CN1865443A - Phosphoenolpyruvate carboxylase gene of watergrass and its coded protein and uses - Google Patents

Abstract

The invention discloses a barnyard grass phosphoenolpyruvate carboxylase gene and coded protein and appliance, which is one ribotide sequence in the following sequences: 1) sequent graph SEQ ID No.:1;2) DNA sequence hybrid ribotide sequence limited by SEQ ID No.:1. the coded gene protein is one of SEQ ID No.:2 in the 1) sequence graph; 2) SEQ ID No.:2 in the 2) sequence graph with two amino residue sequence replaced, lost or added form one to ten to improve plant photosynthesis efficiency.

Description

The phosphoric acid enol pyruvic acid carboxylase gene of barnyard grass grass and proteins encoded thereof and application

Technical field

The present invention relates to plant gene and proteins encoded thereof and application, particularly relate to a phosphoric acid enol pyruvic acid carboxylase gene and proteins encoded thereof and its application in improving photosynthesis of plant efficient.

Background technology

On vegitabilia's photosynthetic carbon assimilation mode C is arranged ₃Approach and C ₄The differentiation of approach.Since the existence of photorespiration, C ₃Quite a few is consumed approach institute fixed carbon by photorespiration, and the formation of biomass reduces more than 50%.C ₄Fixation of C O in the photosynthetic pathway ₂Phosphoric acid enol pyruvic acid carboxylase (PEPCase) to CO ₂Avidity considerably beyond C ₃Carboxydismutase in the photosynthetic pathway (RuBPase), and phosphoric acid enol pyruvic acid carboxylase has concentrated CO ₂Mechanism, CO ₂The rising of concentration helps the carboxylation activity of bifunctional enzyme RuBPase, suppresses it and adds oxygen activity, thereby effectively reduce the photorespiration of plant.Therefore, C ₄Plant has photosynthetic rate height, CO ₂Compensation point is low, does not almost have advantages such as photorespiration, particularly under conditions such as high light, high temperature, arid, and C ₄Plant has the high advantage of obvious growth, moisture and nutrient utilization, and photosynthetic efficiency compares C ₃Plant exceeds two times, and biological yield is also higher.But the plant on the earth more than 95% comprises that many important crops and cash crop such as paddy rice, wheat, spinach, tobacco etc. all are C ₃Plant, (21% O under the atmospheric environment of routine ₂, 0.035% CO ₂), its photosynthetic efficiency compares C ₄Plant is low by 40%, and makes stomatal closure in high temperature, arid, CO ₂Under the situation that concentration reduces, photosynthetic efficiency is then lower.Having only the minority torrid zone and subtropics grass such as corn, sugarcane, Chinese sorghum and barnyard grass grass etc. is C ₄Plant, the strategy with the photorespiration of tackling.

Many C ₃C in the plant ₃Photosynthetic pathway and C ₄Photosynthetic pathway is also deposited, under the varying environment condition or under the artificial induction, and its C ₄Photosynthetic enzyme is active can be improved greatly, shows C ₄The key of photosynthetic pathway does not lie in the cell compartment structure, and is C ₄Photosynthetic key enzyme, as PEPC, PPDK, NADP-Me etc.Therefore, with C ₄The photosynthetic pathway key gene imports C ₃In the plant, might improve C ₃The photosynthetic efficiency of plant.

Phosphoric acid enol pyruvic acid carboxylase (PEPC) is present in all and can carries out in the photosynthetic organism, the pepc gene of many higher plants such as corn (Z.mays), Chinese sorghum (S.vulgare), paddy rice, chrysanthemum Chrysanthemum plants such as (Flaveria) is cloned success in succession, and its sequence signature and structure have been carried out detailed parsing.

Studies have shown that the PEPC of higher plant is encoded by a bit of multigene family.A plurality of members in this family are at different plants such as corn, and Chinese sorghum is identified among ice plant and the Flaveria trinervia etc.There is multiple isozyme form: C in the ppc gene ₄Photosynthetic type, C ₃Photosynthetic type, CAM type and non-green type etc., even the ppc gene in every kind of plant has 1-3 type at least, found 5 types in corn, is present in respectively in leaf and the root tissue.C ₄Photosynthetic type pepc gene exists with single copy form in corn and Chinese sorghum, then exists with the multiple copied form in the Flaceria platymiscium, and its expression is mainly carried out in mesophyll cell, and regulated and control by light, has tissue specificity.

C ₄The PEPC multigene family member's of plant primary structure is similar substantially.Because C ₄The PEPC of form is at C ₄The vital role that plays in the photosynthesis, a large amount of research at present mainly concentrates on C ₄On the PEPC of form.To the ppc gene studies more be plants such as corn, Chinese sorghum and Flaveria genus, find equally that by sequence alignment the ppc gene that is present in the same type of different plants has higher homology, the C of 2 corns and 1 Chinese sorghum ₄The homology of type ppc gene complete sequence is 57.9%, and the transcriptional domain homology is 80.3%.The homology that shows same type ppc gene between different plant species is higher.

