CN108165577A

CN108165577A - Turn the photosynthetic key genes of C4 and improve the photosynthetic method of C3 plant

Info

Publication number: CN108165577A
Application number: CN201810025625.1A
Authority: CN
Inventors: 路铁刚; 张治国; 崔学安; 吴金霞; 孙学辉; 谷晓峰; 保罗·奎克威廉
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2018-01-11
Filing date: 2018-01-11
Publication date: 2018-06-15

Abstract

Turn the photosynthetic key genes of C4 the invention discloses one kind and improve the photosynthetic method of C3 plant.The present invention provides a kind of enhancing photosynthesis of plant and/or plant products and/or the method for improving phytomass are improved, including：Using different startup substrategies, 5 photosynthetic key genes of C4 are imported into recipient plant, obtain genetically modified plants；Compared with the recipient plant, photosynthesis enhancing and/or output increased and/or biomass improve the genetically modified plants.It is successfully formulated using the method for the present invention and provides the material that photosynthetic efficiency significantly improves, big spoke improves the Biomass and yield of plant, and without subsidiary other unfavorable characters.The present invention is worth for cultivating C4 rice materials with great production application.

Description

Turn the photosynthetic key genes of C4 and improve the photosynthetic method of C3 plant

Technical field

The invention belongs to photosynthesis of plant technical field of improvement, are related to one kind and turn the photosynthetic key gene raising C3 plants of C4 Photosynthetic method, more particularly to using polygenes conversion system, transformation obtains a carrier containing 5 photosynthesis genes, profit These genes are driven with different promoters, initiative can significantly improve the method for the rice material of photosynthetic efficiency.

Background technology

With industrialization, the development of urbanization and the increase of population, China's grain demand will be in that rigidity increases, and plough Reduction, shortage of water resources, climate change etc. become increasingly conspicuous to the constraint of grain-production, and China's grain supply and demand will be chronically at " tight Balance " state, Ensuring Food Safety face a severe challenge.The Plant-type Breeding and heterosis utilization of crops are China's grain The raising of crop yield ensures that the fast development of national economy is made that tremendous contribution, but grain yield further improves difficulty It spends increasing.In current Ensuring Food Safety as under the background of China's long-term national policy, how realizing cereal crops yield water Flat lasting promotion is a difficult problem to lie across in Chinese society economy, agricultural science and technology forward march.Crop harvest index is One of leading indicator of crop yield is weighed, the harvest index of rice, wheat is all close to 0.5 at present, it means that both works The grain yield of object accounts for nearly the 50% of all biological amount, and the large span raising for realizing grain yield by traditional breeding method can not Energy.

Fortunately, there are other plants (C4 plants, such as corn, sorghum and sugarcane), they photosynthetic in the world Efficiency is higher by 50% than C3 plant.Using the means of transgenosis, theoretically conversion can significantly increase C4 photosynthesis.

Although the overexpression research of the enzyme and transport protein of C4 approach in C3 plant has made great progress, in C3 plant The technology of the specific region high level expression C4 path enzymes of blade is also very ripe, due to photosynthetical system be a complexity, the division of labor it is bright Thin system, the photosynthetic efficiency of C3 plant can not be significantly improved by turning individual gene.Some research shows that, high level expression C4 Gene pairs C3 plant photosynthetic efficiency improves and the effect of physiological character improvement is limited, sometimes even unfavorable.The surveys such as Agarie The transgenic paddy rice strain of 50 times higher than control of PEPC activity is determined, the result is that its photosynthetic rate slightly below compares.The hairs such as Ku Now partly turn PEPC rice plants and show undesirable economical character, such as plant height is downgraded, blade narrows, infertility.And attempt by PEPC, NADP-ME, PPDK, NADP-MDH are all transferred in rice, to form the experiment of a complete C4 cycle also with mistake Lose and come to an end, this turn polygenes rice equally show growth it is suppressed, illustrate to be established in vivo is an invalid cycle, to C4 Distinctive CO₂Concentrating mechanism is not contributed.

Invention content

In view of the deficiencies of the prior art, present invention employs different startup substrategies, and 5 photosynthetic key genes of C4 are led Enter rice.

First, it is claimed it is a kind of enhancing photosynthesis of plant and/or improve plant products and/or improve plant The method of biomass.

Enhancing photosynthesis of plant and/or raising plant products and/or raising phytomass provided by the present invention Method (or the genetically modified plants method improved to cultivate photosynthesis enhancing and/or output increased and/or biomass), tool Body may include following steps：5 photosynthetic key genes of C4 are imported into recipient plant, obtain genetically modified plants；The transgenosis Compared with the recipient plant, photosynthesis enhancing and/or output increased and/or biomass improve plant.

Wherein, the photosynthetic key genes of 5 C4 are encoding gene, the PEP of phosphopyruvic acid bidifly enzyme (ZmPPDK1) The encoding gene of carboxylase (ZmPEPC1), the encoding gene of malic dehydrogenase (ZmMDH6), NADP- malate dehydrogenases (ZmME2) encoding gene and the encoding gene of carbonic anhydrase (ZmCA1).

In the genetically modified plants, the encoding gene of the phosphopyruvic acid bidifly enzyme (ZmPPDK1) is by the phosphoric acid The endogenesis promoter driving transcription of the encoding gene of pyruvic acid bidifly enzyme (ZmPPDK1)；The PEP carboxylases (ZmPEPC1) Encoding gene by the PEP carboxylases (ZmPEPC1) encoding gene endogenesis promoter drive transcription；The malic acid The encoding gene of dehydrogenase (ZmMDH6) is driven by 35CaMVS promoters and transcribed；The NADP- malate dehydrogenases (ZmME2) Encoding gene is driven by Ubiqutin promoters and transcribed；The encoding gene of the carbonic anhydrase (ZmCA1) is started by OsActin Son driving transcription.

Wherein, the photosynthetic key genes of 5 C4 are imported in the recipient plant by the form of recombinant vector.Institute Recombinant vector is stated as hereafter any recombinant vector.

In the above-mentioned methods, the recombinant vector is imported into the recipient plant concretely：By using Ti-plasmids, Ri The conventional biology methods such as plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated convert Plant cell or tissue, and the plant tissue of conversion is cultivated into plant.

Second, a kind of claimed recombinant vector or the recombinant bacterium containing the recombinant vector.

The recombinant vector is the ring containing DNA fragmentation 1, DNA fragmentation 2, DNA fragmentation 3, DNA fragmentation 4 and DNA fragmentation 5 Form quality grain.

The DNA fragmentation 1 is from above swimming over to the downstream encoding gene containing phosphopyruvic acid bidifly enzyme (ZmPPDK1) successively The encoding gene of endogenesis promoter, phosphopyruvic acid bidifly enzyme (ZmPPDK1).The DNA fragmentation 2 is from above swimming over to downstream successively The endogenesis promoter of encoding gene containing PEP carboxylases (ZmPEPC1), the encoding gene of PEP carboxylases (ZmPEPC1).Institute State DNA fragmentation 3 from above swim over to downstream successively containing 35CaMVS promoters, malic dehydrogenase (ZmMDH6) encoding gene. The DNA fragmentation 4 from above swim over to downstream successively containing Ubiqutin promoters, NADP-malate dehydrogenase (ZmME2) coding base Cause.The DNA fragmentation 5 from above swim over to downstream successively containing OsActin promoters, carbonic anhydrase (ZmCA1) encoding gene.

Further, the recombinant vector is by the DNA fragmentation 1, DNA fragmentation 2, the DNA fragmentation 3, described DNA fragmentation 4 and the DNA fragmentation 5 are inserted into restriction enzyme site the Hind III and Sma of pCambia1300 carriers after being sequentially connected with The recombinant plasmid obtained after between I.

Previously described phosphopyruvic acid bidifly enzyme (ZmPPDK1) can be following any shown protein：

(A1) amino acid sequence is the protein of SEQ ID No.11；

(A2) by the amino acid sequence shown in SEQ ID No.11 by one or several amino acid residues substitution and/or Lack and or add and have the protein of identical function；

(A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, 90% with Above, more than 85% or more than 80% homology and the protein with identical function；

(A4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (A1)-(A3) Albumen.

Previously described PEP carboxylases (ZmPEPC1) can be following any shown protein：

(B1) amino acid sequence is the protein of SEQ ID No.12；

(B2) by the amino acid sequence shown in SEQ ID No.12 by one or several amino acid residues substitution and/or Lack and or add and have the protein of identical function；

(B3) with (B1)-(B2) in any limited amino acid sequence have more than 99%, more than 95%, 90% with Above, more than 85% or more than 80% homology and the protein with identical function；

(B4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (B1)-(B3) Albumen.

Previously described malic dehydrogenase (ZmMDH6) can be following any shown protein：

(C1) amino acid sequence is the protein of SEQ ID No.13；

(C2) by the amino acid sequence shown in SEQ ID No.13 by one or several amino acid residues substitution and/or Lack and or add and have the protein of identical function；

(C3) with (C1)-(C2) in any limited amino acid sequence have more than 99%, more than 95%, 90% with Above, more than 85% or more than 80% homology and the protein with identical function；

(C4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (C1)-(C3) Albumen.

Previously described NADP- malate dehydrogenases (ZmME2) can be following any shown protein：

(D1) amino acid sequence is the protein of SEQ ID No.14；

(D2) by the amino acid sequence shown in SEQ ID No.14 by one or several amino acid residues substitution and/or Lack and or add and have the protein of identical function；

(D3) with (D1)-(D2) in any limited amino acid sequence have more than 99%, more than 95%, 90% with Above, more than 85% or more than 80% homology and the protein with identical function；

(D4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (D1)-(D3) Albumen.

Previously described carbonic anhydrase (ZmCA1) can be following any shown protein：

(E1) amino acid sequence is the protein of SEQ ID No.15；

(E2) by the amino acid sequence shown in SEQ ID No.15 by one or several amino acid residues substitution and/or Lack and or add and have the protein of identical function；

(E3) with (E1)-(E2) in any limited amino acid sequence have more than 99%, more than 95%, 90% with Above, more than 85% or more than 80% homology and the protein with identical function；

(E4) N-terminal of any limited protein and/or C-terminal connect the fusion obtained after label in (E1)-(E3) Albumen.

The encoding gene of previously described phosphopyruvic acid bidifly enzyme (ZmPPDK1) can be following any the DNA points Son：

(a1) DNA molecular shown in SEQ ID No.2；

(a2) hybridize under strict conditions with (a1) DNA molecular limited and encode the phosphopyruvic acid bidifly enzyme (ZmPPDK1) DNA molecular；

(a3) with (a1)-(a2) in any restriction DNA sequence dna have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the DNA molecular of the coding phosphopyruvic acid bidifly enzyme (ZmPPDK1).

The encoding gene of previously described PEP carboxylases (ZmPEPC1) can be following any DNA molecular：

(b1) DNA molecular shown in SEQ ID No.4；

(b2) hybridize under strict conditions with (b1) DNA molecular limited and encode the PEP carboxylases (ZmPEPC1) DNA molecular；

(b3) with (b1)-(b2) in any restriction DNA sequence dna have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the DNA molecular of the coding PEP carboxylases (ZmPEPC1).

