WO2023087761A1 - 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用 - Google Patents

大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用 Download PDF

Info

Publication number
WO2023087761A1
WO2023087761A1 PCT/CN2022/106929 CN2022106929W WO2023087761A1 WO 2023087761 A1 WO2023087761 A1 WO 2023087761A1 CN 2022106929 W CN2022106929 W CN 2022106929W WO 2023087761 A1 WO2023087761 A1 WO 2023087761A1
Authority
WO
WIPO (PCT)
Prior art keywords
soybean
gmga3ox1
gibberellin
hydroxylase
seq
Prior art date
Application number
PCT/CN2022/106929
Other languages
English (en)
French (fr)
Inventor
喻德跃
黄方
胡德洲
王慧
杨中义
陆少奇
李霄
Original Assignee
南京农业大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京农业大学 filed Critical 南京农业大学
Priority to US18/031,159 priority Critical patent/US20230313151A1/en
Publication of WO2023087761A1 publication Critical patent/WO2023087761A1/zh

Links

Images

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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/20—Brassicaceae, e.g. canola, broccoli or rucola
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A01H6/542—Glycine max [soybean]
    • 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)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/11—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with 2-oxoglutarate as one donor, and incorporation of one atom each of oxygen into both donors (1.14.11)
    • C12Y114/11015—Gibberellin 3-beta-dioxygenase (1.14.11.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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 relates to the application of soybean gibberellin 3 ⁇ -hydroxylase coding gene GmGA3ox1, which belongs to the field of genetic engineering.
  • Soybean yield-related traits are complex quantitative traits controlled by multiple genes, and their traits are the result of the interaction of genotype and environment. There are many factors affecting soybean yield, including growth morphological traits, photosynthetic physiological traits, and yield components. Among them, the yield components of soybean include 100-seed weight, number of pods per plant, number of grains per plant, yield per plant, etc.
  • Gibberellin (Gibberellin, GA) is a plant hormone that plays a vital role in the growth and development of plants. Many studies have reported that GA is involved in the regulation of seed germination, leaf extension, flowering and fruit development, as well as the regulation of environmental signals, such as daylight length and so on. At the cellular level, GA also stimulates cell elongation and cell division. In addition, the results of the functional analysis of the currently cloned crop yield and yield-related trait genes showed that multiple yield-related genes were mainly related to GA synthesis or its regulatory pathways.
  • GmGA3ox1 is a soybean gibberellin 3 ⁇ -hydroxylase (gibberellin 3 ⁇ -hydroxylase, GA3ox).
  • GA3ox is the key rate-limiting enzyme in the GA synthesis pathway, responsible for the synthesis of biologically active gibberellins, and is a key factor in regulating the synthesis of active GA.
  • GA3ox gene there is no report on the function of GA3ox gene in soybean.
  • the purpose of the present invention is to disclose the application of soybean gibberellin 3 ⁇ -hydroxylase gene GmGA3ox1 genetic engineering to improve grain weight.
  • Tissue expression and GUS staining analysis show that GmGA3ox1 is mainly expressed in soybean leaves and stems, and almost not expressed in other tissues.
  • the expression level of GmGA3ox1 gene was positively correlated with soybean grain weight.
  • subcellular localization analysis showed that GmGA3ox1 was localized in the cell membrane and cytoplasmic matrix.
  • the gene can be introduced into Arabidopsis thaliana and soybean as a target gene, and the grain weight of the transgenic Arabidopsis and the transgenic soybean is increased.
  • the nucleotide sequence shown in SEQ ID NO.1 is specifically:
  • GmGA3ox1 can complement the low grain weight phenotype of the atga3ox1 mutant.
  • overexpressing the excellent haplotype of this gene can significantly increase the grain weight of soybean.
  • soybean GmGA3ox1 gene positively regulates the grain weight of transgenic Arabidopsis.
  • GmGA3ox1 was mainly expressed in soybean leaves and stems, and showed a positive correlation with soybean grain weight.
  • Subcellular localization analysis proved that GmGA3ox1 is a protein localized to cell membrane and cytoplasmic matrix.
  • Overexpression of GmGA3ox1 found that the gene positively regulates the grain weight of transgenic Arabidopsis. It can be seen that GmGA3ox1 can be used as a target to regulate soybean grain weight and be used for soybean yield modification.
  • A GmGA3ox1 pro (-1957): GUS staining of GUS transgenic Arabidopsis;
  • B GmGA3ox1 pro (-1005): GUS transgenic Arabidopsis GUS staining of Arabidopsis;
  • C GmGA3ox1 pro (-487): GUS transgenic Arabidopsis;
  • GmGA3ox1 overexpression in atga3ox1 mutants increases Arabidopsis grain weight;
  • FIG. 7 The superior haplotype of overexpressing GmGA3ox1 increased the grain weight of soybean;
  • A Seed phenotype of Williams82 (W82) and two GmGA3ox1-OE transgenic lines;
  • B Schematic diagram of soybean seed length, width and height
  • G W82 and 2 GmGA3ox1-OE transgenic lines
  • the grain height of the strain, N 10; the error bars represent ⁇ SEM, and the statistical analysis adopts two-tailed t-test, **: P ⁇ 0.01,
  • FIG. 8 PCR detection of transgenic Arabidopsis and soybean;
  • A GmGA3ox1 pro (-1957): detection of GUS transgenic Arabidopsis, the target fragment size is 1957bp; M: Marker DL2000, P: positive plasmid, ck: wild Arabidopsis Types, 1-6: different GmGA3ox1 pro (-1957): GUS transgenic plants, H 2 O: blank control;
  • B GmGA3ox1 pro (-1005): detection of GUS transgenic Arabidopsis, target fragment size 1005bp, M: Marker DL2000, P: positive plasmid, ck: Arabidopsis wild type, 1-6: different GmGA3ox1 pro (-1005): GUS transgenic plants, H 2 O: blank control;
  • C GmGA3ox1 pro (-487): GUS transgenic Arabidopsis detection, target fragment size 487bp, M: Marker DL2000, P: positive plasmi
  • RNA Kit Total RNA Kit (Tiangen, Beijing, China).
  • RNA obtained is a template, and the reverse transcription kit (TaKaRa) provided by Japan TaKaRa Company Primer Script TM RT reagent kit, Japan) instructions for reverse transcription, after obtaining the first strand of cDNA, PCR amplification, PCR program is as follows: PCR program is as follows: 95 °C pre-denaturation for 3 minutes, 95 °C denaturation for 15 seconds, 60 °C Anneal for 15 seconds, extend at 72°C for 90 seconds, a total of 35 cycles, and finally keep at 72°C for 5 minutes, and then keep the temperature at 12°C to obtain the cDNA of Nannong 1138-2.
  • TaKaRa reverse transcription kit
  • PCR program is as follows: 95 °C pre-denaturation for 3 minutes, 95 °C denaturation for 15 seconds, 60 °C Anneal for 15 seconds, extend at 72°C for 90 seconds, a total of 35 cycles, and finally keep at 72°C for 5 minutes, and then keep the temperature at 12°
  • the gene (Glyma.07g033800, GeneID: 100795921) corresponding to GmGA3ox1 was found, and specific primers were designed according to the nucleotide sequence provided by the database.
