basis.
PPDK catalysis CO
2the generation of primary receptor phosphoenolpyruvic acid (PEP), this reaction is C
4rate-limiting step (Usuda, 1984) in pathways metabolism is another enzyme receiving much concern in C4 photosynthetic pathway process.Ishimaru etc. (1998) proceed to the ppdk of corn in Arabidopis thaliana and potato, and the PPDK activity of transfer-gen plant improves 4 and 5.4 times than contrast respectively.The plant ppdk of crassulacean acid section is proceeded to tobacco by Sheriff etc. (1998), and the seed number in each capsule of transgene tobacco is more than wild-type, and capsule is also heavier.The ppdk of corn is proceeded to paddy rice by Ku etc. (2000), and transgenic paddy rice field shows as 30-35%.Fukayama etc. (2001) are after ppdk Introduced into Rice, and the expression of ppdk has 40% plant more than 40 times, to reach 1/2 level of corn higher than original seed.More than research all successfully proceeds to ppdk in the C3 plants such as tobacco, potato, Arabidopis thaliana and paddy rice, but the photosynthetic physiological characteristics of these transfer-gen plants does not all have significantly to change.Ji Benhua etc. (2004) find to spray NaHSO
3can increase transfer-gen plant photosynthetic rate with ATP, its mechanism may be: turn C
4in trans-genetic hybrid rice, PPDK activation Pyr generation PEP is endoergic reaction, needs the participation of ATP, and detailed mechanism need further further investigation.Therefore, clone newly, the C4 type ppdk gene with good function is significant for the Photosynthetic Efficiency of improvement C3 plant.
Summary of the invention
The object of this invention is to provide two kinases (PPDK) genes of a kind of phosphopyruvate, in another aspect of this invention, also relate to a kind of two kinases (PPDK) genes of phosphopyruvate that can improve Wheat Photosynthesis effect.In order to realize object of the present invention, intend adopting following technical scheme:
One aspect of the present invention relates to two kinases (PPDK) genes of a kind of phosphopyruvate, it is characterized in that the nucleotides sequence of described gene is classified SEQIDNO.1 as, or has the gene order of 95% above homology with SEQIDNO.1.
The present invention also relates to the two kinases (PPDK) of a kind of phosphopyruvate on the other hand, and the aminoacid sequence of the described two kinases (PPDK) of phosphopyruvate is the corresponding aminoacid sequence of SEQIDNO.1 nucleotide sequence.
The present invention also relates to a kind of carrier on the other hand, and described carrier contains two kinases (PPDK) gene nucleotide series of above-mentioned phosphopyruvate.
In another aspect of this invention, the invention still further relates to two kinases (PPDK) genes of above-mentioned phosphopyruvate, the two kinases (PPDK) of phosphopyruvate or carrier and improving the active application of Wheat Photosynthesis.
In a preferred embodiment of the present invention, described carrier imports in wheat by particle gun mediated method, agrobacterium-mediated transformation or pollen tube passage method.
Novel ppdk gene provided by the invention can be in wheat stably express and heredity, photosynthetic efficiency obviously improves compared with acceptor material, for utilizing the Photosynthetic Efficiency of the high light efficiency improvement of genes of C4 type C3 crop, the high light efficiency transgenic wheat kind that seed selection yield level increases substantially provides important gene source and parent material to support.
Accompanying drawing explanation
Fig. 1: expression vector p3301-ppdk vector construction schematic diagram;
Fig. 2: transgenic wheat and the unconverted variation that contrasts Net Photosynthetic Rate under different illumination intensity, wherein: CK: unconverted control group; PK: transgenic line;
Fig. 3: flowering period transgenic wheat and the daily variation of control group Net Photosynthetic Rate, wherein: CK: unconverted control group; PK: transgenic line.
Embodiment
Embodiment 1
Experiment is extracted test kit, sepharose DNA with RNA, and to reclaim test kit be all Time Inc. purchased from sky, MLV reverse transcription test kit is purchased from Promega company, LA-TaqDNA polysaccharase, pMD-19T cloning vector, intestinal bacteria (Escherichiacoli) bacterial strain DH5 α competent cell are prepared test kit and are TaKaRa company product, plant expression vector pCOMBIA3301 is for being purchased product, restriction enzyme is MBI company product, penbritin is Amersco company product, and all the other reagent are import or domestic analytical pure.
According to corn pyruvate phosphate two kinases (PPDK) gene cDNA sequence (GenBank accession number: BT054438) of having reported, use a pair of Auele Specific Primer of Primer5.0 software design, 5' end respectively adds two protection bases.PK
1: 5 ’ – GCagatctTCGGCTCCCTCTCCCCTTGCTCCAT – 3 ', containing Bgl II restriction enzyme site, PK
2: 5 ’ – GGttataaCACATCCACCAGCAGCAGGCAATCC – 3 ', containing Aan I restriction enzyme site.Primer is synthetic by Shanghai Sangon company.The total RNA of corn extracts and cDNA is synthetic carries out with reference to the RNA of Promega company extraction test kit and MLV reverse transcription test kit specification sheets.