The encoding gene of previously described malic dehydrogenase (ZmMDH6) can be following any DNA molecular：

(c1) DNA molecular shown in SEQ ID No.6；

(c2) hybridize under strict conditions with (c1) DNA molecular limited and encode the malic dehydrogenase (ZmMDH6) DNA molecular；

(c3) with (c1)-(c2) in any restriction DNA sequence dna have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the DNA molecular of the coding malic dehydrogenase (ZmMDH6).

The encoding gene of previously described NADP- malate dehydrogenases (ZmME2) can be following any DNA molecular：

(d1) DNA molecular shown in SEQ ID No.8；

(d2) hybridize under strict conditions with (d1) DNA molecular limited and encode the NADP- malate dehydrogenases (ZmME2) DNA molecular；

(d3) with (d1)-(d2) in any restriction DNA sequence dna have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the DNA molecular of the coding NADP- malate dehydrogenases (ZmME2).

The encoding gene of previously described carbonic anhydrase (ZmCA1) can be following any DNA molecular：

(e1) DNA molecular shown in SEQ ID No.10；

(e2) hybridize under strict conditions with (e1) DNA molecular limited and encode the carbonic anhydrase (ZmCA1) DNA molecular；

(e3) with (e1)-(e2) in any restriction DNA sequence dna have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the DNA molecular of the coding carbonic anhydrase (ZmCA1).

Above-mentioned stringent condition can be with 6 × SSC, and the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC, It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.

The nucleotide sequence of the endogenesis promoter of the encoding gene of previously described phosphopyruvic acid bidifly enzyme (ZmPPDK1) As shown in SEQ ID No.1.The nucleotide of the endogenesis promoter of the encoding gene of previously described PEP carboxylases (ZmPEPC1) Sequence is as shown in SEQ ID No.3.The nucleotide sequence of previously described 35CaMVS promoters is as shown in SEQ ID No.5. The nucleotide sequence of previously described Ubiqutin promoters is as shown in SEQ ID No.7.Previously described OsActin promoters Nucleotide sequence as shown in SEQ ID No.9.

Further, the nucleotides sequence of the DNA fragmentation 1 is classified as SEQ ID No.1 and SEQ ID No.2 is directly suitable The sequence obtained after secondary connection.The nucleotides sequence of the DNA fragmentation 2 is classified as SEQ ID No.3 and SEQ ID No.4 is direct The sequence obtained after being sequentially connected with.The nucleotides sequence of the DNA fragmentation 3 is classified as SEQ ID No.5 and SEQ ID No.6 is straight Connect the sequence obtained after being sequentially connected with.The nucleotides sequence of the DNA fragmentation 4 is classified as SEQ ID No.7 and SEQ ID No.8 The sequence obtained after being directly sequentially connected with.The nucleotides sequence of the DNA fragmentation 5 is classified as SEQ ID No.9 and SEQ ID The sequence that No.10 is obtained after being directly sequentially connected with.

In one embodiment of the invention, the recombinant vector is specially by 10 sequences shown in SEQ ID No.1-10 Row be directly sequentially connected with after be inserted into pCambia1300 carriers " GGCCAGTGCCAAGCTT " and The recombinant plasmid obtained after at " CCCGGGCTGCAAGTTCAA ".

Third, the claimed recombinant vector or recombinant bacterium are in enhancing photosynthesis of plant and/or improve plant Application in produce amount and/or raising phytomass.

In the present invention, the plant is C3 plant.Specifically, the C3 plant can be rice；More specifically, such as water Rice varieties Nipponbare (Oryza sativa L.ssp.Japonica).

In the present invention, the enhancing photosynthesis of plant (or photosynthesis enhancing) can specifically be presented as that raising is planted Object photosynthetic rate.The raising plant products (or described output increased), which can specifically be presented as, improves vegetable seeds mass of 1000 kernel.

The present invention using different promoters drive 5 photosynthetic pathway key protein encoding gene, 5 gene sources in Corn (Zea mays.L), respectively phosphopyruvic acid bidifly enzyme (ZmPPDK1), PEP carboxylases (ZmPEPC1), malic acid take off Hydrogen enzyme (ZmMDH6), NADP- malate dehydrogenases (ZmME2), carbonic anhydrase (ZmCA1).Different promoters be respectively 35CaMV S, Ubiqutin, OsActin, ZmPPDK1 promoter, ZmPEPC1 promoters.This multigene carrier is named as MGTV (Multi-gene-transformation-vector) carrier.By in MGTV vector introduction C3 plant rice, initiative has been provided The material that photosynthetic efficiency significantly improves, big spoke improve the biomass of plant, and without subsidiary other unfavorable characters.The present invention is right It is worth in cultivating C4 rice materials with great production application.

Description of the drawings

Fig. 1 is the structure flow chart of MGTV carriers.

Fig. 2 is unloaded control and turns the T1 of polygenes positive strain for plant phenotype.PCambia1300 is planted for unloaded control Strain；MGTV-OV4 is turns polygenes positive strain OV4.

Specific embodiment

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Quantitative test in following embodiments, is respectively provided with three repeated experiments, and results are averaged.

Rice varieties Nipponbare (Oryza sativa L.ssp.Japonica), also known as wild type, are represented with WT：China Paddy rice cross breeding breeding center (China, Changsha).PCambia1300 carriers, pCambia1302 carriers, pCambia1390 carriers： PCAMBIA companies.Agrobacterium AGL1：Chinese plasmid vector strain cell pnca gene collection.

Embodiment 1, the acquisition of MGTV vector constructions and transgenic paddy rice and Function Identification

First, the clone of 5 photosynthetic elements and multigene carrier structure

C4 plant Zea mays have higher photosynthetic efficiency, biological yield etc. than C3 plant rice, and main cause is that C4 plants Object has the photosynthetic cycle approach of C4, and the present invention has cloned these photosynthetic pathway key genes from corn：Phosphopyruvic acid bidifly Enzyme (ZmPPDK1), PEP carboxylases (ZmPEPC1), malic dehydrogenase (ZmMDH6), NADP- malate dehydrogenases (ZmME2), Carbonic anhydrase (ZmCA1).It is driven using different promoters, wherein ZmPPDK1 promoters driving ZmPPDK1, ZmPEPC1 start Son driving ZmPEPC1,35CaMV S driving ZmMDH6, Ubiqutin promoters drive ZmME2, the driving of OsACTIN promoters ZmCA1。

Encoding gene (CDS) sequence of phosphopyruvic acid bidifly enzyme (ZmPPDK1) encodes SEQ as shown in SEQ ID No.2 Protein shown in ID No.11.Encoding gene (CDS) sequence of PEP carboxylases (ZmPEPC1) is compiled as shown in SEQ ID No.4 Protein shown in code SEQ ID No.12.Encoding gene (CDS) sequence such as SEQ ID of malic dehydrogenase (ZmMDH6) Shown in No.6, protein shown in SEQ ID No.13 is encoded.Encoding gene (CDS) sequence of NADP- malate dehydrogenases (ZmME2) As shown in SEQ ID No.8, protein shown in SEQ ID No.14 is encoded.The encoding gene (CDS) of carbonic anhydrase (ZmCA1) Sequence encodes protein shown in SEQ ID No.15 as shown in SEQ ID No.10.

The nucleotide sequence of ZmPPDK1 promoters is as shown in SEQ ID No.1.The nucleotide sequence of ZmPEPC1 promoters As shown in SEQ ID No.3.The nucleotide sequence of 35CaMVS promoters is as shown in SEQ ID No.5.Ubiqutin promoters Nucleotide sequence as shown in SEQ ID No.7.The nucleotide sequence of OsActin promoters is as shown in SEQ ID No.9.

1st, the encoder block (CDS) and promoter sequence of 5 photosynthesis genes elements are expanded respectively first.

Using TOYOBO KOD high-fidelity enzymatic amplifications, operated to specifications, reaction system is as shown in table 1.

Table 1 expands the encoder block (CDS) of 5 photosynthesis genes elements and the reaction system of promoter sequence

The correct amplified band gel extraction to molecular weight, -20 DEG C of preservation products.

2nd, splicing obtains 5 complete element sequences respectively.

Because for expanding the F ends primer of every set of pieces CDS, added with the 15-18bp sequences being overlapped with promoter, thus can The promoter and CDS of every set of pieces are spliced into a PCR product using fusion DNA vaccine.Meanwhile in order to which next step is 5 In element recombination to purpose carrier, this wheel PCR amplification needs to increase overlap in respective F ends primer, and amplification system is such as Shown in table 2.

Table 2 splices the amplification system for obtaining 5 complete element sequences respectively

Using the efficient recombinase In-Fusion HD Cloning Kit (article No. 639648) of Clontech, using each 15-18bp repetitive sequences, element 1 between element is to the repetitive sequence between element 5 and pCambia1300 carriers, according to table Correspondence ratio shown in 3 carries out recombining reaction, conversion, identification, sequencing, is finally completed the structure of carrier.

3 element 1 of table is to element 5 and the reaction ratio of pCambia1300 carriers

Element number	Clip size	Aequum	Volume
				Element 1	4844bp	216ng	1.5μl
Element 2	4913bp	219ng	1.5μl
				Element 3	1837bp	82ng	0.5μl
Element 4	3982bp	177ng	1μl
				Element 5	2784bp	124ng	1μl
PCambia1300 carriers	8958bp	200ng	2μl
							2.5 μ l recombinases are mended to 10 μ l

Each primer sequence referred to above is specifically as shown in table 4.

Primer used in 4 vector construction of table

By 900 to being grown on screening and culturing medium more, clone carries out PCR identifications, and it is completely correct to obtain 6 recombinations Clone.Sequence verifications are carried out to this 5 clones, it is found that carrier construction is consistent with expected sequence, has no base mutation, it can be with For next step rice transformation.This multigene carrier is named as MGTV (Multi-gene-transformation- Vector) carrier is shown in Fig. 1.

The structure description of MGTV carriers：10 sequences shown in SEQ ID No.1-10 are spliced into 5 elements respectively, so Afterwards using in-between coincidence sequence, 5 elements are recombinated simultaneously to pCambia1300 carriers in the case where recombinating enzyme effect At " GGCCAGTGCCAAGCTT " and " CCCGGGCTGCAAGTTCAA " (between restriction enzyme site Hind III and SmaI) after Obtained recombinant plasmid.

In addition, the present invention also construct following 5 for turn monogenic recombinant vector：

(1) pCambia1300-PPDK1 carriers：2 sequences shown in SEQ ID No.1 and SEQ ID No.2 are direct It is inserted into after being sequentially connected at " GGCCAGTGCCAAGCTT " and " CCCGGGCTGCAAGTTCAA " of pCambia1300 carriers The recombinant plasmid obtained afterwards.

