CN110184293B - A method for increasing plant biomass or yield by increasing photosynthetic efficiency - Google Patents
A method for increasing plant biomass or yield by increasing photosynthetic efficiency Download PDFInfo
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- CN110184293B CN110184293B CN201910348836.3A CN201910348836A CN110184293B CN 110184293 B CN110184293 B CN 110184293B CN 201910348836 A CN201910348836 A CN 201910348836A CN 110184293 B CN110184293 B CN 110184293B
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8221—Transit peptides
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8269—Photosynthesis
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
- C12Y101/99014—Glycolate dehydrogenase (1.1.99.14)
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- C12Y203/00—Acyltransferases (2.3)
- C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
- C12Y203/03009—Malate synthase (2.3.3.9)
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- Molecular Biology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
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- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
Disclosed is a method for increasing plant biomass or yield by improving photosynthetic efficiency by overexpressing a Glycolate Dehydrogenase (GDH) gene and a Malate Synthase (MS) gene in plant chloroplasts. The present invention provides a method for improving plant photosynthetic efficiency by overexpressing GDH gene and MS gene, and further improving biomass or yield, wherein the biomass or yield is increased by 3% -50% in the case of overexpressing MS gene, and the overexpression of Escherichia coli-derived GDH in plant chloroplast is compared with the overexpression of chlorella-derived GDH in plant chloroplast.
Description
(I) technical field
The present invention relates to a method for increasing plant yield, and more particularly, to a method for increasing plant yield using photosynthetic efficiency.
(II) background of the invention
The increase in the number of human beings and the improvement of living standard require more food and feed to be consumed, which requires more food to be harvested on a limited land. Therefore, it is very important to breed new high-yielding plant varieties.
The photosynthesis products of the whole plant are all derived from enzyme-catalyzed CO2Is converted into organic carbon compounds. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is a carboxylase in the Calvin cycle (Calvin-benson (cb) cycle). Due to RubisCO and CO2Or O2All can react, RubisCO and O2The reaction produces glycolic acid phosphate, entering the photorespiration cycle, which results in the waste of fixed carbon and nitrogen in the plant. This process releases approximately 29GT of fresh assimilated carbon into the atmosphere each year, worldwide (Anav a, axialroduction:a review.Rev Geophys 2015,53:785-818.)。
In order to reduce the loss caused by light respiration and improve the photosynthetic efficiency of plants, the common method at present is to recover CO in glycolic acid by a new light respiration branch2Thereby achieving the purpose of reducing light respiration and improving photosynthetic efficiency (Peterhansel C, Blume C, Offermann S.Photorespiratory bypass: how can the work].Journal of Experimental Botany,2013,64(3):709-715.)。
Glycolate Dehydrogenase (GDH) can convert glycolate into glyoxylate. The glycolate dehydrogenase currently used for plant transgenic research and application is mainly derived from lower plant green algae (Chlamydomonas reinhardtii) or Escherichia coli. Glycolate dehydrogenase in green algae is encoded by one gene, while glycolate dehydrogenase in large bacillus is composed of D, E, F three subunits encoded by 3 genes, respectively. It has been reported that by overexpressing fusion genes encoding genes for D, E, F subunits in potato, DEFP fusion protein expression in plants is increased, and sugars such as glucose, fructose, and sucrose are multiplied, and biomass is also significantly increased (Nolke G, Houdel M, Kreuzaler F, et al. the expression of a recombinant glucose dehydrogenase in protein fertilizer to Plant and tube yield [ J ] Plant Biotechnology Journal,2014,12(6): 734-. However, since there is a significant difference in the function and activity between glycolate dehydrogenases derived from Escherichia coli and those derived from green algae, there is a large difference in the expression in transgenic plants.
Malate Synthase (MS) catalyzes the conversion of acetyl-CoA and glyoxylate into malate and CoA. The malate synthase participates in the glyoxylate cycle and is widely present in different plants. It has been reported that the overexpression of GDH from green algae and the MS from pumpkin (C.maxima) in tobacco can increase the photosynthetic efficiency and biomass (PF South, AP Cavanagh, HW Liu, equivalent. synthetic glycerol metabolism pathway protein yield and productivity in the field, Science,2019:363(6422) and eat 9077).