First attempts changeing C ₄The mosaic gene that the ppc gene uses is the C with corn ₄The cDNA of special PEPC (ppccDNA) be fused to Nicotiana phimbaginifolia chlorophyll a/b binding protein gene (Cab) 5 ' with constitute on 3 ' the flanking sequence, after this mosaic gene imported tobacco, the specific activity non-transgenic plant of PEPC had improved 2.2 times in the leaf.Equally, such as Cab, the C under the control of strong promoters such as rbcS promotor and cauliflower mosaic virus 35S promoter ₄The expression of the cDNA of enzyme also obtains to make the activity of PEPC, PPDK and NADP-MDH to improve 2-5 result doubly.For improving C ₄The expression level of enzyme, can in the gene of introducing, comprise sequence with enhanser effect, chalcone synthase genes by detecting parsley 5 ' end non-coding region (UTR) back finds that its existence can make the expression level of Ppc gene under 35S promoter of C.glutamicum improve.The active highest level of PEPC reaches 5 times of non-transgenic plant.Corn C ₄The promotor of specific gene, as Ppc, Pdk can improve the expression level of reporter gene in paddy rice greatly, and the mode that can carry out as in corn that microtubule is special, mesophyll cell is special and rely on light is expressed.This result of study shows that paddy rice has high level expression C ₄The regulatory factor that specific gene is permitted, and hint that the importing of complete corn gene can cause C in the paddy rice leaf ₄The high expression level of enzyme.1999, (Ku MSB, Agarie S such as Ku, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, MiyaoM, Matsuoka M.High-level expression of maize phosphoenolpyruvate carboxylasein transgenic rice plants. Nature Biotech., 1999,17,76-80.) at first with C ₄The C of plant corn ₄Type phosphoric acid enol pyruvic acid carboxylase gene (comprising promotor and terminator sequence) is by the agriculture bacillus mediated C that imported ₃The plant paddy rice, and realized effective expression, make the activity stabilized raising of phosphoric acid enol pyruvic acid carboxylase (PEPCase) in the rice leaf, the PEPC specific activity of 85% transfer-gen plant contrasts high 2-30 doubly, 15% comparison according to high 30-110 doubly, even be higher than corn 2-3 doubly even doubly than the high 2-3 of corn, reach 12% of blade soluble protein.(Ku MSB such as Ku, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuoka M.High-level expression of maizephosphoenolpyruvate carboxylase in transgenic rice plants. Nature Biotech., 1999,17,76-80) transgenic rice plant of the overexpression corn PEPC of Huo Deing can significantly weaken oxygen to photosynthetic restraining effect, under same atmospheric condition, the photosynthetic rate comparison is according to having improved 2 times.It is reported that this transgenic paddy rice can increase production 35%.(Jiao Demao, Kuang Tingyun .2003. such as Li Xia change the pepc gene paddy rice and have elementary CO people such as Jiao Demao ₂The physilogical characteristics of concentrating mechanism. Chinese science (C collects), 33,33-39; Big late, Jiao Demao, yellow snow wait .2001. to change the photosynthetic physiological characteristics of pepc gene paddy rice clearly. Botany Gazette, and 43,657-660.) detected the physical signs of changeing Ppc trans-genetic hybrid rice plant, the result shows, compares with adjoining tree, the light saturation rate improves 55%, CO ₂Compensation point descends 27%.Relatively, the photosynthetic capacity of changeing the pepc gene paddy rice significantly strengthens under the natural condition of Nanjing, and single plant yield has increased 14%-22%, shows that the Photosynthetic Productivity by engineered method raising crop has bright prospects.

Advantage weeds barnyard grass grass (Echinochloa crusgalli) in the rice field is a typical C ₄Photosynthetic type plant, its ground, underground part growth vigor far are more than paddy rice, and result of study shows that the barnyard grass straw or like vegetable can adapt to environment in the water well, can adapt to extremely arid, hot conditions again.C with other dry farming ₄Crop corn, Chinese sorghum and millet are compared, and have characteristics such as photosynthetic rate height, PEPCase content height.Therefore it is that a kind of the development changes the PEPCase gene to transform C ₃The classic wild resource donor of crop photosynthesis efficient.What up to the present, report was more is with C such as corn, Chinese sorghums ₄Genes such as the ppc of farm crop, pdk change over to as C such as tobacco, paddy rice and wheats ₃Plant, but the rare report that the photosynthetic gene of wild plant resource is succeedd and utilized.Relevant to the barnyard grass grass resource development and use and clone's research of photosynthetic function gene still belong to blank in the world.

Paddy rice has kept in its evolutionary process and is suitable in the paddy field growth and needs higher relatively temperature simultaneously and the sufficient sunlight irradiation.And the growing environment of high temperature and high light intensity C just ₄Plant-growth institute is necessary.Therefore the growth of paddy rice is easy to be subjected to paddy field C ₄The influence of weeds, the barnyard grass grass has formed intensive with paddy rice with this understanding just and has contended with, and the photosynthetic efficiency that therefore improves farm crop such as paddy rice has important and practical meanings.

Summary of the invention

The purpose of this invention is to provide a phosphoric acid enol pyruvic acid carboxylase gene and a proteins encoded thereof that derives from the barnyard grass grass.

Phosphoric acid enol pyruvic acid carboxylase gene provided by the present invention, name is called Eppc, derives from Echinochloa barnyard grass grass (Echinochloa crusgalli), and it can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 1 by 2886 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence from 5 ' end 1-2886 bit base.

The albumen (PEPC) that phosphoric acid enol pyruvic acid carboxylase gene of the present invention is coded is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein with raising photosynthesis of plant efficient.

SEQ ID № in the sequence table: 2 are made up of 961 amino-acid residues.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Another object of the present invention provides a kind of method that improves photosynthesis of plant efficient.

The method of raising photosynthesis of plant efficient provided by the present invention is with the phosphoric acid enol pyruvic acid carboxylase gene importing explant of described barnyard grass grass, obtains the plant that photosynthetic efficiency improves.

The phosphoric acid enol pyruvic acid carboxylase gene of described barnyard grass grass can be by containing described barnyard grass grass the plant expression vector of phosphoric acid enol pyruvic acid carboxylase gene import explant; The carrier that sets out that is used to make up described plant expression vector can be any one double base agrobacterium vector or can be used for carrier of plant micropellet bombardment etc., as pCAMBIA1301, pBI121, pCAMBIA1300 etc., wherein, pCAMBIA1301 is the carrier that preferably sets out.

