CN111153973B - Application of over-expressed GhCBL2 gene in promotion of accumulation of soluble sugar in cotton leaves - Google Patents
Application of over-expressed GhCBL2 gene in promotion of accumulation of soluble sugar in cotton leaves Download PDFInfo
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Abstract
The invention relates to application of an over-expression GhCBL2 gene in promoting accumulation of soluble sugar in cotton leaves, belonging to the technical field of plant genetic engineering. The invention provides application of a GhCBL2 gene with a nucleotide sequence shown as SEQ ID No.1 or a GhCBL2 protein with an amino acid sequence shown as SEQ ID No.2 in promoting accumulation of soluble sugar in cotton leaves. The GhCBL2 gene cloned in cotton is constructed on an over-expression vector pKGWFFS7.0, and the content of soluble sugar in cotton leaves is obviously improved by means of agrobacterium-mediated genetic transformation, so that the resistance of the cotton to abiotic stress is improved, and an ideal way can be provided for cultivating new water-saving and high-yield cotton varieties.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to application of an over-expressed GhCBL2 gene in promoting accumulation of soluble sugar in cotton leaves.
Background
The cotton is used as an important economic crop, and the transgenic technology is used as a quick and effective method for cotton genetic improvement, thereby bringing a huge development space for cotton genetic breeding. The insect-resistant cotton cultivated by the genetic engineering technology becomes the most successful transgenic crop popularized in China, effectively improves the resistance of the cotton to lepidoptera pests, particularly cotton bollworms, recovers great economic loss, greatly reduces the use of pesticides, and plays an important role in protecting the environment, reducing the poisoning risk of people and livestock, improving the cotton planting benefit and the like.
Calcium ions are used as an important second messenger and are widely involved in various aspects of plant growth and development and stress response, wherein CBLs are used as a plant-specific calcium receptor, protein kinases CIPKs which specifically interact with CBLs are activated to phosphorylate downstream target proteins, and the decoding and downstream response of cytoplasmic calcium ions are realized by regulating the activity of the target proteins. The research of the CBL participating in the regulation and control path mainly has the aspects of regulating and controlling the growth and development of plants, ion transport, hormone signal integration, active oxygen steady state, response to adversity stress and the like. However, the use of CBL for soluble sugar accumulation in cotton has not been disclosed.
Disclosure of Invention
The invention aims to provide application of a GhCBL2 gene in promoting accumulation of soluble sugar in cotton leaves. The GhCBL2 gene cloned in cotton is constructed on an over-expression vector pKGWFFS7.0, and the content of soluble sugar in cotton leaves is obviously improved by means of agrobacterium-mediated genetic transformation, so that the resistance of the cotton to abiotic stress is improved, and an ideal way can be provided for cultivating new water-saving and high-yield cotton varieties.
The invention provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in promoting accumulation of soluble sugar in cotton leaves.
The invention also provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in culturing cotton with high soluble sugar accumulation in leaves.
The invention also provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in cultivation of cotton with strong stress resistance.
The invention also provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in cultivation of cotton with strong drought resistance.
The invention also provides application of the pKKWFFS7.0 vector containing the GhCBL2 gene with the nucleotide sequence shown as SEQ ID NO.1 in promoting accumulation of soluble sugar in cotton leaves.
The invention also provides application of agrobacterium tumefaciens containing the GhCBL2 gene with the nucleotide sequence shown as SEQ ID NO.1 in promoting the accumulation of soluble sugar in cotton leaves.
The invention provides application of a GhCBL2 gene in promoting accumulation of soluble sugar in cotton leaves. The GhCBL2 gene cloned in cotton is constructed on an over-expression vector pKGWFFS7.0, and the content of soluble sugar in cotton leaves is obviously improved by means of agrobacterium-mediated genetic transformation, so that the resistance of the cotton to abiotic stress is improved, and an ideal way can be provided for cultivating new water-saving and high-yield cotton varieties.
Drawings
FIG. 1 is a detection diagram of GhCBL2 overexpression cotton molecules provided by the invention;
FIG. 2 is a map of pK2GW7-GhCBL2 overexpression plasmid vector provided by the invention;
FIG. 3 is a Southern hybridization pectin map of the transgenic material provided by the present invention;
FIG. 4 is a graph for measuring the sugar content in the GhCBL2 over-expressed cotton provided by the invention.
