CN116676320A - Gene GhBEL1 for regulating cotton growth and development - Google Patents
Gene GhBEL1 for regulating cotton growth and development Download PDFInfo
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- 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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- 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
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
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- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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Abstract
The invention discloses a gene GhBEL1 for regulating cotton growth and development, which belongs to the technical field of genetic engineering, and the nucleotide sequence of the gene is shown in SEQ ID NO:1, and also discloses the application of the gene in improving the growth and development of upland cotton. According to the invention, the target gene is cloned, a VIGS silencing vector of the target gene is constructed, and as a result, the result shows that GhBEL1 gene silencing causes the plant height of upland cotton to be shortened, the bud and flowering time to be delayed, and the growth and development of upland cotton are affected. The GhBEL1 gene is proved to positively regulate the growth and development of cotton, promote the phenotype growth and development of cotton such as flowering, bud emergence, plant height and the like, and provide important gene resources for cultivating upland cotton early-maturing high-yield high-quality germplasm.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a gene GhBEL1 for regulating cotton growth and development.
Background
Cotton is one of the most important commercial crops and plays an indispensable role in daily life and industry. As global climate warms, temperature becomes one of the important reasons currently considered for crop cultivation. In the northwest inland region of one of the cotton-planted regions, the temperature at the seedling stage is substantially continued at around 20 ℃, however, the number of high-temperature days increases due to climate change. The high temperature in a certain range has promotion effect on the growth and development of cotton, but if the temperature is too high or the temperature in a seedling stage is high, the plant height, the stem thickness, the leaf number, the leaf area, the net photosynthetic rate and the flowering time in the seedling stage, the boll yield, the ginned cotton yield, the seed cotton yield and the cotton fiber quality are affected, so that the yield of the cotton is reduced. Research shows that in the cotton growing area in the IQ area in 1985-2021, the temperature is increased to increase the temperature Wen Rishu, and the accumulated temperature is increased to lead to different degrees of advance of sowing, seedling emergence, three leaves and five leaves of cotton in the area, and different degrees of delay of bud, bell cracking and boll opening periods; so that the cotton growth and development are obviously affected by temperature. Therefore, it is important to identify the genes which have influence on the growth and development of cotton, clarify the regulation mechanism and create the cotton germplasm resource with early maturity and high yield.
Disclosure of Invention
The invention aims to provide a gene GhBEL1 for regulating cotton growth and development, so as to solve the problems in the prior art, and the invention proves that the GhBEL1 gene positively regulates cotton growth and development, promotes the phenotype characters of flowering, bud emergence, plant height and the like of cotton, and provides important gene resources for cultivating upland cotton early-maturing high-yield high-quality germplasm.
In order to achieve the above object, the present invention provides the following solutions:
the invention provides a gene GhBEL1 for regulating and controlling the growth and development of upland cotton, the nucleotide sequence of which is shown in SEQ ID NO: 1.
The invention also provides a primer group for amplifying the gene GhBEL1, and the nucleotide sequence of the primer group is shown as SEQ ID NO: 2-3.
The invention also provides a recombinant vector comprising the gene GhBEL1.
The invention also provides a recombinant bacterium, which comprises the recombinant vector.
The invention also provides an application of the gene GhBEL1 or the recombinant vector or the recombinant bacterium in improving the growth and development of upland cotton.
The invention also provides an application of the gene GhBEL1 or the recombinant vector or the recombinant bacterium in improving flowering traits of upland cotton.
The invention also provides an application of the gene GhBEL1 or the recombinant vector or the recombinant bacterium in improving the property of upland cotton buds.
The invention also provides an application of the gene GhBEL1 or the recombinant vector or the recombinant bacterium in improving upland cotton plant height traits.