When using Eppc to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as general living plain gene Ubiquitn promotor (pUbi), leaf texture specific expressed 1, the specific promoter of 5-diphosphoribulose carboxylase small ylidene gene promotor rbcS, corn phosphoric acid enol pyruvic acid carboxylase gene, cauliflower mosaic virus (CAMV) 35S promoter etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Be the carrier that sets out with pCAMBIA1301, structure to contain general living plain gene Ubiquitin1 promotor (pUb1) and barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene plant expression vector be pUbi-Eppc.

Be the carrier that sets out with pCAMBIA1301, structure contain leaf texture specific expressed 1, the plant expression vector of 5-diphosphoribulose carboxylase small ylidene gene rbcS promotor and barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene is pRbcS-Eppc.

Be the carrier that sets out with pCAMBIA1301, the plant expression vector of the specific promoter that contains the corn phosphoric acid enol pyruvic acid carboxylase gene of structure and barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene is pZMp-Eppc.

Carry Eppe of the present invention plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.By the plant transformed host both can be C such as paddy rice, wheat, tobacco, soybean, Arabidopis thaliana ₃Plant.

The Eppc of barnyard grass grass of the present invention is transformed at first improving its photosynthetic rate paddy rice, and then make it better adapt to the living environment of high temperature and high light intensity.At C ₄In the plant population, from the angle of origin and evolution, barnyard grass grass and other C ₄Type crop (corn, Chinese sorghum, millet etc.) is compared, belong to the same gang plant nearer with the paddy rice sibship, might be close by its regulatory mechanism, help after the transgenosis high reactivity in paddy rice and express about the relevant photosynthetic enzyme gene of regulatory mechanism of photosynthetic enzyme gene with paddy rice.The phosphoric acid enol pyruvic acid carboxylase gene Eppc that derives from the barnyard grass grass of the present invention can significantly improve photosynthesis of plants efficient, will improve farm crop, particularly C ₃Play an important role in the output of farm crop (as paddy rice).

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the structure schema of the plant expression vector pUbi-Eppc of Eppc

Fig. 1 a is the physical map of plasmid vector pUC-Pubi

Fig. 1 b is the physical map of carrier pCAMBIA1301

Fig. 2 cuts qualification result for the enzyme of the plant expression vector pUbi-Eppc of Eppc

Fig. 3 is the structure schema of the plant expression vector pRbcS-Eppc of Eppc

Fig. 3 a is the physical map of carrier pRGN

Fig. 4 cuts qualification result for the enzyme of the plant expression vector pRbcS-Eppc of Eppc

Fig. 5 is the structure schema of the plant expression vector pZMp-Eppc of Eppc

Fig. 5 a is the physical map of carrier PUCm-T-ZMp

Fig. 6 cuts qualification result for the enzyme of the plant expression vector pZMp-Eppc of Eppc

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and it is synthetic that the primer is given birth to the worker by Shanghai.

The clone of embodiment 1, barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene Eppc

1, the extraction of the total RNA of barnyard grass grass and eDNA's is synthetic

Get the greenery of the barnyard grass grass plant behind the illumination 4h, use Trizol test kit (Gibeco company) and reference reagent box specification sheets to extract total RNA, concrete grammar is: the barnyard grass blade of grass sheet that clip 1g is fresh, put and grind to form lyophilized powder in the liquid nitrogen rapidly, in the income 1.5mL centrifuge tube and add 1mL Trizol reagent, abundant mixing, room temperature are placed behind the 5min 4 ℃, the centrifugal 10min of 12000g goes to supernatant in another centrifuge tube, room temperature is placed 5min, add the 0.2mL chloroform again, shaking 15sec, room temperature is placed 5min, 4 ℃, the centrifugal 15min of 12000g, supernatant is changed in another centrifuge tube, add the 0.5mL Virahol, room temperature is placed 10min, 4 ℃, the centrifugal 10min of 12000g, abandon supernatant, add 75% ethanol 1mL, washing precipitation, 4 ℃, the centrifugal 5min of 7500g, after will precipitating drying at last, be dissolved in the aqua sterilisa that DEPC handles, obtain total RNA of barnyard grass blade of grass sheet.Total RNA with this barnyard grass blade of grass sheet is a template, uses ImProm-II ^TMCDNA is synthesized in ReverseTranscriptase test kit (promega company) reverse transcription, and reaction system is: RNA 1 μ g, oligo (dT) ₁₅0.5 μ g uses the aqua sterilisa of handling through DEPC to complement to 5 μ L.70 ℃, 5min, 4 ℃ again, rapid ice bath behind the 5min.Add ImProm-II then respectively ^TM5 * Reaction Buffer, 4 μ L, MgCl ₂(25mM) 2.4 μ L, dNTP mix (10mM) 1 μ L, rRNasin Ribonuclease Inhibitor 20u, ImProm-II ^TM Reverse Transcriptase 1 μ L uses the aqua sterilisa of handling through DEPC to supply reaction system to 20 μ L, fully mixing.Reaction conditions is: 25 ℃ of 5min, and 42 ℃ of 60min, 70 ℃ of 15min, 4 ℃ of preservations obtain the cDNA of barnyard grass grass.