However, we have found that the E.coli-derived GDH gene is better than the overexpressed green alga-derived GDH gene in terms of the effect of increasing the biomass or yield of plants due to the difference in function and activity between the E.coli-derived GDH and the green alga-derived GDH. Under the premise of simultaneously over-expressing the MS gene, the biomass or yield of the GDH derived from escherichia coli over-expressed in the plant chloroplast is increased by 3% -50% compared with the GDH derived from green algae over-expressed in the plant chloroplast. Therefore, overexpression of E.coli-derived GDH in plant chloroplasts together with another MS is the best method to increase plant biomass or yield by improving photosynthetic efficiency.
Disclosure of the invention
The present invention aims to provide a method for reducing plant light respiration, increasing plant photosynthetic efficiency, increasing plant biomass or yield by overexpressing in plant chloroplasts GDH of E.coli origin and MS of any origin. The invention solves the problem that the co-use of a high-activity GDH gene and an MS gene is used for optimizing the plant light respiration path, and transgenic plants with more increased biomass or yield are obtained.
The technical scheme adopted by the invention is as follows:
the present invention provides a method for increasing plant biomass or yield by improving photosynthetic efficiency by overexpressing a Glycolate Dehydrogenase (GDH) gene and a Malate Synthase (MS) gene in plant chloroplasts. Preferably, the gene of the glycolate dehydrogenase GDH is derived from escherichia coli, the GDH consists of three independent subunits (the nucleotide sequence is 3126522bp-3128021bp in GenBank: CP029238.1, the coded amino acid is GenBank: QBP03082.1, 3125470bp-3126522bp in GenBank: CP029238.1, the coded amino acid is GenBank: QBP03081.1, the nucleotide sequence is 3124236 bp-3125459 bp in GenBank: CP029238.1, and the coded amino acid is GenBank: QBP03080.1), and more preferably, the gene sequence of the GDH is shown as 125bp-4009bp in SEQ ID NO.1 and 48-1333 in SEQ ID NO. 2.
The malic acid synthase MS gene is derived from prokaryotes or eukaryotes, such as the MS gene shown in Table 1, preferably the nucleotide sequence of the MS gene is shown as SEQ ID NO.3, SEQ ID NO.4(11bp-1852bp) or SEQ ID NO.5, and the amino acid sequence is shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8.
Table 1: malate Synthase (MS) genes from different species
Further, overexpression of the GDH gene and the MS gene in chloroplast is mediated by a signal peptide, and the mediation is to fuse a chloroplast signal peptide sequence to the N terminal of the GDH or MS protein; the signal peptide is derived from plant RuBisCO small subunit (RbcS) or phosphoglucomutase transfer peptide sequence, and preferably the amino acid sequence of the signal peptide is shown as SEQ ID NO.9 or SEQ ID NO. 10.
Further, the GDH gene and the MS gene are overexpressed in chloroplasts, and also comprise promoters, wherein the promoters are derived from eukaryotes or prokaryotes and can also be obtained by artificial synthesis; preferably, the promoter is a constitutive promoter or a specific promoter, and more preferably, the nucleotide sequence of the promoter is a UBI promoter (shown by 4879bp-6876bp in GenBank: KR 297238.1), an Act1 promoter (shown by 2428bp-3797bp in GenBank: AY 452735.1) or a 35S promoter (shown by 848bp-1628bp in GenBank: MG 719235.1).
Further, the GDH gene and the MS gene also comprise a terminator which is derived from eukaryote or prokaryote and can also be obtained by artificial synthesis in the chloroplast overexpression process, and preferably, the nucleotide sequence of the terminator is shown as SEQ ID NO.11 or SEQ ID NO. 12.
The overexpression can be realized by a molecular polymerization method or a hybrid polymerization method; the molecular polymerization is that the expression frames of the GDH gene and the MS gene are constructed on the T-DNA of the same vector, and the T-DNA is transferred into the genome of a receptor plant by a transgenic method, so that the GDH gene and the MS gene are simultaneously over-expressed in a target plant. The hybrid polymerization refers to obtaining plants respectively overexpressing GDH and MS genes in chloroplasts, and then hybridizing the plants respectively expressing the GDH and MS genes by using a traditional breeding method to obtain plants overexpressing the GDH and MS genes simultaneously.