  • the primer sequence is shown in SEQ ID NO.3 (atgccttctc tctcgaagc ct ) and SEQ ID NO.4 (ctaaatatct agattgaag), the gene was amplified from the gene coding region (CDS) sequence of soybean variety Nannong 1138-2, and after PCR cloning, the PCR product was tapped and purified, ligated and transformed, and positive Monoclonal sequencing, after sequencing, obtain the CDS sequence of the soybean GmGA3ox1 gene with the length of 1137bp with the complete coding region, wherein the coding region sequence is shown in SEQ ID NO.1, the size is 1137bp (Fig. 1), nucleotide sequence and amino acid sequence ( SEQ ID NO.1 and SEQ ID NO.2).
  • amino acid sequence shown in SEQ ID NO.2 is specifically:
  • GmGA3ox1 was mainly expressed in soybean leaf and stem tissues, with lower expression levels in roots, stems, flowers, pods and seeds (Fig. 2).
  • the PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 90 seconds, a total of 35 cycles, and finally incubation at 72°C for 5 minutes, followed by constant temperature at 4°C, and the length was obtained after sequencing.
  • the promoter sequence of GmGA3ox1 gene is 1957bp, 1005bp and 487bp.
  • the sequences of the promoters of the above three GmGA3ox1 genes with different lengths were inserted into the pCAMBIA1381Z vector, and then the constructed vectors were transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method.
  • Roots , stems, leaves , flowers , Pods and seeds were stained with GUS.
  • the kit used for the staining was from Beijing Suo Laibao Technology Co., Ltd., the product number is G3060.
  • specific primers are used to perform PCR detection on the extracted DNA fragments, and the detection primers are SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12 , SEQ ID NO.13 and SEQ ID NO.14, the PCR detection gel map is shown in Figure 8.
  • GUS staining results showed that the three types of GmGA3ox1 gene promoters all had high activity, and the tissues with the highest activity of these three types of promoters were all located in leaves, followed by stems, flowers, seeds, pods and roots ( Figure 4) .
  • RNA of leaves of soybean variety Nannong 1138-2 was used as a template, and the first strand of cDNA was synthesized by reverse transcription, followed by PCR amplification.
  • SEQ ID NO.15(cctctagaat gccttctctc tccgaagcct) and SEQ ID NO.16( ccgctcgaga atatctagat tgaag) the PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 90 seconds, a total of 35 cycles, and a final incubation at 72°C for 5 minutes, followed by constant temperature at 4°C , the CDS sequence of soybean GmGA3ox1 gene without stop codon was obtained after sequencing.
  • the CDS sequence of soybean GmGA3ox1 gene without stop codon was inserted into pFGC5941 expression vector containing GFP tag.
  • the pFGC5941 vector has a 35S promoter, which can strongly induce the expression of the target gene GmGA3ox1 in the recipient.
  • the constructed vector was transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method.
  • the empty pFGC5941 vector was also transformed into EHA105 as a control.
  • the total RNA of leaves of soybean variety Nannong 1138-2 was used as a template, and the first strand of cDNA was synthesized by reverse transcription, followed by PCR amplification.
  • the primer sequence is shown in SEQ ID NO.17 (gtcgacggta tcgataagct tatgccttct ctctccgaag cc), SEQ ID NO .18 (cgctctagaa ctagtggatc cctaaatatc tagattgaag cccac).
  • the PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 90 seconds, a total of 35 cycles, and a final incubation at 72°C for 5 minutes, followed by constant temperature at 4°C.
  • the length of the complete coding region is the CDS sequence of the soybean GmGA3ox1 gene with a length of 1137bp.
  • the total RNA of the leaves of the soybean landrace Jurong lentils was used as a template, and after the first strand of cDNA was synthesized by reverse transcription, PCR amplification was carried out.
  • the primer sequences are shown in SEQ ID NO.19 (ggatatgttg aaactttcct ttgc) and SEQ ID NO. 20 (atgcatgatc aatcatttac).
  • the PCR program is as follows: pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 15 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 120 seconds, a total of 35 cycles, and finally incubation at 72°C for 5 minutes, followed by constant temperature at 4°C, and 4187bp were obtained after sequencing The soybean GmGA3ox1 genome sequence.
  • the overexpression vector for transforming Arabidopsis thaliana When constructing the overexpression vector for transforming Arabidopsis thaliana, insert the CDS sequence of the GmGA3ox1 gene amplified from the leaves of Nannong 1138-2 into the pBI121 expression vector to obtain the pBI121-GmGA3ox1 plant overexpression vector.
  • the plant transformation vector pBI121 contains 35S Strong promoter, can strongly induce the expression of the target gene GmGA3ox1 in the recipient.
  • the vector was then transformed into Agrobacterium tumefaciens strain EHA105 by freeze-thaw method.
  • the 4187bp genome sequence of GmGA3ox1 was amplified from the soybean landrace Jurong lentil.
  • This 4187bp includes 1554bp promoter and 5'UTR region, 1899bp intron and exon region and 539bp 3'UTR region, this 4187bp fragment is connected to the pCAMBIA3301 vector that has removed CaMV 35S promoter, obtains pCAMBIA3301-GmGA3ox1 plant overexpression vector.
  • This vector was transformed into Agrobacterium tumefaciens strain EHA105.
  • Transform Arabidopsis thaliana by the flower-dipping method soak the flower buds of the atga3ox1 mutant Arabidopsis thaliana in step 2) Agrobacterium tumefaciens strain EHA105 containing pBI121-GmGA3ox1, and infect for 30 seconds to 1 minute. Wrap the plants with plastic wrap or fresh-keeping bags to maintain humidity, and cultivate them in the dark for 24 hours. Afterwards, the growth status of the plants can be seen, and after a new batch of inflorescences grow out, they will be transformed again. The Arabidopsis was placed under normal conditions to continue growing, and the seeds were harvested.
  • the grain weight of three 35S:GmGA3ox1/atga3ox1 transgenic lines was significantly larger than that of atga3ox1 mutant plants and slightly higher than that of Col-0 plants (Fig. 6D), proving that overexpression of GmGA3ox1 gene in atga3ox1 mutants can increase grain weight.
  • the soybean cultivar Williams 82 was transformed by the cotyledonary node transformation method, and the soybean leaf axils grown for 5 to 6 days were wounded, and then the vectors containing pCAMBIA3301-GmGA3ox1 obtained in step 2) were inoculated into the wounds of the soybean leaf axils. Placed at 25°C for co-cultivation for 4-5 days. Then they were washed with sterilized ultrapure water and Wish-Liquid respectively, placed in SIM medium without glufosinate, and cultured under light at 26°C for 15 days to induce germination. After 15 days, it was changed to the SIM medium added with 6 mg/L glufosinate.
  • the expression level of GmGA3ox1 in the two transgenic soybean lines was significantly higher than that in the control W82 plant ( Figure 7C) (primers are SEQ ID NO.23, SEQ ID NO.24), proving that this promoter can Effectively drives the expression of GmGA3ox1.
  • the 100-grain weight and grain length of the two GmGA3ox1 transgenic soybean lines were significantly greater than those of the W82 plant, and the grain width and grain height were not significantly different from those of the W82 plant (A, B, D-G in Fig. 7), proving the superiority of overexpressing GmGA3ox1 Haplotype genes can increase the grain weight and grain length of transgenic soybean.