2, the clone of barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene Eppc

To several C such as sugarcane, corn, wheat, paddy rice ₃And C ₄The cDNA sequence of the phosphoric acid enol pyruvic acid carboxylase gene of grass (ppc) is carried out the homology comparison, and comparison result shows that 5 ' end and the 3 ' terminal sequence of ppc is all more conservative.Therefore, the cDNA full length sequence according to the phosphoric acid enol pyruvic acid carboxylase gene of the sugarcane of having reported designs a pair of primer, and primer sequence is:

P1:5 '-ACT TCTAGAATGGCGTCCGAGCGGCACC-3 ' (band underscore base is a restriction enzyme Xba I recognition site)

P2:5 '-GTA GGTACCCTAGCCGGTGTTCTGCA-3 ' (band underscore base is a restriction enzyme Kpn I recognition site)

The barnyard grass grass cDNA that obtains with step 1 is a template, under the guiding of primer P1 and primer P2, carries out pcr amplification.50 μ L PCR reaction systems are: 2 * GC damping fluid (TaKaRa company), 25 μ L, dNTPmix 16 μ L, P1 1 μ L (5 μ M), P2 1 μ L (5 μ M), LA Taq archaeal dna polymerase (TaKaRa company) 2.5U, the about 1 μ g of template DNA uses the aqua sterilisa of handling through DEPC to complement to 50 μ L.The PCR reaction conditions is: 94 ℃ of 5min of elder generation; 94 ℃ of 30s then, 70 ℃ of 30s, 70 ℃ of 3min, totally 30 circulations, last 70 ℃ of 10min.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis detect, downcut the purpose band of about 3000bp, reclaim DNA with PCR product purification test kit (the vast Imtech in Beijing).The DNA that reclaims is connected with carrier pUCm-T (worker is given birth in Shanghai) with T4DNA ligase enzyme (Promega company), to connect product Transformed E .coli DH5 α competent cell, get 200 μ L bacterium liquid and 4 μ L IPTG and 40 μ L X-gal (20mg/mL) and carry out blue hickie screening at the LB flat board, after growing single bacterium colony, the some hickie bacterium colonies of picking upgrading grain, the plasmid DNA of being carried is carried out enzyme with restriction enzyme Xba I and Kpn I cut evaluation, can produce the positive clone of about 2900bp and 2700bp band, positive colony is delivered to Shanghai bio-engineering corporation carry out sequencing, sequencing result shows that pcr amplification product length is 2906bp, its open reading frame (ORF) is from 5 ' end 1-2886 bit base, has SEQ ID № in the sequence table: 1 nucleotide sequence, coding has SEQ ID № in the sequence table: the protein of 2 amino acid residue sequence, with this sequence called after Eppc, will contain the recombinant plasmid vector called after PUCm-Eppc of Eppc.On the NCBI website, the nucleotide sequence of this gene is carried out homology search with blast search software, the result shows all that in GenBank, EMBL, DDBJ and PDB database this sequence is the phosphoric acid enol pyruvic acid carboxylase gene sequence, with the portion C of Clustal W software to filtering out ₃, C ₄Plant carries out the multiple compare of analysis of nucleotide sequence, wherein the higher plant of homology is millet (Setariaitalica), Chinese sorghum (Sorgham bicolor), corn (Zea mays) and paddy rice (Oryza stativa) successively, and the homology of corresponding homology segment is respectively 94.8%, 93.2%, 93.0% and 89.7%.The dna fragmentation that proof is cloned is the full-length cDNA of the phosphoenolpyruvic acid carboxylation gene of barnyard grass grass.Screen several typical C again ₃, C ₄The multi-form PEPCase protein sequence utilization ClustalW software of plant carries out the multiple compare of analysis of aminoacid sequence, the root type of result and corn, Chinese sorghum, the C of millet and paddy rice ₃Type PEPCase homology is high, can reach 96.5%, 96.4%, 96.4% and 94.3% respectively, with the C of millet, corn, sugarcane and Chinese sorghum ₄The homology of type PEPCase is respectively 82.2%, 79.1%, 77.1%, 76.6%.

The functional verification of embodiment 2, barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene Eppc

One, the structure that contains the plant expression vector of Eppc

1, the structure of plant expression vector pUbi-Eppc

Contain Eppc plant expression vector pUbi-Eppc the structure flow process as shown in Figure 1, concrete grammar is as follows: the plasmid vector pUC-Pubi (its physical map as shown in Figure 1a) that contains Ubiquitin1 promotor and NOS terminator earlier with restriction enzyme Hind III and EcoR I double digestion, enzyme is cut product carry out the detection of 1.0% agarose gel electrophoresis, reclaim the purpose band that size is about 2.0kb, to reclaim fragment with (its physical map is shown in Fig. 1 b through same enzyme double digestion carrier pCAMBIA1301, available from Australian CAMBIA company) connect, to connect product transformed into escherichia coli DH5a competent cell, after blue hickie screening, obtain intermediate carrier, called after pCAMBIA1301-pUbiFPF1; With restriction enzyme Xba I and Kpn I carrier pCAMBIA1301-pUbiFPF1 is carried out double digestion again, enzyme is cut product carry out the detection of 0.8% agarose gel electrophoresis, reclaim size and be about 12, the big fragment that contains the NOS terminator of 000kb, with this fragment called after pCAMBIA1301-NOS-Ter, with same enzyme the carrier PUCm-Eppc that embodiment 1 makes up is carried out double digestion, enzyme is cut product carry out the detection of 0.8% agarose gel electrophoresis, reclaim the goal gene Eppc fragment that size is about 2.9kb, Eppc fragment with 2.9Kb is connected with pCAMBIA1301-NOS-Ter then, to connect product transformed into escherichia coli DH5a competent cell, after screening, obtain intermediate carrier, called after pCAMBIA1301-Ecppc-NOS-Ter, use restriction enzyme Xba I single endonuclease digestion pCAMBIA1301-Ecppc-NOS-Ter and pUC-Pubi at last, connect behind the dephosphorylation, the Ubiquitin1 promotor is connected into Eppc front end among the pCAMBIA1301-Ecppc-NOS-Ter, to connect product transformed into escherichia coli DH5a competent cell, it is carried out the white screening of indigo plant, select positive monoclonal upgrading grain and carry out enzyme and cut evaluation, enzyme is cut qualification result, and (swimming lane 1 is 1kb DNA Ladder as shown in Figure 2, swimming lane 2 is institute's upgrading grain, swimming lane 3 is the plasmid through Sal I and Kpn I double digestion, swimming lane 4 is the plasmid through Xba I and Kpn I double digestion, swimming lane 5 is the plasmid through Hind III and Kpn I double digestion), can obtain size about 16 through Hind III and SalI double digestion, the positive colony for the forward connection of 000kb band is with this positive colony plasmid called after pUbi-Eppc.

