CN119265207A - A gene for regulating plant height of upland cotton and its application - Google Patents
A gene for regulating plant height of upland cotton and its application Download PDFInfo
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Abstract
The invention belongs to the technical fields of plant molecular biology and genetic breeding, and particularly relates to a gene for regulating and controlling upland cotton plant height and application thereof, wherein the gene is named GhLHCB, the length of the gene is 1353 and bp, the nucleotide sequence of the gene is shown as SEQ ID No.1, the amino acid sequence of the encoded protein of the gene is shown as a sequence table SEQ ID No.2, and the invention also provides an application method of the gene in cotton molecular breeding, and the accurate regulation and control of cotton plant height are realized by regulating and controlling the expression level of the gene.
Description
Technical Field
The invention belongs to the technical fields of plant molecular biology and genetic breeding, and particularly relates to a gene for regulating and controlling upland cotton plant height and application thereof.
Background
Cotton, malvaceae (Malvaceae) cotton (Gossypium) plants, are one of the most important fiber crops in the world and are also the most important commercial crops in our country. Upland cotton (Gossypium hirsutum l.) is used as the main cultivar of cotton, and its plant height is an important agronomic trait affecting cotton yield, quality and lodging resistance. With the development of molecular technology and the construction of genetic linkage maps, the genetic control mechanism of cotton plant height is studied more deeply. However, the genes and their specific functions related to cotton plant height regulatory sites are still not completely understood at present, and the ability to optimize cotton plant height by molecular breeding means is limited.
Dwarfing is the subject of the green revolution, and has better effects on rice and wheat, but the plant height dwarfing of cotton is still in progress. The shorter variety has excellent lodging resistance, can meet the growth conditions of close planting, can meet the mechanized requirements at the same stage, and can use the applied fertilizer for forming and maturing fruits instead of overgrowth, thereby improving the utilization efficiency of the fertilizer. Cotton is used as an infinitely growing crop, natural plants are tall and big, the control is needed by manpower and pesticide means, however, the manpower control can generate high manpower cost, which is unfavorable for the generation of higher economic benefits, the chemical means is used for controlling the cotton plants, certain pollution is brought, chemical inhibitors are sprayed to control the cotton plants to cause the conditions of falling resistance of the cotton or rotting of cotton bolls and the like when the cotton plants meet adverse conditions in the later period, the yield of the cotton is further reduced, and the economic benefits of the cotton are further unfavorable for the promotion, so that the chemical control means can be widely applied and popularized at the current stage, but the chemical control means still have great hidden danger and defect. In addition, the phenomena of plant height and plant type change, crop yield and quality improvement and the like in rice, wheat and other cereal crop breeding and research provide infinite possibility for improving cotton fiber quality and yield by reducing plant height.
Under ideal conditions, cotton plants should have proper plant height, namely, controlled to be 0.8-0.9 m and good comprehensive properties in natural conditions, but the requirements are hardly met at the present stage, so that breeding is required by a breeding means, and excellent sites and genes are key resources and important basis in the breeding process. The genetic basis of plant height formation can be analyzed by intensive research and excavation of excellent allele sites or genes related to plant height. The excellent SNP locus unfolding analysis is a genetic basis for biological directional improvement by means of genetic engineering technology in the modern breeding process, and can provide good cotton gene resources for deep research of cotton genetic mechanism.
In the prior studies, although a plurality of QTL (Quantitative Trait Loci) related to cotton plant height are found, the positioning of the QTL is often not accurate enough, and large genetic background differences exist among different studies, so that the repeatability and reliability of the QTL are low. Therefore, it is needed to identify more accurate and stable plant height regulation site genes, and define specific functions and action mechanisms of the genes, so as to provide theoretical basis and technical support for cotton molecular breeding.
