CN103805618A

CN103805618A - C4 type phosphoenolpyruvate carboxylase (PEPC) gene of corn and application thereof in wheat

Info

Publication number: CN103805618A
Application number: CN201410064062.9A
Authority: CN
Inventors: 许为钢; 王玉民; 王会伟; 张磊; 李艳; 齐学礼; 胡琳
Original assignee: Wheat Research Institute Henan Academy Of Agricultural Sciences
Current assignee: Wheat Research Institute Henan Academy Of Agricultural Sciences
Priority date: 2014-02-25
Filing date: 2014-02-25
Publication date: 2014-05-21

Abstract

The invention discloses a C4 type phosphoenolpyruvate carboxylase (PEPC) gene of corn and an application thereof in wheat. The novel PEPC gene provided by the invention can be stably expressed and inherited in wheat, and the photosynthetic efficiency is remarkably improved compared with that of an acceptor material, thereby providing an important gene source and parent material support for improving the photosynthetic efficiency of C3 crop by means of a C4 type high photosynthetic efficiency gene and breeding high photosynthetic efficiency transgenetic wheat variety with the level of output greatly improved.

Description

Corn C 4 type phosphoric acid enol pyruvic acid carboxylase (PEPC) gene and the application in wheat thereof

Technical field

The present invention relates to biology field, be specifically related to a kind of corn C 4 type phosphoric acid enol pyruvic acid carboxylase (PEPC) gene and 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.

Phosphoric acid enol pyruvic acid carboxylase PEPC is C ₄one of key enzyme of photosynthetic pathway.At present, many species are as corn (Z.mays) (Richard et al, 1989), Chinese sorghum (S.vulgare) (Lepiniec et al, 1993), soybean (G.max) (Sugimoto et al, 1992), barnyard grass (Echinochloa crusgalli) (Zhang Guifang etc., 2005), chrysanthemum Chrysanthemum (Flaveria) (Westhoff et al, 1997; Matsuoka et al, 1989), blue-green algae (Cyanobacterium) (Luinenburg et al, 1992; Katagiri et al, 1985), the pepc of the plant such as intestinal bacteria (E.coli) (Hisatake et al, 1984) clones successfully in succession.Hudspeth etc. (1992) have reported the earliest corn pepc cDNA have been proceeded in tobacco, and the transfer-gen plant PEPC specific activity contrast of acquisition has improved 2 times.Gehlin etc. (1996) imports the pepc of intestinal bacteria and chrysanthemum Chrysanthemum in potato subsequently, obtained the active transfer-gen plant that improves 2～12 times of PEPC, but transfer-gen plant does not show obvious variation aspect photosynthesis characteristics.The state Ku of university of Washington, DC in 1999 etc. are successfully by the full-length gene Introduced into Rice of corn pepc, obtain PEPC active with corn quite even higher than the transgenic rice plant of corn, and transfer-gen plant has significantly reduced oxygen to photosynthetic restraining effect.Subsequently, domestic and international many scholars have carried out C ₃do the research of object height light efficiency genetically engineered, and obtained remarkable effect.Japan north japonica rice variety Kitaake morning that Jiao Demao etc. (2001) turn corn pepc from U.S.'s introduction breeds evaluation by the generation, obtain high for genetic stability transfer-gen plant, compared with original seed, the raising of transfer-gen plant PEPC activity is stabilized in 20 times of left and right, the saturated photosynthetic rate of light improves 50%, CO ₂compensation point declines 27%, shows at high light intensity or CO ₂under the condition being restricted, there is stronger CO ₂assimilative capacity and higher efficiency of energy conversion, the ability of fast light inhibition and photooxidation resistant strengthens, and single plant yield improves 10%-30%.Wang De is just waiting (2002) research to show, turns the Major Economic indexs such as the effective fringe of individual plant of corn pepc paddy rice, every total grain panicle number, thousand seed weight and single plant yield and improves 14.9%, 5.7%, 1.3% and 13.9% than original parent respectively.From Chinese sorghum, clone after pepc full-length gene continue (1993) such as Lepiniec, Zhang Fang etc. (2003) clone a kind of novel pepc from different sorghum varieties, and by it Introduced into Rice, obtain PEPC activity and contrasted the transfer-gen plant that improves 26 times, the CO of transfer-gen plant ₂compensation point and photorespiration rate significantly reduce, and the saturated photosynthetic rate of light and carboxylation efficiency improve, and demonstrate C ₄the Photosynthetic Characteristics of plant.OAA or MA feed original seed paddy rice and transgenic paddy rice for Ji Benhua etc. (2004), from different structure level, all can see that OAA or MA put the promoter action of oxygen and be obviously greater than original seed paddy rice to turning pepc Rice Photosynthesis, if the PEPC activity that can further improve in existing high-yield variety is described, can further improve potential photosynthesis.