2, the structure of plant expression vector pRbcS-Eppc

Contain Eppc plant expression vector pRbcS-Eppc the structure flow process as shown in Figure 3, concrete grammar is as follows: with restriction enzyme PstI carrier pCAMBI1301 is carried out enzyme earlier and cut, segment reclaims the back dephosphorylation and handles, dephosphorylized endonuclease bamhi is connected with the carrier pRGN that contains RuBPCase gene small subunit promotor (its physical map is shown in Fig. 3 a) that cuts through the same enzyme enzyme and handle through dephosphorylation, to connect product transformed into escherichia coli DH5a competent cell, after screening, obtain intermediate carrier, called after pCB-PRbcS, use restriction enzyme HindIII and Xba I double digestion plasmid vector pCB-PRbcS then, enzyme is cut product carry out the detection of 0.8% agarose gel electrophoresis, reclaim the RuBPCase gene small subunit promoter fragment that size is about 2.7kb, then with this promoter fragment and being connected with the carrier pCAMBIA1301-Ecppc-NOS-Ter of Xba I double digestion that step 1 makes up through Hind III, to connect product transformed into escherichia coli DH5a competent cell, it is carried out the white screening of indigo plant, select positive monoclonal upgrading grain and carry out enzyme and cut evaluation, enzyme is cut qualification result, and (swimming lane 1 is the plasmid through Xba I and Kpn I double digestion as shown in Figure 4, swimming lane 2 is the plasmid through Xba I and Hind III double digestion, swimming lane 3 is the plasmid through Hind III and EcoR I double digestion, swimming lane 4 is the plasmid through Hind III and EcoR V double digestion, swimming lane 5 is DL15,000 DNAMarker, swimming lane 6 is institute's upgrading grain), can obtain the positive colony that is connected for forward of the about 2.4kb band of size through HInd III and EcoRV double digestion, with this positive colony plasmid called after pRbcS-Eppc.

3, the structure of plant expression vector pZMp-Eppc

Contain Eppc plant expression vector pZMp-Eppc the structure flow process as shown in Figure 5, concrete grammar is as follows: earlier with restriction enzyme Hind III and Xba I the carrier PUCm-T-ZMp (its physical map is shown in Fig. 5 a) of the promotor that contains the corn phosphoric acid enol pyruvic acid carboxylase gene is carried out enzyme and cut, enzyme is cut product carry out the detection of 0.8% agarose gel electrophoresis, reclaim the promoter sequence fragment that size is about the corn phosphoric acid enol pyruvic acid carboxylase gene of 1.3kb, then this promoter fragment is connected with the carrier pCAMBIA1301-Ecppc-NOS-Ter through the same enzyme double digestion that step 1 makes up, to connect product transformed into escherichia coli DH5a competent cell, it is carried out blue hickie screening, selecting positive monoclonal upgrading grain carries out enzyme and cuts evaluation, enzyme is cut qualification result, and (swimming lane 1 is DL15 as shown in Figure 6,000DNA Marker, swimming lane 2 is institute's upgrading grain, swimming lane 3 is the plasmid through Xba I and Kpn I double digestion, swimming lane 4 is the plasmid through Hind III and Xba I double digestion, swimming lane 5 is the plasmid through Hind III and Kpn I double digestion, and swimming lane 6 is 1kb DNA Ladder).

Two, the acquisition of Eppc transfer-gen plant and photosynthetic functional verification thereof

Vegetable material: in spend No. 8 paddy rice and upland rice 65

Plant expression vector pUbi-Eppc, pRbcS-Eppc, the pZMp-Eppc agrobacterium-mediated transformation rice transformation of the Eppc that step 1 is made up obtains the T that barnyard grass grass Eppc is changeed in 219 strains respectively ₀For plant (breaking up the transfer-gen plant that obtains by callus).30 strain transfer-gen plants are wherein carried out photosynthetic performance identify, the result as shown in Table 1 and Table 2, the plant that photosynthetic rate is higher than contrast 30% accounted for for 23.33% (amounting to 7 strains), the plant that photosynthetic rate is the highest is higher than contrast (not transfer-gen plant) 45%.Show that changeing Eppc can significantly improve C ₃The photosynthetic rate of crop, transpiration rate and water use efficiency.Simultaneously, to changing the paddy rice of corn pepc gene over to, measure altogether 5 individual plants (strain number: ZH65, ZH65-1, ZH65-2, ZM-2, ZM-3), the photosynthetic rate of wherein having only 1 strain (ZH65) is a little more than contrast, all the other all are lower than contrast.