Disclosure of Invention
The invention aims to provide a gene for regulating and controlling the plant height of upland cotton and application thereof, and the gene can obviously influence the plant height character of cotton. Meanwhile, the invention also provides an application method of the gene in cotton molecular breeding, and accurate regulation and control of cotton plant height are realized by regulating and controlling the expression level of the gene.
In order to achieve the above purpose, the invention adopts the following technical scheme:
A gene for regulating and controlling the plant height of upland cotton is named GhLHCB, the length of the gene is 1353bp, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
The gene coding protein for regulating and controlling upland cotton plant height is tetraploid cotton gene and has the amino acid sequence shown in SEQ ID No. 2.
An application of a gene GhLHCB for regulating and controlling the plant height of upland cotton in regulating and controlling the plant height of upland cotton, wherein the regulation and control is regulation and control for increasing the plant height or regulation and control for reducing the plant height.
Furthermore, the regulation of the increased plant height can inhibit the expression of the gene GhLHCB by a gene editing technology (such as CRISPR/Cas 9) or reduce the expression level by an RNAi (RNA interference) technology, so that the increase of the cotton plant height is realized.
Further, the inhibition is the silencing of the gene regulating the plant height of upland cotton.
Furthermore, the gene silencing for regulating and controlling the upland cotton plant height is realized by adopting a virus-induced gene silencing technology, and the virus-induced gene silencing technology comprises the following steps:
The gene for regulating and controlling the upland cotton plant height is inserted into a viral vector TRV2 and then is transferred into agrobacterium, and the agrobacterium carrying the viral vector infects cotyledons of upland cotton seedlings, and the upland cotton plant with gene GhLHCB for silencing is obtained after cultivation.
The regulation of the plant height is reduced, and the expression level of the gene GhLHCB is improved by a gene over-expression technology.
A vector comprising the gene for regulating a plant height of upland cotton according to claim 1.
A primer pair for amplifying upland cotton plant height trait gene GhLHCB comprises a forward primer F5'-ATGGCTTCAATGCTTAGCTCAGGAAC-3' and a reverse primer R5'-TTACGATCCAGGCACAAACTTGGTAG-3'.
The invention has the advantages that:
1. The invention utilizes high throughput sequencing technology and Quantitative Trait Locus (QTL) mapping method to screen SNP (Single Nucleotide Polymorphism) loci closely related to plant height traits in upland cotton natural population, and further identifies cotton plant height regulation locus gene GhLHCB3 with length of 1353bp through fine positioning and functional verification, the nucleotide sequence of the gene is shown as SEQ ID No.1, the amino acid sequence of the encoded protein is shown as SEQ ID No.2 of a sequence table, and new gene resources are provided for cotton molecular breeding;
2. According to the invention, through silencing GhLHCB genes in cotton, the plant heights of the cotton semi-dwarf material Ari1327 and the normal material Ari971 are obviously increased;
3. the invention provides a cotton plant height regulation method based on a gene GhLHCB, which provides technical support for cotton breeding practice.
Drawings
FIG. 1 is a cloning map of the GhLHCB gene DNA sequence according to the present invention, in which M represents DNA MARKER and the red arrow indicates the size of the gene band of interest.
FIG. 2 is a graph showing the result of fluorescence quantification of GhLHCB gene in the present invention. In the figure, ari971 and Ari1327 represent normal plant height material and semi-dwarf material, respectively. Error bars represent the mean of three biological replicates (±sem,) for each sample.
FIG. 3 is a phenotype analysis of a gene silencing plant in accordance with the present invention. Wherein A, B: ghLHCB3 gene silencing VIGS plant height data diagram. C. Figure GhLHCB phenotype comparison of gene-silenced VIGS plants. In the figure, ari971 and Ari1327 represent normal plant height material and semi-dwarf material, respectively.
