Gene for regulating and controlling plant height of upland cotton and application thereof

Abstract

The invention belongs to the technical field of plant molecular biology and genetic breeding, and particularly relates to a gene for regulating and controlling the plant height of upland cotton and application of the gene, the gene is named as GhLHCB3, the length of the gene is 1353 bp, the nucleotide sequence of the gene is shown as SEQ ID No.1, the amino acid sequence of encoded protein of the gene is shown as SEQ ID No.2 in a sequence table, and the nucleotide sequence of the encoded protein of the gene is shown as SEQ ID No.2 in the sequence table. The invention further provides an application method of the gene in molecular breeding of cotton, and accurate regulation and control of the plant height of the cotton are realized by regulating and controlling the expression level of the gene.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology and genetic breeding, and in particular relates to a gene for regulating plant height of upland cotton and an application thereof. Background Art

[0002] Cotton, a plant of the genus Gossypium in the family Malvaceae, is one of the most important fiber crops in the world and the most important cash crop in my country. As the main cultivated cotton species, plant height of upland cotton (Gossypium hirsutum L.) is an important agronomic trait that affects cotton yield, quality and lodging resistance. With the development of molecular technology and the construction of genetic linkage maps, the genetic regulation mechanism of cotton plant height has been studied more deeply. However, the genes and their specific functions of cotton plant height regulatory sites are still not fully understood, which limits the ability to optimize cotton plant height through molecular breeding methods.

[0003] Dwarfing, as the theme of the Green Revolution, has achieved good results in rice and wheat, but the dwarfing of cotton plant height is still in progress. Varieties with shorter plant height have superior lodging resistance and can meet the growth conditions of dense planting. At the same time, they can also adapt to the current mechanization needs, and use the applied fertilizer for fruit formation and maturity rather than leggy growth, thereby improving the utilization efficiency of fertilizer. As a crop with unlimited growth, cotton naturally has tall plants and needs to be controlled by manpower and pesticides. However, manpower control will incur high labor costs, which is not conducive to the generation of higher economic benefits; using chemical means to control will bring certain pollution. At the same time, spraying chemical inhibitors to control cotton plants will cause the cotton's resistance to stress to decrease or cause the cotton bolls to rot if they encounter adversity in the later stage, thereby reducing the cotton yield, which is not conducive to improving the economic benefits of cotton. Therefore, even if chemical control methods to adjust plant height have been widely used and promoted at this stage, they still have huge hidden dangers and disadvantages. In addition, the phenomenon of changes in plant height and plant type that improve crop yield and quality in the breeding and research of cereal crops such as rice and wheat provides unlimited possibilities for improving cotton fiber quality and yield by reducing plant height.

[0004] Ideally, cotton plants should have an appropriate plant height under natural conditions, that is, controlled at 0.8-0.9 meters and have good comprehensive traits, but it is difficult to meet the demand at this stage, so it is necessary to select and breed through breeding methods, and excellent loci and genes are key resources and important basis in the breeding process. Through in-depth research and exploration of excellent alleles or genes related to plant height, the genetic basis of plant height formation can be analyzed. Analysis of excellent SNP loci is the genetic basis for biological directional improvement with the help of genetic engineering technology in modern breeding, which can provide good cotton gene resources for in-depth research on cotton genetic mechanisms.

[0005] In existing studies, although several QTLs (Quantitative Trait Loci) related to cotton plant height have been found, the positioning of these QTLs is often not precise enough, and there are large differences in genetic backgrounds between different studies, resulting in low repeatability and reliability of QTLs. Therefore, it is urgent to identify more accurate and stable plant height regulatory loci and clarify their specific functions and mechanisms of action to provide a theoretical basis and technical support for cotton molecular breeding. Summary of the invention

[0006] The purpose of the present invention is to provide a gene for regulating plant height of upland cotton and its application, wherein the gene can significantly affect the plant height trait of cotton. At the same time, the present invention also provides an application method of the gene in cotton molecular breeding, and by regulating the expression level of the gene, the precise regulation of cotton plant height is achieved.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A gene for regulating plant height of upland cotton, the gene is named GhLHCB3, the gene length is 1353 bp, and the nucleotide sequence of the gene is shown in SEQ ID No.1.

[0009] A protein encoded by a gene regulating plant height of upland cotton. The gene encoding the protein is a tetraploid cotton gene, and the amino acid sequence of the encoded protein is shown in SEQ ID No. 2 in the sequence table.

[0010] A gene GhLHCB3 for regulating plant height of upland cotton is used in regulating plant height of upland cotton, wherein the regulation is to increase plant height or to reduce plant height.