The invention discloses the following technical effects:
the invention adopts transcriptome data to mine candidate genes related to the environmental temperature, and discovers that the GhBEL1 gene can respond to high temperature stress, and the expression quantity of the gene is obviously reduced under the high temperature stress. In order to further determine the influence of the GhBEL1 gene on the growth and development of upland cotton, cloning the target gene and constructing a VIGS silencing vector of the target gene, and the result shows that the GhBEL1 gene silencing causes the plant height of upland cotton to be shortened, and the bud and flowering time to be delayed, so that the growth and development of upland cotton are influenced. The GhBEL1 gene is proved to positively regulate the growth and development of cotton, promote the phenotype characters of flowering, bud emergence, plant height and the like of cotton, and provide important gene resources for cultivating upland cotton early-maturing high-yield high-quality germplasm.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a map of the vectors pGM-T and TRV;
FIG. 2 shows the results of PCR amplification, bacterial liquid PCR and double digestion of the target gene; a: PCR amplification of GhBEL1 gene; b: bacterial liquid PCR of GhBEL 1; c: detecting positive plasmids by double enzyme digestion;
FIG. 3 shows the sequencing results of the clone of GhBEL 1;
FIG. 4 is a graph showing the detection of silencing efficiency of a gene of interest; a: TRV PDS positive control phenotype; b: detecting GhBEL1 gene silencing efficiency;
FIG. 5 is a graph showing the phenotype of GhBEL 1-silenced plants and their effect on plant height, bud time and flowering time;
FIG. 6 is an RNA-seq analysis of the GhBEL1 gene.
Detailed Description
Various exemplary embodiments of the invention will now be described in detail, which should not be considered as limiting the invention, but rather as more detailed descriptions of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in this disclosure, it is understood that each intermediate value between the upper and lower limits of the ranges is also specifically disclosed. Every smaller range between any stated value or stated range, and any other stated value or intermediate value within the stated range, is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the invention described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from consideration of the specification of the present invention. The specification and examples of the present invention are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are intended to be inclusive and mean an inclusion, but not limited to.
In the invention, cotton seeds 7, 14 and 21d are placed in two environments of 20 ℃ (control temperature, control temperature, CT) and 30 ℃ (moderate High temperature, HT) respectively for three different time periods after normal emergence by taking cotton 113 in upland cotton variety as a study object, and the treatments are named 7, 14, 21CT and 7, 14 and 21HT respectively. And then placing the plant at room temperature to observe morphological characteristics of the plant, and comparing the change trend of physiological indexes of the stem tip and the young leaf in CT and HT environments. RNA-seq was performed on stem tips of four samples of 14CT, 14HT, 21CT, and 21HT, candidate genes related to the ambient temperature were mined by using their transcriptome data, and it was found that GhBEL1 had an opposite trend in expression level after temperature treatment of 14d and 21d, and that the expression level of GhBEL1 gene was significantly decreased at a high temperature of 30℃compared with a temperature of 20℃ (see FIG. 6), and it was found that the expression level was consistent with the transcriptome level by qRT-PCR verification. Therefore, it is hypothesized that the gene is likely to regulate plant growth in response to ambient temperature. In order to further determine the influence of the GhBEL1 gene on the growth and development of plants, the target gene is cloned, and a VIGS silencing vector of the target gene is constructed. The experiment is as follows:
1 test materials and reagents
1.1 test materials
The material used in this test was cotton-in-cotton 113 (ZM 113), and seeds were provided by cotton institute, national academy of agricultural sciences.
1.2 test reagents
The reagents used in this study are shown in Table 1:
table 1 reagents used in the experiments
1.3 Strain and vector
GV3101 Agrobacterium competent cells and pGM-T cloning vector (VT 302-02) were purchased from Shanghai Weidi Biotechnology Co., ltd and Tiangen Biotechnology Co., ltd, respectively, and the VIGS vector systems (TRV 156, TRV192 and TRV-GhPDS) for gene silencing were given away by the cotton institute transgenic subject group of China department of agriculture, pGM-T and TRV vector maps are shown in FIG. 1.