Abstract

本发明公开了大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用。SEQIDNO.1所示大豆赤霉素3β-羟化酶基因GmGA3ox1在基因工程改造拟南芥和大豆粒重的应用。在拟南芥中，过表达GmGA3ox1能互补atga3ox1突变体低粒重的表型。在大豆中，过量表达该基因的优异单倍型，能够显著提高大豆的粒重。

Description

大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用
本申请要求于2021年11月17日提交中国专利局、申请号为“202111360441.9”、发明名称为“大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用，属于基因工程领域。
背景技术
大豆起源于我国，双子叶植物，是重要的油料和粮食作物，为人类和动物提供丰富的蛋白和充分的油脂，具有重要的经济价值。大豆在我国国民经济发展及国际油料作物生产中均占有重要地位。作为一种重要的作物，大豆的产量一直是人们关注的重点。培育高产、优质的大豆品种一直是育种家的首要目标之一。大豆产量的构成可以从两个方面解析，一种是产量由生物量和收获指数组成，二是由单位面积株数、单株荚数、每荚粒数、百粒重构成。大豆产量相关性状是复杂的数量性状，受多基因控制，其性状表现是基因型与环境共同作用的结果。大豆产量影响因素较多，包括生长形态性状、光合生理性状以及产量组分等。其中，大豆的产量组分包括百粒重、单株荚数、单株粒数、单株产量等。
赤霉素(gibberellin，GA)是一种植物激素，对植物的生长发育过程起着至关重要的作用。很多的研究报道表明GA参与调节种子萌发、叶片扩展、开花和果实发育，以及调节环境信号，如日照长度等。在细胞水平上，GA还会刺激细胞伸长和细胞分裂。另外，目前克隆的作物产量及产量相关性状基因功能分析结果表明，多个产量相关基因主要与GA合成或与其调控途径相关。GmGA3ox1是一种大豆赤霉素3β-羟化酶(gibberellin 3β-hydroxylase，GA3ox)。在植物体内，GA3ox是GA合成途径中的关键限速酶，负责合成具有生物活性的赤霉素，是调节活性GA合成的关键因子。然而在大豆中还没有GA3ox基因功能的报道。
发明内容
本发明的目的在于公开大豆赤霉素3β-羟化酶基因GmGA3ox1基因工程改良粒重的应用，组织表达及GUS染色分析表明GmGA3ox1主要在大豆叶片和茎中表达，在其他组织里几乎不表达。GmGA3ox1基因表达量与大豆粒重呈现正相关 关系。此外，亚细胞定位分析表明GmGA3ox1定位在细胞膜及细胞质基质中。该基因可作为目的基因导入拟南芥和大豆中，表现为提高转基因拟南芥和转基因大豆的粒重。
本发明的目的可通过以下技术方案实现：
SEQ ID NO.1所示大豆赤霉素3β-羟化酶基因GmGA3ox1在基因工程改造拟南芥和大豆粒重的应用。SEQ ID NO.1所示核苷酸序列具体为：
Figure PCTCN2022106929-appb-000001
在拟南芥中，过表达GmGA3ox1能互补atga3ox1突变体低粒重的表型。在大豆中，过量表达该基因的优异单倍型，能够显著提高大豆的粒重。
有益效果
我们发现大豆GmGA3ox1基因正调控转基因拟南芥的粒重。通过表达量分析，证明GmGA3ox1主要在大豆叶片和茎中表达，与大豆粒重呈现正相关关系。 亚细胞定位分析证明GmGA3ox1为细胞膜及细胞质基质定位蛋白。过量表达GmGA3ox1发现该基因正调控转基因拟南芥的粒重。由此可见，GmGA3ox1可以作为调节大豆粒重的靶点，用于大豆的产量改造。
附图说明
图1.PCR克隆GmGA3ox1琼脂糖凝胶电泳图；目的片段大小1137bp，Marker：DL2000Plus；
图2.GmGA3ox1组织表达模式，N＝3；
图3.GmGA3ox1的表达量与大豆粒重呈正相关关系；
图4.GmGA3ox1转基因拟南芥各组织的GUS染色，N＝3；(A)GmGA3ox1 pro(-1957):GUS转基因拟南芥的GUS染色；(B)GmGA3ox1 pro(-1005):GUS转基因拟南芥的GUS染色；(C)GmGA3ox1 pro(-487):GUS转基因拟南芥；
图5.GmGA3ox1蛋白的亚细胞定位分析；GFP，GFP荧光；Light，明场；Merge，融合蛋白；35S:GFP，空载对照；35S:GmGA3ox1-GFP，带GFP标签的GmGA3ox1蛋白；比例尺：20μm；
图6.atga3ox1突变体中GmGA3ox1的过量表达提高了拟南芥的粒重；(A)4周大的拟南芥野生型(Col-0)、atga3ox1突变体和三个35S:GmGA3ox1/atga3ox1转基因株系的莲座叶表型；(B)7周大的Col-0、atga3ox1突变体和三个35S:GmGA3ox1/atga3ox1转基因株系的成熟植株表型。(C)Col-0、atga3ox1突变体和三个35S:GmGA3ox1/atga3ox1转基因株系的种子表型；比例尺＝1mm；(D)atga3ox1突变体和三个35S:GmGA3ox1/atga3ox1转基因株系的千粒重比较，N＝9；误差线表示±SEM；统计分析采用双尾t检验；**：P<0.01，***：P<0.001；
