Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
  • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
  • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

• the invention belongs to the field of plant bioengineering and plant improvement genetic engineering. Specifically, the present invention relates to isolation of a cotton fiber-specific expression gene RDL1 cDNA and construction of an overexpression vector, and a method for promoting superior traits such as seed enlargement and fiber growth of a transgenic crop by transferring the RDL1 gene.
• Background technique
• Crop seeds are important raw materials in agriculture and industry such as grain, cotton and oil. Their traits are directly related to the quality of seeds and the quality of their processed products. These traits primarily include the size of the seed volume, the weight of the seed, the length of the seed fiber (for the use of seed fibers;) and/or the strength of the seed fiber.
• Cotton is an important economic crop, and cotton fiber is an important raw material for the textile industry. In 2006, the world produced a total of 25.22 million tons of cotton, of which China's output was 6.73 million tons.
• the cotton textile industry is increasingly demanding the quality of cotton fibres, such as longer fibres, stronger hardness, finer fibres and more tidy fibres, so increasing the quality and yield of cotton is essential.
• Cotton fiber development is a highly programmed process, and improving the quality of cotton fiber is a major goal of cotton breeding research.
• Cotton fiber is a single-cell fiber formed by the differentiation and development of ovule epidermal cells. Its development process can be divided into four stages: the initial stage of fiber development, the elongation stage, the secondary wall thickening stage and the mature stage, in which the elongation period and The secondary wall thickening period has overlapping time domains. In these four periods, the morphological structure of fibroblasts is accompanied by important physiological and biochemical processes, and a large number of genes are involved in the regulation of fiber development. It is important to study the expression and regulation of these genes.
• GhRDL1 Gossypium hirsutum RD22-likel
• GhRDL1 Arabidopsis thaliana RD22-likel
• the GhRDL1 protein contains a plant-specific BURP domain protein at the C-terminus, and this function has been studied less.
• the object of the present invention is to use the plant RDL1 gene, especially the cotton RDL1 gene, to improve the traits of crop seeds, thereby improving the quality of crop seeds.
• a plant RDL1 gene or an encoded RDL1 protein thereof for improving the seed trait of a crop.
• the plant RDL1 gene is a cotton RDL1 gene.
• the sequence of the plant RDL1 gene is selected from the group consisting of:
• sequence of the RDL1 protein is selected from the group consisting of:
• the improvement of the crop seed trait comprises: increased seed volume, increased seed weight, seed fiber growth, and/or increased seed fiber strength.
• the crop is a dicot or a monocot.
• the crop is selected from the group consisting of: a gramineous crop, a Malvaceae cotton crop, a Brassica Brassica crop, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably cotton. Or rapeseed, most preferably cotton.
• a vector comprising a plant RDL1 gene.
• the vector contains the cotton RDL1 gene.
• the sequence of the RDL1 gene is selected from the group consisting of: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or a DNA sequence defined under (a) under stringent conditions
• Such DNA molecules include molecules or fragments thereof that are highly homologous to the cotton RDL1 or Arabidopsis RD22 gene or fragments thereof.
• the vector is selected from the group consisting of: a bacterial plasmid, a bacteriophage, a yeast plasmid, a plant cell virus, or a mammalian cell virus, preferably pEGFP-1, pCAMBIA1300, pCAMBIA2301, or pBI121 o.
• a genetically engineered host cell is provided, the host cell comprising a vector of the invention.
• the host cell is selected from the group consisting of a prokaryotic cell, a lower eukaryotic cell or a higher eukaryotic cell, preferably a bacterial cell, a yeast cell or a plant cell, more preferably Escherichia coli, Streptomyces, Agrobacterium, yeast, Agrobacterium is most preferred.
• a method of preparing a transgenic crop comprising:
• the seed of the transgenic crop has an improved trait.
• the RDL1 gene is a cotton RDL1 gene.
• the host cell is Agrobacterium.
• the crop is selected from the group consisting of: gramineous crops, Malvaceae, Brassica, Brassica, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably Cotton or canola, most preferably cotton.
• gramineous crops Malvaceae, Brassica, Brassica, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably Cotton or canola, most preferably cotton.
• the improved trait comprises: increased seed volume, increased seed weight, increased seed fiber, and/or increased seed fiber strength.
• the crop is cotton.