Table 1 changes the paddy rice Net Photosynthetic Rate of Ubiquitin and Rubisco promotor control commentaries on classics barnyard grass grass Eppc gene over to

Different promoters	Scope	Be lower than contrast	Increase 10-20 %	Increase 20-30%	Increase more than 30%	Remarks:
							Ubiquitin Rubisco strain number	Number of individuals frequency (%) number of individuals frequency (%) Ubiquitin Rubisco	3 16.67 1 8.33 Uzh8-1 Uzh8-4 ULan-3 rzh8-1	4 22.22 4 33.33 Uzh8 Uzh8-3 Uzh8-15 Ushp1 rzh8-4q rzh8-6 rzh8-12 rzh8-13	8 44.44 1 8.33 Ushp2 Uzh8-2 Uzh8-5 Uzh8-9 Uzh8-12 Uzh8-14 Uzh8-17 ULan-4 rzh8-2	3 16.67 4 33.33 Ulan-1 Ulan-2 Uzh8-13 rzh8-3 rzh8-4 rzh8-5 rzh8-14	The contrast photosynthetic rate is 18.28 (μ molm -2·S -)

Table 2 changes paddy rice Net Photosynthetic Rate Pn, stomatal conductance Gs, the intercellular CO of barnyard grass grass Eppc gene ₂Concentration C i, transpiration rate E and each frequency zones of water use efficiency WUE (Pn/Trmmol) are on average planted variation tendency

Different promoters	The feature classification	Be lower than contrast %	Increase 10-20%	Increase 20-30%	Increase more than 30%
						Ubiquitin	Pn Gs Ci E WUE	15.31 0.21 209.8 4.58 3.68	20.81 0.27 207.29 5.32 3.94	22.89 0.30 209.83 5.96 3.85	25.81 0.311 200.9 6.40 4.00
Rubisco	Pn Gs Ci E WUE	18.1 0.21 202.6 5.15 3.51	21.09 0.23 229.24 4.68 4.55	22.8 0.27 212 4.59 4.96	25.22 0.30 202.08 5.23 4.88

Annotate: 1. the μ molm-2S-1 of unit of transpiration rate E; 2. the unit of stomatal conductance Gs is mmolm-2S-1; 3. the unit of Net Photosynthetic Rate Pn is μ molm-2S-14. intercellular CO ₂The unit of concentration C i is ppm (mg/L).

Sequence table

<160>2

<210>1

<211>2886

<212>DNA

＜213〉Echinochloa barnyard grass grass (Echinochloa crusgalli)

