Ways to dramatically increase rice production

Abstract

A transgenic rice plant contains a recombinant DNA construct in its genome, the construct comprising a first nucleic acid having a sequence of a first Golden 2-like transcription factor (GLK) gene operably linked to its native promoter and 5' untranslated region (5' untranslated region, 5' UTR), and a second nucleic acid having a sequence of a second GLK gene operably linked to its native promoter and 5' UTR, the second GLK gene being different from the first GLK gene. The first GLK gene and the second GLK gene are both from C4 plants, and compared to untransformed wild-type rice plants, the transgenic rice plant increases shoot biomass by 65-106% and grain yield by 50-95%. A method for producing a transgenic rice plant and a recombinant DNA construct that can be used in the method are also provided.

Description

Background Art

[0001] An expanding population requires increased crop yields. To meet this demand, recent efforts have focused on improving the efficiency of photosynthesis in crops such as rice. From the early 1960s to 2000, a series of initiatives aimed at increasing global agricultural yields, the so-called Green Revolution, led to a roughly doubling of rice production. However, yield increases in recent years have been less dramatic.

[0002] Since more than 90% of crop biomass comes from photosynthesis, increasing photosynthesis in staple crops such as rice could significantly increase yields.

[0003] Chloroplasts play an important role in photosynthesis and are also the site of plant hormone synthesis that regulates plant growth, development, and stress tolerance. A pair of Golden 2-like (GLK) transcription factor genes are known to regulate chloroplast development in higher plants such as maize (a C4 plant) and rice (a C3 plant). Through evolution, the GLK genes from maize, especially GLK2, may have acquired newer and stronger functions compared to the GLK genes in rice.

[0004] Since maize photosynthesizes more efficiently than rice, transferring two maize GLK genes into the rice genome could potentially enhance photosynthesis in transgenic rice plants, thereby increasing rice yield.

[0005] Recently, it was reported that the transformation of either of the two maize GLK genes controlled by the maize ubiquitin promoter, especially the maize GLK2 gene, increased rice yield by 16-40%. However, the use of the maize ubiquitin promoter, a strong constitutive promoter, to drive the expression of the maize GLK genes in transgenic rice resulted in smaller rice seeds.

[0006] There remains a strong need to increase rice production to meet the needs of a growing population. Summary of the invention

[0007] In order to meet the increasing demand for food of the growing population, a transgenic rice plant is provided, which comprises in its genome a recombinant DNA construct, the construct comprising a first nucleic acid having the sequence of a first Golden 2-like transcription factor (GLK) gene operably linked to its native promoter and 5' untranslated region (5'UTR), and a second nucleic acid having the sequence of a second GLK gene operably linked to its native promoter and 5'UTR, the second GLK gene being different from the first GLK gene. The first GLK gene and the second GLK gene are both heterologous, i.e. not from rice, and the transgenic rice plant has a significant, i.e. at least 50% increase in shoot biomass (65-106%) and in grain yield (50-95%) compared to a non-transformed wild-type rice plant.

[0008] A method for producing the above-mentioned genetically modified rice plant is also provided.

[0009] A recombinant DNA construct that can be used in the method is further disclosed. The recombinant DNA construct comprises a first nucleic acid sequence, the first nucleic acid sequence comprises a first GLK gene, the first GLK gene is operably linked to its natural promoter and 5'UTR, and a second nucleic acid sequence, the second nucleic acid sequence comprises a second GLK gene, the second GLK gene is operably linked to its natural promoter and 5'UTR, and the second GLK gene is different from the first GLK gene. The first GLK gene and the second GLK gene are from a C4 plant, such as corn.

[0010] The details of several specific embodiments of the present invention are set forth in the following description and accompanying drawings. All features, objects, and advantages of the present invention will be apparent from the description and accompanying drawings, as well as from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following description refers to the accompanying drawings, in which:

[0012] Figure 1Schematic diagram of five maize GLK constructs used for Agrobacterium-mediated rice transformation to compare their effects on gene expression and plant growth and yield. pUbi: ubiquitin promoter; p35S: 35S promoter; Zm: Zea mays (maize); pZmG1::ZmG1: maize GLK1 construct with its own promoter and 5'UTR; pZmG2::ZmG2: maize GLK2 construct with its own promoter and 5'UTR; HygR: hygromycin resistance gene; ZmG1: maize GLK1 gene; ZmG2: maize GLK2 gene; 35ST: 35S terminator; NOST: nopaline synthase (nos) terminator; pro: promoter; LB: left border; and RB: right border.

[0013] Figure 2A Shown are graphs of the relative gene expression of ZmG1 (left) and ZmG2 (right) in different maize tissues, as determined by quantitative real-time polymerase chain reaction (qRT-PCR) of total RNA isolated from young leaves (YL) of 9-day-old seedlings, mature leaves (ML) of 2-month-old plants, and young stems (S), male inflorescences (T), and roots (R). Values ​​are mean ± standard deviation (SD) of three replicates and normalized to 17S rRNA expression.

[0014] Figure 2B Figure 2 shows the relative gene expression of ZmG1 (left) and ZmG2 (right) determined by qRT-PCR of total RNA from wild-type (WT) and transgenic rice plants. Figure 1 Labeled transgenic constructs. Total RNA was isolated from mature leaves (L), stems (S), florets (F), and roots (R). Heterogeneous combinations of pZmG2::ZmG2 plants and isogeneous combinations of the other four transgenic plants are shown; the isogeneous combination of pZmG2::ZmG2 plants died after two months of cultivation due to high gene expression. Values ​​are mean ± standard deviation (SD) of three replicates and normalized to 17S rRNA expression.

[0015] Figure 2C Figure 1 contains the expression levels of OsGLK1 and OsGLK2 in different tissues of WT and transgenic rice plants measured by qRT-PCR. Figure 2BValues ​​are mean ± standard deviation (SD) of three replicates and normalized to the expression of 17S rRNA.

[0016] Figure 2D Shown is a Western blot analysis of GLK protein in young maize leaves, mature WT rice flag leaves, and transgenic rice flag leaves using an antibody against a common peptide of rice and maize GLK proteins. The same amount of total leaf protein (20 μg) was added to each lane. RbcL was used as an internal control, and maize leaf protein was used as a positive control for GLK. M: molecular weight marker.

[0017] Figure 3A Shown are photos of WT and transgenic rice plants at 4 months old. Transgenic markers were as above. Figure 1 shown.

[0018] Figure 3B The bar graph shows the chlorophyll content per leaf area in flag leaves of wild-type and transgenic rice plants as shown, expressed in μg / cm 2 Representation. The relative chlorophyll content per leaf area is shown above the graph, expressed as a percentage relative to wild-type plants, which were set as 100%. Statistical significance relative to WT (*P<0.05; **P<0.01; ***P<0.001) is shown. Data = mean ± SD, n = 4 (each sample obtained from leaf parts of three different plants).

[0019] Figure 3C The bar graph shows the chlorophyll content per 10 grains in the florets of the indicated wild-type and transgenic rice plants, expressed in μg / 10 grains. Figure 3B as shown in the legend.

[0020] Figure 3D Shown is a bar graph of ribulose bisphosphate carboxylase (rubisco) content analyzed by enzyme activity (upper figure) and total soluble protein content per leaf area in flag leaves (lower figure). Statistical tests were based on paired t-tests. Asterisks indicate the degree of statistical significance compared to WT (*P<0.05; **P<0.01; ***P<0.001). Data = mean ± SD, n = 4 (each sample was from leaf parts of three different plants). Transgenic plants are as described above.

[0021] Figure 4 Shown are the light-saturated photosynthetic rates per leaf area in flag leaves of 4-month-old WT and GLK transgenic rice plants, expressed in μmol / m 2 / s. The figure shows the range of values, the quartiles from 25% to 75% (boxes), and the mean value (horizontal line). The measurement condition was 2000 μmol / m 2 / s PPFD, 30°C, and 400 μL / L CO2. Statistical test was based on Wilcoxon-Mann-Whitney U-test for nonparametric comparison of two groups. Asterisks indicate statistical significance compared to WT (**P<0.01, n=6). Constructs were as described above.

[0022] Figure 5A Shown is the total shoot biomass dry weight / plant of WT and five GLK transgenic rice plants. Plants were grown in a greenhouse from May to September. Statistical tests were as above for Figure 4 Asterisks indicate the degree of statistical significance (*P<0.05; **P<0.01; ***P<0.001, n=9). Transgenic constructs were Figure 1 shown.

[0023] Figure 5B Figure 2 shows the total number of panicles per plant in WT and five GLK gene transgenic rice plants. Figure 5A as shown in the legend.

[0024] Figure 5C The total grain weight of each plant of WT and the above five GLK gene transgenic rice plants is shown. Figure 5A as shown in the legend.

[0025] Figure 5D Shown are the dry weights of 1000 grains per plant for WT and five GLK transgenic rice plants. Figure 5A as shown in the legend.

[0026] Fig. 6A Shown are the results of enrichment analysis of Gene Ontology (GO) annotation entries of biological program categories of differentially expressed genes in young shoots of transgenic plants pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1 / pZmG2::ZmG2 relative to WT. The colored grids represent biological programs that are upregulated (left) or downregulated (right).

[0027] Figure 6B Shown is the enrichment analysis of GO annotation terms for differentially expressed genes in shoots of transgenic plants (labeled above) relative to WT. Colored boxes indicate biological programs that are upregulated (left panel) or downregulated (right panel). DETAILED DESCRIPTION

[0028] As described above, a transgenic rice plant is provided, wherein the genome of the transgenic rice plant contains two different heterologous GLK genes, each gene under the control of its native promoter and 5'UTR. For example, both GLK genes can be from C4 plants. Exemplary C4 plant sources of the GLK genes include, but are not limited to, Zea mays L. (corn), Sorghum bicolor L. (sorghum), Saccharum officinarum L. (sugarcane), and Setaria italica L. (millet).

