WO2013063794A1

WO2013063794A1 - A growth regulatory factor gene grf2 derived from brassica napus and the use thereof

Info

Publication number: WO2013063794A1
Application number: PCT/CN2011/081793
Authority: WO
Inventors: Hanzhong Wang; Wei Hua; Jing Liu; Guihua Liu; Xinfa Wang; Qing Yang
Original assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Current assignee: Oil Crops Research Institute of Chinese Academy of Agriculture Sciences
Priority date: 2011-11-04
Filing date: 2011-11-04
Publication date: 2013-05-10
Anticipated expiration: 2014-05-04

Abstract

The current invention provides the sequence of the GRF2 gene and protein from Brassica napus, and the use of GRF2 to increase seed oil content and thousand kernel weight in plants.

Description

A Growth Regulatory Factor gene GRF2 derived from Brassica napus and the use thereof

FIELD OF THE INVENTION

The present invention relates to the field of plant genetic engineering, more particularly to growth regulatory factor GRF2 genes and proteins derived from Brassica napus, as well as to the use of the growth regulatory factor GRF2 genes and proteins derived from Brassica napus.

BACKGROUND OF THE INVENTION

The production of oil crops not only plays an important role in the national economic and social development, but is also one of the main factors that promote the sustainable development of agriculture in China. The annual yield of vegetable oils in China can only meet 1/2-2/3 of the domestic needs at present, and has to heavily rely on imports. For instance, China has imported more than six million tons of vegetable oils in 2005, and twenty four million tons of soybeans. China is becoming the largest importing country of oil-comprising materials. With the increase in both the population and the improvement of the people's living standard, the amount of edible vegetable oils consumed in China is expected to increase to 25 million tons in 2010. The gap between the actual annual yield and the expected domestic consumption will be considerably wide according to the current production scale. Considering food safety and increase in farmers' income, it is important to increase the yield of oil crops per unit area by a larger margin. The most direct measure for increasing the annual yield of vegetable oils in China is to substantially extend the planting area of oil crops. However, it is impractical to further considerably extend the planting area of oil crops, due to limited area of arable land in China. Therefore, one should increase the unit yield of oil crops or the contents of oils and proteins in seeds to achieve high production and high efficacy. Moreover, vegetable oils are good sources for many important raw materials in industry, and thus have larger social significance in terms of crop oils for energy source and oils for industrial raw materials.

Rape {Brassica napus) is one of the main oil crops in China, and rapeseed oil accounts for about 40% in edible oils in China. The annual planting area of the rape is seven million hectares in China. The annual production of rapeseeds is more than ten million tons. Both the annual planting area and the annual total production of rapeseeds stand in the first place in the world, accounting for 1/3 of the annual planting area and the annual total production in the world, respectively. Nowadays, through the effort of breeders in several generations, the unit production of rape has been greatly increased by conventional breeding approaches, in particular, from more than 35 kg in 1950s to more than 150 kg at present, i.e., increased by more than 4 times. However, it is very difficult to further increase the unit production by conventional breeding approaches. Since the oil content of the rape seeds in China is lower than that in other countries, with a general survey on quality of the rape seeds in Yangtze River basin showing that the oil content of commercial rape seeds in 2004 is 39.1%, which is lower than that of the commercial rape seeds from Canada (42.6%) in the same year by 3.5 percent, thus, increasing the oil content of the rape in China by a large margin not only becomes a main object of rape breeding, but also is one of the most effective approaches for solving the problem of increasing need for rapeseed oil in China. The oil content is a quantitative trait for rape seeds, the heredity of which is controlled by minor polygenes and is significantly affected by enviromental factors, which results in some difficulties in the conventional breeding. Nowadays, more and more efforts have focused on the isolation of functional genes associated with the oil content, and use of these genes in modification of the relevant genetically engineered varieties, in order to improve the quality of the seeds and increase the oil content fundamentally. It is a major task to find and clone oil content-associated genes.

The biosynthetic pathways of fatty acids as well as oils and fats in plants have been relatively well delineated. A large number of the relevant genes from various species have been isolated and identified, and the researches have also shown that the biosynthesis pathways of fatty acids as well as oils and fats in different species are substantially identical (Lung and Weselake, 2006, Lipids 41 : 1073; Snyder et al., 2009, New Biotechnol 26: 11). The main precursors for synthesis of oils and fats in seeds include acetyl coenzyme A, NAD(P)H, and ATP. The sources of and regulation on these synthesis precursors have direct effects on the rate and quantity of oil accumulation (Voelker, 2001 , Annu Rev Plant Physiol Plant Mol Biol 52:335). While the sources of these precursors have been intensively investigated, there are still many disagreements on their actual sources, due to the complexity and redundancy in the metabolic pathway (Rawsthorne, 2002, Prog Lipid Res 41 : 182). Acetyl coenzyme A is an initial substrate in synthesis of the carbon skeleton of all fatty acids, and also is an important intermediate product in the metabolism in various cells, that is, it is both synthesized and consumed in large amount in cells. Pyruvate dehydrogenase, acetyl coenzyme A synthetase, and carnitine acetyltransferase in chloroplast, as well as cytoplasmic ATP citrate lyase, may be involved in synthesis of acetyl coenzyme A (Neuhaus and Emes, 2000, Annu Rev Plant Physiol Plant Mol Biol 51 : 1 11). It has been demonstrated by Schwender et al.(2004, Nature 432:779) that not all of pyruvic acid required for oils and fats synthesis in rape is provided by the glycolysis pathway as stated previously, instead the ribulose bisphosphate carboxylase (Rubisco) functions in the rape seed independent of Calvin cycle (the dark reaction in the photosynthesis) and yields more fatty acids from the product of photosynthesis in the ovule, which suggests that C02 may play an important role as a precursor in synthesis of oils and fats. It is generally believed that in the light, ATP and NAD(P)H required for synthesis of fatty acids is provided by the light reactions (Ruuska et al, 2004, Plant Physiol 136:2700). Yet there are some contradictories, for example, Eastmond and Rawsthorne (1998, J Exp Bot 49: 1 105) demonstrated ATP and NAD(P)H required for synthesis of fatty acids is provided insufficiently from the light reactions and still needs to be supplied from the cytoplasm. In tissues in the dark and in the absence of chloroplast, the pentose phosphate oxidation pathway may be the most probable source of the reductive NAD(P)H; but it has also been demonstrated in a research that this pathway together with other pathways such as glycolysis can supply enough reductive power (Schwender et al, 2003, J Biol Chem 278:29442). Recently, the results from researches on Brassica napus and Arabidopsis thaliana have shown that contribution percentage of photosynthesis in the seed to the oil content is about 40% (Goffman et al, 2005, Plant Physiol 138:2269; Wakao et al, 2008, Plant Physiol 146:277). On the other hand, it is reported that inhibition of the pentose phosphate pathway leads to the increased oil content in the seed, showing that compared with providing the reductant NADPH for fatty acid synthesis, increasing carbon sources by inhibition of the pentose phosphate pathway can increase the content of oils and fats more effectively (Wakao et al, 2008, Plant Physiol 146:277).

