CN114561401B - Upland cotton GhEXO2 and application thereof in aspect of regulating and controlling plant types - Google Patents
Upland cotton GhEXO2 and application thereof in aspect of regulating and controlling plant types Download PDFInfo
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- CN114561401B CN114561401B CN202210222828.6A CN202210222828A CN114561401B CN 114561401 B CN114561401 B CN 114561401B CN 202210222828 A CN202210222828 A CN 202210222828A CN 114561401 B CN114561401 B CN 114561401B
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- 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
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- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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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- 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
Abstract
The invention discloses a cotton GhEXO2 gene, the nucleotide sequence of the coding protein is shown as SEQ ID NO. 2, and the amino acid sequence is shown as SEQ ID NO. 1. The invention belongs to the technical field of plant gene functions, and particularly provides a cotton GhEXO2 gene obtained through first cloning, expression characteristic analysis is carried out, and the function of the gene is verified in plants (arabidopsis and cotton) through a transgenic technology, so that the method has important practical significance for further understanding the EXO function and cultivating an ideal cotton plant type.
Description
Technical Field
The invention belongs to the technical field of cotton germplasm resource cultivation and molecular biology, and particularly relates to cotton GhEXO2 (Gh_A04G 0976) and application thereof in regulating and controlling leaf development and then affecting plant type.
Background
The ideal plant type is the morphological characteristic of high product quality. The ideal plant types required by different crops are not identical, but the plant heights, leaf shapes, she Zi, leaf colors, leaf distribution and the relation between tillers and main stems are mostly related. Leaves are the main photosynthetic organs of plants, have a significant impact on growth and development, and have a surprisingly diverse shape due to differences in cell expansion rates. The flexibility of the leaf shape is largely dependent on the size and arrangement of the leaf margin growths.
However, physiological functions produced by different leaf shapes of plants are rarely reported. In 2003, exodium (EXO) gene was first reported in arabidopsis, and is highly expressed in embryo and in the part where cell division is vigorous, especially in apical meristem and young leaves, and overexpression of the gene can promote plant growth, i.e., rosette leaves become large.
The function and application of the upland cotton homologous gene GhEXO2 in regulating and controlling the development of upland cotton leaves are not reported in the prior art, and how the gene function is in cotton is not reported, so that the research on the function of EXO in the development of cotton leaves has important significance in exploring the development mechanism of cotton leaves and developing ideal plant types.
Disclosure of Invention
Aiming at the situation, in order to overcome the defects of the prior art, the invention provides the upland cotton GhEXO which is obtained by cloning for the first time, carries out expression characteristic analysis, verifies the function of the gene in plants (arabidopsis and cotton) by a transgenic technology, and has important practical significance for further understanding the EXO function and cultivating the ideal cotton plant type and the application of the upland cotton GhEXO in the aspect of regulating and controlling the plant type.
The technical scheme adopted by the invention is as follows: the invention discloses upland cotton GhXO 2 (Gh_A04G 0976), wherein the CDS sequence of a GhXO 2 gene is shown in a sequence table SEQ No. 2, and the amino acid sequence of the GhXO 2 gene is shown in a sequence table SEQ No. 1.
The invention also discloses an application of the upland cotton GhEXO2 in the aspect of regulating the plant type, and the application of the GhEXO2 gene is to promote the development of upland cotton leaves so as to influence the plant type.
In the scheme, the qRT-PCR technology is utilized to analyze the expression conditions of cell elongation related genes AtAGP4, atEXP5, atKCS1 and Atdelta-TIP in GhEXO2 transgenic plants and WT plants, and the cell elongation is enhanced by regulating and controlling the expression of the AtAGP4, atEXP5, atKCS1 and Atdelta-TIP genes, so that the shape of the GhEXO2 transgenic plant leaves is improved.
Further, the AtAGP4, atKCS1 and Atdelta-TIP were each up-regulated 2-8-fold in 3 transgenic lines, and the AtEXP5 expression level was up-regulated 15-20-fold.
Further, the VIGS technology is utilized to silence and verify that the cotton GhEXO2 gene regulates the cotton leaf size and plant height.
To further investigate the role of GhEXO2 in cotton leaf shape regulation, the function of GhEXO2 in cotton was verified by knocking out its expression by virus-induced gene silencing (VIGS) using pTRV system.
The application of the upland cotton GhEXO2 and the strain type regulation and control aspect adopts the structure, and the beneficial effects obtained by the invention are as follows:
1. according to the invention, through the use of an over-expression vector and a VIGS vector, the gene GhEXO2 is over-expressed in arabidopsis, the leaf area of transgenic arabidopsis is larger than that of wild arabidopsis, the leaf area of the transgenic arabidopsis is more than twice that of the wild arabidopsis, the GhEXO2 gene is silenced in a cotton plant 24 by using VIGS, and the leaf of a silenced plant is obviously smaller than that of a cotton plant 24 in a control group, so that the application of the GhEXO2 in regulating and controlling the development of cotton leaves is proved.