1.4 preparation of solutions
a. The preparation of the medium is shown in Table 2:
table 2 preparation of culture medium
Note that: the culture medium is sterilized at 121deg.C for 20min
b.100mL 50 xTAE buffer: 24.2g Tris+3.72gNa 2 EDTA·2H 2 O+5.71mL glacial acetic acid;
50mg/mL rifampicin (Rif) and 20mg/mL Acetosyringone (AS): dissolving 5g of Rif powder and 2g of acetosyringone powder in 100mL of DMSO solution respectively, filtering and sterilizing with 0.22 filter membrane respectively, packaging, and preserving at-20deg.C;
d.0.5M MES: 10.65g MES powder was dissolved in 50mL ddH 2 In O, regulating the pH value to 5.6 by NaOH, then fixing the volume to 100mL, filtering and sterilizing by using a filter membrane of 0.22, packaging, and preserving at 4 ℃;
e.1M MgCl 2 : with 100mL ddH 2 O-solution 9.521g MgCl 2 Powder (amplified heat), 0.22 filter membrane for sterilization, and storing at 4deg.C;
f.500mL of heavy suspension: 10mL 0.5M MES+1ml 20mg/mLAS+5mL of 1M MgCl2, sterile ddH 2 O was made up to 500mL.
1.5 use of instruments
The main instrument is as follows: visible spectrophotometers, high-speed refrigerated centrifuges, water baths, electrophoresis apparatuses, artificial climate boxes, gel cutting apparatuses, ultra-low temperature refrigerators, fluorescence quantitative apparatuses, gradient PCR apparatuses, sterilization pans, electronic balances, microwave ovens, table-type constant temperature shaking tables, ice-making machines, ultra-clean benches, biochemical incubators, ultra-micro spectrophotometers, ultra-pure water apparatuses and mini vortex mixing apparatuses.
2 test method
2.1 primer design
Cloning primers, qRT-PCR primers, and VIGS silencing primers (the fragment size of the VIGS silencing Primer is 300-500 bp) of the GhBEL1 gene were designed by using NCBI Primer-BLAST (https:// www.ncbi.nlm.nih.gov/tools/Primer-BLAST /), and the Primer sequences are shown in Table 3:
TABLE 3 list of primers used in this experiment
2.2 extraction of Total DNA and RNA
2.2.1DNA extraction
a. Putting the apical meristem of the plant and liquid nitrogen into a mortar, fully grinding the apical meristem of the plant and the liquid nitrogen into powder, and immediately filling the powder into a 1.5mL centrifuge tube;
b. rapidly adding 400 mu L of GPS buffer solution and 10 mu L of RNaseA into the centrifuge tube, carrying out vortex oscillation, and placing in a water bath kettle at 65 ℃ for 15min;
c. after the water bath is finished, adding 100 mu LGPA buffer into the centrifuge tube, oscillating for 1min, and centrifuging for 5min under the condition of 12000 rpm;
d. transferring the supernatant to a filter column CS, centrifuging at 12000rpm for 1min, and transferring the supernatant to a new centrifuge tube;
e. adding equal amount of absolute ethyl alcohol, mixing, transferring to an adsorption column CR2, centrifuging at 12000rpm for 1min, and discarding the waste liquid;
f. adding 550 mu L deproteinized solution RD into CR2, centrifuging for 1min, and pouring out waste liquid;
g. adding 700 mu L of the rinse PW into CR2, centrifuging for 1min, and pouring out waste liquid;
h. repeating the step g, and centrifuging for 2min;
i. placing CR2 into a new centrifuge tube, and airing for 5-10min at room temperature;
j. 70 mu L of elution buffer TB is added into the middle of the CR2 membrane, and the mixture is placed for 3 to 5 minutes and centrifuged for 2 minutes to obtain DNA solution, and the DNA solution is split-packed and stored at the temperature of minus 20 ℃.
k. In order to ensure the accuracy of the subsequent experiments, the obtained DNA solution was checked for extraction quality, content and purity by ultra-Micro spectrophotometry (Micro Drop) and 1% agarose gel electrophoresis.
2.2.2RNA extraction and reverse transcription
RNA is extracted according to the instruction of the polysaccharide polyphenol plant total RNA extraction kit.