[0029] In one embodiment, the first GLK gene is Zea mays GLK1, and the second GLK gene is Zea mays GLK2, also known as G2. More specifically, the protein expressed by the GLK1 gene has an amino acid sequence as shown in SEQ ID NO:3, and the protein expressed by the GLK2 gene has an amino acid sequence as shown in SEQ ID NO:6.

[0030] In addition, the native promoter and 5'UTR of the first GLK gene may have the sequence shown in SEQ ID NO:1, and the native promoter and 5'UTR of the second GLK gene may have the sequence shown in SEQ ID NO:4.

[0031] As described above, the shoot growth and grain yield of the transgenic rice plants were significantly increased, ie, at least 50%, compared to the non-transformed wild-type rice plants.

[0032] In one embodiment, the increase in shoot growth in pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants, as measured by biomass change, can range from 65% to 106% compared to non-transformed wild-type rice plants, such as the rice variety Tainong 67 (TNG67).

[0033] In the same embodiment, the grain yield of the pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants can be 50% to 95% higher than that of the non-transformed wild-type rice.

[0034] The Summary of the Invention section also mentions a method for producing the above-mentioned transgenic plants, which comprises (i) introducing a recombinant DNA construct into a host rice plant, wherein the construct comprises a first GLK gene operably linked to its native promoter and 5'UTR and a second GLK gene operably linked to its native promoter and 5'UTR, and (ii) identifying the transgenic rice plant, wherein the transgenic rice plant has at least 50% increased shoot biomass and grain yield compared to a non-transformed wild-type rice plant, such as TNG67.

[0035] In the method, the first GLK gene and the second GLK gene are different from each other and are heterologous to rice. In one embodiment, the first GLK gene and the second GLK gene are from C4 plants, such as corn, sorghum, sugarcane, and millet. In a specific method, the first GLK gene is Zea mays GLK1, and the second GLK gene is Zea mays GLK2.

[0036] In one embodiment, the recombinant DNA construct can express a protein having an amino acid sequence as shown in SEQ ID NO:3, ie, Zea mays GLK1, and a protein having an amino acid sequence as shown in SEQ ID NO:6, ie, Zea mays GLK2.

[0037] The expression of GLK1 and GLK2 can be controlled by the natural promoter and 5'UTR having the sequence shown in SEQ ID NO: 1 and the natural promoter and 5'UTR having the sequence shown in SEQ ID NO: 4, respectively.

[0038] The identified transgenic rice plants have significantly higher grain yields compared to untransformed wild-type rice plants, such as TNG67. For example, pZmG1::ZmG1 / pZmG2::ZmG2 transgenic rice plants can be seen to have grain yields increased by 50% to 95% compared to the untransformed wild-type rice plants.

[0039] The shoot biomass of the transgenic rice plants was also significantly higher than that of the untransformed wild-type rice plants. The shoot mass of the exemplary pZmG1::ZmG1 / pZmG2::ZmG2 transgenic rice plants was 65% to 106% higher than that of the untransformed wild-type rice plants.

[0040] Next, the recombinant DNA construct mentioned in the above summary of the invention is discussed. The recombinant DNA construct comprises a first GLK gene operably linked to its native promoter and 5'UTR, and a second GLK gene operably linked to its native promoter and 5'UTR. The second GLK gene is different from the first GLK gene, and the two GLK genes are from a C4 plant. Therefore, they are heterologous to rice.

[0041] In an exemplary recombinant DNA construct, the first GLK gene is Zea mays GLK1 and the second GLK gene is Zea mays GLK2.

[0042] A specific recombinant DNA construct comprises (i) a first GLK gene encoding the amino acid sequence shown in SEQ ID NO:3, which is controlled by its native promoter / 5'UTR having the sequence shown in SEQ ID NO:1, and (ii) a second GLK gene encoding the amino acid sequence shown in SEQ ID NO:6, which is controlled by its native promoter / 5'UTR having the sequence shown in SEQ ID NO:4.

[0043] In a specific recombinant DNA construct, the first GLK gene is encoded by a nucleic acid having a sequence as shown in SEQ ID NO:2, and the second GLK gene is encoded by a nucleic acid having a sequence as shown in SEQ ID NO:5.

[0044] Without further elaboration, it is believed that those skilled in the art can utilize the present invention to the maximum extent based on the disclosure herein. Therefore, the following specific examples are to be interpreted as merely illustrative and not limiting the remainder of the present invention in any way whatsoever. All publications and patent documents cited herein are incorporated by reference in their entirety.

[0045] Example 1: Maize GLK construct for Agrobacterium-mediated rice transformation

[0046] Five maize GLK constructs were prepared by standard techniques as described in Example 7 below, including Figure 1 The genes and regulatory sequences shown in .

[0047] Rice (Oryza sativa cv. TNG67) callus induction, co-cultivation with Agrobacterium, hygromycin selection of transformed callus, and plant regeneration were performed according to the methods of Yeh et al. 2015 (Rice 8:36-48). All positive transgenic seedlings were transplanted into soil and cultured in a greenhouse for molecular, physiological, and anatomical analyses. The transgene insertion position was determined by thermal asymmetric interlaced (TAIL)-PCR. The conditions of TAIL-PCR were previously described in Liu et al. 2005 (Methods Mol. Biol. 286:341-348) and et al., 2009 (Plant Cell 21: 2163-2178). Genotyping primers designed based on the 3' and 5' regions flanking the transgenic insertion site determined by Tail-PCR were used to screen transgenic plants with the same genotype combination by genotyping PCR.

[0048] Example 2: Performance of the Maize GLK Construct in Transgenic Rice Plants

[0049] The expression of the maize GLK gene was determined by qRT-PCR and Western blot analysis, as described in Example 7 below. Figures 2A-2D as shown in .

[0050] The ubiquitin and 35S promoters overdrive the expression of the maize GLK gene in all transgenic rice tissues. A large amount of GLK protein was detected in the leaves of pZmUbi::ZmG1 transgenic plants, reflecting the high expression of ZmG1 in leaves. Figure 2B .

[0051] The growth and development of the heterozygous pZmG2::ZmG2 plants were delayed by one month, while the isogenic pZmG2::ZmG2 seedlings died after two months of cultivation, indicating that the high expression of maize GLK2 in pZmG2::ZmG2 plants inhibited the growth and development of the plants.

[0052] In all studied tissues of pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants, the expression of ZmG1 was promoted and that of ZmG2 was repressed, providing evidence for the coordinate expression of these two genes in pZmG1::ZmG1 / pZmG2::ZmG2 transgenic rice.

[0053] The expression levels of endogenous rice GLK1 and GLK2 genes were generally reduced in rice plants transformed with maize GLK genes.

[0054] Example 3: Growth and chloroplast characterization in transgenic rice plants.

[0055] Flowering and fruiting were delayed by one month in pZmG2::ZmG2 (heterogeneous combination) rice plants compared to all other tested rice plants. Figure 3A .

[0056] Transmission electron microscopy (TEM) analysis showed that the expression of the maize GLK gene in rice induced the development of large chloroplasts in the mesophyll (M) and bundle sheath (BS) cells of all five transgenic rice lines. Similar to WT, the M and BS chloroplast structures of all GLK transgenic rice plants had complete granule structures and thylakoid membranes.

[0057] Compared with WT, the chlorophyll content in flag leaves and florets of all five GLK gene-transfected plants increased by 18-39% and 10-127%, respectively, and the ribulose bisphosphate carboxylase (Rubisco) activity and protein content in flag leaves increased by 27-28% and 21-28%, respectively. Figure 3B ,3C , and 3D. These observations are consistent with increased chloroplast development and the expression of associated genes.

[0058] Obviously, all photosynthetic functions of these five transgenic rice lines were significantly enhanced, especially in the pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants.

[0059] Example 4: Light-saturated photosynthetic rate

[0060] Flag leaves were analyzed to measure photosynthesis because they contribute more to grain growth than other leaves. All five GLK transgenic plants showed a 5-11% increase in flag leaf photosynthesis compared to WT. Figure 4 The highest photosynthetic rate was observed in the pZmG1::ZmG1 / pZmG2::ZmG2 transgenic rice plants.

[0061] Example 5: Growth traits and grain yield

[0062] All five GLK transgenic plants had increased total shoot biomass (109-180%) and total ear number per plant (137-224%) compared to WT (100%), mainly due to the production of additional tillers. Figure 5A and 5B .

[0063] The total grain weight per plant of transgenic rice plants with pZmG1::ZmG1 and pZmG2::ZmG2 (heterogeneous combinations) increased by 51% and 47%, respectively, with a modest decrease in the weight of 1000 grains, i.e., 1-10%. Figure 5C and 5D .

[0064] Surprisingly, the total grain weight per plant was increased by up to 95% and by an average of 70% in the pZmG1::ZmG1 / pZmG2::ZmG2 rice plants. Figure 5C .

[0065] pZmUbi::ZmG1 plants did not show a significant increase in total grain yield due to a 15% reduction in grain weight. Thus, although the constitutive ubiquitin promoter increased biomass (see Figure 5A ), but it leads to reduced reproductive growth.

[0066] Without being bound by theory, it is believed that high expression of maize GLK1 driven by the strong maize ubiquitin promoter may inhibit reproductive growth and development.

[0067] Example 6: Transcriptome Analysis of Transgenic Rice Plants

[0068] Total RNA was isolated from shoots and roots of 4-day-old seedlings of WT, pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants as described previously. See Liu et al. 2013 (PNAS 110:3979-3984). Transcriptome analysis was also performed as described by Liu et al. (2013). The results are shown in Fig. 6A and 6B .