2. By further utilizing fluorescence quantification, the expression level of the related genes (AtAGP 4, atEXP5, atKCS1 and Atdelta-TIP) for positively regulating cell elongation in transgenic Arabidopsis thaliana is obviously higher than that of control wild type Arabidopsis thaliana, so that the gene is presumed to promote the expression of the related genes of cell elongation such as the AtAGP4, atEXP5, atKCS1 and Atdelta-TIP genes, and the like, so that the leaves of plants are enlarged.
Drawings
FIG. 1 is the amino acid sequence of GhEXO2 of the present scheme;
FIG. 2 is the GhEXO2 nucleotide sequence of the scheme;
FIG. 3 is a graph showing the leaf phenotype and interaction analysis of GhEXO2 transgenic Arabidopsis thaliana in this scheme;
fig. 4 is a schematic diagram of the present approach to verify that the GhEXO2 regulates leaf development and plant height using VIGS technology.
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate and not to limit the invention to the embodiments of the invention and, together with the description, serve to explain the principles of the invention and to enable a person skilled in the art to make and use the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in figures 1-2, the cotton upland GhXO 2 (Gh_A04G 0976) provided by the invention has the CDS sequence of the GhXO 2 gene shown in SEQ No. 2 in a sequence table, and the amino acid sequence of the GhXO 2 gene shown in SEQ No. 1 in the sequence table.
Example 1, as shown in fig. 3, the GhEXO2 transgenic Arabidopsis thaliana leaf phenotype identification assay and possibly regulatory network assay:
in order to understand the biological function of GhEXO2, ghEXO2 was ectopic overexpressed in Arabidopsis by constructing an overexpression vector.
The primer list is shown in table 1 below:
primer list 1
The phenotype was observed in the seedling stage, and the GhEXO2 transgenic strain was found to have enhanced vegetative growth as compared with the WT plant, and leaf area was measured, and the GhEXO2 transgenic strain was found to have significantly larger leaf area than the WT, which is twice as much as the WT plant (shown in FIGS. 3A and B).
In addition, the relative expression level of GhEXO2 in GhEXO2 transgenic plants is higher (shown in figures 3C and D) through qRT-PCR and semi-quantitative RT-PCR analysis, which shows that the gene plays a role in the transcription level.
The expression conditions of cell elongation related genes AtAGP4, atEXP5, atKCS1 and Atdelta-TIP in GhEXO2 transgenic plants and WT plants are analyzed by using qRT-PCR technology.
The results showed that the expression levels of all cell elongation related genes were significantly up-regulated in 3 independent GhEXO2 transgenic lines compared to WT (FIGS. 3E, F, G and H), with AtAGP4, atKCS1 and Atdelta-TIP up-regulated 2-8 fold in 3 transgenic lines. The expression level of AtEXP5 is up-regulated 15-20 times.
The results show that GhEXO2 can enhance cell elongation by regulating the expression of AtAGP4, atEXP5, atKCS1 and Atdelta-TIP genes, thereby improving the leaf shape of GhEXO2 transgenic plants.
Example 2, as shown in fig. 4, silencing using VIGS technology verifies that cotton GhEXO2 gene regulates cotton leaf size and plant height:
to further investigate the role of GhEXO2 in cotton leaf shape regulation, the function of GhEXO2 in cotton was verified by knocking out its expression by virus-induced gene silencing (VIGS) using pTRV system.
As predicted, the expression of GhEXO2 was significantly inhibited, vegetative growth was reduced (fig. 4A), and leaf size was altered (fig. 4B) in the silenced plants compared to the control.
Statistical analysis found that Leaf Area Index (LAI) of each of the three independent lines of the GhEXO 2-silenced plants was significantly lower than the control (fig. 4E). qRT-PCR analysis showed a significant decrease in the expression of GhEXO2 in the silent strain observed compared to the control, indicating that GhEXO2 was effectively silenced in the VIGS strain (fig. 4C).
To gain insight into the mechanisms by which leaf morphology of GhEXO 2-silenced plants changes, we performed histological microscopic analysis of GhEXO 2-silenced plants. From microscopic image results of the GhEXO 2-silenced plant leaves, it was found that the leaf morphology was small and the number was greater than that of the control leaves, whereas the control leaves were large in cell morphology but small in number, indicating that silencing GhEXO2 inhibited cell elongation, thereby reducing leaf morphology (FIG. 4D).