Reverse transcription (synthesis of the first strand of cDNA):
a. the RNA was removed from the-80℃refrigerator and thawed on ice as required, and 5 XFastKing-RT Supermix reagent and RNase-Free ddH were removed in the-20℃refrigerator 2 O is melted on ice and is gently mixed;
b. the reaction system:
TABLE 4 reaction system
c. The reaction procedure:
table 5 reaction procedure
d. After the reaction, the purity and concentration of cDNA were measured, and the cDNA was packaged and stored at-20 ℃.
2.3 amplification of fragments of interest and ligation and transformation of cloning vectors
2.3.1 amplification of fragments of interest
The cDNA of Zhongcotton 113 (ZM 113) is used as a template, and 2 xPro Taq premix (containing dye) kit is used for amplifying the target gene, and the system is shown in Table 6:
TABLE 6 amplification System
After the reaction solution is added according to the system, the mixture is gently mixed, centrifuged for a short time, and the reaction is carried out according to the reaction procedure of Table 7:
table 7 reaction procedure
After completion of the reaction, the size of the target gene was determined to be appropriate by agarose gel electrophoresis at 1.8%.
Amplifying the full length of the GhBEL1 gene from upland cotton by adopting a PCR method, wherein the nucleotide sequence of the gene is shown as SEQ ID NO: 1.
Cloning to obtain the gene sequence of GhBEL1 (the sequence comprises 5'UTR, 3' UTR and CDS sequence, SEQ ID NO: 1):
ATCAACTTTTGATCAGTATCTCAGAAGCTGGTGGGCAAGACAAGTATACACAAAGGACCAAAAAGAAGATTAAAAGAAAAAGAAAGAAAAACTGGTTTTCATGGCTAGAGAATCGTGTGAAGATAAATCAAGGAATATTGTGTCATCCACTGGATTTTGCTACTCAGATGTTTCATCTGGTAACTCAACCATGCAAACCCATCTGGTGAATCAGATCCAAGGCTTTGAATCCAACCCAGAGATCTTCAACTTAACAACAGGCATGGAGATGATAGGGTTTGCCAAGAACCTACAGCAACAACAAGGTGACACCAACAACATGATCATGTGGAAAGGGTTCTTCAACAAACATGGAAACAACCCAGGAGCAGCAGGCCCTTCTTCTCCTTCTTCTTCCAAGCCAATCAATGAGTCAACCACTGATTTCTATCAACATGAATTCCACAAACCTGAGTTCACAACTGGGATATCTGAAACCAGTACTGGGAATCTAATAGTTGGACCTGAATCAGCTCCATGGCAAGAAAACAGGTTGCTTGTTGATGATTCTTCTTTTAGGTGTGTGTTCCCTTGTGAAGGGAACGAGAGACCAAGTCAAGGTCTTTCGCTCTCTCTTTCATCAAGTAATCCTACTAGTATCGGGTTACAGTCTTTTGAACTAAGACAAACAAGCCATAGTAACCATGATGACCAGCCAGATGATATGAGGTTTATTGGTTCAAGTTCAAGAGATGGGTTCTTTGGAAAGCCGGCAATTGTTCAACAACATGAAGGTCAGTTCCAAATAAGGGGTTCCAGGTACTTGAATGCTGCTCAAGAACTTTTGAATGAATTTTGCAGCTTGGGAACAAAGCAATTGGATACATCAAAGCAAAAGCAAACCCAAAAGACCACCAAACAGTGGGATGATGACGATGGAGGAGCTAGCTCTTCAAGGAAGCAATCACTATATTCCCTCGATTTCACTGAGTTGCAGAGAAGAAAAACCAAGATGCTTTCAACGCTGGAAGAGGTGGACAGAAGATATAAGATCTACTGTGATCAAATGAAAGCCGTAGTATCATCGTTCGAAGCAGTGGCTGGTACTGGGGCAGCATCAGTGTATTCAGCTTTAGCCTCTAAAGCCATGTCAAGACATTTTAGATGCTTAAGAGATGGGATTGTGAGTCAGATTCAAGCTACAAGGAAAGCCATGGGAGAAAAAGACACAGTTGCACCAGGCACAACGAAAGGCGAAACACCAAGGCTAAGAATACTCGACCAAGCTTTAAGGCAACAAAGGGCATTTCAACAGATGAGCATGATGGAGAGTCATCCGTGGAGACCCCAAAGAGGCTTACCAGAAAGATCTGTTTCAGTTCTTCGAGCTTGGCTGTTTGAACATTTCCTTCACCCGTACCCTAGCGATGTTGATAAACATATCTTAGCCCGCCAAACCGGCCTCTCAAGAAGCCAGGTATCGAATTGGTTCATCAATGCCAGGGTGAGGCTATGGAAACCAATGGTGGAGGAAATGTACTTGGAAGAAACAAAGGAGCACGAAAACAACATGGTTTCCTCAGATAATGGAGGTACTGATGGTGGTGATGATAATAACAATAATGACCGGTTAAATATTCCACTGGCTGATCAGAAACCCACCCCGGACCAGCTCGTTCGAGTTGACTCGGAGTGTCTATCTTCCATTATCACTACTAACCCGGAAAAAAACGATGGGAAAAGTGGTACCAAAACCCTTGAAAACCAGCACTTGCATCATCATCATCATCATCATCATCAACAACAACAAAGTTTCGGAACCTATGGTGCCACTGCCATGGAACTGGACTTCGCTTCCTACGGTGATCACATGGCGGGTGGTGGGGTACCTTATCATAACGCTAATCAAAGCTTCAACGGTGGAGGAGGTGTGTCATTGACATTAGGGTTGCAACAACATGGTGGGAGTGGCGTGAGCTTAGCTTTCTCGCCTGCAACACAAACTTCACTTTTTTACCGTAGAGACCACATAGAAGATTGTCAGCCAGTTCAGTACTCACTTTTAGACAACGAGGGACAACATTTGCCTTACAGGAACTTGATGGGGGCACAATTGCTTCATGATTTGGCCGGATAGCAAAGCAAAAGAAGGAAAAAAAAGGTCAAAATCGATCAGTTTGTTGGTGGGGTTCATTAGAGCTCTCATCAAATGATGAAAATGGCAGTAGATAAAGTAG。
2.3.2 recovery of the gel of the fragments of interest
a. Adding 500 mu LBL of balance liquid into CB2 column (the column is placed in a collecting pipe), centrifuging for 1min (the rotating speed and the temperature are 12000rpm and 4 ℃), and pouring out waste liquid in the collecting pipe;
b. recovering a target strip rubber block on a rubber cutting table (the ultraviolet irradiation time is not too long during rubber cutting), placing the rubber block in a 1.5mL centrifuge tube for weighing, adding a PC reagent (100 mu L of PC reagent is added for every 0.1 g) into the centrifuge tube, treating the rubber block in a water bath kettle at 50 ℃ for 10min (turning up and down during the period), placing the rubber block at room temperature for cooling, adding the rubber block into a balanced CB2 column, centrifuging the rubber block for 1min (the conditions are consistent with those in the step a and the subsequent centrifuging conditions are consistent), and pouring out waste liquid;
c. adding 600 mu L of PW solution added with absolute ethyl alcohol into CB2, standing for 2min at room temperature, centrifuging for 1min, and pouring out waste liquid;
d. after repeating step c, centrifuging for 2min, standing at room temperature for 5min, placing CB2 into a new 1.5mL centrifuge tube, and adding ddH with pH=7.0-8.5 at the center of the adsorption column membrane (note not to be stamped to the membrane) 2 O, standing for 2min, centrifuging for 2min to obtain DNA solution of target fragment, detecting its concentration and purity with Micro spectrophotometer (Micro Drop), packaging, and storing at-20deg.C.