(2) pCambia1300-PEPC1 carriers：2 sequences shown in SEQ ID No.3 and SEQ ID No.4 are direct It is inserted into after being sequentially connected at " GGCCAGTGCCAAGCTT " and " CCCGGGCTGCAAGTTCAA " of pCambia1300 carriers The recombinant plasmid obtained afterwards.

(3) pCambia1300-MDH6 carriers：2 sequences shown in SEQ ID No.5 and SEQ ID No.6 are directly suitable After being inserted into after secondary connection at " GGCCAGTGCCAAGCTT " and " CCCGGGCTGCAAGTTCAA " of pCambia1300 carriers Obtained recombinant plasmid.

(4) pCambia1300-ME2 carriers：2 sequences shown in SEQ ID No.7 and SEQ ID No.8 are directly suitable After being inserted into after secondary connection at " GGCCAGTGCCAAGCTT " and " CCCGGGCTGCAAGTTCAA " of pCambia1300 carriers Obtained recombinant plasmid.

(5) pCambia1300-CA1 carriers：2 sequences shown in SEQ ID No.9 and SEQ ID No.10 are directly suitable After being inserted into after secondary connection at " GGCCAGTGCCAAGCTT " and " CCCGGGCTGCAAGTTCAA " of pCambia1300 carriers Obtained recombinant plasmid.

2nd, the initiative and identification of transgenic line

1st, the recombinant plasmid MGTV of step 1 structure is imported into Agrobacterium AGL1, obtains recombinational agrobacterium.

2nd, the recombinational agrobacterium that step 1 is taken to obtain is resuspended in liquid and co-cultures culture medium (YEP fluid nutrient mediums+100mg/ L acetosyringones, pH5.2), obtain OD_600nm=0.14 bacterium solution.

3rd, the embryo callus of Nipponbare is taken, 30min is impregnated with the bacterium solution that step 2 obtains, then through co-culturing, sieving It selects, take root, strong sprout, obtaining T₀For plant.

4、T₀T is obtained for plant selfing₁For seed, T₁It is T for the plant that seed grows up to₁For plant.

5th, above-mentioned conversion is carried out with pCambia1300 carriers, obtains turning empty carrier plant.

6th, with pCambia1300-PPDK1, pCambia1300-PEPC1, pCambia1300-MDH6, PCambia1300-ME2 and pCambia1300-CA1 carriers carry out above-mentioned conversion, obtain 5 kinds and turn single-gene plant.

With primer sequence amplification regeneration plant DNA corresponding in table 2, electrophoresis determines positive strain.Turn from identification is positive 4 are randomly choosed in polygenes strain, number is OV-1, OV-2, OV-3 and OV-4 respectively.

3rd, photosynthetic parameters measure

4 transgenic positive systems that Nipponbare, the step 2 for taking growth conditions consistent obtain (OV-1, OV-2, OV-3 and OV-4), the unloaded T1 for compareing, turning individual gene, in full-bloom stage, utilizes LI-COR6400 to carry out the survey of photosynthetic parameters for plant It is fixed, and other economical characters shown in statistical form 5.

The result shows that it compareed with zero load, wild control, turn individual gene (PPDK1, PEPC1, MDH6, ME2, CA1) phase Than big degree of convergence improves photosynthetic efficiency to 4 positive strains (OV-1, OV-2, OV-3 and OV-4), and then improves to some extent Mass of 1000 kernel, and that unfavorable economical character phenotype is not presented.See Fig. 2 and table 5.

5 transgenosis of table is compared with each economical character of empty map

Note：* show compared with empty map, significant difference (P<0.05).

<110>Biological Technology institute, Chinese Academy of Agricultural Sciences

<120>Turn the photosynthetic key genes of C4 and improve the photosynthetic method of C3 plant