图7.过量表达GmGA3ox1的优异单倍型提高了大豆的粒重；(A)Williams82(W82)和2个GmGA3ox1-OE转基因株系的种子表型；(B)大豆种子长、宽、高示意图(C)GmGA3ox1在W82和2个GmGA3ox1-OE转基因株系中的相对表达水平，N＝3；(D)W82和2个GmGA3ox1-OE转基因株系的百粒重，N＝10；(E)W82和2个GmGA3ox1-OE转基因株系的粒长，N＝10；(F)W82和2个GmGA3ox1-OE转基因株系的粒宽，N＝10；(G)W82和2个GmGA3ox1-OE转基因株系的粒高，N＝10；误差线表示±SEM，统计分析采用双尾t检验，**：P<0.01，***：P<0.001，ns：不显著；
图8.转基因拟南芥、大豆PCR检测；(A)GmGA3ox1 pro(-1957):GUS转基因拟 南芥检测，目的片段大小1957bp；M：Marker DL2000，P：阳性质粒，ck：拟南芥野生型，1-6：不同的GmGA3ox1 pro(-1957):GUS转基因植株，H 2O：空白对照；(B)GmGA3ox1 pro(-1005):GUS转基因拟南芥检测，目的片段大小1005bp，M：Marker DL2000，P：阳性质粒，ck：拟南芥野生型，1-6：不同的GmGA3ox1 pro(-1005):GUS转基因植株，H 2O：空白对照；(C)GmGA3ox1 pro(-487):GUS转基因拟南芥检测，目的片段大小487bp，M：Marker DL2000，P：阳性质粒，ck：拟南芥野生型，1-6：不同的GmGA3ox1 pro(-487):GUS转基因植株，H 2O：空白对照；(D)拟南芥atga3ox1突变体的PCR检测，设定特定的扩增时间，在设定的时间内atga3ox1突变体不能扩增出2000bp大小的片段，而野生型植株Col-0能扩增出2000bp大小的片段，M：Marker DL2000，Col-0：拟南芥野生型Col-0，atga3ox1：拟南芥atga3ox1突变体，H 2O：空白对照；(E)GmGA3ox1转基因拟南芥的PCR检测，目的片段大小1137bp，M：Marker DL2000，Col-0：拟南芥野生型Col-0，atga3ox1：拟南芥atga3ox1突变体，H 2O：空白对照，1-6：不同的35S:GmGA3ox1/atga3ox1转基因植株；(F)GmGA3ox1转基因大豆的PCR检测，目的片段大小800bp，M：Trans5K DNA Marker，P：阳性质粒，ck：野生型大豆W82，H 2O：空白对照，1-6：不同的GmGA3ox1转基因植株。
具体实施方式
下面结合附图及实施例对本发明做进一步说明。
下述实施例中所用方法如无特别说明，均为常规方法。
实施例1
1)大豆赤霉素3β-羟化酶基因GmGA3ox1的克隆
以大豆品种南农1138-2为取材对象，取其叶片，用研钵研碎，加入盛有裂解液的1.5mL EP管，充分振荡后，移至1.5mL EP管中，抽提总RNA(Total RNA Kit(天根，北京，中国)。用甲醛变性胶电泳鉴定总RNA质量，分光光度计测定RNA含量。以获得的总RNA为模板，按照日本TaKaRa公司提供的反转录试剂盒(TaKaRa Primer Script TM RT reagent kit，日本)的说明书进行反转录，得到cDNA第一链后，进行PCR扩增，PCR程序如下PCR程序如下：95℃预变性3分钟，95℃变性15秒，60℃退火15秒，72℃延伸90秒，共35个循环，最后72℃保温5分钟，随后12℃恒温，获得南农1138-2的cDNA。
从NCBI数据库和Phytozome v12大豆数据库，找到GmGA3ox1所对应的基 因(Glyma.07g033800，GeneID:100795921)，根据数据库提供的核苷酸序列设计特异引物引物，引物序列见SEQ ID NO.3(atgccttctc tctccgaagc ct)和SEQ ID NO.4(ctaaatatct agattgaag)，从大豆品种南农1138-2的基因编码区(CDS)序列扩增基因，PCR克隆后随后进行PCR产物割胶纯化、连接及转化工作，挑取阳性单克隆测序，测序后获得具有完整编码区的长度为1137bp的大豆GmGA3ox1基因的CDS序列，其中编码区序列见SEQ ID NO.1，大小为1137bp(图1)，核苷酸序列和氨基酸序列(SEQ ID NO.1和SEQ ID NO.2)。
SEQ ID NO.2所示氨基酸序列具体为：
Figure PCTCN2022106929-appb-000002
2)GmGA3ox1的组织表达分析
为了鉴定GmGA3ox1在不同组织中的表达水平，采集大豆品种南农1138-2不同发育阶段的根、茎、叶、花、荚和种子：根、茎和叶为V4期；成熟花为R2期；种子和荚在开花后15天采集。样品于液氮速冻后-80℃保存。总RNA的提取同步骤1)。以上述各组织取样获得的总RNA为模板，反转为cDNA。GmGA3ox1荧光定量引物序列见SEQ ID NO.5(agtccacatg tcaagttggt gg)和SEQ ID NO.6(cggttaggct ttctgcgtct a)，检测GmGA3ox1基因在各组织的表达量变化以大豆组成内参基因Tubulin作为内部参照，引物序列见SEQ ID NO.7(cctcgttcga attcgctttt tg)和SEQ ID NO.8(caactgtctt gtcacttggc at)，进行实时荧光定量PCR反应(Real-time RT-PCR)。