[0011] Furthermore, the regulation of increasing plant height can achieve an increase in cotton plant height by inhibiting the expression of gene GhLHCB3 through gene editing technology (such as CRISPR / Cas9) or reducing its expression level using RNAi (RNA interference) technology.

[0012] Furthermore, the inhibition is to silence the gene that regulates plant height of upland cotton.

[0013] Furthermore, the gene silencing for regulating plant height of upland cotton is achieved by using virus-induced gene silencing technology; the steps of the virus-induced gene silencing technology are as follows:

[0014] The gene regulating plant height of upland cotton is inserted into the virus vector TRV2 and then transferred into Agrobacterium; the Agrobacterium carrying the virus vector infects the cotyledons of upland cotton seedlings, and after cultivation, upland cotton plants with silenced gene GhLHCB3 are obtained.

[0015] The regulation of reducing plant height is achieved by increasing the expression level of the gene GhLHCB3 through gene overexpression technology.

[0016] A vector comprising the gene for regulating plant height of upland cotton as claimed in claim 1.

[0017] A primer pair for amplifying a plant height trait gene GhLHCB3 of upland cotton comprises a forward primer F: 5'-ATGGCTTCAATGCTTAGCTCAGGAAC-3' and a reverse primer R: 5'-TTACGATCCAGGCACAAACTTGGTAG-3'.

[0018] The present invention has the advantages that:

[0019] 1. The present invention uses high-throughput sequencing technology and quantitative trait loci (QTL) mapping methods to screen SNP (Single Nucleotide Polymorphism) sites closely associated with plant height traits in natural populations of upland cotton, and through further fine positioning and functional verification, identifies the cotton plant height regulatory site gene GhLHCB3, which is 1353 bp in length, and the nucleotide sequence of the gene is shown in SEQ ID No. 1, and the amino acid sequence of the encoded protein is shown in the sequence table SEQ ID No. 2, providing a new gene resource for cotton molecular breeding;

[0020] 2. The present invention found that the height of the cotton semi-dwarf material Ari1327 and the normal material Ari971 increased significantly by silencing the GhLHCB3 gene in cotton;

[0021] 3. The present invention provides a cotton plant height regulation method based on the gene GhLHCB3, providing technical support for cotton breeding practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a cloning diagram of the GhLHCB3 gene DNA sequence in the present invention. In the diagram, M represents DNA Marker and the red arrow indicates the size of the target gene band.

[0023] Figure 2 1 is a fluorescence quantitative result diagram of the GhLHCB3 gene in the present invention. In the figure, Ari971 and Ari1327 represent normal plant height materials and semi-dwarf materials, respectively. The error bars represent the mean value (±SEM,) of three biological replicates of each sample.

[0024] Figure 3 This is the phenotypic analysis of the gene silenced plants in the present invention. Among them, A and B: VIGS plant height data diagrams of GhLHCB3 gene silenced plants. C and D: VIGS plant phenotype comparison diagrams of GhLHCB3 gene silenced plants. In the figure, Ari971 and Ari1327 represent normal plant height materials and semi-dwarf materials, respectively. DETAILED DESCRIPTION

[0025] The biomaterial upland cotton lines Ari971 and Ari1327 in the examples have been published in the literature "Wang Xinkun, Sun Junling, Pan Zhao'e, Zhang Chao, He Shoupu, Jia Yinhua, Zhou Zhongli, Tang Canming, Du Xiongming. Study on the dwarfing mechanism of upland cotton dwarf mutant Ari1327 [J]. Cotton Science, 2012, 24(4): 293-302. https: / / doi.org / 10.11963 / cs120402", and the public can obtain them from the National Cotton Germplasm Medium-term Bank (Anyang) of the Cotton Research Institute of the Chinese Academy of Agricultural Sciences.

[0026] Example 1

[0027] A gene for regulating plant height of upland cotton and its application, comprising the following processes:

[0028] (1) Gene identification

[0029] The present invention uses high-throughput sequencing technology and quantitative trait loci (QTL) mapping methods to screen SNP (Single Nucleotide Polymorphism) sites closely associated with plant height traits in natural populations of upland cotton. Through further fine positioning and functional verification, the cotton plant height regulation site gene GhLHCB3 was identified. The gene is 1353bp in length, and the nucleotide sequence of the gene is shown in SEQ ID No.1.

[0030] Furthermore, the gene encoding the protein is a tetraploid cotton gene, and the amino acid sequence of the encoded protein is shown in the sequence table SEQ ID No. 2.