[0069] Up-regulated differentially expressed genes

[0070] pZmG2::ZmG2 shoots were significantly enriched in Gene Ontology (GO) annotation terms related to biotic / abiotic stimuli and chitin degradation processes (see Fig. 6A ), while the pZmG1::ZmG1 / pZmG2::ZmG2 branches were mainly enriched in the GO annotation terms of lipid transport ( Figure 6B ).

[0071] pZmG1::ZmG1 roots were significantly enriched in GO annotation terms related to transcriptional regulation, while pZmG2::ZmG2 roots were enriched in GO annotation terms related to biotic stress response, including oxylipin biosynthesis (methyl jasmonate biosynthesis pathway), chitin catabolism, and plant defensin metabolism. Apparently, in rice, both maize GLK genes stimulate chloroplast development, but ZmG1 acts more like a transcriptional regulator, while ZmG2 acts more like a defense regulator.

[0072] pZmG1::ZmG1 / pZmG2::ZmG2 roots were enriched in GO annotation terms related to transcriptional regulation (also found in pZmG1::ZmG1 roots), chitin catabolism / plant defensin biosynthesis (also found in pZmG2::ZmG2 roots), and pathways related to photosynthesis activation, gibenin (GA) metabolism, isoprenoid biosynthesis, xylan catabolism, and protein ubiquitination.

[0073] Together, these results suggest that the two maize GLK genes contribute to rice photosynthesis, metabolism, and stress responses in a complementary manner.

[0074] Down-regulation of differentially expressed genes

[0075] pZmG2::ZmG2 roots showed enrichment in down-regulation of genes involved in ion transport, amine biosynthesis, sulfur compound biosynthesis, and aspartic acid family amino acid biosynthesis, while pZmG1::ZmG1 / pZmG2::ZmG2 roots were mainly enriched in down-regulation of amine biosynthesis and ion transport. Fig. 6A and 6B This suggests that when ZmG1 and ZmG2 are co-expressed in rice, ZmG1 may partially counteract the repressive function of ZmG2. This is consistent with the observation that seedlings of pZmG2::ZmG2 plants showed stunted growth in both shoots and roots after germination, and that pZmG2::ZmG2 plants of the isogenic combination with high expression of the ZmG2 gene died after two months of growth.

[0076] Example 7: Materials and Methods

[0077] Promoter and gene selection

[0078] Genomic DNA was extracted from maize leaves (White Crystal, a waxy maize variety) using the cetyl trimethylammonium bromide method. Based on the sequences of ZmG1 (GRMZM2G026833) and ZmG2 (GRMZM2G087804) in the Ensembl plant database (available at plants.ensembl.org / index.html), the maize GLK1 promoter (ZmG1; primers SEQ ID NOs: 9 and 10 in Table 1 below) and G2 promoter (ZmG2; primers SEQ ID NOs: 11 and 12), both of which contain 5'-UTR regions, were cloned by polymerase chain reaction (PCR). Total RNA was extracted from maize embryonic leaves and used to synthesize cDNA as described by Liu et al., PNAS 110: 3979-3984 (2013). ZmG1 and ZmG2 cDNAs were cloned by PCR using specific primers (ZmG1: SEQ ID NOs: 13 and 14; ZmG2: SEQ ID NOs: 15 and 16) shown in Table 1. The sequences of the promoters and full-length cDNAs of ZmG1 and ZmG2 were confirmed by sequencing.

[0079] Table 1. Primer sequences

[0080]

[0081]

[0082] Construction of transformation vector

[0083] Five constructs were prepared to express ZmG1 and ZmG2 genes in rice under the control of maize or constitutive promoters. Maize promoters and full-length cDNAs of these two genes were cloned by PCR and subcloned into transformation vectors pCAMBIA or Geteway (Invitrogen). Specific primers (SEQ ID NOs: 17-24 in Table 1) were used to generate PCR fragments with compatible restriction sites. The 5' and 3' ends of the ZmG1 promoter and cDNA fragment were treated with XbaI / BamHI and BglII, respectively, and the 5' and 3' ends of the ZmG2 promoter and cDNA fragment were treated with HindIII / BamHI and BglII, respectively. The ZmG1 promoter (2134 bp; SEQ ID NO:1) fused to its cDNA (1428 bp; SEQ ID NO:2) and the ZmG2 promoter (1942 bp; SEQ ID NO:4) fused to its cDNA (1386 bp; SEQ ID NO:5) were ligated into an intermediate vector containing the nopaline synthase terminator to obtain ZmG1p::ZmG1 and ZmG2p::ZmG2 fragments, respectively. All fragments in the intermediate vector were confirmed by restriction endonuclease digestion analysis. Subsequently, the ZmG1p::ZmG1 fragment digested with EcoRI and the ZmG2p::ZmG2 fragment digested with HindIIII / EcoRI from the intermediate vector were ligated into the EcoRI and HindIIII / EcoRI sites of pCAMBIA1300 to generate pZmG1::ZmG1 and pZmG2::ZmG2 constructs. In addition, the ZmG1p::ZmG1 fragment cut with EcoRI was cloned into the same restriction enzyme site of the ZmG2p::ZmG2 vector to generate the pZmG1::ZmG1 / pZmG2::ZmG2 construct.

[0084] To express maize G1 and G2 in rice under the control of a constitutive promoter, Gateway entry clones containing ZmG1 or ZmG2 cDNA were transferred to the Gateway donor vector pCAMBIA1302 under the control of the maize ubiquitin promoter or pH2GW7 under the control of the 35S promoter to generate pZmUbi::ZmG1 and p35S::ZmG2 constructs, respectively.

[0085] The pZmUbi::ZmG1, p35S::ZmG2, pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1 / pZmG2::ZmG2 constructs containing the hygromycin phosphotransferase II gene for selection were transfected into Agrobacterium tumefaciens EHA105 strain by electroporation. All constructs were Figure 1 Displayed in chart form.

[0086] Rice transformation

[0087] Rice (Oryza sativa cv. TNG67) callus induction, co-cultivation with Agrobacterium, hygromycin selection of transformed callus, and plant regeneration were performed according to Yeh et al., 2015, Rice 8:36-48. All transgenic positive seedlings were transplanted to soil and subjected to molecular, physiological, and anatomical analyses in the greenhouse.

[0088] Determination of transgene insertion position by thermal asymmetric interlaced (TAIL)-PCR

[0089] TAIL-PCR conditions were as described by Liu et al. (2013) and et al. (2009). The genomic DNA of the T0 transgenic plants was used as template for three consecutive PCR runs using short arbitrary degenerate primers (AD; SEQ ID NOs: 28-30) and three specific nested primers (SP0; SEQ ID NO: 25, SP1; SEQ ID NO: 26 and SP2; SEQ ID NO: 27) to amplify the T-DNA flanking genomic DNA region. Tail-PCR products were purified and sequenced. The PCR product sequences were compared with the temperate japonica rice (Oryza sativa Japonica) group DNA sequences in Ensemble Plants (available at plants.ensembl.org / Multi / Tools / Blast?db=core) by BLAST to determine the insertion site of the transgene on the rice chromosome. The identified sequences were then used to evaluate the conjugation status of the transgenic plants.

[0090] Screening of transgenic plants with homologous genes by genotyping PCR

[0091] Genotyping primers were designed from the 3' and 5' end regions flanking the transgenic insertion site determined by Tail-PCR. Genome-specific primer pairs (GT-F and GT-R; SEQ ID NOs: 31-36) were designed based on the chromosomal DNA sequence across the assumed insertion site. In addition, transgenic-specific SP2 and genome-specific primers GT-R were used to screen transgenic plants with transgenic insertion. DNA was extracted from leaf tissues of T1 transgenic plants using Phire Plant Direct PCR Master Mix (ThermoScientific) according to the operating manual. In the PCR reaction, PCR amplification was performed with two primer sets (genome-specific GT-F and GT-R primers, and transgenic-specific SP2 and genome GT-R primers; Table 1). Due to the absence of transgenes, WT only produced one PCR product from the genome-specific primer pair. Transgenic plants of heterogeneous combinations produced two PCR products by (i) amplifying the undamaged DNA strand of the genomic sequence by the genome-specific primer pair and (ii) amplifying a strand of DNA with transgenes by SP2 and GT-R primers. In contrast, only one PCR product from SP2 and GT-R primers was seen in the isogenic transgenic plants because both DNA strands were disrupted by the transgene insertion at a specific site. After screening, genomic DNA was isolated from young leaves of plants of the positive isogenic combination to further confirm the presence of the maize GLK gene by PCR.

[0092] DNA extraction and PCR

[0093] To detect the maize GLK gene in transgenic rice plants, specific primers (see Table 1, SEQ ID NOs: 37-43) were used in PCR analysis. Genomic DNA was isolated from young leaves of WT and representative transgenic rice plants by the CTAB method described above. PCR amplification of the maize GLK gene was performed in a thermal cycler under the following conditions: 94°C / 5 minutes, 94°C / 30 seconds, 58°C / 30 seconds, and 72°C / 30 seconds for 30 cycles, and finally extension at 72°C / 5 minutes.

[0094] Southern blot analysis

[0095] Preparation of genomic DNA and Southern gel blot analysis were performed as previously described by Yeh et al. (2015). Genomic DNA was isolated from transgenic plants in which the presence of the maize GLK gene was confirmed by genomic PCR, digested with HindIII at 37°C for 16-18 hours, electrophoresed on a 1% agarose gel, and transferred to a nylon membrane for probe hybridization. Random primer DNA labeling was performed using the long-leaved foxglove primer-dUTP probe according to the instructions in the DIG High Prime DNA Labeling and Detection Starter Kit II (Roche).