• a method of producing a crop seed having an improved trait comprising: increasing the expression level of the RDL1 gene in the crop.
• the expression level of the RDL1 gene in the crop is increased by transferring the RDL1 gene into the crop and expressing the gene.
• the method includes the steps of:
• step (iii) regenerating the plant cell, tissue or organ in step (ii), and using the plant to produce a crop seed having a modified trait.
• the improved trait comprises: increasing seed volume, increasing seed weight, increasing seed fibers, and/or increasing seed fiber strength.
• the crop is selected from the group consisting of: a gramineous crop, a Malvaceae cotton crop, a Brassicaceae Brassica crop, preferably cotton, rape, rice, wheat, barley, corn, or sorghum, more preferably Cotton or canola, most preferably cotton.
• the method comprises preparing a seed trait modified transgenic product using the method of the invention and obtaining a seed thereof.
• the method comprises preparing a transgenic crop by the method of the present invention, and hybridizing the transgenic crop with a non-transgenic crop or other transgenic crop to produce a hybrid progeny, the seed being improved
• the hybrid progeny are obtained and their seeds are obtained.
• a transgenic plant comprising a plant RDL1 gene is provided.
• the sequence of the RDL1 gene is selected from the group consisting of: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5; or (; b) under stringent conditions and (a) A defined sequence hybridizes and has a molecule that improves the activity of the crop seed trait.
• DNA molecules include molecules or fragments thereof which are highly homologous to the cotton RDL 1 or Arabidopsis RD22 gene or a fragment thereof.
• the transgenic plant is a gramineous plant, a malvaceae plant or a cruciferous plant, preferably a Malvaceae cotton plant or a Brassicaceae Brassica crop, more preferably the plant is selected from the group consisting of: Cotton, canola, rice, wheat, barley, corn or sorghum.
• a method of producing a plant comprising crossing a transgenic plant of the invention with a non-transgenic plant or other transgenic plant to obtain a hybrid progeny comprising the plant RDL1 gene is provided.
• the transgenic plant for hybridization and the non-transgenic plant or other transgenic plant are classified into the same family or different families of plants, preferably the same family of plants.
• the plant is preferably a gramineous plant, a malvaceae plant or a cruciferous plant, preferably a Malvaceae cotton plant or a Brassicaceae Brassica crop, more preferably the plant is selected from the group consisting of: cotton, rapeseed, rice, wheat , barley, corn or sorghum.
• the hybrid progeny have a stable genetic trait.
• the cotton RDL1 gene or the RDL1 protein encoded thereby is employed.
• Other aspects of the invention will be apparent to those skilled in the art from this disclosure.
• FIG. 1 Construction of the GFP-GhRDL1 transgenic vector.
• FIG. 1 Molecular identification of GFP-GhRDL1 transgenic cotton.
• FIG. 3 GhRDL1 subcellular localization by the GFP-GhRDL1 fusion protein.
• Figure 4 Analysis of 100-grain weight (fiber-containing) of GFP-GhRDL1 transgenic cotton seeds (; T 2 generation;). Among them, A is the comparison of the morphology of the seeds; B is the comparison of the 100-grain weight of the seeds.
• Figure 5 Seed size analysis of GhRDL l transgenic Arabidopsis thaliana.
• Figure 5A is a comparison of the morphology of the seeds;
• the present inventors conducted a long-term and in-depth study on the structure, localization and function of the RDL1 gene, and constructed a transgenic vector containing the RDL1 gene, and obtained plants having improved seed traits by plant transgenic technology, for example, an increase in 100-grain weight and Fiber-grown transgenic cotton, and transgenic Arabidopsis with increased seed volume.
• plant transgenic technology for example, an increase in 100-grain weight and Fiber-grown transgenic cotton, and transgenic Arabidopsis with increased seed volume.
• the RDL1 gene is involved in the traits of crop seeds, and the traits of the seeds of plants expressing the gene can be improved, such as increased volume, increased weight, fiber growth, and increased fiber strength.
• the inventors completed the present invention.
• the present inventors confirmed that the GhRDL1 protein is localized in certain regions of the cell wall by a subcellular localization of the RDL1-GFP fusion protein, and has a certain polarity distribution.