GmGA3ox1主要在大豆叶和茎组织中表达，在根、茎、花、荚和种子中的表达量较低(图2)。
3)GmGA3ox1的表达量与粒重的相关性分析
挑取具有不同粒重的大豆材料39份。种植于温室内。在V4期采集每个大豆 材料的倒三叶叶片，样品于液氮速冻后-80℃保存。总RNA的提取同步骤1)。以上述各组织取样获得的总RNA为模板，反转为cDNA。检测GmGA3ox1基因在各个大豆材料的表达量变化以大豆组成内参基因Tubulin作为内部参照，进行实时荧光定量PCR反应(Real-time RT-PCR)。引物序列见SEQ ID NO.7和SEQ ID NO.8。利用SPSS 20.0软件计算GmGA3ox1的表达量与粒重的相关性。
GmGA3ox1的表达量与粒重的相关性系数r＝0.531，P值为0.0005。表明GmGA3ox1基因的表达量与粒重呈现显著的正相关关系(图3)。
实施例2
1)大豆赤霉素3β-羟化酶基因GmGA3ox1启动子序列的克隆
以大豆品种南农1138-2叶片DNA为模板，进行PCR扩增，引物序列见SEQ ID NO.9(cagctatgac catgattact cacatcgata aacgaggttt g)、SEQ ID NO.10(agtgccaagc ttggctgcag attgtgttga atgcttcgg)、SEQ ID NO.11(cagctatgac catgattact cacgatccca acattcgtg)、SEQ ID NO.12(agtgccaagc ttggctgcag attgtgttga atgcttcgg)、SEQ ID NO.13(cagctatgac catgattacc acctagtgag agagaaagaa gg)和SEQ ID NO.14(agtgccaagc ttggctgcag attgtgttga atgcttcgg)。PCR程序如下：95℃预变性3分钟，95℃变性15秒，60℃退火15秒，72℃延伸90秒，共35个循环，最后72℃保温5分钟，随后4℃恒温，测序后获得长度为1957bp，1005bp和487bp的GmGA3ox1基因启动子序列。
2)GUS染色载体的构建
将上述三种GmGA3ox1基因不同长度启动子的序列插入到pCAMBIA1381Z载体，之后通过冻融法将构建完成的载体转入根癌农杆菌菌株EHA105中。
3)GmGA3ox1的转基因拟南芥GUS染色
利用沾花法转化拟南芥，分别将拟南芥花蕾浸于步骤2)含GmGA3ox1 pro(-1957):GUS，GmGA3ox1 pro(-1005):GUS和GmGA3ox1 pro(-487):GUS的根癌农杆菌菌株EHA105菌液中，侵染30s～1min。用保鲜膜或保鲜袋包裹植株以保持湿度，暗培养24h。之后可视植株生长状况，待新的一批花序长出后再次转化。将拟南芥放于正常条件下继续生长，收获种子。选择三个纯合的T 3代GmGA3ox1 pro(-1957):GUS，GmGA3ox1 pro(-1005):GUS和GmGA3ox1 pro(-487):GUS转基因拟南芥株系的根、茎、叶、花、荚、种子进行GUS染色。染色所使用的试剂盒来自于北京索莱宝科技有限公司，货号为G3060。为检测转基因拟南芥是否为阳性， 分别利用特异性引物对提取的DNA片段进行PCR检测，检测引物为SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11、SEQ ID NO.12、SEQ ID NO.13和SEQ ID NO.14，PCR检测胶图见图8。
GUS染色结果表明，三种类型的GmGA3ox1基因启动子都具有较高的活性，且这三种类型启动子活性最高的组织均位于叶片，其次是茎，花、种子、荚和根(图4)。
实施例3
1)大豆赤霉素3β-羟化酶基因GmGA3ox1的克隆
以大豆品种南农1138-2叶片总RNA为模板，经反转录合成cDNA第一链后，进行PCR扩增，引物序列见SEQ ID NO.15(cctctagaat gccttctctc tccgaagcct)和SEQ ID NO.16(ccgctcgaga atatctagat tgaag)，PCR程序如下：95℃预变性3分钟，95℃变性15秒，60℃退火15秒，72℃延伸90秒，共35个循环，最后72℃保温5分钟，随后4℃恒温，测序后获得不含终止密码子的大豆GmGA3ox1基因的CDS序列。
2)亚细胞定位载体的构建
将不含终止密码子的大豆GmGA3ox1基因的CDS序列插入到含有GFP标签的pFGC5941表达载体。pFGC5941载体具有35S启动子，可强烈诱导目的基因GmGA3ox1在受体中的表达。之后通过冻融法将构建完成的载体转入根癌农杆菌菌株EHA105中。同时将空载pFGC5941载体也转化到EHA105中，作为对照。
3)GmGA3ox1的亚细胞定位
将含有35S:GmGA3ox1-GFP和35S:GFP的菌液分别注射到7～8周大小的本氏烟的叶片中，培养36～48小时后用LeicaTCS SP2型激光共聚焦显微镜观察报告基因GFP的定位情况。GFP信号显示，GmGA3ox1-GFP融合蛋白定位于细胞膜及细胞质基质，而空载定位于整个烟草细胞(图5)。
实施例4基因GmGA3ox1的基因工程应用
1)大豆赤霉素3β-羟化酶基因GmGA3ox1的克隆