[0096] RNA extraction and real-time quantitative PCR (qRT-PCR)

[0097] Total RNA was isolated from leaves of WT and transgenic plants using TRIZOL reagent (Invitrogen) and purified by acidic phenol-chloroform extraction. cDNA synthesis and qRT-PCR were performed as described by Yeh et al. Gene-specific primers for amplifying target genes (SEQ ID NOs: 44-57) and 17S genes (Accession No. X00755; SEQ ID NOs: 58 and 59) as internal controls for normalized expression values ​​are listed in Table 1.

[0098] Transcriptome analysis

[0099] Total RNA was isolated from the branches and roots of 4-day-old seedlings of WT, pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants using TRIZOL reagent (Invitrogen). Sample preparation and transcriptome analysis were completed using the method of Liu et al. Sequencing reads were processed using Tophat (version 2.0.10) and mapped to the rice genome (IRGSP-1.0). Each read was aligned, allowing up to 10 hits. Cufflinks (version 2.1.1) was used to evaluate the expression of each gene, i.e., reads per kilobase exon per million reads mapped, "RPKM"). Genes with RPKM≥1 in at least one sample were considered "expressed" and selected for further analysis. To compare the expression of selected genes in shoots and roots of WT, pZmG1::ZmG1, pZmG2::ZmG2, and pZmG1::ZmG1 / pZmG2::ZmG2 transgenic plants, the upper quartile normalization procedure described in Bullard et al., 2010, BMC Bioinform. 11:94 was used. The nonparametric method of Tarazona et al. (2011, Genome Research 21:2213-2223) was used to identify differentially expressed genes ("DEGs") between the two samples, with the q value (probability of differential expression) set to 0.740. Functional enrichment analysis was performed with the background set of all expressed genes in this study. Fisher's exact test with a false discovery rate ("FDR") < 0.05 was applied together with functional annotations in MapMan (see mapman.gabipd.org). For the functional classification of DEGs, GO analysis was performed using AgriGO V2 software with Fisher’s exact test, FDR ≤ 0.05, to obtain GO annotations based on biological programs.

[0100] GO analysis of DEGs

[0101] To classify the biological functions of DEGs, GO analysis was performed using AgriGO V2 software based on biological processes. Directed acyclic graph (DAG) drawing is a visualization tool used to illustrate important GO annotation entries. DAG indicates the interrelationships between annotation entries based on the nature of GO structure. To investigate the effect of GLK gene on each transgenic rice, up-regulated and down-regulated DEGs from roots and shoots were analyzed using a false discovery rate (FDR) of ≤0.05.

[0102] Protein purification and western blot analysis

[0103] Total soluble protein was extracted from newly mature leaves for SDS-PAGE and Western immunoassay analysis as previously described (see Dai et al., 1994, Planta 192:287-294 and Ku et al., 1999, Nat. Biotechnol. 17:76-80). Antibodies against the large subunit of maize Rubisco were prepared as described by Ku et al. In addition, a partial 681 bp sequence of OsGLK1 shared with the maize GLK gene (SEQ ID NO:7) was cloned into the pET21b vector to produce recombinant protein for the production of anti-GLK antibodies. Antibodies raised against the rice / maize GLK partial peptide were purified by affinity chromatography (Yao-Hong Biotechnology Inc., Taiwan, China).

[0104] Chlorophyll measurement and Rubisco assay

[0105] Leaf segments of flag leaves or florets were collected and extracted with 96% ethanol. Chlorophyll content was calculated from absorbance at wavelengths of 665 nm and 649 nm and expressed as leaf area or fresh weight. Rubisco activity was determined as previously described. See Pyankov et al., 2000, Photosynth. Res. 63: 69-84. Enzyme activity is based on chlorophyll content or leaf area.

[0106] Photosynthesis measurement

[0107] The midsection of the intact flag leaf on the main tiller of four-month-old rice plants grown in a greenhouse during summer flowering was used to measure CO2 and H2O exchange using a CIRAS2 portable photosynthesis system (PP Systems, Amesbury, MA). Measurements were performed between 7:00 and 12:30 a.m. on sunny days at 2000 μmol m -2 s -1Photon flux density, 30°C leaf temperature, 70% relative humidity, and 415 ppm CO2. Steady-state photosynthetic rate (Pn), stomatal conductance (Gs), intercellular carbon dioxide concentration (Ci), and evapotranspiration rate (E) were recorded.

[0108] Transmission Electron Microscope

[0109] Flag leaf samples (1mm x 2mm) were fixed in 0.1M phosphate-citrate buffer containing 1% glutaraldehyde, pH 7.2, 4°C overnight. After rinsing in the buffer 3 times for 20 minutes each time, the samples were fixed in the same buffer containing 1% OsO4 for 2 hours, then rinsed 3 times at room temperature for 20 minutes each time, and the buffer was changed. The samples were dehydrated in an acetone series, embedded in Spurr resin, and sliced ​​with a Lecia Reichert Ultracut S or Lecia EM UC7 ultrathin slicer. Ultrathin sections (70-90nm) were stained with 5% uranyl acetate / 50% methanol and 0.4% lead citrate / 0.1N NaOH. Observations were performed using a FEI G2 Tecnai Spirit Twin transmission electron microscope at 80KV, and images were recorded using a GatanOrius CCD camera.

[0110] Agronomic traits research

[0111] WT and GLK transgenic rice plants were grown in 5-liter pots (one plant / pot) in a greenhouse under natural sunlight between March and August. Plants were watered daily and fertilized weekly. After maturity, seeds were dried at 43°C and shoots at 60°C for two days, and then the total aboveground biomass, total ear number, 1000-grain weight, and total grain weight per plant were analyzed.