• the green fluorescent signal is located at the corners of the cell wall filled with pectin-rich polysaccharides, and the main component of the primary cell wall in the rapid elongation stage of cotton fibers is also pectin. This result suggests that the localization of GhRDL1 may be related to the pectin of the cell wall;
• the fusion protein of BURP and GFP showed similar localization characteristics.
• the inventors used the yeast two-hybrid method to screen the GhEXPAl (Gossypium hirsutum ⁇ -expansinl) protein to bind to the GhRDL1 protein.
• GhEXPAl Gossypium hirsutum ⁇ -expansinl
• the RFP-GhEXPAl fusion protein was co-transformed into Arabidopsis thaliana, and Arabidopsis root cells were observed by confocal microscopy, and fluorescence signals were found to be concentrated on the cell wall and co-localized. Further interactions between the two proteins were verified by Co-IP experiments.
• plant RDL1 gene or “RDL1 gene” is used interchangeably to refer to a gene that is highly homologous to a sequence encoding a cotton RDL1 protein, or a molecule that hybridizes to the gene sequence under stringent conditions, or A family gene molecule highly homologous to the above molecule, the expression of which has a certain improvement effect on the seed trait of the crop, for example, increasing the seed volume, increasing the weight, increasing the fiber, and/or increasing the fiber strength. Also included in the definition are molecules that hybridize to the cotton RDL1 gene sequence under stringent conditions, or family gene molecules that are highly homologous to the above molecules.
• the term "cotton RDL1 gene” refers to a highly homologous sequence encoding an Arabidopsis RD22 protein, which includes a molecule that hybridizes under stringent conditions to the cotton RDL1 gene sequence, or is highly homologous to the above molecule. Family gene molecules, and preferably the genes are specifically highly expressed during elongation of cotton fibers.
• the Upland cotton RDL1 encoding gene (; GhRDL1) encodes a protein containing 335 amino acid residues and a plant-specific BURP domain at the C-terminus.
• NCBI has published sequences of RDL1 and its homologous genes, such as AY072821 [(Li C-H,
• Asian cotton dehydration-inducible protein RD22-like protein 1 (RDL1) "mRNA, RDL1-1 allele, full-length cds]; AY641991 [Wang S Gossypium arboreum dehydration-induced protein RD22-like protein 2 (RDL2) mRNA, RDL2- 2 allele, complete cds, "Asian cotton dehydration-inducible protein RD22-like protein
• RDL2 RDL1-1 homologous gene, full-length cds
• the RDL1 gene of the present invention may be selected from: (a) SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5 (which correspond to AY072821, AY641990, and AY641991, respectively); or (b) under stringent conditions A molecule that hybridizes to the defined sequence of (a) and has activity to improve crop seed traits.
• stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or (2) hybridization Adding a denaturant such as 50% (v/v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc.; or (3) at least 50% identity between the two sequences, Hybridization occurs preferably at 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more.
• the sequence can be the complement of a sequence defined in ( ).
• the full-length nucleotide sequence of the RDL1 gene of the present invention or a fragment thereof can be usually obtained by a PCR amplification method, a recombinant method or a synthetic method.
• primers can be designed in accordance with the disclosed nucleotide sequences, particularly open reading frame sequences, and can be prepared using commercially available cDNA libraries or conventional methods known to those skilled in the art.
• the library is used as a template to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
• the RDL1 gene of the present invention is preferably obtained from cotton, and is highly homologous to the cotton RDL1 gene obtained from other plants (e.g., having 50% or more, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75).
• Other genes above %, above 80%, more preferably above 85%, such as 85%, 90%, 95%, or even 98% sequence identity are also within the equivalent scope of the present invention.
• Methods and tools for aligning sequence identity are also well known in the art, such as BLAST.
• the term "RDL1 protein” refers to a polypeptide having a gene encoded by the RDL1 gene of the present invention, and the definition also includes a variant form of the above polypeptide having a function of improving plant seed traits.
• the proteins of the invention may be naturally purified products, either chemically synthesized or produced recombinantly from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher plant, insect, and mammalian cells;).
• the RDL1 protein of the present invention is preferably encoded by the cotton RDL1 gene or a homologous gene or family gene thereof.
• the sequence of the RDL1 protein of the present invention may be selected from: (a) SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6; or (b) an amino group defined in (a) (a) Derived protein in an acid sequence which has been substituted, deleted or added with one or several amino acids and has activity to improve crop seed traits.