[0031] (2) Functional Verification

[0032] In order to verify the function of gene GhLHCB3 in regulating cotton plant height, the present invention adopts virus-induced gene silencing (VIGS) technology. The gene GhLHCB3 is inserted into the viral vector TRV2 and then transferred into Agrobacterium. Agrobacterium carrying the viral vector infects the cotyledons of upland cotton seedlings, and after cultivation, upland cotton plants with GhLHCB3 silenced are obtained. By comparing the plant height traits of GhLHCB3 silenced plants and wild-type plants, the role of gene GhLHCB3 in regulating cotton plant height is confirmed.

[0033] (3) Application method

[0034] Based on the functional verification results of the gene GhLHCB3, the present invention provides the following two application methods:

[0035] Increase plant height: Inhibit the expression of the GhLHCB3 gene through gene editing technology (such as CRISPR / Cas9), or use RNAi (RNA interference) technology to reduce its expression level, thereby increasing the height of cotton plants.

[0036] Reduce plant height: On the contrary, increasing the expression level of the gene GhLHCB3 through gene overexpression technology can inhibit the vegetative growth of cotton, reduce plant height, and improve the lodging resistance of cotton.

[0037] Experimental example

[0038] Cloning, identification and functional analysis of the full-length sequence of a negatively regulating plant height gene GhLHCB3 in upland cotton

[0039] 1. Extraction and reverse transcription of total RNA from cotton stem apex

[0040] The stem tips of plants in the critical period of plant height growth in the field were taken and quickly ground in liquid nitrogen. The total RNA was extracted using the RNA extraction kit of Novozymes, and the RNA quality and concentration were detected by 1.2% agarose gel electrophoresis and spectrophotometer, and stored at -80°C. Using total RNA as a template, cDNA was synthesized using the reverse transcription kit (AG11728) of Aikerui Biotechnology Co., Ltd. according to the instructions.

[0041] 2. Extraction of Total DNA from Cotton Stem Apex

[0042] The stem tips of plants in the critical period of plant height growth in the field were taken and quickly ground in liquid nitrogen. The total DNA of the stem tips was extracted using the modified CTAB method. The extraction method is as follows:

[0043] 1) Preheat the prepared DNA lysis buffer to 65°C, take 2 g of fresh sample, quickly grind it with liquid nitrogen, and place it in a 2 mL centrifuge tube;

[0044] 2) Add 600 μL of lysis buffer to the centrifuge tube and lyse in a 65°C water bath for 40-50 min, inverting and mixing every 10 min;

[0045] 3) Cool the centrifuge tube to room temperature, add 5 μL RNase, and place in a 37°C water bath for 45 min;

[0046] 4) Add an equal volume of chloroform:isoamyl alcohol (24:1) and mix gently by inverting at least 50 times;

[0047] 5) Precool the centrifuge in advance, 4°C, 12000r / min, 10min;

[0048] 6) Transfer the supernatant to a clean centrifuge tube;

[0049] 7) Add 0.6 times the volume of pre-cooled isopropanol and slowly invert the centrifuge tube until flocculent DNA precipitates appear;

[0050] 8) Use a pipette to remove the DNA flocculent precipitate and transfer it to a clean 1.5 mL centrifuge tube;

[0051] 9) Add 500 μL of 70% ethanol to rinse the DNA twice, then wash it again with anhydrous ethanol, and dry it at room temperature;

[0052] 10) Add an appropriate amount of sterile ultrapure water or TE to dissolve the DNA and store at -20℃.

[0053] 3. Cloning of cotton GhLHCB3 gene

[0054] Using cotton stem tip total DNA as template, gene-specific primers were designed according to the GhLHCB3 gene sequence. The primer sequences were: F: ATGGCTTCAATGCTTAGCTCAGGAAC-3', R: 5'-TACGATCCAGGCACAAACTTGGTAG-3'.

[0055] The GhLHCB3 gene was obtained by PCR amplification using DNA as a template. Figure 1 As shown, the target gene with the corresponding band size was recovered by PCR product.

[0056] Through the above steps, the full-length 1353 bp gene in cotton was obtained, and the coding sequence is shown in SEQ ID NO:1.

[0057] The PCR program is shown in Table 1:

[0058]

[0059] The PCR system is shown in Table 2

[0060] KODOnePCRMaasterMix 25μl Primer F 1μl Primer R 1μl cDNA 2μl ddH2O 21μl

[0061] 4. Expression analysis of cotton GhLHCB3 gene

[0062] The cDNA of the shoot tip of the normal plant material Ari971 and the semi-dwarf material Ari1327 were used as templates and the fluorescence quantitative kit of Nearshore Biotech was used. Fast SYBR qPCR SuperMix, with histone3 as the internal reference gene, forward primer F: 5'-TAGGAACAGGGCTCTTGAGGTA-3' and reverse primer R: 5'-CATTGATGCGGAATCCTTCGAC-3', was used to verify the GhLHCB3 gene fluorescence quantitative PCR. Three technical replicates and three biological replicates were designed for each gene. The relative expression of GhLHCB3 in the normal plant height material Ari971 and the semi-dwarf material Ari1327 was analyzed, and the relative expression was calculated by the 2–ΔΔCT method.