Detailed Description
The invention provides application of a GhCBL2 gene with a nucleotide sequence shown as SEQ ID No.1 or a GhCBL2 protein with an amino acid sequence shown as SEQ ID No.2 in promoting accumulation of soluble sugar in cotton leaves. The GhCBL2 gene from cotton is screened and cloned, and genetic transformation of cotton is carried out, and transgenic plant analysis shows that the gene can remarkably promote the accumulation of soluble sugar in cotton leaves, and has important significance in promoting the accumulation of soluble sugar in plant leaves. The sequence shown in SEQ ID NO.1 is the nucleotide sequence (ATGTTGCAGTGCATAGACGGATTGAAGCATTTATTTGCTTCAGTGCTGCAATGCTGTGATATTGATTTGTACAAGCAATCAAGAGGTCTTGTTGATCCAGAACTTCTTGCAAGAGAGACCGTTTTTAGTGTAAGTGAAATAGAAGCACTTTATGAGCTTTTCAAGAAGATAAGCAGTGCTGTTATAGATGATGGGCTGATCAACAAGGAGGAGTTCCAATTGGCGTTATTCAAAACAAACAAAAAGGAGAGTTTGTTTGCTGATCGGGTATTTGACTTGTTTGATACAAAGCATAATGGAATTCTAGGTTTCGAAGAGTTTGCTCGTGCTCTCTCTGTCTTCCATCCAAATGCGCCCATTGACGATAAGATTGATTTCTCTTTTCAACTATACGATCTCAAGCAGCAAGGTTTTATTGAGAGGCAGGAGGTGAAGCAAATGGTAGTGGCTACCCTAGCTGAATCTGGCATGAACCTTTCAGATGATGTTATAGAAAGTATAATTGACAAGACCTTTGAGGAAGCTGATACAAAACATGACGGGAGGATCGACAAGGAAGAGTGGAGAAGCCTTGTTTTGCGACATCCATCCCTTCTGAAAAATATGACCCTGCAATACCTTAAGGACATCACCACAACGTTCCCAAGCTTTGTTTTCCACTCTCAAGTTGATGATACCTGA) of the cloned GhCBL2 gene, wherein 1-681bp is the coding region (CDS) of the GhCBL2 gene and is also the corresponding amino acid sequence of the GhCBL2 gene, and the sequence shown in SEQ ID NO.2 is the protein sequence (MLQCIDGLKHLFASVLQCCDIDLYKQSRGLVDPELLARETVFSVSEIEALYELFKKISSAVIDDGLINKEEFQLALFKTNKKESLFADRVFDLFDTKHNGILGFEEFARALSVFHPNAPIDDKIDFSFQLYDLKQQGFIERQEVKQMVVATLAESGMNLSDDVIESIIDKTFEEADTKHDGRIDKEEWRSLVLRHPSLLKNMTLQYLKDITTTFPSFVFHSQVDDT) coded by the GhCBL2 gene and codes 226 proteins. Specifically, the invention designs a primer with an attB joint to carry out PCR amplification on the full length of the gene by referring to a cotton genome sequence, wherein the primer sequence is as follows: GhCBL 2-BP-R: GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGTTGCAGTGCATAGACGG (SEQ ID NO.3), GhCBL 2-BP-A: GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGTATCATCAACTTGAG (SEQ ID NO.4), BP recombination reaction is carried out on the clone product, GhCBL2 is constructed on a pDONR221 plasmid, sequencing is carried out on the clone fragment through an M13 sequencing primer on the vector, a plurality of sequencing results and reference sequences are integrated for comparison, and finally the actual sequence of GhCBL2 is determined, the nucleotide sequence of the actual sequence is shown in SEQ ID NO.1, and the protein sequence of the gene is shown in SEQ ID NO. 2.
The invention also provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in culturing cotton with high soluble sugar accumulation in leaves.
The invention also provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in cultivation of cotton with strong stress resistance.
The invention also provides application of GhCBL2 gene with an over-expression nucleotide sequence shown as SEQ ID NO.1 or GhCBL2 protein with an amino acid sequence shown as SEQ ID NO.2 in cultivation of cotton with strong drought resistance.
The invention also provides application of the pKKWFFS7.0 vector containing the GhCBL2 gene with the nucleotide sequence shown as SEQ ID NO.1 in promoting accumulation of soluble sugar in cotton leaves. The invention preferably recombines the target gene GhCBL2 to pK2GW7 expression vector through LR recombination reaction, and the constructed vector is named as pK2GW7-GhCBL2 (the vector map is shown in figure 2). Transforming escherichia coli TOP10 by heat shock, identifying positive clones by PCR, propagating and extracting plasmids, transforming the plasmids into agrobacterium strain EHA105 competence by an electrotransfer method, and storing the clones identified as positive by PCR for later use.
The invention also provides application of agrobacterium tumefaciens containing the GhCBL2 gene with the nucleotide sequence shown as SEQ ID NO.1 in promoting the accumulation of soluble sugar in cotton leaves. The invention adopts a genetic operation means and a 35S promoter to drive, and the GhCBL2 gene is overexpressed in cotton by means of agrobacterium-mediated transformation, a transgenic strain is screened by molecular detection, and the transgenic strain is identified in the aspects of soluble sugar, glucose accumulation and the like, so that the GhCBL2 gene is determined to promote the large accumulation of the soluble sugar.