Furthermore, the inventors measured the number of cells in the same leaf area for both the silenced and control plants under an electron microscope, which showed a significant increase in the number of cells for all three lines of GhEXO2 silencing compared to the control (FIG. 4F). These findings are consistent with previous findings that overexpression of GhEXO2 by Arabidopsis resulted in increased leaf size and leaf area index was higher than that of wild-type Arabidopsis. In addition, as shown in fig. 4A, it was clearly observed that the GhEXO2 gene regulates leaf blades and also regulates cotton plant height.
The methods and apparatus used in the following examples of the present invention are conventional methods and apparatus unless otherwise specified;
the equipment and the reagent are conventional equipment and reagents purchased by reagent companies. In order to make the objects, technical solutions and advantages of the present patent more apparent, the following detailed description of the present patent refers to the field of 'electric digital data processing'. Examples of these preferred embodiments are illustrated in the specific examples. It should be noted that, in order to avoid obscuring the technical solutions of the present invention due to unnecessary details, only the technical solutions and/or processing steps closely related to the solutions according to the present invention are shown in the embodiments, and other details having little relation are omitted.
Database: cottonFGD (https:// CottonFGD. Org /) recalls the GhEXO2 (Gh_A04G 0976) sequence through a database.
Arabidopsis thaliana material: columbia wild type (Columbia, col-0), arabidopsis plants were grown in a greenhouse at a temperature of 21℃under 16h light, 8h dark ambient conditions for transgenic Arabidopsis acquisition.
Cotton material: cotton plants used in VIGS experiments were grown in a greenhouse in the state of zheng in Henan, cotton institute 24 (ZM 24) in upland cotton (Gossypium hihirsutum), at 23 ℃, with 16h illumination, and 8h dark environment.
Reagent: coli competence: trans1-T1 competence (full gold); agrobacteria competence: GV3101 competent (Shanghai, ind.) homologous recombination enzymes, reverse transcription kits, plasmid extraction kits, gene amplification and high fidelity enzymes were purchased from Dalian TaKaRa. Fluorescent quantitation kits are purchased from roche company. Polysaccharide polyphenol plant RNA extraction kit, gum recovery kit and plant DNA extraction kit are purchased from Beijing Tiangen biological company. Nucleic acid dye solution and bacterial solution identification PCR Mix was purchased from Nanjinouzan corporation. Conventional agents such AS antibiotics, surfactants (silwet-77), acetosyringone (AS) and the like are all purchased from Beijing Sophora technologies Co. Primers involved in this experiment were all synthesized by the Optimaceae organism.
Paraffin section: in the experimental process, the same part of the leaf is selected to be soaked in FAA fixing solution, and then alcohol gradient is used for dehydration. The paraffin-embedded tissue was then sectioned into 10 μm thick sections (lycra instruments, wetzlar, germany). Sections were stained with toluidine blue and observed under a lycra M165FC fluorescence stereo microscope (lycra instruments ltd). Each sample was repeated at least three times.
Leaf area index calculation: calculation was performed using imageJ software. The method comprises the following steps: the leaf is cleaned and then stored as JPG format scanning, and the whole picture is scanned to be A4 size during scanning, so that later measurement is facilitated; opening ImageJ software, file-Open, and opening a picture to be measured; a scale is set. Select Analyze- > Set Scale. Inputting a pixel value 2480 with the width of A4 at Distance in pixels, inputting a width 21,Unit of length of standard A4 paper by using a knowndistance, inputting cm, checking a Global check box (the standard is used for all pictures, that is, setting is not needed after a picture is newly opened), and clicking OK; the first step, image-Type-8-bit; secondly, performing background correction and enhancement on an Image by using Image-Adjust-Threshold, and performing adjusted apply; thirdly, checking Area in Analysis-Set Measurements, and then OK; fourth, select show-lines in Analysis-Analyzeparticles, and select Display results, OK, the area of the leaf.
Cell number per unit area assay: cell count registration statistics were performed using imageJ software with the same size field of view under the same magnification.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
The invention and its embodiments have been described above with no limitation, and the actual construction is not limited to the embodiments of the invention as shown in the drawings. In summary, if one of ordinary skill in the art is informed by this disclosure, a structural manner and an embodiment similar to the technical solution should not be creatively devised without departing from the gist of the present invention.