Although the change in oil content may be regulated by merely changing a series of enzymes in the synthesis pathway of fatty acids as well as oils and fats such as ACCase, GPAT, DGAT etc. (Roesler et al, 1997, Plant Physiol 1 13:75, Jain et al, 2000, Biochem Soc Trans 28:958; Weselake et al, 2008, J Exp Bot 59:3543; Zheng et al, 2008, Nature Genet 40:367), some genes are capable of performing a general control over synthesis of fatty acids as well as oils and fats in a more effectively way at an upstream position in the metabolic pathway. Recent studies have shown that the plant embryo development- associated transcription factors regulate the accumulation of the storage products in the process of seed maturation by influencing a variety of enzymes in a series of biochemical pathways including glycolysis, fatty acid metabolism, protein synthesis, etc (Gutierrez et al, 2007, Trends Plant Sci 12:294; Verdier and Thompson 2008, Plant Cell Physiol 49: 1263; Santos et al, 2008, Plant J 54:608). By screening the mutants of the model plant Arabidopsis thaliana, a series of transcription factors regulating accumulation of oils and fats in seeds have been identified, such as LEC1 , LEC2, WRI1, GLABRA2, etc, (Shen et al, 2006, Plant Mol Biol 60:377; Baud et al, 2007, Plant J 50:825; Mu et al, 2008, Plant Physiol 148: 1042). However, the functional redundancy of various transcription factors increases the complexity in investigation of these transcription factors on regulating the oil content, the action mechanisms of some transcription factors are yet unknown (To et al, 2008, Plant Cell 18: 1642; Gao et al, 2009, Plant Cell 21 :54).

Although the aforementioned studies have beneficially explored methods for increasing the oil content theoretically by genetic manipulation of some key enzymes in the metabolic pathway of fatty acids, there is quite some distance from the actual practice. It should be recognized that the oil content in Brassica napus is controlled by interaction of multiple genes, so an increase in oil content will hardly be achieved solely by altering a certain gene or a few genes. In order to achieve desired purpose, it may be necessary to genetically transform multiple coding genes for the trait control, especially regulatory genes, simultaneously, then stably express and pass down said genes in a transgenic plant and offspring thereof. For breeding of an excellent transgenic line, the choice of appropriate functional genes is the step of most importance. Exploration, isolation, and cloning of dominant functional genes are favourite research subjects in various countries over the world all the time.

The applicants of the present invention have identified a gene differentially expressed not only between the parent lines but also between the extremely segregated lines of F2 generation by analyzing the differences in gene expressions between the two parent rape lines as well as between the mixed samples of the segregated lines of F2 generation having significantly different oil-content. Eventually, a gene that is capable of controlling the change in oil content, GRF2, has been obtained by cloning of the full-length gene, construction of an expression vector, and transformation of the same into the model plant Arabidopsis thaliana. Moreover, overexpression of the gene in Arabidopsis thaliana also increases the thousand kernel weight of the seeds. Utilization of the gene into crop breeding can increase the oil yield of an oil crop maximally.

The Arabidopsis GRF2 gene has been described by Kim et al., 2003, Plant J. 36:94. They showed that overexpression of GRF2 in Arabidopsis results in larger leaves and cotyledons. WO2009/034188 describes overexpression of Arabidopsis GRF2 in rice plants under control of the constitutive GOS2 promoter. They observed that the thousand kernel weight (TKW) of the seeds was increased.

The current invention provides methods and means to improve plant seed oil content by increasing the expression of GRF2, as will become apparent from the following description, examples, drawings and claims provided herein.

SUMMARY OF THE INVENTION

In a first embodiment of the invention, a method is provided for increasing oil content, such as seed oil content, in plants, comprising increasing expression of a nucleic acid encoding a GRF2 protein. In a further embodiment, said method is further characterized in that the Thousand Kernel Weight of the seeds of said plants is increased. In a further embodiment, said GRF2 protein has at least 65% sequence identity to SEQ ID No. 2. In a further embodiment, said GRF2 protein has at least 90% sequence identity to SEQ ID No. 2. In yet a further embodiment, said nucleic acid has at least 90% sequence identity to SEQ ID NO. 1.

In yet another embodiment of the invention, a method is provided for increasing seed oil content in plants comprising increasing expression of a nucleic acid encoding a protein with has at least 90% sequence identity to the GRF2 protein from Arabidopsis thaliana. In yet a further embodiment, said nucleic acid has at least 90% sequence identity to the GRF2 coding sequence from Arabidopsis thaliana.

In a further embodiment of the invention, a method is provided for increasing oil content in plants comprising increasing expression of a nucleic acid encoding a GRF2 protein comprising the steps of:

a) providing a plant cell with a chimeric gene comprising the following operably linked nucleic acid molecules:

i) a heterologous plant-expressible promoter;

ii) said nucleic acid encoding a GRF2 protein; and optionally iii) a DNA region involved in transcription termination and polyadenylation;

b) regeneration of said plant cell into a plant.

In a further embodiment, said plant-expressible promoter is a constitutive promoter, and in yet another embodiment, said constitutive promoter is the 35 S promoter. In a further embodiment, said plant-expressible promoter is a seed-specific promoter.

It is a further aspect of the invention to provide methods for increasing seed oil content in seed oil plants, and it is yet another embodiment of the invention to provide methods for increasing seed oil content in Brassicaceae plants. In a further embodiment, plants are provided that are obtained by the methods according to the invention. Another embodiment provides seeds from plants obtained by the methods according to the invention. In yet another embodiment, oil from the seeds of plants obtained by the methods according to the invention are provided.

A further object of the invention is to provide an isolated DNA encoding the Brassica napus GRF2 protein selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, such as an isolated DNA selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7.

The invention further provides a chimeric gene comprising the following operably linked nucleic acid molecules:

a) a heterologous plant-expressible promoter;

b) a nucleic acid encoding a GRF2 protein containing at least 65% sequence identity to SEQ ID No. 2; and optionally

c) a DNA region involved in transcription termination and polyadenylation.

In another embodiment, said GRF2 protein contains at least 90% sequence identity to SEQ ID No. 2, whereas in yet another embodiment said nucleic acid encoding a GRF2 protein contains at least 90% sequence identity to SEQ ID No. 1. In a further embodiment, said plant-expressible promoter is a constitutive promoter, such as the 35S promoter, and in yet a further embodiment, said plant-expressible promoter is a seed-specific promoter.