Detailed Description
Ari971 and Ari1327 in the biological material upland cotton lines of the examples are disclosed in the documents "Wang Xinkun, sun Junling, pan Zhaoe, zhang Chao, he Shoupiao, gu Yinhua, zhou Zhongli, tang Canming, du Xiongming. Dwarf mechanism study [ J ] of upland cotton dwarf mutant Ari1327, cotton theory, 2012,24 (4): 293-302.Https:// doi. Org/10.11963/cs120402", publicly available from the cotton germplasm metaphase library (Anyang) of the national institute of agricultural sciences cotton.
Example 1
A gene for regulating and controlling upland cotton plant height and application thereof comprise the following steps:
(1) Gene identification
The invention utilizes a high-throughput sequencing technology and a Quantitative Trait Locus (QTL) mapping method to screen SNP (Single Nucleotide Polymorphism) loci closely related to plant height traits in natural populations of upland cotton. Through further fine positioning and functional verification, the cotton plant height regulation site gene GhLHCB is identified. The length of the gene is 1353bp, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
Furthermore, the encoding gene of the protein is tetraploid cotton gene, and the amino acid sequence of the encoded protein is shown in a sequence table SEQ ID No. 2.
(2) Functional verification
In order to verify the function of gene GhLHCB in regulating cotton plant height, the invention adopts virus-induced gene silencing (VIGS) technology. Gene GhLHCB was inserted into the viral vector TRV2 and then transferred into Agrobacterium. The agrobacterium carrying the virus vector infects cotyledons of upland cotton seedlings, and a upland cotton plant with gene GhLHCB silenced is obtained after cultivation. The effect of the gene GhLHCB3 in regulating and controlling the cotton plant height is confirmed by comparing the plant height characters of the silenced plant of the gene GhLHCB and the wild plant.
(3) Application method
Based on the functional verification result of the gene GhLHCB, the invention provides the following two application methods:
Increasing plant height by inhibiting expression of gene GhLHCB by gene editing techniques (such as CRISPR/Cas 9) or reducing its expression level by RNAi (RNA interference) techniques.
And conversely, the expression level of the gene GhLHCB is improved by a gene over-expression technology, so that the vegetative growth of cotton can be inhibited, the plant height is reduced, and the lodging resistance of the cotton is improved.
Experimental example
Clone identification and functional analysis of full-length sequence of negative regulation upland cotton plant height gene GhLHCB3
1. Extraction and reverse transcription of total RNA of cotton stem tip
And (3) taking stem tips of plants in a critical period of field plant height growth, rapidly grinding the stem tips in liquid nitrogen, extracting total RNA by using an RNA extraction kit of Novozan company, detecting the quality and concentration of the RNA by using 1.2% agarose gel electrophoresis and a spectrophotometer, and storing at-80 ℃. cDNA synthesis was performed using total RNA as template and Ai Kerui Biotechnology Co., ltd. Reverse transcription kit (AG 11728) according to the instructions.
2. Extraction of total DNA from the tip of cotton stem
The stem tip of the plant in the key period of the field plant height growth is taken and placed in liquid nitrogen for rapid grinding, and the stem tip total DNA is extracted by utilizing an improved CTAB method, and the extraction method is as follows:
1) Preheating the prepared DNA lysate at 65 ℃, taking 2g of fresh sample, quickly grinding with liquid nitrogen, and placing into a 2mL centrifuge tube;
2) Adding 600 mu L of lysate into the centrifuge tube, cracking in a 65 ℃ water bath for 40-50min, and mixing reversely and uniformly every 10min;
3) Cooling the centrifuge tube to room temperature, adding 5 mu L of RNase, and carrying out water bath in a 37 ℃ water bath kettle for 45min;
4) Adding equal volume of chloroform and isoamyl alcohol (24:1), gently reversing and mixing for at least 50 times;
5) Pre-cooling the centrifuge in advance, wherein the temperature is 4 ℃, the speed is 12000r/min, and the speed is 10min;
6) Transferring the supernatant to a clean centrifuge tube;
7) Adding 0.6 times of pre-cooled isopropanol, slowly inverting the centrifuge tube until flocculent DNA precipitation occurs;
8) Picking the DNA flocculent precipitate by a gun head and putting the DNA flocculent precipitate into a clean 1.5mL centrifuge tube;
9) Adding 500 mu L of 70% ethanol, rinsing the DNA twice, washing with absolute ethanol once again, and airing at room temperature;