Claims (2)
1. The application of the upland cotton GhEXO2 gene in promoting the development of upland cotton leaves is characterized in that the nucleotide sequence of the upland cotton GhEXO2 gene is as follows:
atggctacta tttaccacct tgccactctc atgctcttct gctttatcca actcagcttc
tccgagttgg ttcaagagca acccttggtt ttaaagtacc acaacggtcc actcttgaaa
ggcaagatca ccgttaatct tatctggtat ggcaagttta cacccatcca acgctccata
atcgttgatt tcatcaactc tttgaattcc gataaggcca caatggttcc ttccacgtcg
tcgtggtgga aaattactaa aaagtacaag ggtggttctt ctaccttagc tgtaggcaaa
caaatccttc ttgaaaatta cccgctcggt aaattcctta aaaacccaca tttacgggca
ttggccagta agttcaatgg cgttgatacc atcaacgtaa tccttacttc caaagacgtc
gccgtagaag ggttttgtat gaggtgtggg actcacgggt caacccgggt tggacgtggt
cctgttcgtg ggacttatat ttgggtcgga aactcggaga ctcagtgtcc gggtcaatgt
gcttggcctt ttcatcaacc tatttacggt ccacaaaccc cccctttggt tgctcctaac
ggagacgttg gagttgacgg gatgattatt aatttggcta cgcttttggg aaacaccgtt
acgaacccgt ttaacaatgg gtattttcag ggcccggcga atgctccttt ggaagcggtt
tcggcttgta cggggatgtt cggttcgggt tcgtacccgg gatatccggg taaggtgttg
gtggagaaaa gtacaggggc gagttacaat gcgaacggga ttaatgggag gaaatatttg
ttgccggcga tgtgggatcc tcagacgtca acttgtaaga cgttaatg。
2. a method for promoting development of upland cotton leaves, which is characterized by comprising the following steps: over-expressing GhEXO2 gene in upland cotton, wherein the sequence of the GhEXO2 gene is as follows:
atggctacta tttaccacct tgccactctc atgctcttct gctttatcca actcagcttc
tccgagttgg ttcaagagca acccttggtt ttaaagtacc acaacggtcc actcttgaaa
ggcaagatca ccgttaatct tatctggtat ggcaagttta cacccatcca acgctccata
atcgttgatt tcatcaactc tttgaattcc gataaggcca caatggttcc ttccacgtcg
tcgtggtgga aaattactaa aaagtacaag ggtggttctt ctaccttagc tgtaggcaaa
caaatccttc ttgaaaatta cccgctcggt aaattcctta aaaacccaca tttacgggca
ttggccagta agttcaatgg cgttgatacc atcaacgtaa tccttacttc caaagacgtc
gccgtagaag ggttttgtat gaggtgtggg actcacgggt caacccgggt tggacgtggt
cctgttcgtg ggacttatat ttgggtcgga aactcggaga ctcagtgtcc gggtcaatgt
gcttggcctt ttcatcaacc tatttacggt ccacaaaccc cccctttggt tgctcctaac
ggagacgttg gagttgacgg gatgattatt aatttggcta cgcttttggg aaacaccgtt
acgaacccgt ttaacaatgg gtattttcag ggcccggcga atgctccttt ggaagcggtt
tcggcttgta cggggatgtt cggttcgggt tcgtacccgg gatatccggg taaggtgttg
gtggagaaaa gtacaggggc gagttacaat gcgaacggga ttaatgggag gaaatatttg
ttgccggcga tgtgggatcc tcagacgtca acttgtaaga cgttaatg。
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676538A (en) * | 2011-03-16 | 2012-09-19 | 嘉汉林业(广州)有限公司 | WPGS 1 gene or WPGS 2 gene of eucalyptus and over-expression having function of increasing plant biomass thereof |
| WO2020107816A1 (en) * | 2018-11-26 | 2020-06-04 | 中国农业科学院棉花研究所 | Upland cotton transformation event 18c006-10-13 and specific identification method therefor |
| CN113337520A (en) * | 2021-03-31 | 2021-09-03 | 甘肃农业大学 | Upland cotton GhA0749 and GhD0744 transcription factors and application thereof in flowering regulation |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2010259295B2 (en) * | 2009-06-10 | 2015-05-07 | Temasek Life Sciences Laboratory Limited | Virus induced gene silencing (VIGS) for functional analysis of genes in cotton. |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102676538A (en) * | 2011-03-16 | 2012-09-19 | 嘉汉林业(广州)有限公司 | WPGS 1 gene or WPGS 2 gene of eucalyptus and over-expression having function of increasing plant biomass thereof |
| WO2020107816A1 (en) * | 2018-11-26 | 2020-06-04 | 中国农业科学院棉花研究所 | Upland cotton transformation event 18c006-10-13 and specific identification method therefor |
| CN113337520A (en) * | 2021-03-31 | 2021-09-03 | 甘肃农业大学 | Upland cotton GhA0749 and GhD0744 transcription factors and application thereof in flowering regulation |
Non-Patent Citations (1)
| Title |
|---|
| "Identification and Analysis of GhEXO Gene Family Indicated That GhEXO7_At Promotes Plant Growth and Development Through Brassinosteroid Signaling in Cotton (Gossypium hirsutum L.)";Shengdong Li et al.;《Frontiers in Plant Science》;第12卷;第1-18页 * |
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