Further, the method for overexpression of the GDH gene and the MS gene in chloroplasts comprises the following steps: (1) based on a commercial binary vector pCambia1300, replacing hptII (hygromycin resistance) gene of the vector with a glufosinate-ammonium-resistant bar gene (GenBank: MG719235.1(287bp-837bp)) through an XhoI enzyme cutting site, and naming the replaced vector as pCambia 1300-bar; (2) the promoter and the GDH gene were ligated into the pCambia1300-bar vector via EcoRI and KpnI sites to obtain the transition vector pCambia 1300-bar-GDH; (3) the promoter and MS gene were then ligated into the transition vector pCambia1300-bar-GDH via KpnI and HindIII sites to obtain the final vector pCambia1300-bar-GDH-MS, the T-DNA plasmid. (4) Finally, the T-DNA plasmid is transferred into agrobacterium LB4404 by electrotransfer, positive clones are screened out by YEP solid culture medium containing 15 mug/mL tetracycline and 50 mug/mL kanamycin, and plants with reduced light respiration, improved photosynthetic efficiency and improved biomass or yield are obtained by screening.
The plants of the present invention include corn, rice and soybean.
Compared with the prior art, the invention has the following beneficial effects:
the present invention provides a method for improving plant photosynthetic efficiency by overexpressing GDH gene and MS gene, and further increasing biomass or yield, wherein the biomass or yield is increased by 3% -50% in comparison with GDH derived from green algae overexpressed in plant chloroplast, by overexpressing Escherichia coli-derived GDH in plant chloroplast under the premise of simultaneously overexpressing MS gene.
(IV) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
example 1 construction of vectors
In order to construct a transformation vector, a GDH gene derived from Escherichia coli and a corresponding terminator sequence are artificially synthesized, the GDH gene comprises a chloroplast signal peptide, a GDH coding gene and a terminator, the nucleotide sequence is shown as SEQ ID NO.1 (the amino acid sequence of the coding protein is shown as SEQ ID NO. 2), and BamHI and KpnI sites are respectively arranged at the 5 'end and the 3' end.
The rice MS gene is artificially synthesized, and comprises chloroplast signal peptide, an MS coding gene and a terminator, wherein the nucleotide sequence is shown as SEQ ID NO.4, and BamHI and HindIII sites are respectively arranged at the 5 'end and the 3' end.
Meanwhile, 35S promoter sequence (shown by 848bp-1628bp in GenBank: MG 719235.1) of cauliflower mosaic virus (CaMV) is artificially synthesized, and KpnI and BamHI sites are respectively arranged at the 5 'end and the 3' end. Artificially synthesizing a maize Ubi promoter sequence (shown by 4879bp-6876bp in GenBank: KR 297238.1), and respectively arranging EcoRI and BamHI sites at the 5 'end and the 3' end.
In order to construct a binary vector which can be used for transforming plants by the Agrobacterium method, a commercial binary vector pCambia1300 is used as a base, the prior hptII (hygromycin resistance) gene is replaced by a glufosinate-resistant bar gene (GenBank: MG719235.1(287bp-837bp)) through an XhoI enzyme cutting site, and the replaced vector is named as pCambia 1300-bar.
The Ubi promoter and GDH gene were ligated into pCambia1300-bar vector via EcoRI and KpnI sites to obtain the transition vector pCambia 1300-bar-GDH. The 35S promoter and the MS gene were then ligated into the transition vector pCambia1300-bar-GDH via KpnI and HindIII sites to obtain the T-DNA plasmid pCambia 1300-bar-GDH-MS.
As a control, a chlorella-derived CrGDH gene comprising a chloroplast signal peptide, a CrGDH coding gene and a terminator was artificially synthesized, and the nucleotide sequence was shown in SEQ ID NO.13, with BamHI and KpnI sites provided at the 5 'and 3' ends, respectively.
The Ubi promoter and the CrGDH gene were ligated into the pCambia1300-bar vector via EcoRI and KpnI sites to obtain the transition vector pCambia 1300-bar-CrGDH. The 35S promoter and MS gene were ligated into the transition vector pCambia1300-bar-CrGDH through KpnI and HindIII sites to obtain the T-DNA plasmid pCambia 1300-bar-CrGDH-MS.