10 Adding proper amount of sterile ultrapure water or TE to dissolve DNA, and preserving at-20 ℃.
3. Cloning of cotton GhLHCB gene
The total DNA of the cotton stem tip is used as a template, and a gene specific primer is designed according to GhLHCB gene sequences, wherein the primer sequences are F: ATGGCTTCAATGCTTAGCTCAGGAAC-3', R:5'-TACGATCCAGGCACAAACTTGGTAG-3'.
The GhLHCB genes are obtained by PCR amplification by taking DNA as a template, the result is shown in figure 1, and the target genes with the corresponding band sizes are obtained and recovered by PCR products.
Through the steps, the full length 1353bp of the gene in cotton is obtained, and the coding sequence is shown as SEQ ID NO. 1.
The PCR procedure is shown in table 1:
the PCR system is shown in Table 2
| KODOnePCRMaasterMix | 25μl |
| Primer F | 1μl |
| Primer R | 1μl |
| cDNA | 2μl |
| ddH2O | 21μl |
4. Cotton GhLHCB gene expression analysis
Respectively taking stem tip cDNA of normal plant height material Ari971 and semi-dwarf material Ari1327 as templates, and adopting fluorescent quantitative kit of coastal biotechnology companyFast SYBR qPCR SuperMix, carrying out GhLHCB gene fluorescence quantitative PCR verification by taking Histone3 as an internal reference gene, a forward primer F5'-TAGGAACAGGGCTCTTGAGGTA-3' and a reverse primer R5'-CATTGATGCGGAATCCTTCGAC-3', and designing 3 technical repeats and 3 biological repeats for each gene. And analyzing the relative expression quantity of GhLHCB3 in the normal plant height material Ari971 and the semi-dwarf material Ari1327, and calculating the relative expression quantity according to a 2-delta CT method.
As shown in FIG. 2, ghLHCB gene expression level in semi-short stalk material Ari1327 is extremely higher than that of normal plant height material Ari971.
Phenotypic analysis of TRV2: ghLHCB3 silenced lines
The vector systems used in this experiment were TRV1 (auxiliary vector), TRV2 (empty vector) and TRV2:: CLA1
(Positive control) VIGS primers were designed on the http:// VIGS. Solgenomics. Net/website according to the GhLHCB gene CDS sequence, the primer sequences were as follows:
VIGS-GhLHCB3-F:gtgagtaaggttaccgaattcCTACCTAGGCAACCCCAACCT
VIGS-GhLHCB3-R:cgtgagctcggtaccggatccAAGAGATTTTCCAGTGGACCTTTG
The GhLHCB gene is amplified by the primer, and EcoRI and BamHI sites are utilized to construct a TRV2 vector, so as to obtain a TRV2: ghLHCB3 recombinant plasmid. The recombinant plasmid was transferred into Agrobacterium GV3101 to obtain a positive clone. Inoculating the agrobacteria LBA4404 containing TRV1, TRV2 and TRA2:: CLA1 and TRV 2::: ghLHCB to liquid LB medium containing 50 mug/mLKan + and 50 mug/mL Rif + respectively, and culturing at 28 ℃ and 200r/min overnight to logarithmic phase. Before cotton leaf injection, the cells were well suspended with an equal volume of heavy suspension (200. Mu. Mol/LAS+10mmol/L MgCl 2 +1 mmol/LMES) and adjusted to OD 600=1.5 and allowed to stand at room temperature for 3h. At inoculation, TRV2: ghLHCB3, empty vector TRV2 and positive control TRV 2:CLA 1 were mixed with auxiliary vector TRV1 in equal volumes, and two cotyledons of cotton were injected with a 1mL syringe. Covered with plastic film and treated with a dark light at 25 ℃ for 24 hours. And then grow normally in a 23-25 ℃ and 16h light artificial climate chamber.