• the variant forms include (but are not limited to): one or more (typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid deletions, insertions and/or substitutions, and addition of one or several at the C-terminus and/or N-terminus (usually Within 20, preferably less than 10, more preferably less than 5; amino acids.
• amino acids typically 1-50, preferably 1-30, more preferably 1-20, optimally 1-10, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
• amino acid deletions amino acid deletions, insertions and/or substitutions
• addition of one or several at the C-terminus and/or N-terminus usually Within 20, preferably less than 10, more preferably less than 5; amino acids.
• the RDL1 protein of the present invention may or may not include an initial methionine residue and is still improved. Activity of crop seed traits.
• the random mutagenesis can be carried out by irradiation or exposure to a mutagen, and the protein in (b) above can also be obtained by site-directed mutagenesis or other known molecular biology techniques.
• the transgenic plant can be constructed using a coding sequence encoding the protein, and whether the seed trait in the transgenic plant is improved to screen and identify the resulting protein (see, for example, the method of the present invention;).
• the protein of the invention may be glycosylated, or may be non-glycosylated, depending on the host used in the recombinant production protocol.
• the term also encompasses active fragments and active derivatives of the RDL1 protein.
• Variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, sequences encoded by sequences that hybridize to the RDL1 protein coding sequence under high or low stringency conditions.
• Other polypeptides, such as fusion proteins comprising the RDL1 protein or a fragment thereof, can also be used in the present invention.
• the present invention also encompasses soluble fragments of the RDL1 protein.
• the fragment has at least about 10 contiguous amino acids of the RDL1 protein sequence, typically at least about 30 contiguous amino acids, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100.
• a contiguous amino acid is preferably at least about 10 contiguous amino acids of the RDL1 protein sequence.
• Crop refers to a plant of economic value in agriculture, agriculture, such as grain, cotton, oil, etc., whose economic value is mainly embodied in the seed of the plant.
• Crops include, but are not limited to, dicotyledonous plants or monocotyledons.
• Preferred monocot plants are grasses, more preferably rice, wheat, barley, corn, sorghum, and the like.
• Preferred dicot plants include, but are not limited to, Malvaceae, Brassicaceae, and the like, more preferably cotton, oilseed, etc., most preferably cotton.
• the properties of the seed include, but are not limited to, seed volume, seed weight, seed fiber length, and/or strength of seed fibers, and the like.
• the improvement of the seed trait refers to an increase in seed volume, an increase in seed weight, an increase in seed fiber, and/or an increase in seed fiber strength, as compared with the seed produced by the unmodified plant.
• seed finger or “hundred grain weight” are used interchangeably and refer to the weight per 100 seeds, which reflects the seed size and fullness of the seed.
• Also provided in the invention is a method of producing a crop seed having improved traits, the method comprising:
• the expression level of the RDL1 gene (preferably the cotton RDL1 gene;) in the crop is high, that is, the improvement is achieved by increasing the RDL1 gene expression level or the RDL1 protein content in the crop.
• a transgenic method can be employed, which generally includes the steps of constructing a vector into which the RDL1 gene is transferred, transferring to a crop, and breeding.
• the present invention also relates to a vector comprising the RDL1 gene, and a host cell genetically engineered using the vector, and a transgenic plant which highly expresses RDL1 by transgene.
• the coding sequences of the present invention can be used to express or produce recombinant RDL1 proteins by conventional recombinant DNA techniques (Science, 1984; 224: 1431). Generally there are the following steps:
• vector and “recombinant expression vector” are used interchangeably and refer to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art. In general, any plasmid and vector can be used as long as it can replicate and stabilize in the host.
• An important feature of expression vectors is that they typically contain an origin of replication, a promoter, a marker gene, and a translational control element.
• expression vectors containing the RDL1 coding sequence and appropriate transcription/translation control signals can be used to construct expression vectors containing the RDL1 coding sequence and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
• the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
• the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
• pEGFP-1, pCAMBIA1300, pCAMBIA2301, or pBI121 or the like is preferably used.
• the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
• selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
• Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
• the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
• Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like.
• Agrobacterium is preferably used as a host cell.
• transcription will be enhanced if an enhancer sequence is inserted into the vector.