The invention further relates to the use of the chimeric genes or of the isolated DNA according to the invention to increase plant seed oil content, or to increase plant seed oil content and Thousand Kernel Weight.

The invention further provides methods for producing oil, comprising harvesting seeds from the plants according to the invention and extracting the oil from said seeds. In a further aspect, the invention provides a method of producing food or feed such as oil, meal, grain, starch, flour or protein, or an industrial product such as biofuel, fiber, industrial chemicals, a pharmaceutical or a neutraceutical, comprising obtaining the plant according to the invention or a part thereof, and preparing the food, feed, or industrial product from the plant or part thereof.

BRIEF DESCRIPFION OF THE DRAWINGS

Figure 1. Schematic representation of an expression vector for BnGRF2 transgenic Arabidopsis thaliana.

Figure 2. Detection of BnGRF2 in the Tl generation of transgenic Arabidopsis thaliana with PCR. Lanes 1 , 2, 3, 5, 6, and 11 correspond to the transgenic plants, and WT corresponds to the wild type control.

Figure 3. Comparison of the leaves between the transgenic plant and the wild type control, wherein the leaves of the transgenic plant are shown in the left panel (A), and those of the wild type control in the right panel (B).

Figure 4. Comparison of the seed sizes between the transgenic plant and the wild type control, wherein the seeds of the transgenic strain are shown in the left panel (A), and those of the wild type control in the right panel (B).

DETAILED DESCRIPTION

The current invention is based on the finding that overexpression of GRF2 in Arabidopsis results in increased seed oil content and increased Thousand Kernel Weight.

In a first embodiment of the invention, a method is provided for increasing oil content, such as seed oil content, in plants, comprising increasing expression of a nucleic acid encoding a GRF2 protein. In a further embodiment, said method is further characterized in that the Thousand Kernel Weight of the seeds of said plants is increased. In yet a further embodiment, said GRF2 protein has at least 65% sequence identity to SEQ ID No. 2. In yet a further embodiment, said GRF2 protein has at least 90% sequence identity to SEQ ID No. 2. In yet a further embodiment, said nucleic acid has at least 90% sequence identity to SEQ ID NO. 1. In yet another embodiment of the invention, said GRF2 protein has at least 90% sequence identity to the GRF2 protein from Arabidopsis thaliana. In yet a further embodiment, said nucleic acid has at least 90% sequence identity to the GRF2 coding sequence from Arabidopsis thaliana.

The methods and means described herein are believed to be suitable for all plant cells and plants, both dicotyledonous and monocotyledonous plant cells and plants including but not limited to cotton, Brassica, oilseed rape, wheat, corn or maize, barley, sunflowers, rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce, tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry. Especially suited are oil producing plants such as rapeseed (Brassica spp.), flax (Linum usitatissimum), safflower (Carthamus tinctorius), sunflower (Helianthus annuus), maize or corn (Zea mays), soybean (Glycine max), mustard (Brassica spp. and Sinapis alba), crambe(Crambe abyssinica), eruca (Eruca saiva), oil palm (Elaeis guineeis), cottonseed (Gossypium spp ), groundnut (Arachis hypogaea), coconut (Cocus nucifera), castor bean (Ricinus communis), coriander (Coriandrum sativum), squash (Cucurbita maxima), Brazil nut (Bertholletia excelsa) or jojoba (Simmondsia chinensis) gold-of-pleasure (Camelina sativa), purging nut (Jatropha curcas), Echium spp., calendula (Calendula officinalis), olive (Olea europaea), wheat (Triticum spp.), oat (Avena spp.), rye (Secale cereale), rice (Oryza sativa), Lesquerella spp., Cuphea spp., meadow foam (Limnanthes alba), avocado (Persea Americana), hazelnut (Corylus), sesame (Sesamum indicum), safflower (Carthamus tinctorius), tung tree (Aleurites fordiij, poppy (Papaver somniferum) tobacco (Nicotiana spp.).

The methods and means described herein can also be used in algae such as Scenedesmus dimorphus, Euglena gracilis, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetraselmis chui, Tetraselmis suecica, Isochrysis galbana, Nannochloropsis salina, Botryococcus braunii, Dunaliella tertiolecta, Nannochloris spp. or Spirulina spp. As used herein, a "Brassica plant" is a plant which belongs to one of the species Brassica napus, Brassica rapa (or campestris), or Brassica juncea. Alternatively, the plant can belong to a species originating from intercrossing of these Brassica species, such as B. napocampestris, or of an artificial crossing of one of these Brassica species with another species of the Cruciferacea. A Brassica oilseed plant refers to any one of the species Brassica napus, Brassica rapa (or campestris), Brassica carinata, Brassica nigra or Brassica juncea .

An increase in seed oil content can be an increase with at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 50%. Said increase is an increase with respect to levels as obtained in control plants.

"Thousand Kernel Weight" as used herein, also "TKW", weight in grams of 1000 seeds. An increased TKW may result from an increase in seed size and/or an increase in seed weight.

An increase in seed oil content can be an increase with at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%. Said increase is an increase with respect to levels as obtained in control plants.

The "control plant" as used herein is generally a plant of the same species which has wild-type levels of GRF2. "Wild-type levels of GRF2" as used herein refers to the typical levels of GRF2 protein in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a nucleic acid molecule encoding GRF2, or with a nucleic acid activating expression of the endogenous GRF2, such as a T-DNA activation tag or a promoter with stronger activity than the endogenous promoter.

The GRF2 protein is the Growth Regulating Factor 2 transcription activator. GRF2 is one of the members of the GRF gene family. GRF proteins are characterized in that they contain (1) a QLQ domain (InterPro accession IPR014978) containing a conserved Glutamine-Leucine-Glutamine, and (2) a WRC domain (InterPro accession IPRO 14977), which contains a putative nuclear localization signal consisting of many basic amino acids, and a zinc-finger motif consisting of the conserved spacing of three Cysteine and one Histidine residues (C₃H motif). A consensus amino acid sequence of the QLQ and of the WRC domain of Arabidopsis GRF sequences has been described by Kim et al., 2003, Plant J. 36:94, and are depicted in SEQ ID No. 9 and SEQ ID No. 10, respectively. Examples of GRF2 proteins are Arabidopsis GRF2 (At4g37740), and Brassica napus GRF2 (SEQ ID No. 2). A GRF2 protein is a protein that contains at least 50%, or at least 55%), or at least 60%>, or at least 65%, or at least 70%, or at least 75%, or at least 80%>, or at least 85%>, or at least 90%>, or at least 95%, or at least 98%, or at least 99% sequence identity any of the GRF2 proteins as described above. Preferably, said GRF protein contains the consensus QLQ sequence of SEQ ID No. 9, and the consensus WRC domain of SEQ ID No. 10, such as, for example, AA 145-180 of SEQ ID No. 2 (QLQ domain) or AA 208-251 of SEQ ID No. 2 (WRC domain).