Detecting the silencing efficiency of GhCSLD genes in cotton, when a albino phenotype appears in a TRV2:CLA1 positive plant, extracting RNA of CK, TRV2, TRV2:CLA1 positive and TRV2: ghLHCB3 cotton tender leaves, reversely transcribing the RNA into cDNA, detecting the gene silencing efficiency through qRT-PCR, and selecting a plant with high silencing efficiency for subsequent phenotypic observation.
Claims (9)
1. A gene for regulating and controlling the plant height of upland cotton is characterized in that the gene is named GhLHCB < 3 >, the length of the gene is 1353 < bp >, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.
2. The protein encoded by the gene for regulating and controlling the plant height of upland cotton according to claim 1, wherein the encoding gene of the protein is a tetraploid cotton gene, and the amino acid sequence of the encoded protein is shown in a sequence table SEQ ID No. 2.
3. The method of claim 1, wherein the upland cotton plant height control gene GhLHCB3 is used for increasing plant height control or decreasing plant height control.
4. The method of claim 3, wherein the increase in plant height is achieved by inhibiting the expression of gene GhLHCB by gene editing techniques or by reducing the expression level by RNAi techniques.
5. The method of claim 4, wherein said inhibiting is silencing a gene that regulates cotton plant height.
6. The method of claim 5, wherein the gene silencing of upland cotton plant height is achieved by a virus-induced gene silencing technique comprising the steps of:
The gene for regulating and controlling the upland cotton plant height is inserted into a viral vector TRV2 and then is transferred into agrobacterium, and the agrobacterium carrying the viral vector infects cotyledons of upland cotton seedlings, and the upland cotton plant with gene GhLHCB for silencing is obtained after cultivation.
7. The method of claim 3, wherein the reduction of plant height is achieved by increasing the expression level of gene GhLHCB by gene overexpression.
8. A vector comprising the gene for controlling upland cotton plant height according to claim 1.
9. A primer pair for amplifying upland cotton plant height trait gene GhLHCB comprises a forward primer F5'-ATGGCTTCAATGCTTAGCTCAGGAAC-3' and a reverse primer R5'-TTACGATCCAGGCACAAACTTGGTAG-3'.
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| CN119464564A (en) * | 2025-01-16 | 2025-02-18 | 三亚中国农业科学院国家南繁研究院 | SNP molecular marker associated with upland cotton plant height and first fruit branch height and application thereof |
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| US20060288439A1 (en) * | 2005-06-13 | 2006-12-21 | Government Of Malaysia As Represented By The Ministry Of Science Technology And Innovation | Leaf-specific chlorophyll A/B binding protein promoter |
| CN108396031A (en) * | 2018-02-11 | 2018-08-14 | 中国农业科学院棉花研究所 | Regulate and control gene and its application of upland cotton plant height |
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| US20060288439A1 (en) * | 2005-06-13 | 2006-12-21 | Government Of Malaysia As Represented By The Ministry Of Science Technology And Innovation | Leaf-specific chlorophyll A/B binding protein promoter |
| CN108396031A (en) * | 2018-02-11 | 2018-08-14 | 中国农业科学院棉花研究所 | Regulate and control gene and its application of upland cotton plant height |
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| CHENHUI MA ET AL.,: ""Mapping of dwarfing QTL of Ari1327, a semidwarf mutant of upland cotton"", 《BMC PLANT BIOLOGY》, vol. 22, no. 5, 31 December 2022 (2022-12-31), pages 7 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN119464564A (en) * | 2025-01-16 | 2025-02-18 | 三亚中国农业科学院国家南繁研究院 | SNP molecular marker associated with upland cotton plant height and first fruit branch height and application thereof |
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