WO2021051883A1 - Gène ghubx de résistance de fibre de coton upland et son utilisation - Google Patents

Gène ghubx de résistance de fibre de coton upland et son utilisation Download PDF

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WO2021051883A1
WO2021051883A1 PCT/CN2020/093854 CN2020093854W WO2021051883A1 WO 2021051883 A1 WO2021051883 A1 WO 2021051883A1 CN 2020093854 W CN2020093854 W CN 2020093854W WO 2021051883 A1 WO2021051883 A1 WO 2021051883A1
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gene
ghubx
fiber
strength
seq
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PCT/CN2020/093854
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English (en)
Chinese (zh)
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张天真
臧毅浩
胡艳
王洋坤
宁志怨
方磊
朱协飞
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南京农业大学
浙江大学
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Priority to AU2020348612A priority Critical patent/AU2020348612A1/en
Publication of WO2021051883A1 publication Critical patent/WO2021051883A1/fr

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    • 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
    • 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

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  • the invention relates to an upland cotton fiber strength gene (GhUBX), which is a gene sequence obtained from upland cotton Prema and belongs to the field of biotechnology application.
  • GhUBX upland cotton fiber strength gene
  • Cotton is an important economic crop, which is widely grown worldwide, and cotton fiber, as a natural fiber, is an important raw material for the textile industry. Although the different uses of cotton fiber require different varieties, the fiber quality is the only constant important selection condition.
  • the measurement standards of cotton fiber mainly include: fiber length, breaking strength, elongation, micronaire value, etc.
  • the breaking strength of cotton fiber is an important index to evaluate the quality of cotton fiber, and it also determines whether the cotton fiber can be processed. The main conditions.
  • the definition of fiber strength is the breaking load of a single fiber divided by the cross-sectional area of a single fiber, that is, the strongest force that a unit fiber cross-sectional area can withstand, and it is a measure of the relative gravitational force of cotton fibers.
  • the cloth woven from fiber materials with good fiber strength is strong and durable. Therefore, with the innovation of textile technology, the requirements for the quality of cotton fibers are getting higher and higher, especially for the strength of cotton fibers. Therefore, how to improve the strength of cotton fiber has become the main goal of current breeding work. Previous studies have shown that there are different ways of expressing fiber strength, and the factors that affect different ones are also different.
  • the zero-gauge ratio strength is mainly affected by the morphology of cotton fiber, and mainly depends on the cellulose content, fiber polymerization degree, and fiber supramolecular structure, which are jointly affected by the three factors.
  • the 3.2mm gauge ratio strength depends on the zero gauge ratio strength and the number of reverse spirals of cotton fiber.
  • the 3.2mm gauge ratio strength and fiber fineness jointly determine the strength of the single fiber (Yao Mu et al., 1998; Liu Jihua, 1989). It can be seen that the fiber breaking strength is mainly determined by the supramolecular structure and the deposition of cellulose.
  • ubiquitination modification in eukaryotic cells involves a series of reactions of ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3.
  • E1 adheres to the Cys residue at the tail of the ubiquitin molecule to activate ubiquitin
  • E2 enzyme and some different types of E3 Enzymes recognize the target protein together and modify it for ubiquitination.
  • the target protein can be modified by monoubiquitination and polyubiquitination.
  • the ubiquitination of substrates by E1, E2, and E3 can form several different ubiquitination substrates.
  • Some substrate proteins can only be monoubiquitinated, such as H2B; some substrate proteins have multiple lysine residues, which will be monoubiquitinated at multiple sites under suitable conditions; and some proteins are monoubiquitinated in a single lysine.
  • the amino acid site will form a polyubiquitin chain, which can be divided into single, mixed and dendritic structures according to the different lysine sites connected to the ubiquitin chain.
  • the purpose of the present invention is to provide a cotton UBX-Domain Containing10 gene (GhUBX) sequence and its genome sequence in upland cotton 86-1, Prema and Raymond cotton; design a pair of specific primers for GhUBX Detect its temporal and spatial expression in upland cotton Prema, 86-1; and use this gene as a target gene to verify the transgene function through genetic engineering methods, so that it can be used to cultivate new germplasm lines and apply in production.
  • GhUBX cotton UBX-Domain Containing10 gene
  • the fiber strength gene GhUBX from tetraploid upland cotton characterized in that the nucleotide sequence of this gene in tetraploid upland cotton (G. hirsutum) Prema is shown in SEQ ID NO.1, in tetraploid upland cotton (G. hirsutum)
  • SEQ ID NO.1 in tetraploid upland cotton (G. hirsutum)
  • the nucleotide sequence of the gene in .hirsutum)86-1 is shown in SEQ ID NO.2.
  • the cotton UBX gene (GhUBX) of the present invention has a 6-bp InDel difference between Prema (high-strength fiber) and 86-1 (low-strength fiber).
  • the 6-bp difference is an SSR (CCTCCG), and the InDel is missing in the fiber high-strength parent Prema.
  • the number of SSR motifs in the GhUBX gene sequence in different upland cotton varieties is different.
  • the overexpression vector is preferably obtained by cloning the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO. 1 into the eGFP4 expression vector Sma I and BamH I by means of gene recombination.
  • the said application preferably uses genetic engineering means to overexpress the tetraploid upland cotton fiber strength gene GhUBX shown in SEQ ID NO.1 to increase the degree of fiber helix and fiber strength; or to inhibit SEQ ID NO.
  • the overexpression vector of the present invention is used in increasing the degree of cotton fiber helix and improving the strength of cotton fiber.