Aspect wheat, Chen Xuqing etc. (2004) first obtained PEPC enzymic activity high expression level transgenosis T0 for wheat, Net Photosynthetic Rate has improved 39% compared with acceptor, has reached 19 μ molCO ₂m ^-2s ^-1left and right, but do not exceed the photosynthetic rate of existing wheat breed.Zhang Bin etc. (2007) import wheat with agrobacterium-mediated transformation by barnyard grass pepc cDNA, have obtained the T that photosynthetic rate increases ₁for Transgenic plant of wheat.Zhang Qingchen etc. (2010) are by corn total length pepc, and Li Yan etc. (2009) import corn pepc cDNA total length in common wheat, and transfer-gen plant PEPC enzyme is lived and Net Photosynthetic Rate all has raising in various degree.

Therefore, clone newly, the C4 type pepc 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 a kind of phosphoric acid enol pyruvic acid carboxylase (PEPC) gene, in another aspect of this invention, also relate to a kind of phosphoric acid enol pyruvic acid carboxylase (PEPC) gene 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 a kind of phosphoric acid enol pyruvic acid carboxylase (PEPC) gene, it is characterized in that the nucleotides sequence of described gene is classified SEQ ID NO.1 as, or has the gene order of 95% above homology with SEQ ID NO.1.

The present invention also relates to a kind of phosphoric acid enol pyruvic acid carboxylase (PEPC) on the other hand, and the aminoacid sequence of described phosphoric acid enol pyruvic acid carboxylase (PEPC) is the corresponding aminoacid sequence of SEQ ID NO.1 nucleotide sequence.

The present invention also relates to a kind of carrier on the other hand, and described carrier contains above-mentioned phosphoric acid enol pyruvic acid carboxylase (PEPC) gene nucleotide series.

In another aspect of this invention, the invention still further relates to above-mentioned phosphoric acid enol pyruvic acid carboxylase (PEPC) gene, phosphoric acid enol pyruvic acid carboxylase (PEPC) 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 pepc 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-pc vector construction schematic diagram;

Fig. 2: transgenic wheat and the unconverted variation that contrasts Net Photosynthetic Rate under different illumination intensity, wherein: WT: all wheats 19 of unconverted contrast; PC: transgenic line 08T (1)-15-1;

Fig. 3: transgenic wheat and the unconverted variation that contrasts Net Photosynthetic Rate under differing temps, wherein: WT: all wheats 19 of unconverted contrast; PC: transgenic line 08T (1)-15-1;

Fig. 4: different CO ₂transgenic wheat and the unconverted variation that contrasts Net Photosynthetic Rate under concentration, wherein: WT: all wheats 19 of unconverted contrast; PC: transgenic line 08T (1)-15-1;

Fig. 5: flowering period transgenic wheat with contrast Net Photosynthetic Rate daily variation, wherein: WT: unconverted contrast week wheat 19; PC: transgenic line 08T (1)-15-1.

Embodiment

Embodiment 1

1, the cloning and expression vector construction of high light efficiency pepc gene

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 (Escherichia coli) bacterial strain DH5 α competent cell are prepared test kit and are TaKaRa company product, plant expression vector pCOMBIA3301 is for preserving in this laboratory, restriction enzyme is MBI company product, penbritin is Amersco company product, and all the other reagent are import or domestic analytical pure.