<400>1

atggcgtccg agcggcacca gtcgatcgac gcgcagctgc ggctgctggc gccgggcaag 60

gtctccgagg acgacaagct cgtcgagtac gacgccctcc tcgtcgaccg cttcctcgac 120

atcctacagg acctgcacgg cccgcacctc cgcgaattcg tgcaggagtg ctacgagctg 180

tcggcggagt acgagaacga ccgcgacgag gcgcggctcg gcgagctcgg gagcaagctc 240

accagcctgc ccccggggga gtccatcgtc gtcgccagct ccttctcgca catgctcaac 300

ctcgccaacc tcgccgagga agtgcagatc gcgcaccgcc gccggatcaa gctcaagcgc 360

ggggacttcg ccgacgaggc ctcggcgccc accgagtccg acatcgagga gacgctcaag 420

cgcctcgtct cgcagctcgg caagtcgcgc gaggaggtct tcgacgcgct caagaatcag 480

accgtcgacc tcgtcttcac ggcgcaccct acgcagtccg tcaggaggtc cctgctccag 540

aagcacggca ggatccggaa ttgcctgagg cagctgtatg ccaaggacat cactgctgat 600

gacaagcagg agcttgatga ggctcttcag agggagattc aggctgcttt cagaactgat 660

gaaatccgca gaacccctcc cactcctcaa gatgaaatgc gtgctggaat gagttacttc 720

catgaaacta tatggaaggg tgtaccaaaa ttcttgcgtc gtattgacac tgctctgaaa 780

aatattggga tcaatgagcg tctcccttac aatgcccctc ttattcagtt ctcttcctgg 840

atgggtggtg atcgtgatgg aaatccaaga gttacaccgg aggttacacg ggatgtgtgc 900

ttgttggcga gaatgatggc tgctaacctg tacttctctc agatagaaga tctaatgttt 960

gagctctcta tgtggcgctg cagtgatgaa cttcggatcc gtgcagatga tttacatcgg 1020

tctacaaaaa gggctgcaga gcactatata gaattctgga agcaagttcc tccaaatgaa 1080

ccttatcgtg tcatacttgg tgatgtcagg gataaattgt attatacacg agaacgttct 1140

cgtcatttgt tgacaactgg aatttctgag attcctgagg atgcaacttt tacgaatgtt 1200

gaacagtttc tggaacctct tgagctctgt tatagatcat tatgtgcctg tggtgacaaa 1260

cctatagctg acggaagtct ccttgatttc ttgcgtcaag tatcaacttt cgggcttgct 1320

cttgtgaaac tcgacatcag gcaggaatct gatcggcaca ctgacgtcct tgattcaata 1380

accacacatc ctggaattgg atcctatgct gagtggtcgg aggagaaacg ccaggattgg 1440

ctgttgtctg aactgagggg caagcgtcca ttgtttggtt ctgatcttcc tctgactgaa 1500

gagactgctg atgttttggg cacatttcat gtcctcgcag agctcccaac agattgcttt 1560

ggcgcgtata tcatctcgat ggcaactgcc ccgtctgatg tgcttgctgt cgagcttttg 1620

cagcgtgagt gccatgtaaa acagccactg agagttgttc cactctttga gaaacttgca 1680

gatcttgaag cagccccagc agccgtagca cgactctttt ctattgactg gtacatgaat 1740

aggattaatg gcaagcagga ggtgatgatt ggatactcag actctggtaa agacgctgga 1800

cgtctctctg caacatggca aatgtataaa gcacaagagg agctgatcaa ggtggcaaag 1860

cattatggag taaagttgac aatgtttcac ggaaggggtg gaactgttgg cagaggaggt 1920

ggtcccactc atctggccat attatctcag ccaccagaca ctatacatgg atcacttcgt 1980

gtaacagtac aaggtgaggt tattgagcac tcctttggag aggagctctt gtgctttagg 2040

actttgcaac gctacactgc agctaccctt gagcatggca tgcatcctcc aatttcccca 2100

aagccagaat ggcgtgctct gatggatgaa atggctattg tggcaaccaa agaatatcga 2160

tcaattgtct tccaagaacc acgctttgtc gaatacttcg ggtcggccac acctgagact 2220

gaatatggta ggatgaatat tgatagtcgt ccatcgaaga ggaggcctag tgggggaata 2280

gaatcgctcc gtgcaattcc atggatcttt gcttggacac agacaaggtt ccatccccct 2340

gtttggctgg gatttggtgc agcgttcaag catatcatgc agaaggacat caggaacacc 2400

catactctga aagaaatgta caatgagtgg ccattctcca gggtaactct tgacttgctt 2460

gagatggttt tcgccaaggg agacccggga atcgcagctg tatacgacaa attgctagtt 2520

gctgatgatc tgcaatcctt cggagagcag ctgaggaaga actatgagga gacaaaagag 2580

ctactccttc aggttgctgg tcacaaggac gtccttgaag gcgatcctta cctgaagcag 2640

cgtctgcgcc tgcgtgagtc gtacatcacc accctgaacg tatgccaggc gtacaccctg 2700

aagcggattc gcgaccccag cttccaggtg agcccgcagc cggccctgtc caaggagttc 2760

gttgacgaga gccagcctgc ggagctggtg cgactgaacc ctgagagcga gtacgcgccg 2820

ggcctggaga acacgctgat cctgaccatg aagggcattg ccgccggcat gcagaacacc 2880

ggctag 2886

<210>2

<211>961

<212>PRT

＜213〉Echinochloa barnyard grass grass (Echiochloa crusgalli)