Carry out pcr amplification take the total cDNA of corn as template, PCR reaction system: 10-50ng/ul genomic templates; The each 0.5 μ l of the PK1 of 10 μ M and PK2; 2.5 μ l10 × ReactionBuffer; 4 μ ldNTPmix (2.5mM); 0.5 μ lLA – Taq enzyme, adds water to 25 μ l.Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of sex change 45s, 65 ℃ of renaturation 30s, 72 ℃ are extended 2.5min, 30 circulations; Then 72 ℃ are extended 10min.PCR product detects with 0.8% agarose gel electrophoresis, reclaim Kit with sepharose DNA and reclaim 3kb object fragment, be connected on pMD19-T carrier by T/A cloning, transform bacillus coli DH 5 alpha, be coated with dull and stereotyped incubated overnight, picking positive colony is entrusted the order-checking of Beijing Bo Shang biotech firm, be verified as correct clone's called after pMD19-ppdk, recombinant plasmid pMD19 – ppdk is checked order by the precious biological company limited in Dalian, sequencing result shows that answer print segment length is 3004Bp, comprise the open reading frame that a 2916Bp is long, its nucleotides sequence is classified SEQIDNO.1 as, the protein (aminoacid sequence is SEQIDNO.2) of 971 amino-acid residues of its coding.PMD19-ppdk and binary expression vector pCOMBIA3301 are used respectively to Bgl II and Aan I double digestion double digestion, agarose gel electrophoresis detects, cut glue and reclaim 3kb object band and expression vector fragment, 16 ℃ of connections are spent the night, transform bacillus coli DH 5 alpha, be accredited as correct clone's called after p3301-ppdk(Fig. 1 through PCR and double digestion).
2, high light efficiency ppdk gene imports all wheats 19 and PCR evaluation
Take wheat breed week wheat 19 rataria as acceptor material.Take away and spend the fringe middle part of rear 12-14d, immature seed of the same size, with 70% alcohol surface sterilization 1min, aseptic water washing 3 times is afterwards with 0.1% the HgCl2 10min that sterilizes, aseptic water washing 3-4 time.Under aseptic condition, strip out rataria (1mm left and right), in MS inducing culture (MS+2mg/L2,4-D+0.4% agar+3% sucrose, pH6.2) upper dark cultivation after 4-5d, be placed in height and ooze the upper osmotic treated 4-6h of substratum (MS+2mg/L2,4-D+0.7% agar+N.F,USP MANNITOL+3% sucrose, pH5.8), after the particle gun microparticle bombardment that utilization contains p3301-ppdk carrier, ooze on substratum and continue to process 16h at height, go to calli induction media.Cultivate and after 2 weeks, go to careless fourth phosphine resistance screening regeneration culture medium (1/2MS+0.5mg/LNAA+2.0mg/LKT+0.4% agar+3% sucrose+4%PPT, pH6.2), 2 generations of step sizing, 2 weeks per generations, resistance seedling through screening goes to root media (1/2MS+0.2mg/LNAA+0.4% agar+3% sucrose, pH6.5), in, when more healthy and stronger to regrowth root system, can carry out hardening 1-2 days.Take root, after strong sprout, move in flowerpot, finally wash away the substratum residue that root system carries and just can transplant engagement alms bowl, obtain regeneration plant 125 strains.
Extract the genomic dna of all regeneration plants, designed a pair of Auele Specific Primer AKL according to the ppdk gene order of having cloned
2: 5 ’ – GATTGTCGTTTCCCGCCTTCAGT – 3 ' and AKR
25 ’ – ATGGTCTTGTTGCCCTCGCTCTT – 3 '.Amplification program is: 94 ℃ of 3min, and 94 ℃ of 30s, 57 ℃ of 30s, 72 ℃ of 60s, 29 circulations, it is 938bp that 72 ℃ of 10minPCR detect clip size.Utilize this primer pair regeneration plant to carry out PCR detection, to identify positive plant.PCR reaction system is: 10-50ng/ul genomic templates; The each 0.5 μ l of the AKL2 of 10 μ M and AKR2; 2.5 μ l10 × ReactionBuffer; 4 μ ldNTPmix (2.5mM); 0.5 μ lTaq enzyme, adds water to 25 μ l.PCR product detects through 1% agarose gel electrophoresis.There are 146 strains can amplify the object band of 938bp, are accredited as positive plant.
3, transgenic wheat photosynthetic physiological characteristics is analyzed
Utilize CIRAS-2 type photosynthesis measurement system, the variation of the Net Photosynthetic Rate of transgenic wheat material and unconverted contrast (all wheats 19) under analysis different illumination intensity.Result shows (Fig. 2), and the Net Photosynthetic Rate that increases transgenic wheat and unconverted contrast boot leaf along with light intensity all shows as increase gradually, low light intensity (PPFD<500 μ mol/m
2/ s) storeroom Net Photosynthetic Rate difference is not obvious under condition, as PPFD>500 μ mol/m
2when/s, the Net Photosynthetic Rate of storeroom starts to occur difference, and along with light intensity increases gradually, storeroom Net Photosynthetic Rate difference show as gradually and increase.Transgenic wheat is compared with unconverted contrast, and transgenic wheat utilizes the ability of high light intensity significantly to increase, and the light saturation point of all wheats 19 of unconverted contrast is 1465 μ mol/m
2/ s, transgenic line light saturation point reaches 1505 μ mol/m
2/ s, apparently higher than contrast.