  • the antisense expression vector of the present invention is used for thickening the secondary wall of cotton fiber and improving the strength of cotton fiber.
  • GhUBX protein is a key protein in the ubiquitination pathway and is closely related to the degradation of key proteins during cotton fiber development. Observing the mature fibers of GhUBX transgenic plants by scanning electron microscopy ( Figure 4) and transmission electron microscopy ( Figure 5), we found that: compared with the transgenic receptor W0, the secondary wall of the overexpression plants became thinner, and the fibers of the antisense expressing plants became thinner. The green wall became thicker, compared with the control, the fiber breaking strength of the transgenic plants increased by 6.4-11.4%, and most of the transgenic plants showed significant differences (Table 2).
  • the gene cloned in the present invention is more similar to plant UBX10 in terms of structure, and has not been reported in cotton. Through the sequence alignment obtained from the parent, the difference is mainly in the N-terminal short repeat sequence (SSR) ( Figure 7), and there are also differences in individual amino acids. The gene structure has been comprehensively displayed and analyzed for the first time.
  • SSR N-terminal short repeat sequence
  • Figure 1 Tissue expression analysis, quantitative PCR to detect the spatiotemporal distribution of cotton fiber strength gene (GhUBX) expression in different cotton tissues (roots, stems, leaves, 15 days, 20 days, and 25 days after flowering).
  • GhUBX cotton fiber strength gene
  • FIG. 2 Quantitative PCR detection of GhUBX gene transcription level expression in cotton plants overexpressing GhUBX gene and antisense GhUBX gene, of which 120, 141, 145, and 153 are overexpression plants, W0 is the control group, and 159, 163, 177, and 181 are antisense Righteous plant.
  • the samples are fibers at 15 days, 20 days, and 25 days after flowering.
  • Figure 3 A and B are the Western Blot detection of overexpression transgenic cotton plants with UBX gene and antisense UBX vector transgenic cotton. Among them, 120, 141, 145, and 153 are different lines of overexpression plants, W0 is the control group, 159, 163, 177 and 181 are different lines of antisense plants.
  • C and D are the gray-scale scanning statistical results of Western Blot hybridization bands. The samples are fibers at 15 days, 20 days, and 25 days after flowering. ⁇ -actin is an internal reference protein. After Student’s t-test test, *p ⁇ 0.05; **p ⁇ 0.01.
  • FIG. 4 Electron microscopic examination of transgenic fibers, showing the spiral situation of over-expression and antisense transgenic cotton fibers.
  • the scanning electron microscope model is GEMINI 300, the magnification is 200 times, and the scale is 50 ⁇ m.
  • FIG. 5 Transmission electron microscopy of the transgenic fiber resin section, showing the thickening of the secondary wall of the mature fiber of overexpression and antisense transgenic.
  • A 120, 141, 145, 153 are different strains of overexpression plants
  • W0 is the control group
  • B 159, 163, 177, 181 are different strains of antisense plants
  • histogram of cell wall thickness statistics As shown in (C), both overexpression and antisense show significant differences compared with wild-type W0.
  • the sample is a naturally mature dry fiber. After Student’s t-test test, *p ⁇ 0.05; **p ⁇ 0.01.
  • the electron microscope model is Hitachi H-9500 scale bar is 0.5 ⁇ m.
  • the GhUBX amino acid sequences in prema and 86-1 are listed. Compared with the amino acid sequence of 86-1, the amino acid sequence of prema has two amino acids missing because the N-terminal short repeat sequence (SSR) has six bases missing. Base sequence, these six bases encode two amino acids alanine (Ala) and serine (Ser), so the length of the amino acid sequence prema is 470 amino acids, but 86-1 is 472 amino acids.
  • SSR N-terminal short repeat sequence
  • the gene consists of 4 exons and 3 introns, with a total length of 1,413 bp.
  • the gene consists of UBA-like, UAS, and UBX domains ( Figure 7).
  • eGFP4 is a traditional plant binary expression vector, and its promoter is 35S promoter. Sma I and BamH I digested the vector and ligated it with the PCR product of the GhUBX recombination primer at 37°C to obtain the eGFP4 vector containing the complete expression fragment of the GhUBX gene.
  • the recombinant primer F 5'-GAACGATAGGGTACCCCCCCGGGATGGTTGATGTAACCGATAAATTGG-3' (SEQ ID NO.3), R: 5'-GCCCTTGCTCACCATGGATCCGTTTAGCTCCACAAAGAGGCTGG-3' (SEQ ID NO.4), and perform PCR with the T vector plasmid containing the 1410bp target fragment as a template ;
  • the eGFP4 expression vector was digested with Sma I and BamH I, and then cut through gel running, and then inactivated at 85°C for 15 minutes and placed on ice for later use. Perform recombination according to the requirements of the kit system, transfer the recombined plasmid into E. coli competent DH5 ⁇ , and select bacteria for detection after 12°C.
  • PBI121 is a traditional plant binary expression vector, and its promoter is 35S promoter.
  • the antisense plant expression vector is constructed with a specific fragment of 745bp-1,171bp in the sequence of SEQ NO ID.1 of the present invention with a length of 426 bp. The specific process is as follows:
  • the amplified fragment is a 426bp fragment at 745bp-1,171bp.
  • the amplified product was digested with EcoR V and Sac I, and small fragments were recovered; the pBI 121 expression vector was digested with Sma I and Sac I, the Gus gene was excised, and the large fragment (approximately 13 kb) was recovered. Since EcoR V and Sma I are both blunt ends, the 426bp target fragment and the large fragment of the pBI 121 expression vector recovered by restriction digestion are ligated to construct the antisense fragment into the pBI 121 vector.
  • Agrobacterium-mediated transformation of cotton was carried out for functional verification, and 35S promoter overexpression transgenic plants and 35S promoter antisense transgenic plants were obtained respectively.
  • Four overexpression and four antisense transgenic plants have been identified by transgenic molecules ( Figure 2).
  • the average fiber strength of the overexpression and antisense transgenic plants was stronger than that of the control, so that the function of the gene and cotton fiber strength was directly verified.
  • 120, 141, 149, and 153 are overexpression materials, 159, 163, 177, and 181 are antisense materials, and w0 is a control.