2.3.3 ligation and transformation of the fragment of interest with the cloning vector pGM-T
a. Taking out pGM-T vector from the ultralow temperature refrigerator at-80 ℃ and melting on ice;
b. the reaction solution was added to a 1.5mL centrifuge tube according to tables 4-6 (the whole procedure was completed on ice):
table 8 connection system
Note that: the volume of the target fragment was calculated from the molar ratio of vector to target fragment=1:3-1:8.
c. The added components were gently mixed, centrifuged briefly and overnight at 16 ℃.
2.3.4TOP 10 E.coli transformation
a. Preparing a flat plate:
preparing an LB solid culture medium by referring to the method of 1.4, preparing a required culture dish, and sterilizing at 121 ℃ for 20min; placing in an ultra-clean workbench, adding IPTG, X-Gal and Amp into the culture medium when the isothermal temperature is reduced to about 55deg.C + The final concentration is 100 mug/mL, 125 mug/mL and 100 mug/mL, after fully and evenly mixing, the mixture is poured into a sterilized culture dish and inverted for 3 hours at 37 ℃ for standby;
b. transformation
1. TOP10 competent cells were removed from the-80℃ultra-low temperature refrigerator, thawed on ice, split into two tubes, 5. Mu.l ligation product was added, flicked and mixed well, and the reaction was performed in the order of tables 4-7 (whole procedure was completed on ice):
TABLE 9 reaction time
2. After the ice bath is finished for 3min, 250 mu L of LB liquid medium without antibiotics is added (preheating at 37 ℃) and shaking culture is carried out for 45min (37 ℃,150 rpm);
3. after the completion of the culture, 100. Mu.L of the solution (200-fold dilution) was aspirated, and the mixture was spread uniformly on a solid medium using a spreading bar, sealed, and cultured for 12 hours (37 ℃ C., darkness);
after 4.12h, blue and white bacterial plaques appear on the plate, and a white single colony is picked up by a toothpick until the antibiotic Amp is added + In 5mL LB liquid medium, shaking culture is carried out at 37 ℃ for 12-16h (200 rpm);
5. positive was detected by bacterial liquid PCR and plasmids were extracted.
2.3.5 extraction of plasmids
a. Adding 500 mu L of balancing solution BL into an adsorption column CP3, centrifuging for 1min under the condition of 12000rpm, and discarding waste liquid;
b. centrifuging 5mL of bacterial liquid for 1min under the condition of 12000mL, collecting bacterial cells, and sucking the supernatant as much as possible;
c. 250. Mu.L of solution P1 (RNase A was added), and the bacteria were thoroughly suspended by pipetting;
d. adding 250 μl of solution P2, rapidly and gently turning over 7 times, and keeping the duration of time not longer than 5min;
e. adding 350 mu L of solution P3, rapidly and gently turning over for 7 times, and centrifuging for 10min;
f. transferring the supernatant to CP3, centrifuging for 45sec, and discarding the waste liquid;
g. adding 600 μl of the rinse PW, centrifuging at 12000rpm for 45sec, and pouring out the waste liquid;
h. repeating the step g, and centrifuging at 12000rpm for 2min;
i. ddH of 60 μlph=7.5 2 Adding O into the middle of CP3 membrane, standing for 2min, centrifuging for 2min to obtain plasmid solution, packaging, and preserving at-20deg.C.
j. The plasmid was used as a template for PCR detection positive and was sent to the company for sequencing (Shanghai Biotechnology).
2.4 construction and transformation of silencing vectors
2.4.1 silencing vector constructs
PCR amplification was performed using the positive plasmid sequenced in 2.3.5 as a template, restriction enzyme cleavage sites (EcoR I and Xho I) and primers with protective bases were added, and fragments of the GhBEL1, ghCDF3 and GhZAT10 genes were inserted into the TRV-156 vector by double cleavage of the PCR product and the TRV-156 vector to form TRV: ghBEL1, TRV: ghCDF3 and TRV: ZAT10 constructs, the cleavage system being as shown in Table 10:
table 10 enzyme digestion System
After the system is finished, reacting for 3 hours at 37 ℃, and adding 10×loading Buffer to stop the reaction; and (5) recovering enzyme cutting products by using the glue. Then connecting the target fragment with a silencing vector; transferring to colibacillus for propagation, culturing at 37 deg.C for 12 hr, picking up bacteria, vibration culturing, PCR and dual-enzyme digestion of plasmid, and sequencing.