According to existing corn C ₄type pepc gene cDNA sequence (GenBank accession number: X15238), utilizes a pair of Auele Specific Primer of Primer5.0 software design, and upstream primer is: P1:5'-GCAGATCTGCTCCACCCATCTCGCTTCTGTG-3'; Wherein contain Bgl II restriction enzyme site, downstream primer is: P2:5'-GGCACGTGGCCGCCTAGCCAGGGTTCTGCAT-3'.Wherein, containing Pml I restriction enzyme site, 5' end respectively adds two protection bases.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 P1 of 10 μ M and P2; 2.5 μ l10 × Reaction Buffer; 4 μ l dNTP mix (2.5mM); 0.5 μ l LA – Taq enzyme, adds water to 25 μ l.Response procedures: 94 ℃ of denaturation 3min; 94 ℃ of sex change 45s, 68 ℃ 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-pc, clone's pepc full length gene 3014bp, its open reading frame is 2955bp, the protein (its aminoacid sequence is SEQ ID NO.1) of 985 amino-acid residues of coding of deriving.PMD19-pc and binary expression vector pCOMBIA3301 are used respectively to Bgl II and Pml I 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-pc(Fig. 1 through PCR and double digestion).

2, high light efficiency pepc 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-pc 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/L NAA+2.0mg/L KT+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/L NAA+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 PC1:5 '-TGGAAGTGCGTGTCTAAGTT-3 ' and PC2:5 '-ATGCCCAGGTGCGTGTTGAA-3 ' according to the pepc gene order of having cloned, amplification program be 94 ℃ 4 minutes; 94 ℃ 30 seconds, 57 ℃ 45 seconds, 72 ℃ 45 seconds, 42 circulations; 72 ℃ 10 minutes.Object fragment length is 680bp., 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 PC1 of 10 μ M and PC2; 2.5 μ l10 × Reaction Buffer; 4 μ l dNTP mix (2.5mM); 0.5 μ l Taq enzyme, adds water to 25 μ l.PCR product detects through 1% agarose gel electrophoresis.There are 178 strains can amplify the object band of 680bp, are accredited as positive plant.

3, transgenic wheat photosynthetic physiological characteristics is analyzed

(1) photosynthetic dynamic analysis

Utilize CIRAS-1 type photosynthesis measurement system, lower transgenic wheat material 08T (the 1)-15-1 of different illumination intensity (PPFD) and the not Net Photosynthetic Rate variation of all wheats 19 of transgenic wheat are analyzed, result shows (Fig. 2), along with light intensity increases, the Net Photosynthetic Rate of transgenic wheat and not transgenic wheat all increases gradually, but at low light intensity (PPFD<500 μ molm ^-2s ^-1) descend both Net Photosynthetic Rate no significant differences, as PPFD>500 μ molm ^-2s ^-1time, both Net Photosynthetic Rate start to occur difference, and along with light intensity increases gradually, both Net Photosynthetic Rate difference increases gradually.Research simultaneously finds, transgenic wheat is compared with transgenic wheat not, and transgenic wheat utilizes the ability of high light intensity significantly to strengthen, and the light saturation point that contrasts all wheats 19 is about 1400 μ molm ^-2s ^-1, saturated photosynthetic rate is about 24.7 μ molCO ₂m ^-2s ^-1, the light saturation point of transgenic wheat material 08T (1)-15-1 is about 1700 μ molm ^-2s ^-1, contrast has improved 21.4%, and saturated photosynthetic rate is about 30.9 μ molCO ₂m ^-2s ^-1, contrast has improved 25.1%.

Utilize CIRAS-1 type photosynthesis measurement system analyzed 15 ℃ to 40 ℃ between transgenic wheat material 08T (1)-15-1 and not transgenic wheat (all wheats 19) Net Photosynthetic Rate change, result shows (Fig. 3), in transgenic wheat and not transgenic wheat, the relation of temperature and Net Photosynthetic Rate all shows as unimodal curve, the Net Photosynthetic Rate optimum temperuture that wherein contrasts all wheats 19 is 25 ℃ of left and right, the Net Photosynthetic Rate optimum temperuture of transgenic line 08T (1)-15-1 is 30 ℃ of left and right, and contrast has improved 5 ℃.Research is simultaneously found, in trial temperature range, the Net Photosynthetic Rate of transgenic wheat all has raising in various degree compared with the control, and transgenic wheat has stronger adaptive faculty to high temperature, in the time of 30 ℃, the Net Photosynthetic Rate of transgenic wheat is 28.5 μ molCO2m ^-2s ^-1, contrast all wheats 19 and improved approximately 15.8%; In the time of 35 ℃, the Net Photosynthetic Rate of transfer-gen plant is 25.4 μ molCO ₂m ^-2s ^-1, contrast all wheats 19 and improved approximately 11.4%; In the time of 40 ℃, the Net Photosynthetic Rate of transfer-gen plant is 21.9 μ molCO ₂m ^-2s ^-1, contrast has improved approximately 29.5%.