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  • Health & Medical Sciences (AREA)
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  • Life Sciences & Earth Sciences (AREA)
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  • Engineering & Computer Science (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Wood Science & Technology (AREA)
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  • Gastroenterology & Hepatology (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

L'invention concerne le gène de résistance de fibre GhUBX à partir de coton upland tétraploïde (Gossypium hirsutum), la séquence nucléotidique de ce gène dans le coton upland tétraploïde Prema étant telle que représentée dans SEQ ID NO. 1 et la séquence nucléotidique de ce gène dans le coton upland tétraploïde 86-1 est telle que représentée dans SEQ ID NO. 2. L'invention concerne également le gène de résistance de fibre GhUBX tel que présenté dans SEQ ID NO. 1 et l'utilisation d'un vecteur de super-expression et d'un vecteur d'expression antisens du gène dans l'amélioration de la résistance de fibres de coton. Le vecteur de super-expression peut être utilisé pour augmenter le degré de spirale des fibres de coton et améliorer la résistance des fibres de coton. Le vecteur d'expression antisens peut être utilisé pour épaissir les parois secondaires des fibres de coton et améliorer la résistance des fibres de coton.
PCT/CN2020/093854 2019-09-16 2020-06-02 Gène ghubx de résistance de fibre de coton upland et son utilisation WO2021051883A1 (fr)

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CN110551734A (zh) * 2019-09-16 2019-12-10 南京农业大学 陆地棉纤维强度基因GhUBX及其应用

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DATABASE Nucleotide 18 May 2016 (2016-05-18), ANONYMOUS: "PREDICTED: Gossypium hirsutum plant UBX domain-containing protein 10-like (LOC107950718), mRNA", XP055792653, retrieved from Genbank Database accession no. XM_016885639 *
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WANG YANGKUN: "Fine-mapping of Fiber Strength QTL(qFSD03) and Cloning of Candidate Genes", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, no. 1, 15 January 2019 (2019-01-15), pages 1 - 138, XP055792633, ISSN: 1674-022X *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110551734A (zh) * 2019-09-16 2019-12-10 南京农业大学 陆地棉纤维强度基因GhUBX及其应用
CN110551734B (zh) * 2019-09-16 2022-07-19 南京农业大学 陆地棉纤维强度基因GhUBX及其应用

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