• An enhancer is a cis-acting factor of DNA, usually about 10 to 300 base pairs, acting on a promoter to enhance transcription of the gene. It will be apparent to one of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells.
• the terms "transgenic crop”, “transformant” or “transformed plant” are used interchangeably herein to refer to a cell, an organ or a plant obtained by a conventional transgenic method and which has been transferred into the RDL1 gene of the present invention and stably expresses the RDL1 protein. .
• the transformed plant can be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method.
• methods such as Agrobacterium transformation or gene gun transformation, such as the leaf disc method.
• plants can be regenerated by a conventional method to obtain plants having improved disease resistance.
• the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
• the medium used in the culture may be selected from various conventional media depending on the host cell used.
• the cultivation is carried out under conditions suitable for the growth of the host cell. After the host cell has grown to the appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature conversion or chemical induction;) and the cells are cultured for a further period of time.
• the recombinant polypeptide in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell.
• the recombinant protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
• Advantages of the invention include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration),
• Extraction of cotton RNA by cold phenol method 2 g of material (fibers on the surface of the ovule 9 days after flowering of the upland cotton), ground into powder in liquid nitrogen, transferred to a 50 ml centrifuge tube, and added to 8 1111 extraction buffer (1 13 ⁇ 45'11 ( 1, 50 mM) EDTA, 1% SDS, pH 9.0) and an equal volume of water-saturated phenol: chloroform: isoamyl alcohol (25:24: 1), shake and mix, place on ice for 1 h, mix once every 10 minutes. Centrifuge at 13000 g for 20 minutes at °C.
• the supernatant was transferred to a 1.5 ml Enppendorf tube, and 1/3 volume of 8 M LiCl and volume of NaAC was added and allowed to stand overnight at -20 °C. Centrifuge at 13000 g for 20 minutes at ° C. The supernatant was removed, and the precipitate was washed twice with 1 ml of 70% ethanol, and the resulting precipitate was dried at room temperature and dissolved in 100 to 200 L of DEPC-treated water.
• a pair of specific primers (containing an enzyme cleavage site and a protective base;) were synthesized, and the primer was used to carry out a PCR reaction using RDL1 cDNA as a template:
• the GFP gene used was derived from pEGFP-1 (Clontech, Palo Alto, USA). A suitable restriction site is introduced by PCR. The stop codon for GFP was removed and a non-folding sequence of 6 amino acids (GPGGGG) was added.
• GS 1 5'-CTAGTCTAGAATGGTGAGCAAGGGCGAGGAG-3' (SEQ ID NO: 9)
• TCGTCCATGCC-3' (SEQ ID NO: 10) was used as a primer, pEGFP-1 vector was used as a template, and the GFP coding region was amplified with Pyrobest DNA polymerase (purchased from TAKARA). After the gel was recovered, it was digested with Smal and Ncol. Between the EcoRV and the Ncol site of pET32c, a GFP-32c intermediate vector was constructed.
• the target fragment was amplified with Pyrobest DNA polymerase, and the corresponding cleavage site BamHI and
• the Agrobacterium containing the vector plasmid was cultured on YEB bacterial medium supplemented with kanamycin 50 mg/L, rifampicin 100 mg/L, streptomycin 300 mg/L for 2 to 3 days, and then single colonies were inoculated.
• the YEB liquid medium containing the same antibiotic was cultured in suspension at 28 ° C, 200 rpm / min on a shaker overnight.
• the bacterial solution was centrifuged at 4000 rpm/min for 10 minutes, and the pellet was resuspended in 1/2 MS liquid medium containing 30 g/L of glucose and 100 ⁇ /L of acetosyringone, and the OD 6 (K) value was adjusted to about 0.4 to 0.6. Infected solution is reserved.
• Cotton R15 wild-type upland cotton of tetraploid, as the parent of transgenic was routinely sterilized and placed in 1/2MS0 medium [1/2MS salt (purchased from DUCHEFA M0221) + 5g/L glucose + 7g/L agar powder, pH6.0], germinated in the dark, after 5 ⁇ 7 days, the hypocotyls of the sterile seedlings were cut into segments of about 1.0 cm as the transformed explants.