[0112] Other specific embodiments

[0113] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0114] From the above description, those skilled in the art can easily determine the essential features of the present invention, and can make various changes and modifications to the present invention to adapt to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, other specific embodiments are also within the scope of the appended claims. Sequence Listing <110> Liu Fudong <120> Ways to increase rice production <130> 218048-0285PCT <150> US 63 / 019,672 <151> 2020-05-04 <160> 63 <170> PatentIn version 3.5 <210> 1 <211> 2134 <212> DNA <213> Maize <220> <221> Promoter <222> (1)..(1972) <223> ZMG1 <220> <221> 5'UTR <222> (1973)..(2134) <223> ZMG1 <400> 1 ccaaccgctg catatcttgc acgcggaccg acgacatccg cgacacgcca aaatcaacaa 60 aatcgccggc gcgcaactcc tccgccatca aactactcag cgtctcctca gacgaggcgt 120 cggccgacgg tgaggcggcg gccggcggcg tagcggcagc ggaagaagag gaggtcggca 180 tcgctatgta ccgtcggcgg cggcgggcgg tctgcttcac tcgagccaga tctccggcga 240 acaagagcac ggcgggcaga cgagcagcaa ggcggtgaaa aaggcggcta gggttttcac 300 ggagcgcgga aagtgaagtt caaaaaccgc cgacccccgc ccccttttat agacagggcg 360 cggctcttcg ggaaacccgc aatccaacag ggcgcggcgc ttcgggaaac ccgcaatcca 420 atagtacgct gtcaatcaac ggtcatgtca aaaacccccg cggaaccact tcgagcgagg 480 gccgcgtctc cgccatctag cgacgtcttc ggcaccaggt gactttgtcg aactggtccc 540 tcggagggca aatgttgggg cgaaggcaaa gacgccaccc ttcgctcgag gccttcgctg 600 cagtcgctgg tctgacagag acaaaacggg cagggacacc cttcgctcga ctcggcacca 660 gacgaaggcc tgcgacggcg tcatcccaca gtgcggcctc gtccagctca gaggcccacg 720 tgtgatccgg cccagtgtaa cgggccctgc gtggccgccg cgtgttacgg gcctaatttg 780 taaaggcatc cctgtaatta cagtctgtaa ccctgcttta tgggaatatt ctggggataa 840 cctaggtagc tcagggcaca tgcgtcctta gcacaaggcg ctgggcgctc aggtacctat 900 aaataccctc gcacagtgac cgtgggaggc cagattaaca gagctattgc catctagcgc 960 gtaaccctat taacgtcact gttcaccatt gttggccccc cttgcgagtg agagcaagtc 1020 ccaacattat acatcactct tgtagtttca taaactacaa gagagacata taagttttat 1080 aaataaattt acttttattt ttcctataaa gaaataacca aaacaaaaat atacatctcg 1140 atgagttata caactttata gttgaaaact tttacagctc aattattttt tcttcaaaat 1200 ttgatttgca tagtgtaaaa aaaacattca gcgaatgatt tctctagctg tttgccgagt 1260 gttaaaaaac actcgccaaa atgtttcttt gctgagtgtt ttttagtcga gcgttttttg 1320 tttgacaatt agtaaagagt tttttttttg ttttgttgac tgtggaaaga aaaacaataa 1380 gcaaattatt taacgctcgg caaagggcca gaattgtgaa ctatatatac tgctccgtct 1440 cgaactaggc catcggagct tctgcgagac actgagaatc cacgaatcca gcagcctcca 1500 tgcatgacct ccaagctcaa cgcagacgtg aagttcaggg cagcactcca cgatgtcccg 1560 ttggggaaga cctaatagga ccgccagtag aaaacgtcac tgtactagta cagtctccag 1620 ccgatataca tgcagctgag ccgctgtcat atcttgttct cttcctgttg ttcgccaccg 1680 tctcacgaga tcgagtccac ctctctgccc agagttgcat caacgaaaag aaaaaggaaa 1740 aaaaaccggc ggttccaaaa aagactcggc cacacacagg cacacgctcg ctacgaggcc 1800 acaataattc gcagaagcga ccaagctgca cttgcgctcg cggacaaggc cacccatcta 1860 aaggattcag agagatcttt gcgaggcttg tggtcgagat gccttcgccg ttcgccacaa 1920 atactccccg gagagcgagt acttactggc tcgccatatg gcaccaacag caaaacagcg 1980 aaacagtgca gagacaagct acaacaacct accctaacag gccattcctt ccactgcact 2040 tcattcgatc ctgagctata gctggctgcc tgagctcgct ccaagaagtg tatctatagt 2100 atagacacta ggcttcgtgt ggcgtgctcg agat 2134 <210> 2 <211> 1428 <212> DNA <213> Zea mays <220> <221> CDS <222> (1)..(1428) <223> ZMG1 <400> 2 atg ctt gca gtg tcg ccg tcg ccg gtg cgg tgt gcc gat gcg gag gag 48 Met Leu Ala Val Ser Pro Ser Pro Val Arg Cys Ala Asp Ala Glu Glu 1 5 10 15 tgc ggc gga gga ggc gcc agc aag gaa atg gag gag acc gcc gtc ggg 96 Cys Gly Gly Gly Gly Ala Ser Lys Glu Met Glu Glu Thr Ala Val Gly 20 25 30 cct gtg tcc gac tcg gac ctg gat ttc gac ttc acg gtc gac gac ata 144 Pro Val Ser Asp Ser Asp Leu Asp Phe Asp Phe Thr Val Asp Asp Ile 35 40 45 gac ttc ggg gac ttc ttc ctc agg cta gac gac ggg gat gac gcg ctg 192 Asp Phe Gly Asp Phe Phe Leu Arg Leu Asp Asp Gly Asp Asp Ala Leu 50 55 60 ccg ggc ctc gag gtc gac cct gcc gag atc gtc ttc gct gac ttc gag 240 Pro Gly Leu Glu Val Asp Pro Ala Glu Ile Val Phe Ala Asp Phe Glu 65 70 75 80 gca atc gcc acc gcc ggc ggc gat ggc ggc gtc acg gac cag gag gtg 288 Ala Ile Ala Thr Ala Gly Gly Asp Gly Gly Val Thr Asp Gln Glu Val 85 90 95 ccc agt gtc ctg ccc ttt gcg gac gcg gcg cac ata ggc gcc gtg gat 336 Pro Ser Val Leu Pro Phe Ala Asp Ala Ala His Ile Gly Ala Val Asp 100 105 110 ccg tgt tgt ggt gtc ctt ggc gag gac aac gac gca gcg tgc gca gac 384 Pro Cys Cys Gly Val Leu Gly Glu Asp Asn Asp Ala Ala Cys Ala Asp 115 120 125 gtg gaa gaa ggg aaa ggg gag tgc gac cat gcc gac gag gta gca gcc 432 Val Glu Glu Gly Lys Gly Glu Cys Asp His Ala Asp Glu Val Ala Ala 130 135 140 gcc ggt aat aat aat agc gac tcc ggt gag gcc ggc tgt gga gga gcc 480 Ala Gly Asn Asn Asn Ser Asp Ser Gly Glu Ala Gly Cys Gly Gly Ala 145 150 155 160 ttt gcc ggc gaa aaa tca ccg tcg tcg acg gca tcg tcg tcg cag gag 528 Phe Ala Gly Glu Lys Ser Pro Ser Ser Thr Ala Ser Ser Ser Gln Glu 165 170 175 gct gag agc cgg cgc aag gtg tcc aag aag cac tcc caa ggg aag aag 576 Ala Glu Ser Arg Arg Lys Val Ser Lys Lys His Ser Gln Gly Lys Lys 180 185 190 aaa gca aag gtg gat tgg acg ccg gag ctt cac cgg aga ttc gtt cag 624 Lys Ala Lys Val Asp Trp Thr Pro Glu Leu His Arg Arg Phe Val Gln 195 200 205 gcg gtg gag gag ctg ggc atc gac aag gcg gtg ccg tcc agg atc ctc 672 Ala Val Glu Glu Leu Gly Ile Asp Lys Ala Val Pro Ser Arg Ile Leu 210 215 220 gag atc atg ggg atc gac tcc ctc acg cgg cat aac ata gcc agc cat 720 Glu Ile Met Gly Ile Asp Ser Leu Thr Arg His Asn Ile Ala Ser His 225 230 235 240 ctg cag aag tac cgt tcc cac agg aag cac atg ctt gcg agg gag gtg 768 Leu Gln Lys Tyr Arg Ser His Arg Lys His Met Leu Ala Arg Glu Val 245 250 255 gag gca gcg acg tgg acg acg cac cgg cgg ccg atg tac gct gcc ccc 816 Glu Ala Ala Thr Trp Thr Thr His Arg Arg Pro Met Tyr Ala Ala Pro 260 265 270 agc ggc gcc gtg aag agg ccc gac tct aac gcg tgg acc gtg ccg acc 864 Ser Gly Ala Val Lys Arg Pro Asp Ser Asn Ala Trp Thr Val Pro Thr 275 280 285 atc ggt ttc cct ccg ccg gcg ggg acc cct cct cgt ccg gtg cag cac 912 Ile Gly Phe Pro Pro Pro Ala Gly Thr Pro Pro Arg Pro Val Gln His 290 295 300 ttc ggg agg cca ctg cac gtc tgg ggc cat ccg agt ccg acg cca gcg 960 Phe Gly Arg Pro Leu His Val Trp Gly His Pro Ser Pro Thr Pro Ala 305 310 315 320 gtg gag tca ccc cgg gtg cca atg tgg cct cgg cat ctc gcc ccc cgc 1008 Val Glu Ser Pro Arg Val Pro Met Trp Pro Arg His Leu Ala Pro Arg 325 330 335 gcc ccg ccg ccg ccg ccg tgg gct ccg cca ccg cca gct gac ccg gcg 1056 Ala Pro Pro Pro Pro Pro Trp Ala Pro Pro Pro Pro Ala Asp Pro Ala 340 345 350 tcg ttc tgg cac cat gct tac atg agg ggg cct gct gcc cat atg cca 1104 Ser Phe Trp His His Ala Tyr Met Arg Gly Pro Ala Ala His Met Pro 355 360 365 gac cag gtg gcg gtg act cca tgc gtg gca gtg cca atg gca gca gcg 1152 Asp Gln Val Ala Val Thr Pro Cys Val Ala Val Pro Met Ala Ala Ala 370 375 380 cgt ttc cct gct ca cac gtg agg ggt tct ttg ca tgg ca cct cct 1200 Arg Phe Pro Ala Pro His Val Arg Gly Ser Leu Pro Trp Pro Pro Pro 385,390,395,400 atg tac aga cct ctc gtt cct cca gca ctc gca ggc aag agc cag caa 1248 Put Tyr Arg Pro Leu Val Pro Pro Ala Leu Ala Gly Lys Ser Gln Gln 405 410 415 gac gcg ctg ttt cag cta cag ata cag cca tca agc gag agc ata gat 1296 Asp Ala Leu Phe Gln Leu Gln Ile Gln Pro Ser Ser Glu Ser Ile Asp 420 425 430 gca gca ata ggt gat gtc tta acg aag ccg tgg ctg cg ctg ccc cctc 1344 Ala Ala Ile Gly Asp Val Leu Thr Lys Pro Trp Leu Pro Leu Pro Leu 435 440 445 gga ctg aag ccc cct tcg gta gac agt gtc atg ggc gag ctg cag agg 1392 Gly Leu Lys Pro Pro Ser Val Asp Ser Val Met Gly Glu Leu Gln Arg 450 455 460 caa ggc gta gcg aat gtg ccg caa gct tgt gga tga 1428 Gln Gly Val Ala Asn Val Pro Gln Ala Cys Gly 465 470 475 <210> 3 <211> 475 <212> PRT <213> Maize <400> 3 Met Leu Ala Val Ser Pro Ser Pro Val Arg Cys Ala Asp Ala Glu Glu 1 5 10 15 Cys Gly Gly Gly Gly Ala Ser Lys Glu Met Glu Glu Thr Ala Val Gly 20 25 30 Pro Val Ser Asp Ser Asp Leu Asp Phe Asp Phe Thr Val Asp Asp Ile 35 40 45 Asp Phe Gly Asp Phe Phe Leu Arg Leu Asp Asp Gly Asp Asp Ala Leu 50 55 60 Pro Gly Leu Glu Val Asp Pro Ala Glu Ile Val Phe Ala Asp Phe Glu 65 70 75 80 Ala Ile Ala Thr Ala Gly Gly Asp Gly Gly Val Thr Asp Gln Glu Val 85 90 95 Pro Ser Val Leu Pro Phe Ala Asp Ala Ala His Ile Gly Ala Val Asp 100 105 110 Pro Cys Cys Gly Val Leu Gly Glu Asp Asn Asp Ala Ala Cys Ala Asp 115 120 125 Val Glu Glu Gly Lys Gly Glu Cys Asp His Ala Asp Glu Val Ala Ala 130 135 140 Ala Gly Asn Asn Asn Ser Asp Ser Gly Glu Ala Gly Cys Gly Gly Ala 145 150 155 160 Phe Ala Gly Glu Lys Ser Pro Ser Ser Thr Ala Ser Ser Ser Gln Glu 165 170 175 Ala Glu Ser Arg Arg Lys Val Ser Lys Lys His Ser Gln Gly Lys Lys 180 185 190 Lys Ala Lys Val Asp Trp Thr Pro Glu Leu His Arg Arg Phe Val Gln 195 200 205 Ala Val Glu Glu Leu Gly Ile Asp Lys Ala Val Pro Ser Arg Ile Leu 210 215 220 Glu Ile Met Gly Ile Asp Ser Leu Thr Arg His Asn Ile Ala Ser His 225 230 235 240 Leu Gln Lys Tyr Arg Ser His Arg Lys His Met Leu Ala Arg Glu Val 245 250 255 Glu Ala Ala Thr Trp Thr Thr His Arg Arg Pro Met Tyr Ala Ala Pro 260 265 270 Ser Gly Ala Val Lys Arg Pro Asp Ser Asn Ala Trp Thr Val Pro Thr 275 280 285 Ile Gly Phe Pro Pro Pro Ala Gly Thr Pro Pro Arg Pro Val Gln His 290 295 300 Phe Gly Arg Pro Leu His Val Trp Gly His Pro Ser Pro Thr Pro Ala 305 310 315 320 Val Glu Ser Pro Arg Val Pro Met Trp Pro Arg His Leu Ala Pro Arg 325 330 335 Ala Pro Pro Pro Pro Trp Ala Pro Pro Pro Pro Ala Asp Pro Ala 340 345 350 Ser Phe Trp His His Ala Tyr Met Arg Gly Pro Ala Ala His Met Pro 355 360 365 Asp Gln Val Ala Val Thr Pro Cys Val Ala Val Pro Met Ala Ala Ala 370 375 380 Arg Phe Pro Ala Pro His Val Arg Gly Ser Leu Pro Trp Pro Pro Pro 385 390 395 400 Met Tyr Arg Pro Leu Val Pro Pro Ala Leu Ala Gly Lys Ser Gln Gln 405 410 415 Asp Ala Leu Phe Gln Leu Gln Ile Gln Pro Ser Ser Glu Ser Ile Asp 420 425 430 Ala Ala Ile Gly Asp Val Leu Thr Lys Pro Trp Leu Pro Leu Pro Leu 435 440 445 Gly Leu Lys Pro Pro Ser Val Asp Ser Val Met Gly Glu Leu Gln Arg 450 455 460 Gln Gly Val Ala Asn Val Pro Gln Ala Cys Gly 465 470 475 <210> 4 <211> 1942 <212> DNA <213> Maize <220> <221> Promoter <222> (1)..