Examples of GRF2 proteins are the proteins as depicted in SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, and the GRF2 protein from Arabidopsis thaliana.

The "GRF2 protein from Arabidopsis thaliana" as used herein refers to the protein with accession number At4g37740, wheres the "GRF2 coding sequence from Arabidopsis thaliana" refers to the coding sequence with accession number At4g37740.

Examples of a nucleic acid encoding a GRF2 protein are, for example, the sequences as depicted in SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, and teh GRF2 coding sequence from Arabidopsis thaliana.

Based on the available sequences, the skilled person can isolate genes encoding GRF2 other than the genes mentioned above. Homologous nucleotide sequence may be identified and isolated by hybridization under stringent conditions using as probes identified nucleotide sequences. "High stringency conditions" can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCl, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (100X Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 μg/ml denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0. l x SSC, 0.1% SDS.

"Moderate stringency conditions" refers to conditions equivalent to hybridization in the above described solution but at about 60-62°C. Moderate stringency washing may be done at the hybridization temperature in lx SSC, 0.1% SDS.

"Low stringency" refers to conditions equivalent to hybridization in the above described solution at about 50-52°C. Low stringency washing may be done at the hybridization temperature in 2x SSC, 0.1% SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

Other sequences encoding GRF2 may also be obtained by DNA amplification using oligonucleotides specific for genes encoding GRF2 as primers, such as but not limited to oligonucleotides comprising or consisting of about 20 to about 50 consecutive nucleotides from the known nucleotide sequences or their complement.

As used herein, a GRF2 protein which has "at least 65% sequence identity to SEQ ID No. 2" can be a GRF2 protein with at least 65%, or with at least 70% sequence identity to SEQ ID No. 2 such as, for example, the GRF2 protein of SEQ ID No. 4, or that of SEQ ID No. 6, or that of SEQ ID No. 8, or can be the GRF2 protein from Arabidopsis thaliana.

As used herein, a GRF2 protein which has "at least 90% sequence identity to SEQ ID No. 2" can be a GRF2 protein with 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID No. 2, or can be SEQ ID No. 2 itself. As used herein, a nucleic acid which has "at least 90% sequence identity to SEQ ID NO. 1" can be a nucleic acid with 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID No. 1, or can be SEQ ID No. 1 itself.

Increasing expression of a nucleic acid encoding a GRF2 protein can comprise increasing expression on a whole plant level. Alternatively, increasing expression of a nucleic acid encoding a GRF2 protein can comprise increasing expression in specific plant parts or tissues, such as seeds, or specific seed tissues.

Increasing expression of a nucleic acid encoding a GRF2 protein can conveniently be achieved by heterologously expressing a nucleic acid encoding GRF2. Moreover, expression of the endogenous GRF2 encoding gene can be increased through, for example, T-DNA activation tagging, or by targeted genome engineering technologies in which, for example, the endogenous promoter is modified such that it drives higher levels of expression, or in which the endogenous promoter is replaced with a stronger promoter.

T-DNA activation tagging (Memelink, 2003, Methods Mol Biol. 236:345) is a method to activate endogenous genes by random insertion of a T-DNA carrying promoter or enhancer elements, which can cause transcriptional activation of flanking plant genes. The method can consist of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype.

Targeted genome engineering refers to generate intended and directed modifications into the genome. Such intended modifications can be insertions at specific genomic locations, deletions of specific endogenous sequences, and replacements of endogenous sequences. Targeted genome engineering can be based on homologous recombination. Targeted genome engineering to increase expression of the GRF2 endogene can consist of insertion of a promoter, stronger than the endogenous promoter, in front of the GRF2 coding sequence, or insert an enhancer to increase promoter activity, or insert elements enhancing RNA stability or enhancing translation of the encoded mRNA. In a further embodiment of the invention, a method is provided for increasing oil content, such as seed oil content, in plants comprising increasing expression of a nucleic acid encoding a GRF2 protein comprising the steps of:

i) a heterologous plant-expressible promoter;

b) regeneration of said plant cell into a plant.

Said plant cell can be provided with a chimeric gene using methods well-known in the art. Methods to provide plant cells with a chimeric gene are not deemed critical for the current invention and any method to provide plant cells with a chimeric gene suitable for a particular plant species can be used. Such methods are well known in the art and include Agrobacterium-mediated transformation, particle gun delivery, microinjection, electroporation of intact cells, polyethyleneglycol-mediated protoplast transformation, electroporation of protoplasts, liposome-mediated transformation, silicon-whiskers mediated transformation etc. Said chimeric gene may be stably integrated into the genome of said plant cell, resulting in a transformed plant cell. The transformed plant cells obtained in this way may then be regenerated into mature fertile transformed plants.

The obtained transformed plant, comprising the chimeric gene which comprises said nucleic acid encoding a GRF2 protein, can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert and are also encompassed by the invention.

As used herein, the term "plant-expressible promoter" means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Harpster et al. (1988) Mol Gen Genet. 212(1): 182-90, the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue- specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al. (1996) Plant Cell 8(l): 15-30), stem-specific promoters (Keller et al, (1988) EMBO J. 7(12): 3625-3633), leaf specific promoters (Hudspeth et al. (1989) Plant Mol Biol. 12: 579-589), mesophyl- specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al. (1989) Genes Dev. 3: 1639-1646), tuber-specific promoters (Keil et al. (1989) EMBO J. 8(5): 1323-1330), vascular tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369), stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence zone specific promoters (WO 97/13865) and the like.

A "heterologous promoter" as used herein refers to a promoter which is not normally associated in its natural context with the coding DNA region operably linked to it in the DNA molecules according to the invention.

Constitutive promoters are well known in the art, and include the CaMV35S promoter (Harpster et al. (1988) Mol Gen Genet. 212(1): 182-90), Actin promoters, such as, for example, the promoter from the Rice Actin gene (McElroy et al., 1990, Plant Cell 2: 163), the promoter of the Cassava Vein Mosaic Virus (Verdaguer et al., 1996 Plant Mol. Biol. 31 : 1 129), the GOS promoter (de Pater et al, 1992, Plant J. 2:837), the Histone H3 promoter (Chaubet et al., 1986, Plant Mol Biol 6:253), the Agrobacterium tumefaciens Nopaline Synthase (Nos) promoter (Depicker et al, 1982, J. Mol. Appl. Genet. 1 : 561), or Ubiquitin promoters, such as, for example, the promoter of the maize Ubiquitin-1 gene (Christensen et al, 1992, Plant Mol. Biol. 18:675).