<130> GNCLN180019

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 2000

<212> DNA

<213>Corn（Zea mays L.）

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<212> DNA

<213>Corn（Zea mays L.）

<400> 2

atggcggcat cggtttccag ggccatctgc gtacagaagc cgggctcaaa atgcaccagg 60

gacagggaag cgacctcctt cgcccgccga tcggtcgcag cgccgaggcc cccgcacgcc 120

aaagcccgcc ggcgtcatcc gctccgactc cggcgcggga cggggccaca ttgctcgccg 180

ctgagggccg tcgttgacgc cgcgccgata cagacgacca aaaagagggt gttccacttc 240

ggcaagggca agagcgaggg caacaagacc atgaaggaac tgctgggcgg caagggcgcg 300

aacctggcgg agatggcgag catcgggctg tcggtgccgc cagggttcac ggtgtcgacg 360

gaggcgtgcc agcagtacca ggacgccggg tgcgccctcc ccgcggggct ctgggccgag 420

atcgtcgacg gcctgcagtg ggtggaggag tacatgggcg ccaccctggg cgatccgcag 480

cgcccgctcc tgctctccgt ccgctccggc gccgccgtgt ccatgcccgg catgatggac 540

acggtgctca acctggggct caacgacgaa gtggccgccg ggctggcggc caagagcggg 600

gagcgcttcg cctacgactc cttccgccgc ttcctcgaca tgttcggcaa cgtcgtcatg 660

gacatccccc gctcactgtt cgaagagaag cttgagcaca tgaaggaatc caaggggctg 720

aagaacgaca ccgacctcac ggcctctgac ctcaaagagc tcgtgggtca gtacaaggag 780

gtctacctct cagccaaggg agagccattc ccctcagacc ccaagaagca gctggagcta 840

gcagtgctgg ctgtgttcaa ctcgtgggag agccccaggg ccaagaagta caggagcatc 900

aaccagatca ctggcctcag gggcaccgcc gtgaacgtgc agtgcatggt gttcggcaac 960

atggggaaca cttctggcac cggcgtgctc ttcaccagga accccaacac cggagagaag 1020

aagctgtatg gcgagttcct ggtgaacgct cagggtgagg atgtggttgc cggaataaga 1080

accccagagg accttgacgc catgaagaac ctcatgccac aggcctacga cgagcttgtt 1140

gagaactgca acatcctgga gagccactac aaggaaatgc aggatatcga gttcactgtc 1200

caggaaaaca ggctgtggat gttgcagtgc aggacaggga aacgtacggg caaaagtgcc 1260

gtgaagatcg ccgtggacat ggttaacgag ggccttgttg agccccgctc agcgatcaag 1320

atggtagagc caggccacct ggaccagctt cttcatcctc agtttgagaa cccgtcggcg 1380

tacaaggatc aagtcattgc cactggtctg ccagcctcac ctggggctgc tgtgggccag 1440

gttgtgttca ctgctgaaga tgctgaagca tggcattccc aagggaaagc tgctattctg 1500

gtaagggcgg agaccagccc tgaggacgtt ggtggcatgc acgctgctgt ggggattctt 1560

acagagaggg gtggcatgac ttcccacgct gctgtggtcg cacgttggtg ggggaaatgc 1620

tgcgtctcgg gatgctcagg cattcgcgta aacgatgcgg agaagctcgt gacgatcgga 1680

agccatgtgc tgcgcgaagg tgagtggctg tcgctgaatg ggtcgactgg tgaggtgatc 1740

cttgggaagc agccgctttc cccaccagcc cttagtggtg atctgggaac tttcatggcc 1800

tgggtggatg atgttagaaa gctcaaggtc ctggctaacg ccgatacccc tgatgatgca 1860

ttgactgcgc gaaacaatgg ggcacaagga attggattat gccggacaga gcacatgttc 1920

tttgcttcag acgagaggat taaggctgtc aggcagatga ttatggctcc cacgcttgag 1980

ctgaggcagc aggcgctcga ccgtctcttg acgtatcaga ggtctgactt cgaaggcatt 2040

ttccgtgcta tggatggact cccggtgacc atccgactcc tggaccatcc ttcttacgag 2100

ttccttccag aagggaacat cgaggacatt gtaagtgaat tatgtgctga gacgggagcc 2160

aaccaggagg atgccctcgc gcgaattgaa aagctttcag aagtaaaccc gatgcttggc 2220

ttccgtgggt gcaggcttgg tatatcgtac cctgaattga cagagatgca agcccgggcc 2280

atttttgaag ctgctatagc aatgaccaac cagggtgttc aagtgttccc agagataatg 2340

gttcctcttg ttggaacacc acaggaactg gggcatcaag tgactcttat ccgccaagtt 2400

gctgagaaag tgttcgccaa tgtgggcaag actatcgggt acaaagttgg aacaatgatt 2460

gagatcccca gggcagctct ggtggctgat gagatagcgg agcaggctga attcttctcc 2520

ttcggaacga acgacctgac gcagatgacc tttgggtaca gcagggatga tgtgggaaag 2580

ttcattcccg ttcatcttgc tcagggcatc ctccaacatg accccttcga ggtcctggac 2640

cagaggggag tgggcgagct ggtgaagttt gctacagaga ggggccgcaa agctaggcct 2700

aacttgaagg tgggcatttg tggagaacac ggtggagagc cttcgtctgt ggccttcttc 2760

gcgaaggctg ggctggattt cgtttcttgc tcccctttca gggttccgat tgctaggcta 2820

gctgcagctc aggtgcttgt ctga 2844

<210> 3

<211> 1991

<212> DNA

<213>Corn（Zea mays L.）

<400> 3

tctcttaatc agtatatata attaaaaaac taatttcaca tttgtgttcc taatattttt 60

tacaaataaa tcattgttta attccatttg taataagttt ttattaaaat tgcttttatt 120

tcatttatta taaacattta attgttttaa tcctatttta gttttaattt attgtatcta 180

tttattaata taacgaactt cgataagaaa caaaagcaag gtcaaggtgt tttttcaaag 240

tagttgtgga aaagctgaac cccttttatt caacttttag aagcaggaaa acagaaccaa 300

acagacccta aaaatgtgtg aatttttagc aggttaatta ttcgcatctc tttggtcatg 360

tttaagaggc tggaatagat caactgcaag aacacatagc agagtggata gggggggggg 420

gggggggggg gggggagggt cgtcgtctcc ctatctgacc tctcttctgc attggattgc 480

ctttttcggt actctattta aaacttaaaa gtacaaatga ggtgccggat tgatggagtg 540

atatataagt ttgatgtgtt tttcacataa gtgacaagta ttattgaaag agaacatttg 600

cattgctact gtttgcatat gggaaaattg agaattgtat catgccatgg ccgatcagtt 660

ctttacttag ctcgatgtaa tgcacaatgt tgatagtatg tcgaggatct agcgatgtaa 720

tggtgttagg acacgtggtt agctactaat ataaatgtaa ggtcattcga tggtttttct 780

attttcaatt acctagcatt atctcatttc taattgtgat aacaaatgca ttagaccata 840

attctgtaaa tatgtacatt taagcacaca gtctatattt taaaattctt ctttttgtgt 900

ggatatccca acccaaatcc acctctctct tcaatccgtg catgttcacc gctgccaagt 960

gccaacaaca catcgcatcg tgcatatctt tgttggcttg tgcacggtcg gcgccaatgg 1020

aggagacacc tgtacggtgc ccttggtaga acaacatcct tatccctata tgtatggtgc 1080

ccttcgtaga atgacacccc ttatccctac aatagccatg tatgcatacc aagaattaaa 1140

tatacttttt cttgaaccac aataatttat tatagcggca cttcttgttc aggttgaaca 1200

cttatttgga acaataaaat gccgagttcc taaccacagg ttcacttttt tttttcctta 1260

tcctcctagg aaactaaatt ttaaaatcat aaatttaatt taaatgttaa tggaaacaaa 1320

aaattatcta caaagacgac tcttagccac agccgcctca ctgcaccctc aaccacatcc 1380

tgcaaacaga caccctcgcc acatccctcc agattcttca ctccgatgca gcctacttgc 1440

taacagacgc cctctccaca tcctgcaaag cattcctcca aattcttgcg atcccccgaa 1500

tccagcatta actgctaagg gacgccctct ccacatcctg ctacccaatt agccaacgga 1560

ataacacaag aaggcaggtg agcagtgaca aagcacgtca acagcaccga gccaagccaa 1620

aaaggagcaa ggaggagcaa gcccaagccg cagccgcagc tctccaggtc cccttgcgat 1680

tgccgccagc agtagcagac acccctctcc acatcccctc cggccgctaa cagcagcaag 1740

ccaagccaaa aaggagcctc agccgcagcc ggttccgttg cggttaccgc cgatcacatg 1800

cccaaggccg cgcctttccg aacgccgagg gccgcccgtt cccgtgcaca gccacacaca 1860

cacccgcccg ccaacgactc cccatcccta tttgaaccca cccgcgcact gcattgatca 1920

ccaatcgcat cgcagcagca cgagcagcac gccgtgccgc tccaaccatc tcgcttccgt 1980

gcttagcttc c 1991

<210> 4

<211> 2913

<212> DNA

<213>Corn（Zea mays L.）

<400> 4

atggcgtcga ccaaggcacc cggccctggc gagaagcacc actccatcga cgcgcagctc 60

cgtcagctgg tcccaggcaa ggtctccgag gacgacaagc tcatcgagta cgatgcgctg 120

ctcgtcgacc gcttcctcaa catcctccag gacctccacg ggcccagcct tcgcgaattt 180

gtccaggagt gctacgaggt gtcggccgac tatgagggca aaggagacac gacgaagctg 240

ggcgagctcg gcgccaagct cacggggctg gcccccgccg acgccatcct cgtggcgagc 300

tccatcctgc acatgctcaa cctcgccaac ctggccgagg aggtgcagat cgcgcaccgc 360

cgccgcaaca gcaagctcaa gaaaggtggg ttcgccgacg agggctccgc caccaccgag 420

tccgacatcg aggagacgct caagcgcctc gtgtccgagg tcggcaagtc ccccgaggag 480

gtgttcgagg cgctcaagaa ccagaccgtc gacctcgtct tcaccgcgca tcccacgcag 540

tccgcccgcc gctcgctcct gcagaaaaac gccaggatcc ggaattgtct gacccagctg 600

aatgccaagg acatcactga cgacgacaag caggagctcg atgaggctct gcagagagag 660

atccaagcag ccttcagaac cgatgaaatc aggagggcac aacccacccc ccaggacgaa 720

atgcgctatg ggatgagcta catccatgag actgtatgga agggcgtgcc taagttcttg 780

cgccgtgtgg atacagccct gaagaatatc ggcatcaatg agcgccttcc ctacaatgtt 840

tctctcattc ggttctcttc ttggatgggt ggtgaccgcg atggaaatcc aagagttacc 900

ccggaggtga caagagatgt atgcttgctg gccagaatga tggctgcaaa cttgtacatc 960

gatcagattg aagagctgat gtttgagctc tctatgtggc gctgcaacga tgagcttcgt 1020

gttcgtgccg aagagctcca cagttcgtct ggttccaaag ttaccaagta ttacatagaa 1080

ttctggaagc aaattcctcc aaacgagccc taccgggtga tactaggcca tgtaagggac 1140

aagctgtaca acacacgcga gcgtgctcgc catctgctgg cttctggagt ttctgaaatt 1200

tcagcggaat cgtcatttac cagtatcgaa gagttccttg agccacttga gctgtgctac 1260

aaatcaccgt gtgactgcgg cgacaaggcc atcgcggacg ggagcctcct ggacctcctg 1320

cgccaggttt tcacgttcgg gctctccctg gtgaagctgg acatccggca ggagtcggag 1380

cggcacaccg acgtgatcga cgccatcacc acgcacctcg gcatcgggtc gtaccgcgag 1440

tggtccgagg acaagcggca ggagtggctg ctgtcggagc tgcgaggcaa gcgcccgctg 1500

ctgcccccgg accttcccca gaccgaggag atcgccgacg tcatcggcgc gttccacgtc 1560

ctcgcggagc tcccgcccga cagcttcggc ccctacatca tctccatggc gacggccccc 1620

tcggacgtgc tcgccgtgga gctcctgcag cgcgagtgcg gcgtgcgcca gccgctgccc 1680

gtggtgccgc tgttcgaaag gctggccgac ctgcagtcgg cgcccgcgtc cgtggagcgc 1740

ctcttctcgg tggactggta catggaccgg atcaagggca agcagcaggt catggtcggc 1800

tactccgact ccggcaagga cgccggccgc ctgtccgcgg cgtggcagct gtacagggcg 1860

caggaggaga tggcgcaggt ggccaagcgc tacggcgtca agctcacctt gttccacggc 1920

cgcggaggca ccgtgggcag gggtggcggg cccacgcacc ttgccatcct gtcccagccg 1980

ccggacacca tcaacgggtc catccgtgtg acggtgcagg gcgaggtcat cgagttctgc 2040

ttcggggagg aacacctgtg cttccagact ctgcagcgct tcacggccgc cacgctggag 2100

cacggcatgc acccgccggt ctctcccaag tccgagtggc gcaagctcat ggacgagatg 2160

gcggtcgtgg ccacggagga gtaccgctcc gtcgtcgtca aggaggcgcg cttcgtcgag 2220

tacttcagat cggctacacc ggagaccgag tacgggagga tgaacatcgg cagccggcca 2280

gccaagagga ggcccggcgg cggcatcacg accctgcgcg ccatcccctg gatcttctcg 2340

tggacccaga ccaggttcca cccccccgtg tggctgggag tcggcgccgc attcaagttc 2400

gccatcgaca aggacgtcag gaacttccag gtcctcaaag agatgtacaa cgagtggcca 2460

ttcttcaggg tcaccctgga cctgctggag atggttttcg ccaagggaga ccccggcatt 2520

gccggcttgt atgacgagct gcttgtggcg gaagaactca agccctttgg gaagcagctc 2580

agggacaaat acgtggagac acagcagctt ctcctccaga tcgctgggca caaggatatt 2640

cttgaaggcg atccattcct gaagcagggg ctggtgctgc gcaaccccta catcaccacc 2700

ctgaacgtgt tccaggccta cacgctgaag cggataaggg accccaactt caaggtgacg 2760

ccccagccgc cgctgtccaa ggagttcgcc gacgagaaca agcccgccgg actggtcaag 2820

ctgaacccgg cgagcgagta cccgcccggc ctggaagaca cgctcatcct caccatgaag 2880

ggcatcgccg ccggcatgca gaacactggc tag 2913

<210> 5

<211> 538

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 5

catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga ctggcgaaca 60

gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca acatggtgga 120

gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag accaaagggc 180

aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc attgcccagc 240

tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca aatgccatca 300

ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc ccaaagatgg 360

acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt cttcaaagca 420

agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc actatccttc 480

gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac gggggact 538

<210> 6

<211> 1299

<212> DNA

<213>Corn（Zea mays L.）

<400> 6

atgggccttt caacagttta ctccccggcc ggaccgcgcc tcgtgccggc ccctctcggc 60

cgctgccgct ctgcccagcc ccgccgcccg cgccgagccc cgctcgccac cgtccggtgc 120

tccgtcgacg ccaccaagca ggcgcaggat ggcgtggcga cggcggtcgc gacggaggcg 180

ccggcgtcgc ggaaggagtg cttcggggtc ttctgcacca cctacgacct caaggcggag 240

gacaagacca agtcgtggag gaagctagtg aatgttgctg tgtcaggcgc ggccgggatg 300

atatcaaatc acctgctgtt caaactcgct tctggtgagg ttttcggaca agaccaacca 360

atagcactta agctactcgg ctcagaaaga tcgtttcaag ccctcgaagg tgtagctatg 420

gaactggagg actcgctgta tccactgctg agggaagtca gcattggcat agatccttac 480

gtggtctttc aagatgtaga ttgggccctt cttattggcg ctaagccccg aggtcccggc 540

atggagcgag ctgcgctact ggatatcaat ggccaaatct ttgctgacca ggggaaagca 600

cttaatgcgg tcgcctcgcg gaacgacgaa gtcttagttg tcggaaatcc ctgtaacact 660

aatgcgctga tttgtttgaa aaatgcccca aacataccgg caaaaaactt tcatgcattg 720

acgaggttgg atgaaaatag agcaaagtgc cagctggcac taaaagcagg tgtattttat 780

gacaaagtat caaacgtgac tatttggggg aaccattcga cgactcaggt tcctgatttc 840

ttgaatgcca aaattgatgg aagaccagtg aaagaagtca ttaaggatac caagtggtta 900

gaggaagagt tcaccctcac agttcaaaag cgtggaggtg tgctcatcca aaaatggggc 960

agatcatcag ctgcatcgac cgctgtttcg atagtggatg ctattagatc cctcgtaact 1020

cctaccccag aaggtgactg gttttctaca ggggtttata cgactggaaa tccttatggc 1080

atagcagagg atatcgtgtt cagcatgcca tgcagatcga agggtgatgg tgattacgaa 1140

ctagctagtg atgtgttgat ggacgatttt ctctgggaac ggattaaaaa gagcgaagct 1200

gaattgcttg ctgagaagaa atgcgttgcc catcttacag gagaggggaa tgcattctgt 1260

gatcttccgg aagataccat gctaccagga gaagtgtag 1299

<210> 7

<211> 1993

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 7

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagaaatctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttacttctg cag 1993