(1288) <223> ZMG2 <220> <221> 5'UTR <222> (1289)..(1942) <223> ZMG2 <400> 4 aagcttcagg ttattggata aggtgaggaa aattccatat ttctgaaaaa ccgatgatca 60 aagttagtcc aagtgtctag gtcgcggatc gttcgcgata gcatcggacc gtgcaagcaa 120 agacaccgga ctgttcgacg gcatcagaaa tgtcgcctta gttgattcgg ctaacaatgc 180 ggccgatgca gttgattgga gaatgtcttt gattgtatac ctgcataatc caggtgttag 240 gacagacatg atcatttgac agatagattt caagtatgtt ttaatcgatg atgaacttta 300 tcgctgaact tctagtgatg gcttgcttaa ttgcttgggc cctaacgatg ctttattagc 360 tatggacaaa gtaaatgaag gaatttgtgg tactcatcaa tcggctctaa agatgaagtg 420 gctcttaagg tggtctatgg tttattagcc tgatatgata tcgattgttt taaatactag 480 aaagggtgtg aaatatgtca ggagttcggc gatctgcaat tagttctgag ccccgcgtgc 540 ggaccgtccg gtgtgacacg gagaagaccc gctcatgcag cgagatcgcg gaccgtccgc 600 gctcccgcag agagcaccgc caggagatac atccctaatg tttggctcca aatcggagcc 660 aacactaccc tacatgttct aaattacaat gtgttttagt atttttttta ttgtatagtt 720 tttattatat aacttattat aagtagatga atagtaaaat ctgttggaat atgactctaa 780 tttattttat atatttggaa tatgtcttga aatatgcttt catgtacgcc tataaatata 840 gatctttcat gtaacttgta ttctgcggtt gatagttgat ctgtttcctc gtggtcgttt 900 gctcctccat attggaggga attttcgcat gtaaatcttt gtgtctattt tatttcgtaa 960 caaaatctat agattctaaa aagctaaaac ttattatagt ttagaacgga ggggcagtgg 1020 atataggccg agtggtatgg caccgtaact gtaaataaca tgcatgcatg gagacgacga 1080 cgcatgtaga tatatatcg tagtggggtt ccggaacaat atacaaaaaa catatatgcg 1140 tgtatttaat tattatttgt ggcggctggt gcatgtgaac tactggaata ataatggtga 1200 aattgaggag ggtgcactga ctgagatacg gtagacatgc atagataagc actgcggagc 1260 catgctgcct gcctgcaata atgcttgtgt gtatacagcg acacgacacg agacgagagg 1320 gagggtgaac tgaaagagag acccggctag agctataaag caagtggggg aagagagaga 1380 gagaggagaa ggagaaggag aggtttttat gtatgtgtgg tggacatgcg tcctcctgtc 1440 gtctcgagag agcggaggcc ctaatactcc aatcaccacg caccaaacta gctagctagc 1500 agtcgtcttg cattgtagct agtccattgc tctcatctttc tctctttctt cccccagtcc 1560 tccccctt tcccccttc ggccctctc gctcagctca ctcttcatta agcgagctca 1620 cgtcgttcct cccttcttct tctcttatc cgttgttcaa ttcgttcagc tagccggcca 1680 gagatcgagc atcatctcca tcatcgattc atccatctca tctttctctt cttctattct 1740 atgtcgtcgt cgtcccagat tagatcgaag ctgctagcag tctatccagt ctctagctag 1800 ctagctagat caagcccgca gactatacaa tataatacaa gctagctacg tgcttattat 1860 tgctttatta gtctagaata atcttgatat atacgatcga acatatatct cgatctccac 1920 cgatacacac ccggccctat cc 1942 <210> 5 <211> 1386 <212> DNA <213> Maize <220> <221> CDS <222> (1)..(1386) <223> ZMG2 <400> 5 atg ctt gag gtg tcg acg ctg cgc ggc cct act agc agc ggc agc aag 48 Met Leu Glu Val Ser Thr Leu Arg Gly Pro Thr Ser Ser Gly Ser Lys 1 5 10 15 gcg gag cag cac tgc ggc ggc ggc ggc ggc ttc gtc ggc gac cac cat 96 Ala Glu Gln His Cys Gly Gly Gly Gly Gly Phe Val Gly Asp His His 20 25 30 gtg gtg ttc ccg acg tcc ggc gac tgc ttc gcc atg gtg gac gac aac 144 Val Val Phe Pro Thr Ser Gly Asp Cys Phe Ala Met Val Asp Asp Asn 35 40 45 ctc ctg gac tac atc gac ttc agc tgc gac gtg ccc ttc ttc gac gct 192 Leu Leu Asp Tyr Ile Asp Phe Ser Cys Asp Val Pro Phe Phe Asp Ala 50 55 60 gac ggg gac atc ctc ccc gac ctg gag gta gac acc acg gag ctc ctc 240 Asp Gly Asp Ile Leu Pro Asp Leu Glu Val Asp Thr Thr Glu Leu Leu 65 70 75 80 gcc gag ttc tcg tcc acc cct cct gcg gac gac ctg ctg gca gtg gca 288 Ala Glu Phe Ser Ser Thr Pro Pro Ala Asp Asp Leu Leu Ala Val Ala 85 90 95 gta ttc ggc gcc gac gac cag ccg gcg gcg gca gta gca caa gag aag 336 Val Phe Gly Ala Asp Asp Gln Pro Ala Ala Ala Val Ala Gln Glu Lys 100 105 110 ccg tcg tcg tcg ttg gag caa aca tgt ggt gac gac aaa ggt gta gca 384 Pro Ser Ser Ser Leu Glu Gln Thr Cys Gly Asp Asp Lys Gly Val Ala 115 120 125 gta gcc gcc gcc aga aga aag ctg cag acg acg acg acg acg acg acg 432 Val Ala Ala Ala Arg Arg Lys Leu Gln Thr Thr Thr Thr Thr Thr Thr 130 135 140 acg gag gag gag gat tct tct cct gcc ggg tcc ggg gcc aac aag tcg 480 Thr Glu Glu Glu Asp Ser Ser Pro Ala Gly Ser Gly Ala Asn Lys Ser 145 150 155 160 tcg gcg tcg gca gag ggc cac agc agc aag aag aag tcg gcg ggc aag 528 Ser Ala Ser Ala Glu Gly His Ser Ser Lys Lys Lys Ser Ala Gly Lys 165 170 175 aac tcc aac ggc ggc aag cgc aag gtg aag gtg gac tgg acg ccg gag 576 Asn Ser Asn Gly Gly Lys Arg Lys Val Lys Val Asp Trp Thr Pro Glu 180 185 190 ctg cac cgg cgg ttc gtg cag gcg gtg gag cag ctg ggc atc gac aag 624 Leu His Arg Arg Phe Val Gln Ala Val Glu Gln Leu Gly Ile Asp Lys 195 200 205 gcc gtg ccg tcc agg atc ctg gag atc atg ggc acg gac tgc ctc aca 672 Ala Val Pro Ser Arg Ile Leu Glu Ile Met Gly Thr Asp Cys Leu Thr 210 215 220 agg cac aac att gcc agc cac ctc cag aag tac cgg tcg cac aga aag 720 Arg His Asn Ile Ala Ser His Leu Gln Lys Tyr Arg Ser His Arg Lys 225 230 235 240 cac ctg atg gcg cgg gag gcg gag gcc gcc acc tgg gcg cag aag cgc 768 His Leu Met Ala Arg Glu Ala Glu Ala Ala Thr Trp Ala Gln Lys Arg 245 250 255 cac atg tac gcg ccg cca gct cca agg acg acg acg acg acg gac gcc 816 His Met Tyr Ala Pro Pro Ala Pro Arg Thr Thr Thr Thr Thr Asp Ala 260 265 270 gcc agg ccg ccg tgg gtg gtg ccg acg acc atc ggg ttc ccg ccg ccg 864 Ala Arg Pro Pro Trp Val Val Pro Thr Thr Ile Gly Phe Pro Pro Pro 275 280 285 cgc ttc tgc cgc ccg ctg cac gtg tgg ggc cac ccg ccg ccg cac gcc 912 Arg Phe Cys Arg Pro Leu His Val Trp Gly His Pro Pro Pro His Ala 290 295 300 gcc gcg gct gaa gca gca gcg gcg act ccc atg ctg ccc gtg tgg ccg 960 Ala Ala Ala Glu Ala Ala Ala Ala Thr Pro Met Leu Pro Val Trp Pro 305 310 315 320 cgt cac ctg gcg ccg ccc cgg cac ctg gcg ccg tgg gcg cac ccg acg 1008 Arg His Leu Ala Pro Pro Arg His Leu Ala Pro Trp Ala His Pro Thr 325 330 335 ccg gtg gac ccg gcg ttc tgg cac cag cag tac agc gct gcc agg aaa 1056 Pro Val Asp Pro Ala Phe Trp His Gln Gln Tyr Ser Ala Ala Arg Lys 340 345 350 tgg ggc cca cag gca gcc gcc gtg acg caa ggg acg cca tgc gtg ccg 1104 Trp Gly Pro Gln Ala Ala Ala Val Thr Gln Gly Thr Pro Cys Val Pro 355 360 365 ctg ccg agg ttt ccg gtg cct cac ccc atc tac agc aga ccg gcg atg 1152 Leu Pro Arg Phe Pro Val Pro His Pro Ile Tyr Ser Arg Pro Ala Met 370 375 380 gta cct ccg ccg cca agc acc acc aag cta gct caa ctg cat ctg gag 1200 Val Pro Pro Pro Pro Ser Thr Thr Lys Leu Ala Gln Leu His Leu Glu 385 390 395 400 ctc caa gcg cac ccg tcc aag gag agc atc gac gca gcc atc gga gat 1248 Leu Gln Ala His Pro Ser Lys Glu Ser Ile Asp Ala Ala Ile Gly Asp 405 410 415 gtt tta gtg aag cca tgg ctg ccg ctt cca ctg ggg ctc aag ccg ccg 1296 Val Leu Val Lys Pro Trp Leu Pro Leu Pro Leu Gly Leu Lys Pro Pro 420 425 430 tcg ctc gac agc gtc atg tcg gag ctg cac aag caa ggc gta cca aaa 1344 Ser Leu Asp Ser Val Met Ser Glu Leu His Lys Gln Gly Val Pro Lys 435 440 445 atc cca ccg gcg gct gcc acc acc acc ggc gcc acc gga tga 1386 Ile Pro Pro Ala Ala Ala Thr Thr Thr Gly Ala Thr Gly 450 455 460 <210> 6 <211> 461 <212> PRT <213> Maize <400> 6 Met Leu Glu Val Ser Thr Leu Arg Gly Pro Thr Ser Ser Gly Ser Lys 1 5 10 15 Ala Glu Gln His Cys Gly Gly Gly Gly Gly Phe Val Gly Asp His His 20 25 30 Val Val Phe Pro Thr Ser Gly Asp Cys Phe Ala Met Val Asp Asp Asn 35 40 45 Leu Leu Asp Tyr Ile Asp Phe Ser Cys Asp Val Pro Phe Phe Asp Ala 50 55 60 Asp Gly Asp Ile Leu Pro Asp Leu Glu Val Asp Thr Thr Glu Leu Leu 65 70 75 80 Ala Glu Phe Ser Ser Thr Pro Pro Ala Asp Asp Leu Leu Ala Val Ala 85 90 95 Val Phe Gly Ala Asp Asp Gln Pro Ala Ala Ala Val Ala Gln Glu Lys 100 105 110 Pro Ser Ser Ser Leu Glu Gln Thr Cys Gly Asp Asp Lys Gly Val Ala 115 120 125 Val Ala Ala Ala Arg Arg Lys Leu Gln Thr Thr Thr Thr Thr Thr Thr 130 135 140 Thr Glu Glu Glu Asp Ser Ser Pro Ala Gly Ser Gly Ala Asn Lys Ser 145 150 155 160 Ser Ala Ser Ala Glu Gly His Ser Ser Lys Lys Lys Ser Ala Gly Lys 165 170 175 Asn Ser Asn Gly Gly Lys Arg Lys Val Lys Val Asp Trp Thr Pro Glu 180 185 190 Leu His Arg Arg Phe Val Gln Ala Val Glu Gln Leu Gly Ile Asp Lys 195 200 205 Ala Val Pro Ser Arg Ile Leu Glu Ile Met Gly Thr Asp Cys Leu Thr 210 215 220 Arg His Asn Ile Ala Ser His Leu Gln Lys Tyr Arg Ser His Arg Lys 225 230 235 240 His Leu Met Ala Arg Glu Ala Glu Ala Ala Thr Trp Ala Gln Lys Arg 245 250 255 His Met Tyr Ala Pro Pro Ala Pro Arg Thr Thr Thr Thr Thr Asp Ala 260 265 270 Ala Arg Pro Pro Trp Val Val Pro Thr Thr Ile Gly Phe Pro Pro Pro 275 280 285 Arg Phe Cys Arg Pro Leu His Val Trp Gly His Pro Pro Pro His Ala 290 295 300 Ala Ala Ala Glu Ala Ala Ala Ala Thr Pro Met Leu Pro Val Trp Pro 305 310 315 320 Arg His Leu Ala Pro Pro Arg His Leu Ala Pro Trp Ala His Pro Thr 325 330 335 Pro Val Asp Pro Ala Phe Trp His Gln Gln Tyr Ser