Seed specific promoters are well known in the art, including the USP promoter from Vicia faba described in DE10211617; the promoter sequences described in WO2009/073738; promoters from Brassica napus for seed specific gene expression as described in WO2009/077478; the plant seed specific promoters described in US2007/0022502; the plant seed specific promoters described in WO03/014347; the seed specific promoter described in WO2009/125826; the promoters of the omega_3 fatty acid desaturase family described in WO2006/005807 and the like.

A "transcription termination and polyadenylation region" as used herein is a sequence that drives the cleavage of the nascent R A, whereafter a poly(A) tail is added at the resulting RNA 3' end, functional in plants. Transcription termination and polyadenylation signals functional in plants include, but are not limited to, 3'nos, 3'35S, 3'his and 3'g7.

It is a further aspect of the invention to provide methods for increasing seed oil content in seed oil plants, and it is yet another embodiment of the invention to provide methods for increasing seed oil content in Brassicaceae plants.

"Seed oil plants" as used herein refers to plants producing oil in their seeds. Examples of seed oil plants are Brassica oilseeds (including Brassica napus, Brassica campestris (rapa), Brassica juncea or Brassica carinata), sunflower, safflower, soybean, palm, Jatropha, flax, crambe, camelina, corn, sesame, castor beans.

As used herein, "Brassicaceae plants" are plants which according to the current botanical standard would be classified into the family Brassicaceae (formerly Cruciferaeae). Brassicaceae (Mustard) family members are easy to distinguish. They are annual or perannual plants with alternate leaves without stipules and posses simple inflorescence or branched racemes. The flowers are bilaterally symmetrical and hypogynous. With few exceptions, the flowers have 4 petals (free) alternating with 4 sepals (free) ; 6 stamens (4 long and 2 short), an ovary of 2 united carpels with parital placenta, 2 locular through the formation of a membranous false septum ; fruit is a dehiscent capsule opening by 2 valves. Brassicaceae include inter alia the following genera : Sisymbrium, Descurania, Alliaria, Arabidopsis, Myagrum, Isatis, Bunia, Erysium, Hesperis, Malcolmia, Matthiola, Chorispora, Euclidium, Barbarea, Rorippa, Armoracia, Nasturtium, Dentaria, Cardamine, Cardaminopsis, Arabis, Lunaria, Alyssum, Berteroa, Lobularia, Draba, Erophila, Cochlearia, Camelina, Neslia, Capsella, Hornungia, Thlsapi, Iberis, Lepidium, Cardaria, Coronopus, Subularia, Conringia, Diplotaxis, Brassica, Sinapsis, Eruca, Erucastrum, Coincya, Hirschfeldia, Cakile, Rapistum, Crambe, Enarthrocarpus, Rhaphanus and Clausia.

In a further embodiment, plants are provided that are obtained by the methods according to the invention. Another embodiment provides seeds from plants obtained by the methods according to the invention. In yet another embodiment, oil from the seeds of plants obtained by the methods according to the invention is provided.

A further object of the invention is an isolated DNA encoding the Brassica napus GRF2 protein selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6, and SEQ ID No. 8, such as an isolated DNA selected from theg group consisting of SEQ ID No. 1 , SEQ ID No. 3, SEQ ID No. 5, and SEQ ID No. 7.

a) a heterologous plant-expressible promoter;

c) a DNA region involved in transcription termination and polyadenylation;

In another embodiment, said GRF2 protein contains at least 90% sequence identity to SEQ ID No. 2, whereas in yet another embodiment said nucleic acid encoding a GRF2 protein contains at least 90% sequence identity to SEQ ID No. 1. In a further embodiment, said plant-expressible promoter is a constitutive promoter, such as the 35 S promoter, and in yet a further embodiment, said plant-expressible promoter is a seed-specific promoter.

In another embodiment according to the invention, a plant cell is provided comprising the chimeric gene according to the invention.

Suitable for the invention are all plant cells and plants, both dicotyledonous and monocotyledonous plant cells and plants including but not limited to cotton, Brassica, oilseed rape, wheat, corn or maize, barley, sunflowers, rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce, tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry. Especially suited are oil producing plants such as rapeseed {Brassica spp.), flax (Linum usitatissimum), saffiower (Carthamus tinctorius), sunflower (Helianthus annuus), maize or corn (Zea mays), soybean (Glycine max), mustard (Brassica spp. and Sinapis alba), crambe(Crambe abyssinica), eruca (Eruca saiva), oil palm (Elaeis guineeis), cottonseed (Gossypium spp ), groundnut (Arachis hypogaea), coconut (Cocus nucifera), castor bean (Ricinus communis), coriander (Coriandrum sativum), squash (Cucurbita maxima), Brazil nut (Bertholletia excelsa) or jojoba (Simmondsia chinensis) gold-of-pleasure (Camelina sativa), purging nut (Jatropha curcas), Echium spp., calendula (Calendula officinalis), olive (Olea europaea), wheat (Triticum spp.), oat (Avena spp.), rye (Secale cereale), rice (Oryza sativa), Lesquerella spp., Cuphea spp., meadow foam (Limnanthes alba), avocado (Persea Americana), hazelnut (Corylus), sesame (Sesamum indicum), safflower (Carthamus tinctorius), tung tree (Aleurites fordiij, poppy (Papaver somniferum) tobacco (Nicotiana spp.).

A Brassica is a plant which belongs to one of the species Brassica napus, Brassica rapa (or campestris), or Brassica juncea. Alternatively, the plant can belong to a species originating from intercrossing of these Brassica species, such as B. napocampestris, or of an artificial crossing of one of these Brassica species with another species of the Cruciferacea. A Brassica oilseed plant refers to any one of the species Brassica napus, Brassica rapa (or campestris), Brassica carinata, Brassica nigra or Brassica juncea. The obtained plants according to the invention can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the characteristic of increased expression of a nucleic acid encoding a GRF2 protein according to the invention in other varieties of the same or related plant species, or in hybrid plants. The obtained plants can further be used for creating propagating material. Plants according to the invention can further be used to produce gametes, seeds (including crushed seeds and seed cakes), seed oil, embryos, either zygotic or somatic, progeny or hybrids of plants obtained by methods of the invention.

"Creating propagating material", as used herein, relates to any means know in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin-scaling), sexual reproduction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridization).

As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.

Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R.D.D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR - Basics: From Background to Bench, First Edition, Springer Verlag, Germany.

All patents, patent applications, and publications or public disclosures (including publications on internet) referred to or cited herein are incorporated by reference in their entirety.

SEQUENCES

SEQ ID NO: 1 : Brassica napus GRF2al coding sequence.

SEQ ID NO: 2: Brassica napus GRF2al protein.

SEQ ID NO: 3: Brassica napus GRF2a2 coding sequence.

SEQ ID NO: 4: Brassica napus GRF2a2 protein. SEQ ID NO: 5: Brassica napus GRF2M coding sequence.