Finally, the T-DNA plasmid was transferred to Agrobacterium LB4404 by electrotransfer, and positive clones were selected by YEP solid medium containing 15. mu.g/mL tetracycline and 50. mu.g/mL kanamycin and maintained for the subsequent plant transformation.
YEP solidThe culture medium comprises the following components: beef extract 5g/L, yeast extract 1g/L, peptone 5g/L, sucrose 5g/L, MgSO4·H2O0.5 g/L, solvent is water, pH 7.0.
Example 2 transformation of Rice
The transgenic rice is obtained by adopting the prior art (Luzhong, Gong ancestor Xun (1998) Life sciences 10: 125-. Mature and full 'Xishui-134' seeds are selected to be hulled, and callus is generated by induction and is used as a transformation material. Agrobacterium plates containing plasmids pCambia1300-bar-GDH-MS, pCambia1300-bar-CrGDH-MS and pCambia1300-bar-GDH, respectively, constructed in example 1 were taken. A single colony is selected and inoculated, and agrobacterium for transformation is prepared. The callus to be transformed was placed in Agrobacterium tumefaciens broth with OD600 of 0.6 (Agrobacterium tumefaciens broth preparation: Agrobacterium tumefaciens was inoculated into culture medium with OD600 of 0.6; culture medium composition: 3g/L K2HPO4、1g/LNaH2PO4、1g/LNH4Cl、0.3g/L MgSO4·7H2O、0.15g/L KCl、0.01g/L CaCl2、0.0025g/L FeSO4·7H2O, 5g/L sucrose, 20mg/L acetosyringone, water as solvent, pH 5.8), allowing Agrobacterium to bind to the callus surface, and transferring the callus to co-culture medium (MS +2mg/L2,4-D +30g/L glucose +30g/L sucrose +3g/L agar (sigma 7921) +20mg/L acetosyringone) for co-culture for 2-3 days. The transformed calli were rinsed with sterile water, transferred to selection medium (MS +2mg/L2,4-D +30 g/sucrose +3g/L agar (Sigma 7921) +20mg/L acetosyringone +2mM glyphosate (Sigma)), and cultured for two months with selection (intermediate subculture). Transferring the callus with good growth activity after screening to a pre-differentiation culture medium (MS +0.1g/L inositol +5mg/L ABA +1mg/L NAA +5 mg/L6-BA +20g/L sorbitol +30g/L sucrose +2.5g/L gelrite) for about 20 days, then transferring the pre-differentiated callus to the differentiation culture medium, and irradiating for differentiation and germination for 14 hours every day. After 2-3 weeks, transferring the resistant regenerated plants to a rooting culture medium (1/2MS +0.2mg/L NAA +20g/L sucrose +2.5g/L gelrite), strengthening seedlings and rooting, finally washing the regenerated plants, washing agar, transplanting the washed regenerated plants to a greenhouse, selecting high-yield plants, large seeds or high-energy plants with high biomassCan improve the yield of rice and cultivate new species. Obtaining the transgenic rice plants containing the transformation vectors respectively.
Example 3 transformation of Soybean
The procedure used here to obtain transgenic soybeans is known from the prior art (Deng et al, 1998, Plant Physiology Communications 34: 381-387; Ma et al, 2008, Scientia Agricutural national informa 41: 661-668; Zhou et al, 2001, Journal of northern Agricultural University 32: 313-319). Healthy, full and mature soybeans of "Tianlong No. 1" were selected, sterilized with 80% ethanol for 2 minutes, washed with sterile water, and then sterilized in a desiccator filled with chlorine (generated by reacting 50ml of NaClO with 2ml of concentrated HCl) for 4-6 hours. The sterilized semen glycines is sowed in B5 culture medium in clean bench, and cultured at 25 deg.C for 5 days with optical density of 90-150 μmol photon/m2S level. When the cotyledon turns green and breaks the seed coat, the aseptic bean sprouts grow. The bean sprouts with the hypocotyl removed were cut into five-five pieces in length so that both explants had cotyledons and epicotyls. The explants are cut at about 7-8 of the node of the cotyledon and epicotyl and can be used as the target tissue to be infected.