[0063] like Figure 2 As shown, the expression level of GhLHCB3 gene in the semi-dwarf material Ari1327 was significantly higher than that in the normal plant height material Ari971.

[0064] 5. Phenotypic analysis of TRV2::GhLHCB3 silenced strains

[0065] The vector systems used in this experiment are: TRV1 (helper vector), TRV2 (empty vector) and TRV2::CLA1

[0066] (positive control); VIGS primers were designed based on the CDS sequence of the GhLHCB3 gene on the website http: / / vigs.solgenomics.net / . The primer sequences are as follows:

[0067] VIGS-GhLHCB3-F:gtgagtaaggttaccgaattcCTACCTAGGCAACCCCAACCT

[0068] VIGS-GhLHCB3-R: cgtgagctcggtaccggatccAAGAGATTTTCCAGTGGACCTTTG

[0069] The GhLHCB3 gene was amplified using the above primers and constructed into the TRV2 vector using the EcoRI and BamHI sites to obtain the TRV2::GhLHCB3 recombinant plasmid. The recombinant plasmid was transformed into Agrobacterium GV3101 to obtain positive clones. Agrobacterium LBA4404 containing TRV1, TRV2, TRA2::CLA1 and TRV2::GhLHCB3 were inoculated in a 50 μg / mL Kan medium. + and 50 μg / mL Rif + In liquid LB medium, culture overnight at 28°C and 200r / min until the logarithmic growth phase. Before injecting cotton leaves, fully suspend the bacteria with an equal volume of resuspension solution (200μmol / LAS+10mmol / L MgCl2+1mmol / LMES), adjust it to OD600=1.5, and let it stand at room temperature for 3h. When inoculating, mix equal volumes of TRV2::GhLHCB3, empty vector TRV2 and positive control TRV2::CLA1 with auxiliary vector TRV1, and inject two cotyledons of cotton with a 1mL syringe. Cover with plastic film and treat in dark light at 25°C for 24h. Then grow normally in an artificial climate chamber at 23°C-25°C and 16h light.

[0070] Detection of the silencing efficiency of the GhCSLD3 gene in cotton: When the TRV2::CLA1-positive plants showed an albino phenotype, RNA was extracted from young leaves of CK, TRV2, TRV2:CLA1-positive and TRV2::GhLHCB3 cotton plants and reversed transcribed into cDNA. The gene silencing efficiency was detected by qRT-PCR, and plants with high silencing efficiency were selected for subsequent phenotypic observations.

[0071]

[0072]

Claims

1. A gene for regulating plant height of upland cotton, characterized in that: The gene is named GhLHCB3, the length of the gene is 1353 bp, and the nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The protein encoded by the gene for regulating plant height of upland cotton according to claim 1, characterized in that: The coding gene of the protein is a tetraploid cotton gene, and the amino acid sequence of the coded protein is shown in SEQ ID No. 2 in the sequence table.

3. Use of the gene GhLHCB3 for regulating plant height of upland cotton as claimed in claim 1 in regulating plant height of upland cotton, characterized in that: The regulation is regulation of increasing plant height or regulation of decreasing plant height.

4. The use according to claim 3, characterized in that: The regulation of increasing plant height is achieved by inhibiting the expression of the gene GhLHCB3 through gene editing technology, or reducing its expression level by using RNAi technology.

5. The use according to claim 4, characterized in that: The inhibition is to silence the gene regulating plant height of upland cotton.

6. The use according to claim 5, characterized in that The gene silencing for regulating plant height of upland cotton is achieved by using virus-induced gene silencing technology; the steps of the virus-induced gene silencing technology are as follows: The gene regulating plant height of upland cotton is inserted into the virus vector TRV2 and then transferred into Agrobacterium; the Agrobacterium carrying the virus vector infects the cotyledons of upland cotton seedlings, and after cultivation, upland cotton plants with silenced gene GhLHCB3 are obtained.

7. The use according to claim 3, characterized in that: The regulation of reducing plant height is achieved by increasing the expression level of the gene GhLHCB3 through gene overexpression technology.

8. A carrier, characterized in that Contains the gene for regulating plant height of upland cotton as claimed in claim 1.

9. A primer pair for amplifying the plant height trait gene GhLHCB3 of upland cotton, comprising a forward primer F: 5'-ATGGCTTCAATGCTTAGCTCAGGAAC-3' and a reverse primer R: 5'- TTACGATCCAGGCACAAACTTGGTAG-3'.

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