<210> 8

<211> 1989

<212> DNA

<213>Corn（Zea mays L.）

<400> 8

atgatctccg cgcgcaccgc cgcagcttcc cccgcttccc tgtggaagca gggaggacga 60

agcgagggcg gcggcagctg cgacggatgt cggacttaca ggaacacttt gcggagggcc 120

gcagcagcga aggtgcgtgc cttgccgtcg agggcggtgg aggcggtcaa aatgggctcc 180

gccgcggaga tggagcagga ggaggcggag gtggccgcgg ccgcggccgg cgtcgtcgtc 240

gaggaccact acggcgagga cagcgccacc gaggagctgc ccatcctgcc ctgggccttc 300

tccgtcgcaa gatatgtttc cttcagttgg ttgaaaaaat acgctgagga gtcttatttc 360

tacatcagcg gttacaccct cttgagggat ccacatcaca acaagggtct tgctttcatg 420

gagaaggaga gggatgcaca ccacttgcgg gggctgcttc ctcctgcagt tgtgcctcaa 480

gaactccaaa ttaagaagat catgcacaac ctgcggcagt accaggtccc tttgcagcgc 540

tatatggcca tgatggacct tcaggagagg aacgagaggc ttttctacaa gcttttaatt 600

gataacgtgg aggagctgct tcctgtggtt tatacaccaa ctgtaggtga ggcctgccag 660

aagtatgggt ccatctttcg acaaccacag ggtctgtatg tcagcttgag ggacaagggg 720

aaggtcctag aagttctaag gaactggcca cataggaaca ttcaagttat ctgtgttact 780

gatggtgagg caatcttggg acttggagat ttgggttctc agggaatggg aattcctgta 840

ggcaaacttg ctctatacac tgctcttgga ggagttcgtc catcagcttg tttgcctatc 900

acaatcgatg ttggcacaaa taatgagaaa ctgcttaatg atgagtttta cattggactc 960

cggcaaaaac gtgcaactgg cgaggagtat catgagctta tggaagagtt catggctgct 1020

gttaagcaaa tctacggcga gaaagtcctc attcagtttg aggacttcgc gaatcataat 1080

gcttttaatt tgcttgcaaa atatagcaag agccatcttg ttttcaatga tgacatccag 1140

ggcacagcat cagtggtcct tgcaggtttg ttagcagcac tcaaggtggt tggtaggacc 1200

ttggcagagc acacttattt attccttggt gctggggagg ctggaactgg tattgcagaa 1260

ctcattgctc ttgagatttc aaaacagacg tcggctccaa ttgaagagtg ccgcaagaag 1320

atttggttgg tggactcaaa gggtttgatt gttgactctc gtaaaaactc cgttcagtca 1380

ttcaaaaaac cttgggcaca tgagcatgag cccttgacaa ccttgtacga tgctgttcag 1440

tccatcaaac ctacagttct gattgggacg tctggagttg gaagaacatt cacaaaagaa 1500

gttgttgagg ccatggcttc cttcaatgag aggcctatca tcttttcact gtcaaatcca 1560

acctctcatt ctgaatgtac tgctgaagaa gcatataact ggactcaggt cgtaatattt 1620

gccagtggca gtccatttga ccctgtggag tacgatggga aggtttttgt acctggacag 1680

tcgaacaacg cctacatttt ccctggattt ggcctcggtc ttgttatctc tggagccatc 1740

cgtgtccacg aggacatgct tcttgccgcc tcggaagcac tagctgctca ggccacacag 1800

gagaacttcg acaagggatc aatcttccca cccttcacca acattagaaa gatctctgcg 1860

cgcattgcca gagccgtggc tgcaaaagct tatgaactcg gtttggcgac ccgtctgcct 1920

cccccaaggg acctggtgaa atatgcacag agctgcatgt acactcctgt ctaccgtaac 1980

taccggtag 1989

<210> 9

<211> 1413

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 9

tagctagcat actcgaggtc attcatatgc ttgagaagag agtcgggata gtccaaaata 60

aaacaaaggt aagattacct ggtcaaaagt gaaaacatca gttaaaaggt ggtataaagt 120

aaaatatcgg taataaaagg tggcccaaag tgaaatttac tcttttctac tattataaaa 180

attgaggatg tttttgtcgg tactttgata cgtcattttt gtatgaattg gtttttaagt 240

ttattcgctt ttggaaatgc atatctgtat ttgagtcggg ttttaagttc gtttgctttt 300

gtaaatacag agggatttgt ataagaaata tctttaaaaa aacccatatg ctaatttgac 360

ataatttttg agaaaaatat atattcaggc gaattctcac aatgaacaat aataagatta 420

aaatagcttt cccccgttgc agcgcatggg tattttttct agtaaaaata aaagataaac 480

ttagactcaa aacatttaca aaaacaaccc ctaaagttcc taaagcccaa agtgctatcc 540

acgatccata gcaagcccag cccaacccaa cccaacccaa cccaccccag tccagccaac 600

tggacaatag tctccacacc cccccactat caccgtgagt tgtccgcacg caccgcacgt 660

ctcgcagcca aaaaaaaaaa aagaaagaaa aaaaagaaaa agaaaaaaca gcaggtgggt 720

ccgggtcgtg ggggccggaa acgcgaggag gatcgcgagc cagcgacgag gccggccctc 780

cctccgcttc caaagaaacg ccccccatcg ccactatata catacccccc cctctcctcc 840

catcccccca accctaccac caccaccacc accacctcca cctcctcccc cctcgctgcc 900

ggacgacgag ctcctccccc ctccccctcc gccgccgccg cgccggtaac caccccgccc 960

ctctcctctt tctttctccg tttttttttc cgtctcggtc tcgatctttg gccttggtag 1020

tttgggtggg cgagaggcgg cttcgtgcgc gcccagatcg gtgcgcggga ggggcgggat 1080

ctcgcggctg gggctctcgc cggcgtggat ccggcccgga tctcgcgggg aatggggctc 1140

tcggatgtag atctgcgatc cgccgttgtt gggggagatg atggggggtt taaaatttcc 1200

gccatgctaa acaagatcag gaagagggga aaagggcact atggtttata tttttatata 1260

tttctgctgc ttcgtcaggc ttagatgtgc tagatctttc tttcttcttt ttgtgggtag 1320

aatttgaatc cctcagcatt gttcatcggt agtttttctt ttcatgattt gtgacaaatg 1380

cagcctcgtg cggagctttt ttgtaggtag aag 1413

<210> 10

<211> 1371

<212> DNA

<213>Corn（Zea mays L.）

<400> 10

atgtacacat tgcccgtccg cgccaccaca tccagcatcg tcgccagcct cgccaccccc 60

gcgccgtcct cctcctccgg ctccggctcc ggccgcccca ggcccaggct catccggaac 120

gcccccgtct tcgccgcccc cgccaccgtc gtgggcatgg accccaccgt cgagcgcttg 180

aagagcgggt tccagaagtt caagaccgag gtctatgaca agaagccgga gctgttcgag 240

cctctcaagt ccggccagag ccccaggtac atggtgttcg cctgctccga ctcccgcgtg 300

tgcccgtcgg tgacactggg cctgcagccc ggcgaggcat tcaccgtccg caacatcgcc 360

tccatggtcc caccctacga caagatcaag tacgccggca ccgggtccgc catcgagtac 420

gccgtgtgcg cgctcaaggt gcaggtcatc gtggtcattg gccacagctg ctgcggtggc 480

atcagggcgc tcctctccct caaggacggc gcgcccgaca acttccactt cgtggaggac 540

tgggtcagga tcggcagccc tgccaagaac aaggtgaaga aagagcacgc atcggtgccg 600

ttcgatgacc agtgctccat cctggagaag gaggccgtga acgtgtcgct ccagaacctc 660

aagagctacc ccttcgtcaa ggaagggctg gccggcggga cgctcaagct ggttggcgcc 720

cactacgact tcgtcaaagg gcagttcgtc acatgggagc ctccccagga cgccatcgag 780

cgcttgacga gcgggttcca gcagttcaag gtcaatgtct atgacaagaa gccggagctt 840

ttcgggcctc tcaagtccgg ccaggccccc aagtacatgg tgttcgcttg ctccgactcc 900

cgtgtgtgcc catcggtgac cctgggcctg cagcccggcg aggccttcac cgttcgcaac 960

atagccgcca tggtcccagg ctacgacaag accaagtaca ccggcatcgg gtccgccatc 1020

gagtacgctg tgtgcgctct caaggtggag gtcctcgtgg tcattggcca tagctgctgc 1080

ggtggcatca gggcgctcct ctccctccag gacggcgcac ctgacacctt ccacttcgtc 1140

gaggactggg ttaagatcgg cttcattgcc aagatgaagg taaagaaaga gcacgcctcg 1200

gtgccgttcg atgaccagtg ctccattctc gagaaggagg ccgtgaacgt gtccctggag 1260

aacctcaaga cctacccctt cgtcaaggaa gggcttgcaa atgggaccct caagctgatc 1320

ggcgcccact acgactttgt ctcaggagag ttcctcacat ggaaaaagtg a 1371

<210> 11

<211> 947

<212> PRT

<213>Corn（Zea mays L.）

<400> 11

Met Ala Ala Ser Val Ser Arg Ala Ile Cys Val Gln Lys Pro Gly Ser

1 5 10 15

Lys Cys Thr Arg Asp Arg Glu Ala Thr Ser Phe Ala Arg Arg Ser Val

20 25 30

Ala Ala Pro Arg Pro Pro His Ala Lys Ala Arg Arg Arg His Pro Leu

35 40 45

Arg Leu Arg Arg Gly Thr Gly Pro His Cys Ser Pro Leu Arg Ala Val

50 55 60

Val Asp Ala Ala Pro Ile Gln Thr Thr Lys Lys Arg Val Phe His Phe

65 70 75 80

Gly Lys Gly Lys Ser Glu Gly Asn Lys Thr Met Lys Glu Leu Leu Gly

85 90 95

Gly Lys Gly Ala Asn Leu Ala Glu Met Ala Ser Ile Gly Leu Ser Val

100 105 110

Pro Pro Gly Phe Thr Val Ser Thr Glu Ala Cys Gln Gln Tyr Gln Asp

115 120 125

Ala Gly Cys Ala Leu Pro Ala Gly Leu Trp Ala Glu Ile Val Asp Gly

130 135 140

Leu Gln Trp Val Glu Glu Tyr Met Gly Ala Thr Leu Gly Asp Pro Gln

145 150 155 160

Arg Pro Leu Leu Leu Ser Val Arg Ser Gly Ala Ala Val Ser Met Pro

165 170 175

Gly Met Met Asp Thr Val Leu Asn Leu Gly Leu Asn Asp Glu Val Ala

180 185 190

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

195 200 205

Arg Arg Phe Leu Asp Met Phe Gly Asn Val Val Met Asp Ile Pro Arg

210 215 220

Ser Leu Phe Glu Glu Lys Leu Glu His Met Lys Glu Ser Lys Gly Leu

225 230 235 240

Lys Asn Asp Thr Asp Leu Thr Ala Ser Asp Leu Lys Glu Leu Val Gly

245 250 255

Gln Tyr Lys Glu Val Tyr Leu Ser Ala Lys Gly Glu Pro Phe Pro Ser

260 265 270

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

275 280 285

Trp Glu Ser Pro Arg Ala Lys Lys Tyr Arg Ser Ile Asn Gln Ile Thr

290 295 300

Gly Leu Arg Gly Thr Ala Val Asn Val Gln Cys Met Val Phe Gly Asn

305 310 315 320

Met Gly Asn Thr Ser Gly Thr Gly Val Leu Phe Thr Arg Asn Pro Asn

325 330 335

Thr Gly Glu Lys Lys Leu Tyr Gly Glu Phe Leu Val Asn Ala Gln Gly

340 345 350

Glu Asp Val Val Ala Gly Ile Arg Thr Pro Glu Asp Leu Asp Ala Met

355 360 365

Lys Asn Leu Met Pro Gln Ala Tyr Asp Glu Leu Val Glu Asn Cys Asn

370 375 380

Ile Leu Glu Ser His Tyr Lys Glu Met Gln Asp Ile Glu Phe Thr Val

385 390 395 400

Gln Glu Asn Arg Leu Trp Met Leu Gln Cys Arg Thr Gly Lys Arg Thr

405 410 415

Gly Lys Ser Ala Val Lys Ile Ala Val Asp Met Val Asn Glu Gly Leu