Monoclonal agrobacteria containing the vectors pCambia1300-bar-GDH-MS, pCambia1300-bar-CrGDH-MS and pCambia1300-bar-GDH, constructed by example 1, respectively, were separately cultured for use. The prepared explants are immersed in the agrobacterium suspension and co-cultured for about 30 minutes. Then, the excess cell suspension on the infected tissue was absorbed up with absorbent paper and transferred to 1/10B5 co-culture medium for 3-5 days at 25 ℃ in the dark.
The co-cultured plant tissue was washed with B5 liquid medium to remove excess Agrobacterium, and then placed in B5 solid medium for 5 days at 25 ℃ until it germinated. The induced germ tissue was transferred to B5 screening medium containing 0.1M glyphosate and incubated at 25 ℃ with light for 4 weeks, during which the medium was changed every two weeks. Transferring the selected embryo tissue to B5 solid culture medium, culturing at 25 deg.C, and growing into plantlet. Subsequently, the transgenic plants were transferred to 1/2B5 medium for rooting induction. Finally, the grown plantlets are washed to remove agar and planted in a greenhouse.
Example 4: identification of transgenic Rice
Transgenic rice plants carrying the vectors pCambia1300-bar-GDH-MS (GM), pCambia1300-bar-CrGDH-MS (CGM) and pCambia1300-bar-GDH (G), respectively, were obtained by example 2. The above transgenic plants had increased biomass and yield compared to the non-transgenic control, and the magnitude of increase in biomass or yield was greatest for GM plants. To further identify the performance changes of GM transgenic plants, we evaluated the biomass and seed yield of the transgenic plants described above and the results are shown in table 2 below. The biomass or yield of the GM rice plant is increased by 5-50% compared with that of the CGM and G plants.
TABLE 2
| GM | CGM | G | |
| Magnitude of biomass increase over non-transgenic control | 25％ | 15％ | 9％ |
| Magnitude of increase in yield over non-transgenic controls | 18％ | 12％ | 6％ |
Example 5: identification of transgenic Soybean
Transgenic soybean plants with vectors pCambia1300-bar-GDH-MS (GM), pCambia1300-bar-CrGDH-MS (CGM) and pCambia1300-bar-GDH (G), respectively, were obtained by example 3. The above transgenic plants had increased biomass and yield compared to the non-transgenic control, and the magnitude of increase in biomass or yield was greatest for GM plants. To further identify the performance changes of GM transgenic plants, we evaluated the biomass and seed yield of the transgenic plants described above and the results are shown in table 3 below. The biomass or yield of the GM soybean plant is increased by 5-50% compared with the CGM and G plants.
TABLE 3
| GM | CGM | G | |
| Magnitude of biomass increase over non-transgenic control | 21％ | 12％ | 7％ |
| Magnitude of increase in yield over non-transgenic controls | 14％ | 8％ | 5％ |
Claims (1)
1. A method for increasing plant biomass or yield by increasing photosynthetic efficiency, characterized in that the method is carried out by overexpressing a glycolate dehydrogenase GDH gene of E.coli and a malate synthase MS gene derived from rice in plant chloroplasts; the glycollic dehydrogenase GDH consists of three independent subunits, which are 3126522bp-3128021bp in the GenBank CP029238.1, 3125470bp-3126522bp in the GenBank CP029238.1 and 3124236 bp-3125459 bp in the GenBank CP029238.1 respectively; the nucleotide sequence of the malic synthase MS gene is shown as NCBI Accession Number: CAD 79704; overexpression of the GDH gene and the MS gene in chloroplasts is mediated by a signal peptide, and the signal peptide is derived from a plant RuBisCO small subunit or phosphoglucomutase transit peptide sequence; the GDH gene and the MS gene are overexpressed in chloroplast and also comprise promoters, and the nucleotide sequences of the promoters are UBI promoters, Act1 promoters or 35S promoters; the nucleotide sequence of the UBI promoter is shown as 4879bp-6876bp in a GenBank KR 297238.1; the nucleotide sequence of the Act1 promoter is shown as 2428bp-3797bp in GenBank AY 452735.1; the nucleotide sequence of the 35S promoter is shown as 848bp-1628bp in GenBank: MG 719235.1; the plant is rice or soybean.
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