Ala Ala Arg Lys 340 345 350 Trp Gly Pro Gln Ala Ala Ala Val Thr Gln Gly Thr Pro Cys Val Pro 355 360 365 Leu Pro Arg Phe Pro Val Pro His Pro Ile Tyr Ser Arg Pro Ala Met 370 375 380 Val Pro Pro Pro Pro Ser Thr Thr Lys Leu Ala Gln Leu His Leu Glu 385 390 395 400 Leu Gln Ala His Pro Ser Lys Glu Ser Ile Asp Ala Ala Ile Gly Asp 405 410 415 Val Leu Val Lys Pro Trp Leu Pro Leu Pro Leu Gly Leu Lys Pro Pro 420 425 430 Ser Leu Asp Ser Val Met Ser Glu Leu His Lys Gln Gly Val Pro Lys 435 440 445 Ile Pro Pro Ala Ala Ala Thr Thr Thr Gly Ala Thr Gly 450 455 460 <210> 7 <211> 681 <212> DNA <213> Rice <220> <221> misc_feature <223> Partial sequence of OsGLK1 gene <400> 7 gcgaaggtgg actggacgcc tgagcttcac cggaggttcg tgcaggcggt ggagcagctc 60 ggcatcgaca aggccgtgcc gtcgaggata cttgagatca tggggatcga ctctctcacc 120 cggcacaaca tagccagcca tcttcagaag taccggtcac acagaaaaca catgattgcg 180 agagaggcgg aggcagcgag ttggacccaa cggcggcaga tttacgccgc cggtggaggt 240 gctgttgcga agaggccgga gtccaacgcg tggaccgtgc caaccattgg cttccctcct 300 cctccgccac caccaccatc accggctccg attcaacatt ttgctcgccc gttgcatgtt 360 tggggccacc cgacgatgga cccgtcccga gttccagtgt ggccaccgcg gcacctcgtt 420 ccccgtggcc cggcgccacc atgggttcca ccgccgccgc cgtcggaccc tgctttctgg 480 caccaccctt acatgagggg gccagcacat gtgccaactc aagggacacc ttgcatggcg 540 atgcccatgc cagctgcgag atttcctgct ccaccggtgc caggagttgt cccgtgtcca 600 atgtataggc cattgactcc accagcactg gcgagcaaga atcagcagga cgcacagctt 660 caactccagg ttcaaccatc a 681 <210> 8 <211> 227 <212> PRT <213> Rice <220> <221> misc_feature <223> OsGLK1 gene partial protein sequence <400> 8 Ala Lys Val Asp Trp Thr Pro Glu Leu His Arg Arg Phe Val Gln Ala 1 5 10 15 Val Glu Gln Leu Gly Ile Asp Lys Ala Val Pro Ser Arg Ile Leu Glu 20 25 30 Ile Met Gly Ile Asp Ser Leu Thr Arg His Asn Ile Ala Ser His Leu 35 40 45 Gln Lys Tyr Arg Ser His Arg Lys His Met Ile Ala Arg Glu Ala Glu 50 55 60 Ala Ala Ser Trp Thr Gln Arg Arg Gln Ile Tyr Ala Ala Gly Gly Gly 65 70 75 80 Ala Val Ala Lys Arg Pro Glu Ser Asn Ala Trp Thr Val Pro Thr Ile 85 90 95 Gly Phe Pro Pro Pro Pro Pro Pro Pro Pro Ser Pro Ala Pro Ile Gln 100 105 110 His Phe Ala Arg Pro Leu His Val Trp Gly His Pro Thr Met Asp Pro 115 120 125 Ser Arg Val Pro Val Trp Pro Pro Arg His Leu Val Pro Arg Gly Pro 130 135 140 Ala Pro Pro Trp Val Pro Pro Pro Pro Pro Ser Asp Pro Ala Phe Trp 145 150 155 160 His His Pro Tyr Met Arg Gly Pro Ala His Val Pro Thr Gln Gly Thr 165 170 175 Pro Cys Met Ala Met Pro Met Pro Ala Ala Arg Phe Pro Ala Pro Pro 180 185 190 Val Pro Gly Val Val Pro Cys Pro Met Tyr Arg Pro Leu Thr Pro Pro 195 200 205 Ala Leu Ala Ser Lys Asn Gln Gln Asp Ala Gln Leu Gln Leu Gln Val 210 215 220 Gln Pro Ser 225 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <220> <223> G1 Promoter_F <400> 9 ccaaccctgc atatcttgc 19 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G1 promoter_R <400> 10 ggcgacactg caagcatatc 20 <210> 11 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G2 Promoter_F <400> 11 gctatctcca accacggctc 20 <210> 12 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G2 promoter_R <400> 12 cttgctgccg ctgctagtag 20 <210> 13 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G1 cDNA_F <400> 13 atgcttgcag tgtcgccgtc 20 <210> 14 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> G1 cDNA_R <400> 14 tcatccacaa gcttgcggca c 21 <210> 15 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> G2 cDNA_F <400> 15 atgcttgagg tgtcgacgct gcgc 24 <210> 16 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G2 cDNA_R <400> 16 tcatccggtg gcgccggtgg 20 <210> 17 <211> 27 <212> DNA <213> Artificial sequence <220> <223> G1p_F (XbaI) <400> 17 cgtctagacc aaccgctgca tatcttg 27 <210> 18 <211> 26 <212> DNA <213> Artificial sequence <220> <223> G1p_R (BhI) <400> 18 caggatccgg cgacactgca agcata 26 <210> 19 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G2p_mF (HdIII) <400> 19 tggcttggca catgaagctt 20 <210> 20 <211> 26 <212> DNA <213> Artificial sequence <220> <223> G2p_R (BhI) <400> 20 caggatccac ttgctgccgc tgctag 26 <210> twenty one <211> twenty three <212> DNA <213> Artificial sequence <220> <223> G1_cDNA_F (BglII) <400> twenty one ggagatctat gcttgcagtg tcg 23 <210> twenty two <211> 25 <212> DNA <213> Artificial sequence <220> <223> G1_cDNA_R (BglII) <400> twenty two ggagatcttc atccacaagc ttgcg 25 <210> twenty three <211> twenty three <212> DNA <213> Artificial sequence <220> <223> G2_cDNA_F (BglII) <400> twenty three ggagatctat gcttgaggtg tcg 23 <210> twenty four <211> twenty three <212> DNA <213> Artificial sequence <220> <223> G2_cDNA_F (BglII) <400> twenty four atagatcttc atccggtggc gcc 23 <210> 25 <211> 26 <212> DNA <213> Artificial sequence <220> <223> SP0 <400> 25 tcgagtttct ccataataat gtgtga 26 <210> 26 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> SP1 <400> 26 taatgtgtga gtagttccca gata 24 <210> 27 <211> twenty four <212> DNA <213> Artificial sequence <220> <223> SP2 <400> 27 aatccagtac taaaatccag atcc 24 <210> 28 <211> 16 <212> DNA <213> Artificial sequence <220> <223> AD1 <220> <221> Other features <222> (3) <223> n is a, c, g, or t <220> <221> Other features <222> (11)..(11) <223> n is a, c, g, or t <400> 28 gtncgaswca nawgtt 16 <210> 29 <211> 15 <212> DNA <213> Artificial sequence <220> <223> AD2 <220> <221> Other features <222> (4) <223> n is a, c, g, or t <220> <221> Other features <222> (9)..(9) <223> n is a, c, g, or t <400> 29 gwgnagwanc asaga 15 <210> 30 <211> 15 <212> DNA <213> Artificial sequence <220> <223> AD3 <220> <221> Other features <222> (1) <223> n is a, c, g, or t <400> 30 ntcgastwts gwgtt 15 <210> 31 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> GT_F_G1 <400> 31 cgatcgtgtt ggcacgtgat g 21 <210> 32 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> GT_R_G1 <400> 32 tcgaagagtc aaccttcgag gtc 23 <210> 33 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> GT_F_G2 <400> 33 gccttggagg aagtagaaca gc 22 <210> 34 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> GT_R_G2 <400> 34 gtaggtttgg gaaatctttc agc 23 <210> 35 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> GT_F_G1G2 <400> 35 gtgaccggaa tccctctcaa g 21 <210> 36 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> GT_R_G1G2 <400> 36 gttgcactgt gtgtactagt c 21 <210> 37 <211> 19 <212> DNA <213> Artificial sequence <220> <223> G1p_F <400> 37 ccaaccgctg catatcttg 19 <210> 38 <211> 18 <212> DNA <213> Artificial sequence <220> <223> G1p_R <400> 38 ggcgacactg caagcata 18 <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> G2p_F <400> 39 tggcttggca catgaagctt 20 <210> 40 <211> 19 <212> DNA <213> Artificial sequence <220> <223> G2p_R <400> 40 cacttgctgc cgctgctag 19 <210> 41 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> G1_mF <400> 41 gtcgttctgg caccatgctt ac 22 <210> 42 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> G2_F <400> 42 cgcacagaaa gcacctgatg g 21 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> NOS_R <400> 43 tcatcgcaag accggcaaca 20 <210> 44 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> G1-F1 qPCR <400> 44 gcacataggc gccgtggatc c 21 <210> 45 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> G1-R1 qPCR <400> 45 ttaccggcgg ctgctacctc g 21 <210> 46 <211> 26 <212> DNA <213> Artificial sequence <220> <223> G2-F qPCR <400> 46 gacaaaggtg tagcagtagc cgccgc 26 <210> 47 <211> 26 <212> DNA <213> Artificial sequence <220> <223> G2-R qPCR <400> 47 cgttggagtt cttgcccgcc gacttc 26 <210> 48 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> OsGLK1-F qPCR <400> 48 gatgctggca tggaggtcgt c 21 <210> 49 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> OsGLK1-R qPCR <400> 49 tgcacacctt cgcctccact c 21 <210> 50 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> OsGLK2-F qPCR <400> 50 gtgacgacgg aggattcctc g 21 <210> 51 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> OsGLK2-R qPCR <400> 51 ccgatgacga cgacgacgac g 21 <210> 52 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> OsFRD3_F_exon 1 <400> 52 cgcgctgtcc aaggcctatc tct 23 <210> 53 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> OsFRD3_R_exon 1 <400> 53 gcaacagcat ggggagcatt gg 22 <210> 54 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> OsFRD3_F_exon 6 <400> 54 ggcttggcat aactggagct gc 22 <210> 55 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> OsFRD3_R_exon 6-7 <400> 55 aactcaaagc cccctgggag ag 22 <210> 56 <211> 25 <212> DNA <213> Artificial sequence <220> <223> OsFRD3_F_exon 7 <400> 56 ggtggcatgc tgttaggaag aaccc 25 <210> 57 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> OsFRD3_R_exon 7 <400> 57 gccatagctg ttgggccttg tcg 23 <210> 58 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> 17S rRNA_F <400> 58 gataactcga cggatcgcac gg 22 <210> 59 <211> twenty three <212> DNA <213> Artificial sequence <220> <223> 17S rRNA_R <400> 59 gcctgctgcc ttccttggat gtg <210> 60 <211> 22 <212> DNA <213> The snowstorm <220> <223> OsActin_F <400> 60 atccttgtat gctagcggtc 22 times <210> 61 <211> 21 <212> DNA <213> The snowstorm <220> <223> OsActin_R <400> 61 atccaaccgg snow g <210> 62 <211> 21 <212> DNA <213> The snowstorm <220> <223> HptII_F <400> 62 gacctgatgc agctctcgga g <210> 63 <211> 22 <212> DNA <213> The snowstorm <220> <223> HptII_R <400> 63 tgctccatac aagccacca cg