420 425 430

Val Glu Pro Arg Ser Ala Ile Lys Met Val Glu Pro Gly His Leu Asp

435 440 445

Gln Leu Leu His Pro Gln Phe Glu Asn Pro Ser Ala Tyr Lys Asp Gln

450 455 460

Val Ile Ala Thr Gly Leu Pro Ala Ser Pro Gly Ala Ala Val Gly Gln

465 470 475 480

Val Val Phe Thr Ala Glu Asp Ala Glu Ala Trp His Ser Gln Gly Lys

485 490 495

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

500 505 510

Met His Ala Ala Val Gly Ile Leu Thr Glu Arg Gly Gly Met Thr Ser

515 520 525

His Ala Ala Val Val Ala Arg Trp Trp Gly Lys Cys Cys Val Ser Gly

530 535 540

Cys Ser Gly Ile Arg Val Asn Asp Ala Glu Lys Leu Val Thr Ile Gly

545 550 555 560

Ser His Val Leu Arg Glu Gly Glu Trp Leu Ser Leu Asn Gly Ser Thr

565 570 575

Gly Glu Val Ile Leu Gly Lys Gln Pro Leu Ser Pro Pro Ala Leu Ser

580 585 590

Gly Asp Leu Gly Thr Phe Met Ala Trp Val Asp Asp Val Arg Lys Leu

595 600 605

Lys Val Leu Ala Asn Ala Asp Thr Pro Asp Asp Ala Leu Thr Ala Arg

610 615 620

Asn Asn Gly Ala Gln Gly Ile Gly Leu Cys Arg Thr Glu His Met Phe

625 630 635 640

Phe Ala Ser Asp Glu Arg Ile Lys Ala Val Arg Gln Met Ile Met Ala

645 650 655

Pro Thr Leu Glu Leu Arg Gln Gln Ala Leu Asp Arg Leu Leu Thr Tyr

660 665 670

Gln Arg Ser Asp Phe Glu Gly Ile Phe Arg Ala Met Asp Gly Leu Pro

675 680 685

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

690 695 700

Gly Asn Ile Glu Asp Ile Val Ser Glu Leu Cys Ala Glu Thr Gly Ala

705 710 715 720

Asn Gln Glu Asp Ala Leu Ala Arg Ile Glu Lys Leu Ser Glu Val Asn

725 730 735

Pro Met Leu Gly Phe Arg Gly Cys Arg Leu Gly Ile Ser Tyr Pro Glu

740 745 750

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

755 760 765

Thr Asn Gln Gly Val Gln Val Phe Pro Glu Ile Met Val Pro Leu Val

770 775 780

Gly Thr Pro Gln Glu Leu Gly His Gln Val Thr Leu Ile Arg Gln Val

785 790 795 800

Ala Glu Lys Val Phe Ala Asn Val Gly Lys Thr Ile Gly Tyr Lys Val

805 810 815

Gly Thr Met Ile Glu Ile Pro Arg Ala Ala Leu Val Ala Asp Glu Ile

820 825 830

Ala Glu Gln Ala Glu Phe Phe Ser Phe Gly Thr Asn Asp Leu Thr Gln

835 840 845

Met Thr Phe Gly Tyr Ser Arg Asp Asp Val Gly Lys Phe Ile Pro Val

850 855 860

His Leu Ala Gln Gly Ile Leu Gln His Asp Pro Phe Glu Val Leu Asp

865 870 875 880

Gln Arg Gly Val Gly Glu Leu Val Lys Phe Ala Thr Glu Arg Gly Arg

885 890 895

Lys Ala Arg Pro Asn Leu Lys Val Gly Ile Cys Gly Glu His Gly Gly

900 905 910

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

915 920 925

Ser Cys Ser Pro Phe Arg Val Pro Ile Ala Arg Leu Ala Ala Ala Gln

930 935 940

Val Leu Val

945

<210> 12

<211> 970

<212> PRT

<213>Corn（Zea mays L.）

<400> 12

Met Ala Ser Thr Lys Ala Pro Gly Pro Gly Glu Lys His His Ser Ile

1 5 10 15

Asp Ala Gln Leu Arg Gln Leu Val Pro Gly Lys Val Ser Glu Asp Asp

20 25 30

Lys Leu Ile Glu Tyr Asp Ala Leu Leu Val Asp Arg Phe Leu Asn Ile

35 40 45

Leu Gln Asp Leu His Gly Pro Ser Leu Arg Glu Phe Val Gln Glu Cys

50 55 60

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

65 70 75 80

Gly Glu Leu Gly Ala Lys Leu Thr Gly Leu Ala Pro Ala Asp Ala Ile

85 90 95

Leu Val Ala Ser Ser Ile Leu His Met Leu Asn Leu Ala Asn Leu Ala

100 105 110

Glu Glu Val Gln Ile Ala His Arg Arg Arg Asn Ser Lys Leu Lys Lys

115 120 125

Gly Gly Phe Ala Asp Glu Gly Ser Ala Thr Thr Glu Ser Asp Ile Glu

130 135 140

Glu Thr Leu Lys Arg Leu Val Ser Glu Val Gly Lys Ser Pro Glu Glu

145 150 155 160

Val Phe Glu Ala Leu Lys Asn Gln Thr Val Asp Leu Val Phe Thr Ala

165 170 175

His Pro Thr Gln Ser Ala Arg Arg Ser Leu Leu Gln Lys Asn Ala Arg

180 185 190

Ile Arg Asn Cys Leu Thr Gln Leu Asn Ala Lys Asp Ile Thr Asp Asp

195 200 205

Asp Lys Gln Glu Leu Asp Glu Ala Leu Gln Arg Glu Ile Gln Ala Ala

210 215 220

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

225 230 235 240

Met Arg Tyr Gly Met Ser Tyr Ile His Glu Thr Val Trp Lys Gly Val

245 250 255

Pro Lys Phe Leu Arg Arg Val Asp Thr Ala Leu Lys Asn Ile Gly Ile

260 265 270

Asn Glu Arg Leu Pro Tyr Asn Val Ser Leu Ile Arg Phe Ser Ser Trp

275 280 285

Met Gly Gly Asp Arg Asp Gly Asn Pro Arg Val Thr Pro Glu Val Thr

290 295 300

Arg Asp Val Cys Leu Leu Ala Arg Met Met Ala Ala Asn Leu Tyr Ile

305 310 315 320

Asp Gln Ile Glu Glu Leu Met Phe Glu Leu Ser Met Trp Arg Cys Asn

325 330 335

Asp Glu Leu Arg Val Arg Ala Glu Glu Leu His Ser Ser Ser Gly Ser

340 345 350

Lys Val Thr Lys Tyr Tyr Ile Glu Phe Trp Lys Gln Ile Pro Pro Asn

355 360 365

Glu Pro Tyr Arg Val Ile Leu Gly His Val Arg Asp Lys Leu Tyr Asn

370 375 380

Thr Arg Glu Arg Ala Arg His Leu Leu Ala Ser Gly Val Ser Glu Ile

385 390 395 400

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

405 410 415

Glu Leu Cys Tyr Lys Ser Pro Cys Asp Cys Gly Asp Lys Ala Ile Ala

420 425 430

Asp Gly Ser Leu Leu Asp Leu Leu Arg Gln Val Phe Thr Phe Gly Leu

435 440 445

Ser Leu Val Lys Leu Asp Ile Arg Gln Glu Ser Glu Arg His Thr Asp

450 455 460

Val Ile Asp Ala Ile Thr Thr His Leu Gly Ile Gly Ser Tyr Arg Glu

465 470 475 480

Trp Ser Glu Asp Lys Arg Gln Glu Trp Leu Leu Ser Glu Leu Arg Gly

485 490 495

Lys Arg Pro Leu Leu Pro Pro Asp Leu Pro Gln Thr Glu Glu Ile Ala

500 505 510

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

515 520 525

Phe Gly Pro Tyr Ile Ile Ser Met Ala Thr Ala Pro Ser Asp Val Leu

530 535 540

Ala Val Glu Leu Leu Gln Arg Glu Cys Gly Val Arg Gln Pro Leu Pro

545 550 555 560

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

565 570 575

Ser Val Glu Arg Leu Phe Ser Val Asp Trp Tyr Met Asp Arg Ile Lys

580 585 590

Gly Lys Gln Gln Val Met Val Gly Tyr Ser Asp Ser Gly Lys Asp Ala

595 600 605

Gly Arg Leu Ser Ala Ala Trp Gln Leu Tyr Arg Ala Gln Glu Glu Met

610 615 620

Ala Gln Val Ala Lys Arg Tyr Gly Val Lys Leu Thr Leu Phe His Gly

625 630 635 640

Arg Gly Gly Thr Val Gly Arg Gly Gly Gly Pro Thr His Leu Ala Ile

645 650 655

Leu Ser Gln Pro Pro Asp Thr Ile Asn Gly Ser Ile Arg Val Thr Val

660 665 670

Gln Gly Glu Val Ile Glu Phe Cys Phe Gly Glu Glu His Leu Cys Phe

675 680 685

Gln Thr Leu Gln Arg Phe Thr Ala Ala Thr Leu Glu His Gly Met His

690 695 700

Pro Pro Val Ser Pro Lys Ser Glu Trp Arg Lys Leu Met Asp Glu Met

705 710 715 720

Ala Val Val Ala Thr Glu Glu Tyr Arg Ser Val Val Val Lys Glu Ala

725 730 735

Arg Phe Val Glu Tyr Phe Arg Ser Ala Thr Pro Glu Thr Glu Tyr Gly

740 745 750

Arg Met Asn Ile Gly Ser Arg Pro Ala Lys Arg Arg Pro Gly Gly Gly

755 760 765

Ile Thr Thr Leu Arg Ala Ile Pro Trp Ile Phe Ser Trp Thr Gln Thr

770 775 780

Arg Phe His Pro Pro Val Trp Leu Gly Val Gly Ala Ala Phe Lys Phe

785 790 795 800

Ala Ile Asp Lys Asp Val Arg Asn Phe Gln Val Leu Lys Glu Met Tyr

805 810 815

Asn Glu Trp Pro Phe Phe Arg Val Thr Leu Asp Leu Leu Glu Met Val

820 825 830

Phe Ala Lys Gly Asp Pro Gly Ile Ala Gly Leu Tyr Asp Glu Leu Leu

835 840 845

Val Ala Glu Glu Leu Lys Pro Phe Gly Lys Gln Leu Arg Asp Lys Tyr

850 855 860

Val Glu Thr Gln Gln Leu Leu Leu Gln Ile Ala Gly His Lys Asp Ile

865 870 875 880

Leu Glu Gly Asp Pro Phe Leu Lys Gln Gly Leu Val Leu Arg Asn Pro

885 890 895

Tyr Ile Thr Thr Leu Asn Val Phe Gln Ala Tyr Thr Leu Lys Arg Ile

900 905 910

Arg Asp Pro Asn Phe Lys Val Thr Pro Gln Pro Pro Leu Ser Lys Glu

915 920 925

Phe Ala Asp Glu Asn Lys Pro Ala Gly Leu Val Lys Leu Asn Pro Ala

930 935 940

Ser Glu Tyr Pro Pro Gly Leu Glu Asp Thr Leu Ile Leu Thr Met Lys

945 950 955 960

Gly Ile Ala Ala Gly Met Gln Asn Thr Gly

965 970

<210> 13

<211> 432

<212> PRT

<213>Corn（Zea mays L.）

<400> 13

Met Gly Leu Ser Thr Val Tyr Ser Pro Ala Gly Pro Arg Leu Val Pro

1 5 10 15

Ala Pro Leu Gly Arg Cys Arg Ser Ala Gln Pro Arg Arg Pro Arg Arg

20 25 30

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

35 40 45

Gln Asp Gly Val Ala Thr Ala Val Ala Thr Glu Ala Pro Ala Ser Arg

50 55 60

Lys Glu Cys Phe Gly Val Phe Cys Thr Thr Tyr Asp Leu Lys Ala Glu

65 70 75 80

Asp Lys Thr Lys Ser Trp Arg Lys Leu Val Asn Val Ala Val Ser Gly

85 90 95

Ala Ala Gly Met Ile Ser Asn His Leu Leu Phe Lys Leu Ala Ser Gly

100 105 110

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

115 120 125

Glu Arg Ser Phe Gln Ala Leu Glu Gly Val Ala Met Glu Leu Glu Asp

130 135 140