• 1/2MS0 medium 1/2MS salt (purchased from DUCHEFA M0221) + 5g/L glucose + 7g/L agar powder, pH6.0]
• the explants are immersed in Agrobacterium liquid for 15 to 20 minutes and transferred to co-culture medium.
• MSB1 (MS salt ten B5 organic (an organic mixture containing inositol, nicotinic acid, V B ⁇ n V B6 ) ten 30 g / L glucose ten O.lmg / LKT (cytokinin) ten 0.1 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 12.2 g/L GeMte (pH curing agent), pH 6.0), after 2 days of dark culture at 22 ° C, the explants were transferred to the medium. Callus induction was performed on MSB2 (MSB1 + 500 mg/L cephalosporin deca 80 mg/L kanamycin;).
• Explants were induced by resistant callus, callus proliferation and embryogenic callus induction (medium MSB3: MS salt ten B5 organic ten 30 8 glucose ten 2.5 8 0611 ⁇ 6, pH 6.0), body Cell Embryogenesis (medium MSB4: MS salt ten B5 organic ten 30 g/L glucose ten 1.0 g/L asparagine ten 2.0 g/L glutamine ten 3.0 g/L Gelrite, pH 6.0; MS salt KNO 3 is doubled, NH 4 NO 3 ) is removed, and resistant test tube seedlings are regenerated. When the test tube seedlings grow to 3-4 true leaves, they are transplanted into pots and placed in an artificial climate chamber for growth.
• medium MSB3 MS salt ten B5 organic ten 30 8 glucose ten 2.5 8 0611 ⁇ 6, pH 6.0
• body Cell Embryogenesis (medium MSB4: MS salt ten B5 organic ten 30 g/L glucose ten 1.0 g/L asparagine ten
• Transformation of Arabidopsis plants was carried out using floral dip (Clough and Bent, 1998, Plant J. 16, 735-743).
• the Agrobacterium culture method is the same as above, and the bacterial cells are centrifuged at 4000 rpm/min for 10 minutes. Resuspend in 500 1111 containing 0.02% 81 ⁇ 61 1 ⁇ 77 in 5% sucrose solution. Soak the aerial part of the plant in the bacterial solution for 5 seconds, place it in a plastic basin, moisturize and protect from light for 16 to 24 hours.
• T Q seeds were vernalized at 4 ° C for 2 to 4 days, treated with 20% bleaching water for 15 minutes, and washed with sterile water for 3 to 4 times.
• DNA extraction was performed using a cold phenol method. Take 2 g of cotton young leaves (transgenic cotton with R15 as a receptor), grind it into powder in liquid nitrogen, transfer to a 50 ml centrifuge tube, and add 8 1111 extraction buffer (1
• the PCR was identified using a GFP-specific primer (same as in Example 2) and the template was a pEGFP- ⁇ plasmid.
• the PCR reaction conditions were: pre-denaturation at 94 ° C for 5 minutes; then denaturation at 94 ° C for 30 seconds, renaturation at 56 ° C for 30 seconds, extension at 72 ° C for 1 minute for 35 cycles; last 72 ° C extension for 10 minutes .
• RDL1 transgenic cotton and transgenic female R15 were planted in two places: plant No. 105 and plant No. 117 were planted in Shanghai farm, and plant No. 115 and plant No. 119 were planted in Hainan farm.
• the T 2 plants of all transgenic lines were individually harvested from mature cotton, and the consistency of the collected parts was kept as much as possible.
• 100 seeds were randomly taken from the mature cotton collected from each plant, and 10 or more seeds were used to comb the fibers. The fiber length was measured and the 100 seeds (with or without fiber;) were weighed. The above data is statistically plotted.
• the data of the No. 115 plant and the No. 119 plant planted on the Hainan farm were 5 statistical results. As shown, the No. 1 and No.
• the simultaneously constructed GhRDL1 transgenic vector (without the GUS gene;) was also transferred into Arabidopsis thaliana by Agrobacterium-mediated transformation, and homozygous transgenic positive plants were obtained by resistance screening and RT-PCR.
• the next generation of seeds and the simultaneously collected WT seeds were measured for length and width (; n > 50) under a 20-fold magnification dissection microscope and statistical analysis was performed.
• the length and width of GhRDL1 transgenic Arabidopsis seeds were statistically significantly different from the length and width of WT seeds, respectively (p ⁇ 0.01).

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