SEQ ID NO: 6: Brassica napus GRF2M protein.

SEQ ID NO: 7: Brassica napus GRF2b2 coding sequence.

SEQ ID NO: 8: Brassica napus GRF2b2 protein.

SEQ ID NO: 9: Consensus QLQ domain.

SEQ ID NO: 10: Consensus WRC domain.

SEQ ID NO: 11 : BnGRF2F forward primer.

SEQ ID NO: 12: BnGRF2F reverse primer.

SEQ ID NO: 13: AtGRF2 forward primer.

SEQ ID NO: 14: AtGRF2 reverse primer.

SEQ ID NO: 15: BnGRF2 reverse primer.

EXAMPLES

Example 1. Source of the genes

A high-oil content line zy036 (oil contents thereof being 51%) was crossed with a low-oil content line y817 (oil contents thereof being 35%) in order to establish a population of F₂ generation. A total of 169 F₂ offspring plantlets was used as the starting material. The siliquae (with pod shells and ovules) were collected respectively from each of F2 plants about 25 days post blossom; and the oil content was determined in mature seeds from the individual plants. The siliquae from individual plants having the determined oil content of greater than 47% and of less than 38.5% were weighed and mixed in equal amount of 200 mg respectively to constitute two mixed samples with two extreme oil contents, which samples are coded by H and L, respectively. 11 plants had anoil content of greater than 47% and 1 1 plants had an oil content of less than 38.5%. There was a difference of 11.1% between the average oil contents of the two mixed samples. Genes expressed in the two parents and two mixed samples were assayed. The genes whose expression levels were different between the parents as well as between the mixed samples of F2 generations were identified, from which the genes BnGRF2 originate. Both the parent lines zy036 and Y817 used in the study had been established by the technicians of the biotechnical breeding team in the Oil Crops Research Institute (OCRI) of the Chinese Academy of Agricultural Sciences (CAAS) under the direction of the researcher Wang Hanzhong. The zy036 line is developed by establishing a recurrent selection population consisted of Zhongshuang No.4, Zhongshuang No.7, Zhongshuang No.9, Huashuang No.3, and Youyan No.9, performing recurrent selections of two generations, followed by recurrent selection of the third generation by microspore culture of excellent individual plants therefrom, and eventually conducting high oil content-directed selection. Y817 is a maintenance line for the hybrid cultivar Zhongyou hybrid No.l (Study on techniques about seed production of Zhouyou Hybrid No.l and their utilization, Rural Economy and Science-Technology, (10), 2001).

Example 2. Cloning of the full-length genes

By screening for gene expression difference, it had been found that the expression levels of the GRF2 gene in Brassica napus high-oil-content parent and the mixed population of high-oil-content F2 generations are higher than those in low-oil-content parent and the mixed population of low-oil-content F2 generations. Therefore, the applicants aligned the sequence of Arabidopsis thaliana GRF2 gene to obtain EST sequences of this gene in Brassica napus. Then, the Brassica napus genomic sequences homologous to the EST sequences were found by use of the applicants' own sequencing database, and spliced together. Finally, on the basis of the spliced sequence, primers flanking the coding region in the gene of interest were designed. RT-PCR amplification was performed using the cDNA first strand from the parent zy036 as the template. The amplified fragments were sequenced, to obtain the coding regions of the BnGRF2 genes. Further, the homologous gene AtGRF2 in Arabidopsis thaliana was also amplified. The sequenced fragments are Brassica napus growth regulatory genes BnGRF2al, BnGRF2a2, BnGRF2bl , and BnGRF2b2, the base sequence thereof are the nucleotide sequences shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7. The genes can be expressed, and the sequence of the expressed proteins are the amino acid sequence shown as SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. Example 3. Construction of a transgenic vector, transformation and verification The Brassica napus BnGRF2 genes have been obtained by the method described above. A transgenic Arabidopsis thaliana having increased expression level of the Brassica napus BnGRF2al gene has been obtained, which is characterized by that the transgenic plant shows an increased level of the gene expression as compared to that of a wild type control (non-transgenic plant). The detailed measures are:

PCR8/GW/TOPO plasmid (purchase from Invitrogen Co.), which can be recombined into an expression vector plasmid to construct a gene expression vector plasmid;

an expression vector plasmid pEarleygatelOO (purchase from Invitrogen Co.), comprising a 35S promoter and translation regulatory elements;

a host bacteria (e.g. GV3101, LBA4404, etc.) allowing expression of the introduced gene in a plant, and Agrobacterium (the strain GV3101, purchased from Invitrogen Co.) being preferred in the invention; and

a transgenic Arabidopsis thaliana capable of overexpressing a gene, characterized by the increased expression level of the transgene in the Arabidopsis thaliana.

The method for using the cloned gens comprises the following steps:

1) The cloned Brassica napus gene was ligated into the plasmid PCR8/GW/TOPO, then the Brassica napus gene was transferred further to an expression vector plasmid pEarleygatelOO by taking advantage of the feature that the plasmid PCR8/GW/TOPO can be recombined in vitro into the expression vector plasmid;

2) The expression vector prepared in the step 1) was introduced into Agrobacterium tumefaciens GV3101 and further into Arabidopsis thaliana plants;

3) After identified by PCR and screened with the herbicide (Bar, glyphosate), the positively transgenic plants were grown under normal conditions. Seeds from the transgenic plants were harvested and assayed for oil content. The result showed that the oil content in seeds of the transgenic Arabidopsis thaliana was considerably increased in comparison with that of the control Arabidopsis thaliana.

The term "transgenic plant" used in the present invention refers to a plant which comprises the introduced gene. The method for cloning the Brassica napus gene according to the present invention is the one commonly used in the art, such as, CTAB protocol is used to extract DNA from plant leaves. The methods for extracting mR A are various and have been well-established, such as, TRIzol Reagent Protocol from Invetrogen Co. or Total RNA Extraction from Qiagen Co., all of which are commercially available. cDNA library construction is also a conventional technique in molecular biology. Methods for constructing and transforming the vector according to the present invention into a plant are also those commonly used in the art. The involved plasmids (the entry vector PCR8/GW/TOPO and the expression vector plasmid pEarleygate 100) in the methods, host cells for transformation (e.g., Agrobacterium tumefaciens GV3101), and reagents (sucrose, etc.,) used are commercially available. The method most commonly used for polymorphic analysis of molecular marker is polyacrylamide gel electrophoresis in which the reagents used are commercially available, such as, acrylamide, methylene bisacrylamide, etc.