<400>2

Met Ala Ser Glu Arg His Gln Ser Ile Asp Ala Gln Leu Arg Leu Leu

1 5 10 15

Ala Pro Gly Lys Val Ser Glu Asp Asp Lys Leu Val Glu Tyr Asp Ala

20 25 30

Leu Leu Val Asp Arg Phe Leu Asp Ile Leu Gln Asp Leu His Gly Pro

35 40 45

His Leu Arg Glu Phe Val Gln Glu Cys Tyr Glu Leu Ser Ala Glu Tyr

50 55 60

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

65 70 75 80

Thr Ser Leu Pro Pro Gly Glu Ser Ile Val Val Ala Ser Ser Phe Ser

85 90 95

His Met Leu Asn Leu Ala Asn Leu Ala Glu Glu Val Gln Ile Ala His

100 105 110

Arg Arg Arg Ile Lys Leu Lys Arg Gly Asp Phe Ala Asp Glu Ala Ser

115 120 125

Ala Pro Thr Glu Ser Asp Ile Glu Glu Thr Leu Lys Arg Leu Val Ser

130 135 140

Gln Leu Gly Lys Ser Arg Glu Glu Val Phe Asp Ala Leu Lys Asn Gln

145 150 155 160

Thr Val Asp Leu Val Phe Thr Ala His Pro Thr Gln Ser Val Arg Arg

165 170 175

Ser Leu Leu Gln Lys His Gly Arg Ile Arg Asn Cys Leu Arg Gln Leu

180 185 190

Tyr Ala Lys Asp Ile Thr Ala Asp Asp Lys Gln Glu Leu Asp Glu Ala

195 200 205

Leu Gln Arg Glu Ile Gln Ala Ala Phe Arg Thr Asp Glu Ile Arg Arg

210 215 220

Thr Pro Pro Thr Pro Gln Asp Glu Met Arg Ala Gly Met Ser Tyr Phe

225 230 235 240

His Glu Thr Ile Trp Lys Gly Val Pro Lys Phe Leu Arg Arg Ile Asp

245 250 255

Thr Ala Leu Lys Asn Ile Gly Ile Asn Glu Arg Leu Pro Tyr Asn Ala

260 265 270

Pro Leu Ile Gln Phe Ser Ser Trp Met Gly Gly Asp Arg Asp Gly Asn

275 280 285

Pro Arg Val Thr Pro Glu Val Thr Arg Asp Val Cys Leu Leu Ala Arg

290 295 300

Met Met Ala Ala Asn Leu Tyr Phe Ser Gln Ile Glu Asp Leu Met Phe

305 310 315 320

Glu Leu Ser Met Trp Arg Cys Ser Asp Glu Leu Arg Ile Arg Ala Asp

325 330 335

Asp Leu His Arg Ser Thr Lys Arg Ala Ala Glu His TyrIle Glu Phe

340 345 350

Trp Lys Gln Val Pro Pro Asn Glu Pro Tyr Arg ValIle Leu Gly Asp

355 360 365

Val Arg Asp Lys Leu Tyr Tyr Thr Arg Glu Arg Ser Arg His Leu Leu

370 375 380

Thr Thr Gly Ile Ser Glu Ile Pro Glu Asp Ala Thr Phe Thr Asn Val

385 390 395 400

Glu Gln Phe Leu Glu Pro Leu Glu Leu Cys Tyr Arg Ser Leu Cys Ala

405 410 415

Cys Gly Asp Lys Pro Ile Ala Asp Gly Ser Leu Leu Asp Phe Leu Arg

420 425 430

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

435 440 445

Glu Ser Asp Arg His Thr Asp Val Leu Asp Ser Ile Thr Thr His Pro

450 455 460

Gly Ile Gly Ser Tyr Ala Glu Trp Ser Glu Glu Lys Arg Gln Asp Trp

465 470 475 480

Leu Leu Ser Glu Leu Arg Gly Lys Arg Pro Leu Phe Gly Ser Asp Leu

485 490 495

Pro Leu Thr Glu Glu Thr Ala Asp Val Leu Gly Thr Phe His Val Leu

500 505 510

Ala Glu Leu Pro Thr Asp Cys Phe Gly Ala Tyr Ile Ile Ser Met Ala

515 520 525

Thr Ala Pro Ser Asp Val Leu Ala Val Glu Leu Leu Gln Arg Glu Cys

530 535 540

His Val Lys Gln Pro Leu Arg Val Val Pro Leu Phe Glu Lys Leu Ala

545 550 555 560

Asp Leu Glu Ala Ala Pro Ala Ala Val Ala Arg Leu Phe Ser Ile Gly

565 570 575

Trp Tyr Met Asn Arg Ile Asn Gly Lys Gln Glu Val Met Ile Gly Tyr

580 585 590

Ser Asp Ser Gly Lys Asp Ala Gly Arg Leu Ser Ala Thr Trp Gln Met

595 600 605

Tyr Lys Ala Gln Glu Glu Leu Ile Lys Val Ala Lys His Tyr Gly Val

610 615 620

Lys Leu Thr Met Phe His Gly Arg Gly Gly Thr Val Gly Arg Gly Gly

625 630 635 640

Gly Pro Thr His Leu Ala Ile Leu Ser Gln Pro Pro Asp Thr Ile His

645 650 655

Gly Ser Leu Arg Val Thr Val Gln Gly Glu Val Ile Glu His Ser Phe

660 665 670

Gly Glu Glu Leu Leu Cys Phe Arg Thr Leu Gln Arg Tyr Thr Ala Ala

675 680 685

Thr Leu Glu His Gly Met His Pro Pro Ile Ser Pro Lys Pro Glu Trp

690 695 700

Arg Ala Leu Met Asp Glu Met Ala Ile Val Ala Thr Lys Glu Tyr Arg

705 710 715 720

Ser Ile Val Phe Gln Glu Pro Arg Phe Val Glu Tyr Phe Gly Ser Ala

725 730 735

Thr Pro Glu Thr Glu Tyr Gly Arg Met Asn Ile Asp Ser Arg Pro Ser

740 745 750

Lys Arg Arg Pro Ser Gly Gly Ile Glu Ser Leu Arg Ala Ile Pro Trp

755 760 765

Ile Phe Ala Trp Thr Gln Thr Arg Phe His Pro Pro Val Trp Leu Gly

770 775 780

Phe Gly Ala Ala Phe Lys His Ile Met Gln Lys Asp Ile Arg Asn Thr

785 790 795 800

His Thr Leu Lys Glu Met Tyr Asn Glu Trp Pro Phe Ser Arg Val Thr

805 810 815

Leu Asp Leu Leu Glu Met Val Phe Ala Lys Gly Asp Pro Gly Ile Ala

820 825 830

Ala Val Tyr Asp Lys Leu Leu Val Ala Asp Asp Leu Gln Ser Phe Gly

835 840 845

Glu Gln Leu Arg Lys Asn Tyr Glu Glu Thr Lys Glu Leu Leu Leu Gln

850 855 860

Val Ala Gly His Lys Asp Val Leu Glu Gly Asp Pro Tyr Leu Lys Gln

865 870 875 880

Arg Leu Arg Leu Arg Glu Ser Tyr Ile Thr Thr Leu Asn Val Cys Gln

885 890 895

Ala Tyr Thr Leu Lys Arg Ile Arg Asp Pro Ser Phe Gln Val Ser Pro

900 905 910

Gln Pro Ala Leu Ser Lys Glu Phe Val Asp Glu Ser Gln Pro Ala Glu

915 920 925

Leu Val Arg Leu Asn Pro Glu Ser Glu Tyr Ala Pro Gly Leu Glu Asn

930 935 940

Thr Leu lle Leu Thr Met Lys Gly Ile Ala Ala Gly Met Gln Asn Thr

945 950 955 960

Gly

Claims (10)

1, the phosphoric acid enol pyruvic acid carboxylase gene of barnyard grass grass has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits.

2, the phosphoric acid enol pyruvic acid carboxylase gene of barnyard grass grass according to claim 1 is characterized in that: described gene has SEQ ID № in the sequence table: 1 dna sequence dna.

3, the phosphoric acid enol pyruvic acid carboxylase of barnyard grass grass is the albumen with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 2;

2) with SEQ ID № in the sequence table: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and the protein with raising photosynthesis of plant efficient.

4, contain claim 1 or 2 described expression carrier, transgenic cell line and host bacterium.

5, a kind of method that improves photosynthesis of plant efficient is with the phosphoric acid enol pyruvic acid carboxylase gene importing explant of claim 1 or 2 described barnyard grass grass, obtains the plant that photosynthetic efficiency improves.

6, method according to claim 5 is characterized in that: the phosphoric acid enol pyruvic acid carboxylase gene of described barnyard grass grass can import plant explants by the plant expression vector that contains described barnyard grass grass phosphoric acid enol pyruvic acid carboxylase gene; The carrier that sets out that is used to make up described plant expression vector is pCAMBIA1301, pCAMBIA1300, pBI121.

7, method according to claim 6 is characterized in that: the carrier that sets out that is used to make up described plant expression vector is pCAMBIA1301.

8, method according to claim 7 is characterized in that: described plant expression vector is pUbi-Eppc.

9, method according to claim 7 is characterized in that: described plant expression vector is pRbcS-Eppc

10, method according to claim 7 is characterized in that: described plant expression vector is pZMp-Eppc.

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