分别以大豆品种南农1138-2叶片总RNA为模板，经反转录合成cDNA第一链后，进行PCR扩增，引物序列见SEQ ID NO.17(gtcgacggta tcgataagct tatgccttct ctctccgaag cc)，SEQ ID NO.18(cgctctagaa ctagtggatc cctaaatatc tagattgaag cccac)。PCR程序如下：95℃预变性3分钟，95℃变性15秒，60℃退火15秒，72℃延伸 90秒，共35个循环，最后72℃保温5分钟，随后4℃恒温，测序后获得具有完整编码区的长度为1137bp的大豆GmGA3ox1基因的CDS序列。另外，以大豆地方品种句容扁青豆叶片总RNA为模板，经反转录合成cDNA第一链后，进行PCR扩增，引物序列见SEQ ID NO.19(ggatatgttg aaactttcct ttgc)和SEQ ID NO.20(atgcatgatc aatcatttac)。PCR程序如下：95℃预变性3分钟，95℃变性15秒，60℃退火15秒，72℃延伸120秒，共35个循环，最后72℃保温5分钟，随后4℃恒温，测序后获得4187bp的大豆GmGA3ox1基因组序列。
2)植物表达载体的构建
构建转化拟南芥的过表达载体时，将从南农1138-2叶片中扩增得到的GmGA3ox1基因的CDS序列插入到pBI121表达载体，得到pBI121-GmGA3ox1植物过量表达载体，植物转化载体pBI121含有35S强启动子，可强烈诱导目的基因GmGA3ox1在受体中的表达。然后通过冻融法将载体转入根癌农杆菌菌株EHA105中。
构建转化大豆的过表达载体时，从大豆地方品种句容扁青豆中扩增GmGA3ox1的4187bp基因组序列。这4187bp包括1554bp启动子和5’UTR区域、1899bp内含子和外显子区域以及539bp的3’UTR区域，将此4187bp片段连接到去除了CaMV 35S启动子的pCAMBIA3301载体，得到pCAMBIA3301-GmGA3ox1植物过量表达载体。将此载体转化根癌农杆菌菌株EHA105。
3)转基因拟南芥植株的获得
利用沾花法转化拟南芥，将atga3ox1拟南芥突变体花蕾浸于步骤2)含pBI121-GmGA3ox1的根癌农杆菌菌株EHA105菌液中，侵染30s～1min。用保鲜膜或保鲜袋包裹植株以保持湿度，暗培养24h。之后可视植株生长状况，待新的一批花序长出后再次转化。将拟南芥放于正常条件下继续生长，收获种子。选择三个纯合的T 3代35S:GmGA3ox1/atga3ox1转基因拟南芥株系进行粒重表型的调查(图6)。为检测是否为atga3ox1拟南芥突变体和是否为过表达GmGA3ox1转基因拟南芥，分别利用特异性引物对提取的DNA片段进行PCR检测(SEQ ID NO.21：atgccttctc tctccgaagc ct、SEQ ID NO.22：aagaccggca acaggattca)，PCR检测胶图见图8。
Col-0、atga3ox1突变体和三个35S:GmGA3ox1/atga3ox1转基因株系成熟后，收获种子，并将其在37℃的烘箱中干燥一个星期。种子干燥后，测量Col-0、 atga3ox1突变体和三个35S:GmGA3ox1/atga3ox1转基因株系的千粒重(N＝3)。Col-0株系的千粒重要显著高于atga3ox1突变体植株(图6D)，证明拟南芥AtGA3ox1基因的突变可以显著降低粒重。另外，三个35S:GmGA3ox1/atga3ox1转基因株系的粒重显著大于atga3ox1突变体植株，略高于Col-0植株(图6D)，证明在atga3ox1突变体中过量表达GmGA3ox1基因可以提高粒重。
4)转基因大豆植株的获得
利用子叶节转化法转化大豆栽培品种Williams 82，对生长5～6天的大豆叶腋处进行创伤处理，再将步骤2)获得的分别含pCAMBIA3301-GmGA3ox1载体接种到大豆叶腋的伤口处。置于25℃共培养4～5d。然后分别用灭菌的超纯水和Wish-Liquid清洗，放置不含草丁膦的SIM培养基中，26℃光照培养15d，诱导出芽。15天后更换到加入6mg/L草丁膦的SIM培养基中。而后以15天为周期进行继代，逐步减少草丁膦的剂量。当外植体的芽生长到6cm左右时，转入生根培养基中培养10d左右，诱导出根。待根系生长好，即可移栽。利用特异性引物对提取的DNA片段进行PCR检测(SEQ ID NO.23：agtccacatg tcaagttggt gg、SEQ ID NO.24：ccggcaacag gattcaatct taa)，检测是否为过表达GmGA3ox1的转基因大豆。PCR检测胶图见图8。
获得的转基因大豆植株及其对照播种于花盆中，放置于自然条件下的网室内。收获后将种子在37℃的烘箱中干燥一个星期。种子干燥后，测量W82和两个GmGA3ox1转基因大豆株系的百粒重及粒长、宽、高(N＝10)。两个转基因大豆株系中GmGA3ox1的表达量都要显著高于其在对照W82植株中的表达量(图7C)(引物为SEQ ID NO.23、SEQ ID NO.24)，证明此启动子可以有效的驱动GmGA3ox1的表达。另外，两个GmGA3ox1转基因大豆株系的百粒重、粒长显著大于W82植株，粒宽和粒高和W82植株无明显差异(图7中的A,B,D-G)，证明过量表达GmGA3ox1的优异单倍型基因可以提高转基因大豆的粒重和粒长。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。