Example 4. Analysis on the transgenic Arabidoysis thaliana

The appearances of the foliage and seeds in the transgenic Arabidopsis thaliana plants are significantly different from those in the wild type Arabidopsis thaliana, this is due to overexpression of BnGRF2al gene and AtGRF2 gene in the transgenic Arabidopsis thaliana plants (Figs. 3 and 4). After harvest, the oil content of seeds was determined by pulse nuclear magnetic resonance spectrometer, and the result showed that oil content in the transgenic plants was increased to some different degree in comparison with that in the control. It is suggested from the above results that not only the Brassica napus gene but also the genes from other species having at least 70% homology to the sequence of sucha Brassica napus gene can increase the seed oil content.

The advantage of the present invention is that the Brassica napus BnGRF2 gene according to the present invention is reported for the first time to be associated with seed oil content. It is demonstrated by the experiment in Arabidopsis thaliana that such genes can actually increase both the oil content and thousand kernel weight of the seeds. The results from the study show that the seed oil content of transgenic Arabidopsis thaliana is significantly increased in comparison with that of the wild type (Table 1), with the highest increase by above 20%. The thousand kernel weight can be increased by up to 40%. Such genes can be used in breeding of Brassica napus toward high oil content, that is, such genes are overexpressed in Brassica napus varieties by using the constitutive 35S promoter, thus novel Brassica napus varieties with increased oil content and increased thousand kernel weight can be obtained. This accelerates breeding of Brassica napus toward oil content. Meanwhile, the use of said gene may be extended to breeding of other oil crops, such as soybean, peanut, sesame, etc. to increase the oil yield of the oil crops maximally.

Table 1. Determination of the oil content and the thousand kernel weight in Arabidopsis thaliana overexpressing BnGRF2al gene, AtGRF2 gene.

Example 5. The coding region sequences of BnGRF2 cDNA and AtGRF2 cDNA

The published gene sequences of Arabidopsis in the NCBI database were searched. EST sequences of Brassica napus were BLASTed and aligned with the Brassica napus genomic sequences which had been sequenced by the applicants. Based on the alignment, a pair of primers flanking the coding region of the gene of interest was designed (BnGRF2F: forward primer: (SEQ ID NO. 1 1), reverse primer: (SEQ ID No. 12) and used to amplify the corresponding sequence from cDNA of the Brassica napus leaves. Primers for amplification in Arabidopsis thaliana were as follows: AtGRF2 forward primer: (SEQ ID No. 13), and reverse primer: (SEQ ID No. 14), which were used in direct amplification from cDNA of the Arabidopsis leaves.

1. Extraction of mRNAs from Brassica napus, Arabidopsis thaliana.

RNA extraction (extraction of RNA with TRIZOL TM Kit): grind 100 mg of raw material in liquid nitrogen.

A. Add 1 ml of TRIZOL, and the resultant mixture is left at room temperature (RT, 20- 25°C, same hereinafter) for 5 min.

B. Add 200 μΐ of chloroform, agitate vigorously for 30 s, and the resultant mixture is left at RT for 2 min.

C. The mixture from step B is centrifuged at 4°C at 12000g for 15 min. The resultant supernatant is transferred into a new tube. 500 μΐ of isopropanol is added and mixed well. Then, the resulting mixture is left at RT for 15 min.

D. The resulting mixture is centrifuged at 4°C at 12000g for 15 min. The resultant supernatant is discarded. 1ml of 70% (vol/vol) ethanol is added and mixed well.

E. The resulting mixture is centrifuged at 4°C at 7500g for 7 min. The resultant supernatant is discarded and the RNA pellet is air-dired.

F. The air-dired RNA pellet is dissolved in DEPC-H₂0.

2. Reverse transcription of the first strand cDNA is performed with RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas), with operation following the instruction of the kit used.

3. PCR amplification is performed by using the resulting cDNA as a template, and BnGRF2 and AtGRF2 genes have been obtained. The sequences of BnGRF2 and AtGRF2 genes are as follows: the base sequence of the Brassica napus growth regulatory gene BnGRF2al is the nucleotide sequence shown in SEQ ID NO: 1, that of BnGRF2a2 is shown in SEQ ID NO. 3, that of BnGRF2bl is shown in SEQ ID NO. 5, and that of BnGRF2b2 is shown in SEQ ID NO. 7; the isolated protein sequence is illustrated by the amino acid sequence shown in SEQ ID NO: 2 for BnGRF2al , in SEQ ID NO: 4 for BnGRF2a2, in SEQ ID NO: 6 for BnGRF2bl , and in SEQ ID NO: 8 for BnGRF2b2; as for Arabidopsis AtGRF2 sequences, they are shown in the NCBI database (accession No.: NMJ00614 and NM_125364). Example 6. Construction of a transgenic expression vector and transformation of Arabidoysis thaliana

The gene sequence of BnGRF2al and of Arabidopsis GRF2 obtained by PCR amplification was ligated into the TOPO entry vector (Invitrogen Co.), then the obtained vector was transformed into DH5a competent cells (Invitrogen Co.). Screening of positive colonies with spectinomycin was performed. The plasmid of interest with the forwardly ligated insert was identified by PCR amplification using a vector-specific primer (T7 primer) and a gene-specific primer (the forward primer for the target gene). The plasmid of interest was mini-prepared, then recombined into the vector pEarleygate 100 (Invitrogen Co.), and finally transformed into DH5a competent cells. Screening of the transformant with kanamycin was performed. The presence of the inserted fragment in the transformant was confirmed by PCR amplification using a vector-specific primer (35 S promoter-specific primer) and a gene-specific primer (the reverse primer for the target gene). The schematic map of the resulting plasmid was shown in Fig. 1.

Transformation of Arabidopsis thaliana

Formulation of the reagent

Osmotic medium (1L): 1/2 x Murashige-Skoog; 5% (mass percentage) sucrose; 0.5 g of MES; adjusted with KOH to pH = 5.7, and then supplemented with 10 μΐ of lmg/ml 6- BA (6-benzylaminopurine) stock solution; 200 μΐ of Silwet L-77.

Transformation procedure:

(1) 10ml of the suspension of Agrobacterium transformed with the corresponding plasmid was prepared, then transferred into a large flask for overnight cultivation in the night just before the transformation day. Next day upon use, OD600 of the overnight culture of Agrobacterium should be between 1.2 and 1.6.

(2) The overnight culture was centrifuged at 5000 rpm at room temperature for 15 min.

(3) The supernatant was discarded and the Agrobacterium pellet was resuspended in the corresponding volume of the osmotic media, resulting in OD600 of about 0.8.

(4) A whole plant was immersed into the resulting Agrobacterium suspension for 30 s. (5) The treated plant was grown overnight with avoidance of light and then cultivated normally until producing seed.

Example 7. Screening and confirmation of transgenic Arabidopsis thaliana

Screening of the transformant

The vernalized Arabidopsis seeds were seeded in artificial soil irrigated with the saturated PNS nutrient solution and covered with plastic wrap. Two days later, light was given, and three days later, the plastic wrap was removed.