Ser Leu Tyr Pro Leu Leu Arg Glu Val Ser Ile Gly Ile Asp Pro Tyr

145 150 155 160

Val Val Phe Gln Asp Val Asp Trp Ala Leu Leu Ile Gly Ala Lys Pro

165 170 175

Arg Gly Pro Gly Met Glu Arg Ala Ala Leu Leu Asp Ile Asn Gly Gln

180 185 190

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

195 200 205

Asp Glu Val Leu Val Val Gly Asn Pro Cys Asn Thr Asn Ala Leu Ile

210 215 220

Cys Leu Lys Asn Ala Pro Asn Ile Pro Ala Lys Asn Phe His Ala Leu

225 230 235 240

Thr Arg Leu Asp Glu Asn Arg Ala Lys Cys Gln Leu Ala Leu Lys Ala

245 250 255

Gly Val Phe Tyr Asp Lys Val Ser Asn Val Thr Ile Trp Gly Asn His

260 265 270

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

275 280 285

Pro Val Lys Glu Val Ile Lys Asp Thr Lys Trp Leu Glu Glu Glu Phe

290 295 300

Thr Leu Thr Val Gln Lys Arg Gly Gly Val Leu Ile Gln Lys Trp Gly

305 310 315 320

Arg Ser Ser Ala Ala Ser Thr Ala Val Ser Ile Val Asp Ala Ile Arg

325 330 335

Ser Leu Val Thr Pro Thr Pro Glu Gly Asp Trp Phe Ser Thr Gly Val

340 345 350

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

355 360 365

Met Pro Cys Arg Ser Lys Gly Asp Gly Asp Tyr Glu Leu Ala Ser Asp

370 375 380

Val Leu Met Asp Asp Phe Leu Trp Glu Arg Ile Lys Lys Ser Glu Ala

385 390 395 400

Glu Leu Leu Ala Glu Lys Lys Cys Val Ala His Leu Thr Gly Glu Gly

405 410 415

Asn Ala Phe Cys Asp Leu Pro Glu Asp Thr Met Leu Pro Gly Glu Val

420 425 430

<210> 14

<211> 662

<212> PRT

<213>Corn（Zea mays L.）

<400> 14

Met Ile Ser Ala Arg Thr Ala Ala Ala Ser Pro Ala Ser Leu Trp Lys

1 5 10 15

Gln Gly Gly Arg Ser Glu Gly Gly Gly Ser Cys Asp Gly Cys Arg Thr

20 25 30

Tyr Arg Asn Thr Leu Arg Arg Ala Ala Ala Ala Lys Val Arg Ala Leu

35 40 45

Pro Ser Arg Ala Val Glu Ala Val Lys Met Gly Ser Ala Ala Glu Met

50 55 60

Glu Gln Glu Glu Ala Glu Val Ala Ala Ala Ala Ala Gly Val Val Val

65 70 75 80

Glu Asp His Tyr Gly Glu Asp Ser Ala Thr Glu Glu Leu Pro Ile Leu

85 90 95

Pro Trp Ala Phe Ser Val Ala Arg Tyr Val Ser Phe Ser Trp Leu Lys

100 105 110

Lys Tyr Ala Glu Glu Ser Tyr Phe Tyr Ile Ser Gly Tyr Thr Leu Leu

115 120 125

Arg Asp Pro His His Asn Lys Gly Leu Ala Phe Met Glu Lys Glu Arg

130 135 140

Asp Ala His His Leu Arg Gly Leu Leu Pro Pro Ala Val Val Pro Gln

145 150 155 160

Glu Leu Gln Ile Lys Lys Ile Met His Asn Leu Arg Gln Tyr Gln Val

165 170 175

Pro Leu Gln Arg Tyr Met Ala Met Met Asp Leu Gln Glu Arg Asn Glu

180 185 190

Arg Leu Phe Tyr Lys Leu Leu Ile Asp Asn Val Glu Glu Leu Leu Pro

195 200 205

Val Val Tyr Thr Pro Thr Val Gly Glu Ala Cys Gln Lys Tyr Gly Ser

210 215 220

Ile Phe Arg Gln Pro Gln Gly Leu Tyr Val Ser Leu Arg Asp Lys Gly

225 230 235 240

Lys Val Leu Glu Val Leu Arg Asn Trp Pro His Arg Asn Ile Gln Val

245 250 255

Ile Cys Val Thr Asp Gly Glu Ala Ile Leu Gly Leu Gly Asp Leu Gly

260 265 270

Ser Gln Gly Met Gly Ile Pro Val Gly Lys Leu Ala Leu Tyr Thr Ala

275 280 285

Leu Gly Gly Val Arg Pro Ser Ala Cys Leu Pro Ile Thr Ile Asp Val

290 295 300

Gly Thr Asn Asn Glu Lys Leu Leu Asn Asp Glu Phe Tyr Ile Gly Leu

305 310 315 320

Arg Gln Lys Arg Ala Thr Gly Glu Glu Tyr His Glu Leu Met Glu Glu

325 330 335

Phe Met Ala Ala Val Lys Gln Ile Tyr Gly Glu Lys Val Leu Ile Gln

340 345 350

Phe Glu Asp Phe Ala Asn His Asn Ala Phe Asn Leu Leu Ala Lys Tyr

355 360 365

Ser Lys Ser His Leu Val Phe Asn Asp Asp Ile Gln Gly Thr Ala Ser

370 375 380

Val Val Leu Ala Gly Leu Leu Ala Ala Leu Lys Val Val Gly Arg Thr

385 390 395 400

Leu Ala Glu His Thr Tyr Leu Phe Leu Gly Ala Gly Glu Ala Gly Thr

405 410 415

Gly Ile Ala Glu Leu Ile Ala Leu Glu Ile Ser Lys Gln Thr Ser Ala

420 425 430

Pro Ile Glu Glu Cys Arg Lys Lys Ile Trp Leu Val Asp Ser Lys Gly

435 440 445

Leu Ile Val Asp Ser Arg Lys Asn Ser Val Gln Ser Phe Lys Lys Pro

450 455 460

Trp Ala His Glu His Glu Pro Leu Thr Thr Leu Tyr Asp Ala Val Gln

465 470 475 480

Ser Ile Lys Pro Thr Val Leu Ile Gly Thr Ser Gly Val Gly Arg Thr

485 490 495

Phe Thr Lys Glu Val Val Glu Ala Met Ala Ser Phe Asn Glu Arg Pro

500 505 510

Ile Ile Phe Ser Leu Ser Asn Pro Thr Ser His Ser Glu Cys Thr Ala

515 520 525

Glu Glu Ala Tyr Asn Trp Thr Gln Val Val Ile Phe Ala Ser Gly Ser

530 535 540

Pro Phe Asp Pro Val Glu Tyr Asp Gly Lys Val Phe Val Pro Gly Gln

545 550 555 560

Ser Asn Asn Ala Tyr Ile Phe Pro Gly Phe Gly Leu Gly Leu Val Ile

565 570 575

Ser Gly Ala Ile Arg Val His Glu Asp Met Leu Leu Ala Ala Ser Glu

580 585 590

Ala Leu Ala Ala Gln Ala Thr Gln Glu Asn Phe Asp Lys Gly Ser Ile

595 600 605

Phe Pro Pro Phe Thr Asn Ile Arg Lys Ile Ser Ala Arg Ile Ala Arg

610 615 620

Ala Val Ala Ala Lys Ala Tyr Glu Leu Gly Leu Ala Thr Arg Leu Pro

625 630 635 640

Pro Pro Arg Asp Leu Val Lys Tyr Ala Gln Ser Cys Met Tyr Thr Pro

645 650 655

Val Tyr Arg Asn Tyr Arg

660

<210> 15

<211> 456

<212> PRT

<213>Corn（Zea mays L.）

<400> 15

Met Tyr Thr Leu Pro Val Arg Ala Thr Thr Ser Ser Ile Val Ala Ser

1 5 10 15

Leu Ala Thr Pro Ala Pro Ser Ser Ser Ser Gly Ser Gly Ser Gly Arg

20 25 30

Pro Arg Pro Arg Leu Ile Arg Asn Ala Pro Val Phe Ala Ala Pro Ala

35 40 45

Thr Val Val Gly Met Asp Pro Thr Val Glu Arg Leu Lys Ser Gly Phe

50 55 60

Gln Lys Phe Lys Thr Glu Val Tyr Asp Lys Lys Pro Glu Leu Phe Glu

65 70 75 80

Pro Leu Lys Ser Gly Gln Ser Pro Arg Tyr Met Val Phe Ala Cys Ser

85 90 95

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

100 105 110

Ala Phe Thr Val Arg Asn Ile Ala Ser Met Val Pro Pro Tyr Asp Lys

115 120 125

Ile Lys Tyr Ala Gly Thr Gly Ser Ala Ile Glu Tyr Ala Val Cys Ala

130 135 140

Leu Lys Val Gln Val Ile Val Val Ile Gly His Ser Cys Cys Gly Gly

145 150 155 160

Ile Arg Ala Leu Leu Ser Leu Lys Asp Gly Ala Pro Asp Asn Phe His

165 170 175

Phe Val Glu Asp Trp Val Arg Ile Gly Ser Pro Ala Lys Asn Lys Val

180 185 190

Lys Lys Glu His Ala Ser Val Pro Phe Asp Asp Gln Cys Ser Ile Leu

195 200 205

Glu Lys Glu Ala Val Asn Val Ser Leu Gln Asn Leu Lys Ser Tyr Pro

210 215 220

Phe Val Lys Glu Gly Leu Ala Gly Gly Thr Leu Lys Leu Val Gly Ala

225 230 235 240

His Tyr Asp Phe Val Lys Gly Gln Phe Val Thr Trp Glu Pro Pro Gln

245 250 255

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

260 265 270

Val Tyr Asp Lys Lys Pro Glu Leu Phe Gly Pro Leu Lys Ser Gly Gln

275 280 285

Ala Pro Lys Tyr Met Val Phe Ala Cys Ser Asp Ser Arg Val Cys Pro

290 295 300

Ser Val Thr Leu Gly Leu Gln Pro Gly Glu Ala Phe Thr Val Arg Asn

305 310 315 320

Ile Ala Ala Met Val Pro Gly Tyr Asp Lys Thr Lys Tyr Thr Gly Ile

325 330 335

Gly Ser Ala Ile Glu Tyr Ala Val Cys Ala Leu Lys Val Glu Val Leu

340 345 350

Val Val Ile Gly His Ser Cys Cys Gly Gly Ile Arg Ala Leu Leu Ser

355 360 365

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

370 375 380

Lys Ile Gly Phe Ile Ala Lys Met Lys Val Lys Lys Glu His Ala Ser

385 390 395 400

Val Pro Phe Asp Asp Gln Cys Ser Ile Leu Glu Lys Glu Ala Val Asn

405 410 415

Val Ser Leu Glu Asn Leu Lys Thr Tyr Pro Phe Val Lys Glu Gly Leu

420 425 430

Ala Asn Gly Thr Leu Lys Leu Ile Gly Ala His Tyr Asp Phe Val Ser

435 440 445

Gly Glu Phe Leu Thr Trp Lys Lys

450 455

Claims

1. a kind of method for enhancing photosynthesis of plant and/or improving plant products and/or improving phytomass, including as follows Step：5 photosynthetic key genes of C4 are imported into recipient plant, obtain genetically modified plants；The genetically modified plants with it is described by Body plant is compared, and photosynthesis enhancing and/or output increased and/or biomass improve；

The photosynthetic key genes of 5 C4 are the encoding gene of phosphopyruvic acid bidifly enzyme, encoding gene, the apple of PEP carboxylases Encoding gene, the encoding gene of NADP- malate dehydrogenases and the encoding gene of carbonic anhydrase of tartaric acid dehydrogenase；

In the genetically modified plants, the encoding gene of the phosphopyruvic acid bidifly enzyme is by the phosphopyruvic acid bidifly enzyme The endogenesis promoter driving transcription of encoding gene；The encoding gene of the PEP carboxylases by the PEP carboxylases encoding gene Endogenesis promoter driving transcription；The encoding gene of the malic dehydrogenase is driven by 35CaMVS promoters and transcribed；It is described The encoding gene of NADP- malate dehydrogenases is driven by Ubiqutin promoters and transcribed；The encoding gene of the carbonic anhydrase by The driving transcription of OsActin promoters.