Transgenic line and the photosynthetic rate daily variation result that impinges upon flowering period is shown to (Fig. 3), transgenic line in whole day (6:00-18:00) photosynthetic rate all higher than unconverted contrast.The day photosynthetic rate maximum value of transgenic line and contrast all appears at 10:00, reaches respectively 23.1 and 22.1 μ molCO2m
-2s
-1, transgenic line maximum net photosynthetic rate is apparently higher than contrast.Meanwhile, transgenic line has all shown lunch break phenomenon under high light hot conditions with contrasting at noon, but when lunch break transgenic line photosynthetic rate still higher than unconverted contrast.
The above is the preferred embodiments of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of principle of the present invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
ApplicationProject
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<120>Title: two kinases (PPDK) genes of corn C 4 type phosphopyruvates and the application in wheat thereof
<130>AppFileReference:
<140>CurrentAppNumber:
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Claims (5)
1. two kinases (PPDK) genes of phosphopyruvate, is characterized in that the nucleotides sequence of described gene is classified SEQIDNO.1 as, or have the gene order of 95% above homology with SEQIDNO.1.
2. the two kinases (PPDK) of phosphopyruvate, the aminoacid sequence of the described two kinases (PPDK) of phosphopyruvate is the corresponding aminoacid sequence of SEQIDNO.1 nucleotide sequence, preferred, described nucleotides sequence is classified SEQIDNO.2 as.
3. a carrier, described carrier contains two kinases (PPDK) gene nucleotide series of phosphopyruvate claimed in claim 1.
4. two kinases (PPDK) genes of above-mentioned phosphopyruvate, the two kinases (PPDK) of phosphopyruvate or carrier are improving the active application of Wheat Photosynthesis.
5. application according to claim 4, described carrier imports in wheat by particle gun mediated method, agrobacterium-mediated transformation or pollen tube passage method.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063024A (en) * | 2015-07-24 | 2015-11-18 | 江苏省农业科学院 | Specific SSR (simple sequence repeat) marker for maize pyruvate phosphate dikinase genes and application of specific SSR marker |
| CN108165577A (en) * | 2018-01-11 | 2018-06-15 | 中国农业科学院生物技术研究所 | Turn the photosynthetic key genes of C4 and improve the photosynthetic method of C3 plant |
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| CN1220698A (en) * | 1997-02-10 | 1999-06-23 | 日本烟草产业株式会社 | C4 cycle of pck type |
| CN1608132A (en) * | 2001-10-23 | 2005-04-20 | 日本烟草产业株式会社 | Method of elevating photosynthesis speed of plant by improving pyruvate phosphate dikinase |
| CN102002496A (en) * | 2010-11-15 | 2011-04-06 | 中国科学院遗传与发育生物学研究所 | DNA (Deoxyribonucleic Acid) molecule related to photosynthesis and application thereof |
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2014
- 2014-02-25 CN CN201410064052.5A patent/CN103805616A/en active Pending
Patent Citations (3)
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| CN1220698A (en) * | 1997-02-10 | 1999-06-23 | 日本烟草产业株式会社 | C4 cycle of pck type |
| CN1608132A (en) * | 2001-10-23 | 2005-04-20 | 日本烟草产业株式会社 | Method of elevating photosynthesis speed of plant by improving pyruvate phosphate dikinase |
| CN102002496A (en) * | 2010-11-15 | 2011-04-06 | 中国科学院遗传与发育生物学研究所 | DNA (Deoxyribonucleic Acid) molecule related to photosynthesis and application thereof |
Non-Patent Citations (3)
| Title |
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| GENBANK: "GenBank: GU363532.1", 《GENBANK》 * |
| YU-MIN WANG ET AL.: "Expression of Maize Gene Encoding C4-Pyruvate Orthophosphate Dikinase (PPDK) and C4-Phosphoenolpyruvate Carboxylase (PEPC) in Transgenic Arabidopsis", 《PLANT MOL BIOL REP》 * |
| 陈绪清: "小麦（Triticum aestivum）基因工程研究", 《中国优秀博硕士学位论文全文数据库 (博士) 基础科学辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105063024A (en) * | 2015-07-24 | 2015-11-18 | 江苏省农业科学院 | Specific SSR (simple sequence repeat) marker for maize pyruvate phosphate dikinase genes and application of specific SSR marker |
| CN108165577A (en) * | 2018-01-11 | 2018-06-15 | 中国农业科学院生物技术研究所 | Turn the photosynthetic key genes of C4 and improve the photosynthetic method of C3 plant |
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