Condition in the artificial cultivation chamber was as follows: Relative humidity: 80%, constant temperature of 20-24°C, light intensity of 80-200 umol M2/S, light cycle: 8h of Dark, 16 h of Light. After about one week, the herbicide (glyphosate) was sprayed to screen positive plants.

Identification by PCR

(1) Extraction of total DNA from a transformed plant for PCR.

A. Leaves were cleaned with 70% (vol vol) ethanol and weighed about 100 mg.

B. 600 μΐ of extraction buffer (0.2 M Tris-HCl, 0.25 NaCl, 25mM EDTA, 0.5 % (mass percentage) SDS, pH 7.5) was added and rapid grinding was performed at room temperature.

C. the resulting mixture was vortexed for 5- 10s in a 1.5ml Ependorff tube to homogeneity.

D. the vortexed mixture was centrifuged at 12000 rpm at room temperature for 25 min. The supernatant was collected, equal volume of isopropanol was added, then precipitated at -20 °C overnight.

E. the resulting mixture from step C was centrifuged at 12000 rpm at room temperature for 15 min. DNA pellet was washed by adding 200 μΐ of 70% (vol vol) ethanol.

F. the resultant from step E was centrifuged at 12000 rpm at room temperature for 15 min. Ethanol was discarded. The tube with the DNA pellet was placed inversely on a paper towel until the ethanol was volatilized completely.

G. the extracted DNA pellet in the tube was dissolved in 100 μΐ of sterile water. The DNA concentration was estimated with a spectrometer or by electrophoresis.

H. PCR was performed with the total DNA as a template.

(2) PCR protocol Formulation ratio for the PCR reaction mixture was the same as that in identification of the plasmid by PCR, and based on the sequence of 35S promoter in the plant expression vector and of the reverse primer for the BnGRF2 gene (SEQ ID No. 15). The time and temperature for the 5'→3' reaction were as follows:

94°C 3min

94°C 45s,

59°C 45s

72°C 2min 30s, 30 cycles

72°C 5min

It was shown from the PCR identification that a electrophoretic band with expected size could be amplified from most of transformed plants, while no such band could be obtained from negative control, indicating that the foreign DNA fragment of the gene had been incorporated into the genome of the transgenic Arabidopsis thaliana. The result of this identification of the BnGRF2 gene in transgenic Arabidopsis plants is shown in Fig. 2.

Example 8. Determination of seed oil content and thousand kernel weight of the transgenic Arabidopsis thaliana

The transgenic homozygotic lines were grown at 21-23°C in a greenhouse. The seeds were harvested and then examined for the changes in the oil content and thousand kernel weight (Fig. 4). Results indicated that the seed oil content of transgenic Arabidopsis thaliana was significantly increased in comparison with that of the wild type (Table 1), with the highest increase by above 20%. Moreover, the thousand kernel weight in transgenic Arabidopsis thaliana was also increased to some extent, with the highest increase by up to 40%.

Claims

A method for increasing oil content in plants comprising increasing expression of a nucleic acid encoding a GRF2 protein.

The method according to claiml , wherein said oil content is seed oil content.

The method according to claim 2, further characterized in that the Thousand Kernel Weight of the seeds of said plants is increased.

The method according to any one of claims 1 to 3, wherein said GRF2 protein has at least 65% sequence identity to SEQ ID No. 2.

The method according to any one of claims 1 to 4, wherein said GRF2 protein has at least 90% sequence identity to SEQ ID No. 2.

The method according to any one of claims 1 to 5, wherein said nucleic acid has at least 90% sequence identity to SEQ ID No. 1.

The method according to any one of claims 1 to 4, wherein said GRF2 protein has at least 90% sequence identity to the GRF2 protein from Arabidopsis thaliana.

The method according to any one of claims 1 to 4 or 7, wherein said nucleic acid has at least 90% sequence identity to the GRF2 coding sequence from Arabidopsis thaliana.

The method according to any one of claims 1 to 8, comprising the steps of:

i) a heterologous plant-expressible promoter;

ii) a nucleic acid encoding a GRF2 protein; and optionally iii) a DNA region involved in transcription termination and polyadenylation; b) regeneration of said plant cell into a plant.

10. The method according to claim 9, wherein said plant-expressible promoter is a

constitutive promoter.

11. The method according to claim 10, wherein said constitutive promoter is the 35S promoter.

12. The method according to claim 9, wherein said plant-expressible promoter is a seed- specific promoter.

13. The method according to any one of claims 1 to 12, wherein said plant is a seed oil plant.

14. The method according to claim 13, werein said plant is a Brassicaceae plant.

15. Plants obtained by the methods according to any one of claims 1 to 14.

16. An isolated Brassica napus GRF2 gene encoding a protein selected from the group consisting of SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 6 and SEQ ID No. 8.

17. An isolated Brassica napus GRF2 gene consisting of the nucleotide sequence selected from the group consisting of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5 and SEQ ID No. 7.

18. Chimeric gene comprising the following operably linked nucleic acid molecules: a) a heterologous plant-expressible promoter;

c) a DNA region involved in transcription termination and polyadenylation.

19. The chimeric gene according to claim 18, wherein said GRF2 protein contains at least 90% sequence identity to SEQ ID No. 2.

20. The chimeric gene according to claim 19, wherein said nucleic acid encoding a GRF2 protein contains at least 90% sequence identity to SEQ ID No. 1.

21. The chimeric gene according to any one of claims 18 to 20, wherein said plant- expressible promoter is a constitutive promoter.

22. The chimeric gene according to claim 21, wherein said constitutive promoter is the 35 S promoter.

23. The chimeric gene according to any one of claims 18 to 20, wherein said plant- expressible promoter is a seed-specific promoter.

24. A plant or plant cell comprising the chimeric gene according to any one of claims 18 to 23.

25. Seeds from the plants of claim 15 or 24.

26. Oil from the seeds of claim 25.

27. Use of the isolated DNA according to claim 16 or 17 to increase plant seed oil content.

28. Use of the isolated DNA according to claim 16 or 17 to increase plant seed oil content and Thousand Kernel Weight.

29. Use of the chimeric gene according to any one of claims 18 to 23 to increase plant seed oil content.

30. Use of the chimeric gene according to any one of claims 18 to 23 to increase plant seed oil content and Thousand Kernel Weight.

31. Method for producing oil, comprising harvesting seeds from the plants according to claim 15 or 24 and extracting the oil from said seeds.

32. A method of producing food, feed, or an industrial product comprising

a) obtaining the plant of claim 15 or 24 or a part thereof; and

b) preparing the food, feed or industrial product from the plant or part thereof.

33. The method of claim 32, wherein

a) the food or feed is oil, meal, grain, starch, flour or protein; or

b) the industrial product is biofuel, fiber, industrial chemicals, a pharmaceutical or a nutraceutical.