Claims

1. A method for increasing rice yield, the method comprising: A recombinant DNA construct is introduced into a host rice plant, the construct comprising a first nucleic acid sequence comprising a first Golden 2-like transcription factor ( GLK ) gene, and a second nucleic acid sequence comprising a second nucleic acid sequence operably linked to its native promoter and 5'UTR GLK Gene, the second GLK Genetically different from the first GLK Gene, the first GLK Gene and the second GLK The gene is heterologous; and Identification of transgenic first GLK Gene and second GLK Compared with wild-type rice plants that have not been transformed, the first transgenic rice plants that have been transformed alone GLK Gene or single transfer second GLK Genetically modified rice plants, the first GLK Gene and second GLK Genetically modified rice plants showed increased shoot biomass and grain yield; The first nucleic acid sequence comprises a first coding sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 3, and the second nucleic acid sequence comprises a second coding sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO:

6.

2. The method of claim 1, wherein the first GLK The natural promoter and 5'UTR of the gene have the sequence shown in SEQ ID NO: 1, and the second GLK The native promoter and 5'UTR of the gene have the sequence shown in SEQ ID NO:

4.

3. The method of claim 1, wherein the first coding sequence is SEQ ID NO: 2, and the second coding sequence is SEQ ID NO:

5.

4. The method of claim 1, wherein the grain yield of the transgenic rice plant is increased by 50% to 95% compared to the non-transgenic wild-type rice plant.

5. The method of claim 1, wherein the shoot biomass of the transgenic rice plant is increased by 65% ​​to 106% compared to the non-transgenic wild-type rice plant.

6. A recombinant DNA construct comprising A first nucleic acid sequence comprising a first Golden 2-like transcription factor ( GLK ) gene, and a second nucleic acid sequence comprising a second nucleic acid sequence operably linked to its native promoter and 5'UTR GLK Gene, the second GLK Genetically different from the first GLK Gene, the first of which GLK Gene and the second GLK The gene comes from C4 plants; The first nucleic acid sequence comprises a first coding sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO: 3, and the second nucleic acid sequence comprises a second coding sequence encoding a protein having an amino acid sequence as shown in SEQ ID NO:

6.

7. The recombinant DNA construct of claim 6, wherein the first GLK The natural promoter and 5'UTR of the gene have the sequence shown in SEQ ID NO: 1, and the second GLK The native promoter and 5'UTR of the gene have the sequence shown in SEQ ID NO:

4.

8. The recombinant DNA construct of claim 6, wherein the first coding sequence is SEQ ID NO: 2, and the second coding sequence is SEQ ID NO: 5.

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