Claims (9)

  1. 大豆赤霉素3β-羟化酶或大豆赤霉素3β-羟化酶的编码基因GmGA3ox1在基因工程改造拟南芥粒重、大豆粒重或大豆粒长中的应用；所述大豆赤霉素3β-羟化酶的氨基酸序列如SEQ ID NO.2所示；所述的大豆赤霉素3β-羟化酶编码基因GmGA3ox1序列如SEQ ID NO.1所示。
  2. 根据权利要求1所述的应用，其特征在于，所述基因工程改造拟南芥粒重、大豆粒重或大豆粒长通过在拟南芥或大豆中过表达大豆赤霉素3β-羟化酶或大豆赤霉素3β-羟化酶的编码基因GmGA3ox1实现。
  3. 根据权利要求1或2所述的应用，其特征在于，所述拟南芥包括AtGA3ox1基因突变的拟南芥株系。
  4. 一种提高拟南芥或大豆产量的方法，包括以下步骤：在拟南芥或大豆中过表达大豆赤霉素3β-羟化酶或大豆赤霉素3β-羟化酶的编码基因GmGA3ox1；所述大豆赤霉素3β-羟化酶的氨基酸序列如SEQ ID NO.2所示；所述的大豆赤霉素3β-羟化酶编码基因GmGA3ox1序列如SEQ ID NO.1所示。
  5. 一种检测大豆赤霉素3β-羟化酶的编码基因GmGA3ox1表达量的引物对，所述引物对包括核苷酸如SEQ ID NO.7和SEQ ID NO.8所示的序列。
  6. 一种重组载体，插入有豆赤霉素3β-羟化酶的编码基因GmGA3ox1，所述重组载体的原始载体包括pCAMBIA1381Z载体。
  7. 一种重组菌，包含权利要求7所述的重组载体。
  8. 根据权利要求8所述的重组菌，其特征在于，所述重组菌的原始菌包括根癌农杆菌。
  9. 根据权利要求9所述的重组菌，其特征在于，所述根癌农杆菌包括根癌农杆菌EHA105。
PCT/CN2022/106929 2021-11-17 2022-07-21 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用 WO2023087761A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/031,159 US20230313151A1 (en) 2021-11-17 2022-07-21 Use of Gene Encoding Gibberellin 3Beta-Hydroxylase of Glycine Max, GmGA3ox1