2. according to the method described in claim 1, it is characterized in that：The photosynthetic key genes of 5 C4 are to pass through recombinant vector Form import in the recipient plant.

3. according to the method described in claim 2, it is characterized in that：The recombinant vector is any described in claim 4-8 Recombinant vector.

4. recombinant vector or the recombinant bacterium containing the recombinant vector, it is characterised in that：The recombinant vector is contains DNA fragmentation 1st, the circular plasmids of DNA fragmentation 2, DNA fragmentation 3, DNA fragmentation 4 and DNA fragmentation 5；

The DNA fragmentation 1 is from endogenesis promoter, the phosphorus for above swimming over to encoding gene of the downstream successively containing phosphopyruvic acid bidifly enzyme The encoding gene of sour pyruvic acid bidifly enzyme；

The DNA fragmentation 2 is from endogenesis promoter, the PEP carboxylases for above swimming over to encoding gene of the downstream successively containing PEP carboxylases Encoding gene；

The DNA fragmentation 3 is from above swimming over to the downstream encoding gene containing 35CaMVS promoters, malic dehydrogenase successively；

The DNA fragmentation 4 is from above swimming over to the downstream encoding gene containing Ubiqutin promoters, NADP- malate dehydrogenases successively；

The DNA fragmentation 5 is from above swimming over to the downstream encoding gene containing OsActin promoters, carbonic anhydrase successively.

5. recombinant vector according to claim 4 or recombinant bacterium, it is characterised in that：The recombinant vector is by the DNA Segment 1, the DNA fragmentation 2, the DNA fragmentation 3, the DNA fragmentation 4 and the DNA fragmentation 5 are inserted into after being sequentially connected with The recombinant plasmid obtained after between the Hind III and SmaI of 300 carriers of pCambia.

6. method or recombinant vector or recombinant bacterium according to claim 1-5, it is characterised in that：The phosphopyruvic acid is double Kinases is following any shown protein：

(A1) amino acid sequence is the protein of SEQ ID No.11；

(A2) by substitution of the amino acid sequence shown in SEQ ID No.11 by one or several amino acid residues and/or missing And/or add and have the protein of identical function；

(A3) with (A1)-(A2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；

(A4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (A1)-(A3)；

The PEP carboxylases are following any shown protein：

(B1) amino acid sequence is the protein of SEQ ID No.12；

(B2) by substitution of the amino acid sequence shown in SEQ ID No.12 by one or several amino acid residues and/or missing And/or add and have the protein of identical function；

(B3) with (B1)-(B2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；

(B4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (B1)-(B3)；

The malic dehydrogenase is following any shown protein：

(C1) amino acid sequence is the protein of SEQ ID No.13；

(C2) by substitution of the amino acid sequence shown in SEQ ID No.13 by one or several amino acid residues and/or missing And/or add and have the protein of identical function；

(C3) with (C1)-(C2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；

(C4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (C1)-(C3)；

The NADP- malate dehydrogenases are following any shown protein：

(D1) amino acid sequence is the protein of SEQ ID No.14；

(D2) by substitution of the amino acid sequence shown in SEQ ID No.14 by one or several amino acid residues and/or missing And/or add and have the protein of identical function；

(D3) with (D1)-(D2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；

(D4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (D1)-(D3)；

The carbonic anhydrase is following any shown protein：

(E1) amino acid sequence is the protein of SEQ ID No.15；

(E2) by substitution of the amino acid sequence shown in SEQ ID No.15 by one or several amino acid residues and/or missing And/or add and have the protein of identical function；

(E3) with (E1)-(E2) in any limited amino acid sequence have more than 99%, more than 95%, more than 90%, More than 85% or more than 80% homology and the protein with identical function；

(E4) N-terminal of any limited protein and/or C-terminal connect the fusion protein obtained after label in (E1)-(E3).

7. according to the method any in claim 1-6 or recombinant vector or recombinant bacterium, it is characterised in that：The phosphoric acid third The encoding gene of ketone acid bidifly enzyme is following any DNA molecular：

(a1) DNA molecular shown in SEQ ID No.2；

(a2) hybridize and encode DNA points of the phosphopyruvic acid bidifly enzyme with (a1) DNA molecular limited under strict conditions Son；

(a3) have more than 99%, more than 95%, more than 90%, more than 85% with the DNA sequence dna of any restriction in (a1)-(a2) Or more than 80% homology and the coding phosphopyruvic acid bidifly enzyme DNA molecular；

The encoding gene of the PEP carboxylases is following any DNA molecular：

(b1) DNA molecular shown in SEQ ID No.4；

(b2) hybridize under strict conditions with (b1) DNA molecular limited and encode the DNA molecular of the PEP carboxylases；

(b3) have more than 99%, more than 95%, more than 90%, more than 85% with the DNA sequence dna of any restriction in (b1)-(b2) Or more than 80% homology and the coding PEP carboxylases DNA molecular；

The encoding gene of the malic dehydrogenase is following any DNA molecular：

(c1) DNA molecular shown in SEQ ID No.6；

(c2) hybridize under strict conditions with (c1) DNA molecular limited and encode the DNA molecular of the malic dehydrogenase；

(c3) have more than 99%, more than 95%, more than 90%, more than 85% with the DNA sequence dna of any restriction in (c1)-(c2) Or more than 80% homology and the coding malic dehydrogenase DNA molecular；

The encoding gene of the NADP- malate dehydrogenases is following any DNA molecular：

(d1) DNA molecular shown in SEQ ID No.8；

(d2) hybridize under strict conditions with (d1) DNA molecular limited and encode the DNA molecular of the NADP- malate dehydrogenases；

(d3) have more than 99%, more than 95%, more than 90%, more than 85% with the DNA sequence dna of any restriction in (d1)-(d2) Or more than 80% homology and the coding NADP- malate dehydrogenases DNA molecular；

The encoding gene of the carbonic anhydrase is following any DNA molecular：

(e1) DNA molecular shown in SEQ ID No.10；

(e2) hybridize under strict conditions with (e1) DNA molecular limited and encode the DNA molecular of the carbonic anhydrase；

(e3) have more than 99%, more than 95%, more than 90%, more than 85% with the DNA sequence dna of any restriction in (e1)-(e2) Or more than 80% homology and the coding carbonic anhydrase DNA molecular.

8. according to the method any in claim 1-7 or recombinant vector or recombinant bacterium, it is characterised in that：The phosphoric acid third The nucleotide sequence of the endogenesis promoter of the encoding gene of ketone acid bidifly enzyme is as shown in SEQ ID No.1；

The nucleotide sequence of the endogenesis promoter of the encoding gene of the PEP carboxylases is as shown in SEQ ID No.3；

The nucleotide sequence of the 35CaMVS promoters is as shown in SEQ ID No.5；

The nucleotide sequence of the Ubiqutin promoters is as shown in SEQ ID No.7；

The nucleotide sequence of the OsActin promoters is as shown in SEQ ID No.9.

9. any recombinant vector or recombinant bacterium in enhancing photosynthesis of plant and/or improve plant in claim 4-8 Application in yield and/or raising phytomass.

10. according to the method any in claim 1-9 or application, it is characterised in that：The plant is C3 plant；

Specifically, the C3 plant is rice.