2.4.2 transformation of Agrobacterium GV3101
a. GV3101 competent cells were removed from the-80℃ultra-low temperature refrigerator, thawed on ice, split into two tubes, 2. Mu.L of the sequenced plasmid was aspirated, added to centrifuge tubes, and transformed in the order of Table 11:
TABLE 11 reaction time
b. After the above steps were completed, 350. Mu.L of LB liquid medium (without antibiotics) was added, shaking culture was performed for 2 hours (28 ℃,200 rpm), and the bacterial liquid was uniformly spread on the solid medium (Kan was added) + And Rif antibiotics), in the dark at 28℃for 2 days.
c. After the completion of the culture, single colonies were picked into 5mL of LB liquid medium (Kan was added + And an Rif antibiotic), culturing for 12-16h according to the condition of shaking culture in the step b;
d. after the cultivation is completed, the bacterial liquid is preserved by using 50% glycerol (bacterial liquid: glycerol=1:1), and is preserved at 80 ℃ for standby; and (5) performing bacterial liquid PCR to confirm that the vector is positive.
2.4.3 upland cotton plants VIGS silencing
a. Each culture pot was filled with the same weight of nutrient soil (substrate: vermiculite=1:1), which was immersed until the surface was wet for seed germination. Healthy and active ZM113 seeds are selected and placed in a culture pot with the depth of 10 multiplied by 15cm, the seeds are cultivated in a greenhouse in an artificial climatic chamber, when the seeds grow to 7 days, the cotyledons are immersed in water, and after the soil in the nutrition pot is wetted on the surface, the seeds are stopped to be immersed in water and placed for standby;
b. melting bacterial liquid of a VIGS carrier system and a target gene silencing carrier taken out from-80 ℃ on ice, activating the bacterial liquid according to the quantity of the bacterial liquid: LB liquid culture medium=1:10, and propagating according to the same proportion after the activation is completed;
c. after the bacterial liquid is propagated, centrifuging for 10min at 5000rpm, retaining bacterial cells, pouring out supernatant, suspending the bacterial cells by MMA, and measuring the OD value at 600 wavelength to be about 1.8;
d. after resuspension, placing in darkness for 3-5h, mixing TRV192 with bacterial suspension 1:1 containing TRV156 (TRV: 156, blank group), TRV: ghPDS (positive control), TRV156: ghBEL1 (experimental group), TRV156: ghCDF3 and TRV156: ghZAT10 respectively, fully and uniformly mixing, and then injecting bacterial cells on the back of cotton cotyledon by using a 1mL syringe to fill the whole leaf with bacterial cells;
e. the injected seedlings were left to stand in the dark at 25℃for 24 hours and then cultured under normal growth conditions.
2.5 identification of silenced plants
And after the positive control cotton seedlings whiten, adopting cotton young leaves of an experimental group and a blank group to carry out fluorescent quantitative experiments, and detecting the silencing efficiency.
After the injected positive cotton seedlings are whitened, the cotton seedlings which grow to four weeks old in a blank group and an experimental group are respectively placed in a greenhouse at 25 ℃ to grow, and phenotype observation is performed timely.
3 results and analysis
3.1 construction of silencing vectors and Positive identification of silencing lines
3.1.1 construction of the Gene fragment of interest and silencing vector
The target fragment was amplified using NCBI's blast-primer design and cDNA of Miq 113 as a template using Ai Kerui 2 XPro Taq enzyme, and recovered using a universal DNA purification recovery kit (Tiangen) (FIG. 2A). And (3) connecting the recovered target fragment with pGM-T vector for blue-white screening and extracting plasmid. The target fragment was ligated and cultured with TRV156 vector by double digestion, and positive plasmid was obtained by bacterial liquid PCR (FIG. 2B), double digestion (FIG. 2C) and sequencing (FIG. 3). The target fragment ligated to the vector was obtained by sequencing without base deletion and substitution, and thus sequencing was successful. The positive plasmid was transformed into Agrobacterium by freeze thawing for cultivation.