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111360441.9 2021-11-17
CN202111360441.9A CN114277041B (zh) 2021-11-17 2021-11-17 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用

Publications (1)

Publication Number Publication Date
WO2023087761A1 true WO2023087761A1 (zh) 2023-05-25

Family

ID=80869278

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/106929 WO2023087761A1 (zh) 2021-11-17 2022-07-21 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用

Country Status (3)

Country Link
US (1) US20230313151A1 (zh)
CN (1) CN114277041B (zh)
WO (1) WO2023087761A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114277041B (zh) * 2021-11-17 2023-03-31 南京农业大学 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481176A (zh) * 2021-08-03 2021-10-08 中国农业大学 GA3ox1蛋白在调控苜蓿株型中的应用
CN114277041A (zh) * 2021-11-17 2022-04-05 南京农业大学 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108795954B (zh) * 2018-07-09 2021-05-04 南京农业大学 一个大豆E3泛素连接酶家族基因GmRNF2a的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113481176A (zh) * 2021-08-03 2021-10-08 中国农业大学 GA3ox1蛋白在调控苜蓿株型中的应用
CN114277041A (zh) * 2021-11-17 2022-04-05 南京农业大学 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"Doctoral Dissertation", 1 December 2019, CHINESE ACADEMY OF AGRICULTURAL SCIENCES, CN, article SUN, HAO: "Biological Function Analysis of Gibberellin Synthesis Gene MtGA3ox1 in Medicago Truncatula", pages: 1 - 109, XP009545604, DOI: 10.27630/d.cnki.gznky.2019.000029 *
DATABASE Nucleotide 12 March 2019 (2019-03-12), ANONYMOUS : "PREDICTED: Glycine soja gibberellin 3-beta-dioxygenase 1-like (LOC114420303), mRNA", XP093067278, Database accession no. XM_028386239.1 *
HAN YI-QIANG, SHI YING, GAO YA-MEI, ZHENG DIAN-FENG, DU JI-DAO, ZHANG YU-XIAN, FENG NAI-JIE: "Effects of gibberellins and uniconazole on morphology, photosynthetic physiology and yield of soybean", CHINESE JOURNAL OF OIL CROP SCIENCES, vol. 40, no. 6, 1 January 2018 (2018-01-01), pages 820 - 827, XP093067291, ISSN: 1007-9084, DOI: 10.7505/j.issn.1007-9084.2018.06.011 *

Also Published As

Publication number Publication date
CN114277041A (zh) 2022-04-05
US20230313151A1 (en) 2023-10-05
CN114277041B (zh) 2023-03-31

Similar Documents

Publication Publication Date Title
Kung et al. Generation of hermaphrodite transgenic papaya lines with virus resistance via transformation of somatic embryos derived from adventitious roots of in vitro shoots
CN111187789B (zh) 一种水稻myb转录因子及其重组表达载体的应用
CN107177610B (zh) 一种调控种子大小的拟南芥mpk基因及增加种子大小的方法
WO2020221029A1 (zh) 玉米类受体激酶基因ZmRLK7及其应用
WO2023087761A1 (zh) 大豆赤霉素3β-羟化酶编码基因GmGA3ox1的应用
CN106520723B (zh) 蛋白VvMas、编码基因及其在提高植物耐盐性中的应用
CN114292855A (zh) 一种调控杨树木质部发育的PagARR9基因及其应用
CN110904131A (zh) 一种棉花GhGlu19基因及其在提高棉花产量中的应用
CN115287290B (zh) 组蛋白去甲基化酶基因OsJMJ718及其编码蛋白在调控水稻种子活力中的应用
CN117586982B (zh) 一种棉花谷胱甘肽硫转移酶基因GhGSTU19及其应用
CN116789785B (zh) 长雄蕊野生稻高产、高光效基因FarL1及其应用
CN111944772B (zh) 茄子隐花色素蓝光抑制因子SmBIC1蛋白及编码基因
CN117305266B (zh) 一种与水稻抗逆相关的基因OsBDG1及其编码蛋白的应用
NL2030997B1 (en) Zea mays receptor-like kinase 7 (zmrlk7) gene related to kernel and plant type development of maize and use thereof
CN117802153A (zh) SlBIW基因在调控番茄果实形状中的应用
CN117402890A (zh) 一个簇毛麦转录因子wrky1-v及其所编码的蛋白和应用
CN106939307B (zh) 水稻核苷酸片段hal及含该片段的表达载体与应用
CN117327729A (zh) AtWRKY41基因在提高植物根结线虫病抗性中的应用
CN112410348A (zh) 一种水稻根系构型调控基因OsVST1及其应用
CN117586982A (zh) 一种棉花谷胱甘肽硫转移酶基因GhGSTU19及其应用
CN116444637A (zh) 调控植物叶片衰老和产量相关蛋白GmWRKY100及其编码基因与应用
CN116355870A (zh) 玉米核糖核苷酸还原酶大亚基ZmLSC1基因在植物品种育种中的应用
CN117417950A (zh) 一种水稻分蘖调控基因、突变体及其制备方法和应用
CN116376958A (zh) 加州月桂树UcFatB基因的DNA密码子优化序列在改善植物抗旱上的应用
KR20230154995A (ko) 벼 수확량 관련 단백질과 생체재료 및 양자의 벼 수확량 향상에서의 응용

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22894304

Country of ref document: EP

Kind code of ref document: A1