To transgenic wheat material 08T (1)-15-1 and not Net Photosynthetic Rate and its intercellular CO of transgenic wheat (all wheats 19) boot leaf ₂concentration (C _i) relation analyzes.Result shows (Fig. 4), at different intercellular CO ₂under concentration, the Net Photosynthetic Rate of transgenic wheat all has raising in various degree than transgenic wheat not, and at same intercellular CO ₂concentration 50 μ molmol ^-1to 300 μ molmol ^-1under condition, the Net Photosynthetic Rate of transgenic wheat and not transgenic wheat and intercellular CO ₂concentration is all linear to be increased, the Net Photosynthetic Rate of transgenic wheat material 08T (1)-15-1 and intercellular CO ₂concentration linear equation is y=0.0992x-7.43, R2=0.995; Not Net Photosynthetic Rate and the intercellular CO of transgenic wheat (all wheats 19) ₂concentration linear equation is y=0.0804x-6.20, R2=0.9862.Known by further analysis, the carboxylation efficiency of transgenic wheat and not transgenic wheat is respectively 0.0992 and 0.0804, and transgenic wheat contrasts and improved approximately 23.4%; The CO of transgenic wheat and not transgenic wheat ₂compensation point is respectively 74.9 μ molmol ^-1with 77.1 μ molmol ^-1, transgenic wheat contrasts and has reduced approximately 2.8%; This shows that transgenic wheat has compared with the control stronger assimilation and absorbs CO ₂ability.

(2) Net Photosynthetic Rate daily variation in flowering period

On May 5th, 2011 T ₃ transgenic line 08T (1)-15-1 and on May 2nd, 2012 T ₄fig. 5 is shown in the daily variation of transgenic line 08T (1)-15-1-4 Net Photosynthetic Rate.Between 2 years, transgenic line is identical with contrast Net Photosynthetic Rate daily variation general trend, is all bimodal curve.Transgenic line boot leaf Net Photosynthetic Rate is all higher than contrast during 8:00-16:00, and both maximum values all appear at 10:00 left and right, and wherein transgenic line maximum in 2011 has reached 26.66 μ molm ^-2s ^-1, contrast 22.37 μ molm ^-2s ^-1improve 19.18%; Within 2012, maximum has reached 27.38 μ molm ^-2s ^-1, compared with the latter 23.60 μ molm ^-2s ^-1improve 16.01%.

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: corn C 4 type phosphoric acid enol pyruvic acid carboxylase (PEPC) gene and the application in wheat thereof

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GIGLCRTEHM FFASDERIKA VRQMIMAPTL ELRQQALDRL LPYQRSDFEG IFRAMDGLPV 660

TIRLLDPPLH EFLPEGNIED IVSELMAETG ANQEDALARI GKLSEVNDML GFRGCRLGIS 720

YPELTEDQAR AIFEAAIAMT NQGVQVFPEI DVPLVGTPQE LGHQVTLIRQ VAEKVFANVG 780

KTIGYKVGTM IEIPRAALVA DEIAEQAEFF SFGTNDLTQM TFGYSRDDVG KFIPVYLAQG 840

ILQHDPFEVL DQRGVGELVK FATERGRKAR PNLKVGICGE HGGEPSSVAF FAKAGLDYVS 900

CSPFRVPIAR LAAAQVLV 918

<212>Type:PRT

<211>Length:918

SequenceName:2

SequenceDescription:

Claims

1. phosphoric acid enol pyruvic acid carboxylase (PEPC) gene, is characterized in that the nucleotides sequence of described gene is classified SEQ ID NO.1 as, or has the gene order of 95% above homology with SEQ ID NO.1.

2. a phosphoric acid enol pyruvic acid carboxylase (PEPC), the aminoacid sequence of described phosphoric acid enol pyruvic acid carboxylase (PEPC) is the corresponding aminoacid sequence of SEQ ID NO.1 nucleotide sequence, preferably, described nucleotides sequence is classified SEQ ID NO.2 as.

3. a carrier, described carrier contains phosphoric acid enol pyruvic acid carboxylase claimed in claim 1 (PEPC) gene nucleotide series.

4. above-mentioned phosphoric acid enol pyruvic acid carboxylase (PEPC) gene, phosphoric acid enol pyruvic acid carboxylase (PEPC) 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.