3.1.2 detection of silencing efficiency of the Gene of interest and phenotypic analysis
Based on transcriptome data analysis, candidate genes of which the temperature influences the growth and development of upland cotton are screened. Therefore, the effect of the target gene on upland cotton under temperature stress is studied by using the VIGS technology. We selected the gene GhBEL1 for the VIGS test. Silencing of the gene of interest in cotton 113 in upland cotton by epicutaneous injection under cotyledons revealed that the positive control (TRV: ghPDS) plants began to whiten at day 7 post-infection, and that the positive control whitened significantly at day 12 post-silencing (FIG. 4A) indicating successful Agrobacterium infection of upland cotton. The silencing efficiency of the target gene was detected (B in FIG. 4), and it was found that the expression of GhBEL1 in the TRV: ghBEL1 plant was significantly suppressed, and that the silencing efficiency of the GhBEL1 gene was 66.67% as compared with the control group. Analysis of the expression levels of VIGS-silenced plants and control plants indicated that the gene of interest had been silenced.
3.1.3 phenotypic Effect of appropriate high temperature stress on silenced plants
Four-week old TRV: ghPDS, TRV:156 and TRV: ghBEL1 plants were selected and grown in a 7d greenhouse at 25℃respectively. The plant height, bud and flowering time were then observed (fig. 5), and it was found that after the GhBEL1 gene was silenced, the plant height of upland cotton became shorter, and the bud and flowering time were delayed. In order to study the function of the GhBEL1 gene, the GhBEL1 gene is firstly silenced in upland cotton by utilizing the VIGS technology, and then the plant height, the bud and the flowering time of the GhBEL1 gene are observed, and the plant height of the upland cotton is shortened by 2.05cm, and the bud and the flowering time are respectively delayed by 4.2 days and 1.7 days after the gene is silenced. The GhBEL1 gene is helpful for promoting the cotton to grow and develop in advance of the bud and flowering time of cotton, and the GhBEL1 gene is proved to positively regulate and control the cotton to grow and develop on upland fields.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (8)
1. A gene GhBEL1 for regulating cotton growth and development, which is characterized in that the nucleotide sequence is shown in SEQ ID NO: 1.
2. A primer set for amplifying the gene GhBEL1 according to claim 1, wherein the nucleotide sequence of the primer set is as shown in SEQ ID NO: 2-3.
3. A recombinant vector comprising the gene GhBEL1 as claimed in claim 1.
4. A recombinant bacterium comprising the recombinant vector of claim 3.
5. Use of the gene GhBEL1 according to claim 1 or the recombinant vector according to claim 3 or the recombinant bacterium according to claim 4 for improving the growth and development of upland cotton.
6. Use of the gene GhBEL1 according to claim 1 or the recombinant vector according to claim 3 or the recombinant bacterium according to claim 4 for improving flowering traits of upland cotton.
7. Use of the gene GhBEL1 according to claim 1 or the recombinant vector according to claim 3 or the recombinant bacterium according to claim 4 for improving the bud trait of upland cotton.
8. Use of the gene GhBEL1 according to claim 1 or the recombinant vector according to claim 3 or the recombinant bacterium according to claim 4 for improving upland cotton plant height traits.
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Non-Patent Citations (3)
| Title |
|---|
| NCBI: "NCBI Reference Sequence:XM_041107462.1", 《NCBI》, 25 April 2021 (2021-04-25), pages 1 - 2 * |
| WU ZHENG ET AL.: "AtWuschel promotes formation of the embryogenic callus in Gossypium hirsutum", 《PLOS ONE》, 31 January 2014 (2014-01-31), pages 1 - 8 * |
| 胡时友等: "镁肥施用量对棉花生长发育及产量的影响", 《农村经济与科技》, 31 December 2015 (2015-12-31), pages 59 - 60 * |
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