Specifically, the invention preferably expands agrobacterium EHA105 containing pK2GW7-GhCBL2 plasmid, then transforms T-DNA fragment to cotton hypocotyl by agrobacterium-mediated genetic transformation method, obtains regeneration plant by tissue culture, designs primer to carry out PCR positive identification to regeneration plant, extracts positive plant DNA to carry out Southern hybridization copy number identification, selects RNA of single copy positive plant and carries out reverse transcription to cDNA, designs qRT-PCR primer to carry out expression analysis.
The selected GhCBL2 overexpression strain and a control are planted in a transgenic test field and a culture room and used for determining and analyzing the content of soluble sugar and the like. Different plant lines are respectively planted in a transgenic test field with good conditions, leaves at the same part are selected and are rapidly frozen and stored by liquid nitrogen, the same amount of leaves are weighed under laboratory conditions, the soluble sugar is respectively extracted, the extract is diluted and then the content of the soluble sugar in the different plant lines is determined by adopting an anthrone sulfate colorimetric method, and the result shows that the content of the cotton soluble sugar in the overexpression plant lines can be obviously improved under normal conditions.
Cotton is an important economic crop, but the competition of the cotton and main grain crops to the land gradually leads the main production area of the cotton to develop to the arid and semi-arid Xinjiang and other places, and water resources are insufficient to be the main problem restricting the development of the cotton; similarly, as global climate changes have led to an increasing drought problem, increasing water use efficiency in other crops has also been of great importance. The invention discovers that GhCBL2 can obviously improve the content of cotton soluble sugar through cloning and transgenic identification of cotton GhCBL2, the soluble sugar can be used as an important energy substance, and has great effect on improving abiotic stress resistance of plants, especially drought, so the invention can improve various properties of crops and has wide application value.
The application of the GhCBL2 gene in promoting the accumulation of soluble sugar in cotton leaves will be described in further detail with reference to the following specific examples, and the technical scheme of the present invention includes, but is not limited to, the following examples.
Example 1
Cloning and expression pattern analysis of GhCBL2 gene
Extraction of RNA and obtaining of cDNA
Taking samples of upland cotton strain (YZ1, also called Yunan precocious cotton No.1, from the institute of economic crops of academy of agricultural sciences, Henan province) at different periods in the plant somatic embryogenesis process, extracting total RNA by adopting a guanidine isothiocyanate method, and mixing 2 mu g of total RNA as a template, 1 mu L of 500 mu g/ml oligo-dT (15) primer (purchased from Promega corporation) and DEPC-water, wherein the total volume is 14 mu L; then, denaturation is carried out at 70 ℃ for 5min, and ice quenching is carried out; then 10. mu.L of a mixture containing 5. mu.L of RT buffer, 1.25. mu.L of 10mM dNTP, 1.75. mu.L of DEPC-water, 1. mu.L
A mixture of ribonuclear Inhibitor (available from Promega, usa) and 1 μ L Superscript iii reverse transcriptase (available from Invitrogen, usa); the first chain is synthesized after being bathed for 1h at 42 ℃; after the reaction, Superscript III reverse transcriptase was inactivated by treatment at 70 ℃ for 15 min. Each cDNA was diluted to 200. mu.L and stored at-20 ℃ until use.
Screening and identification of GhCBL2 gene
The GhCBL family was identified and cloned. The upland cotton genome contains 22 GhCBL members, different GhCBLs have obvious expression difference in leaves, 5 GhCBLs with relatively high expression quantity (the average value of FPKM is more than 10) exist, and as the GhCBL from the A and D subgenomic sources is highly consistent in sequence, one of each pair of homologous genes is selected, and 12 GhCBLs in total are selected to represent the whole GhCBL gene family. Soluble sugar content determination shows that the silencing of GhCBL2 obviously reduces the content of soluble sugar in cotton plants, which indicates that GhCBL2 can promote the accumulation of soluble sugar.
And D, carrying out expression profile sequencing analysis on the cDNA sample in the step A, and finding out a sequence with high expression quantity. Extracting the gene sequence (Gh _ A01G0740) from a upland cotton genomic database according to the annotation, and designing a primer sequence for amplifying the gene as follows: the forward primer is 35S-S: CTGACGTAAGGGATGACGC (SEQ ID NO.5), and the reverse primer is GhCBL 2-BP-A: GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGTATCATCAACTTGAG (SEQ ID NO. 4). Carrying out PCR amplification by taking cDNA as a template, wherein the PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30sec at 94 ℃, 30sec at 57 ℃, 1min at 72 ℃ and 28 cycles; extension at 72 ℃ for 5 min. The PCR product was ligated to pDONR by BP reactionTM221 vector (BP enzyme from Invitrogen, USA; 4 hours at RT, pDONR)TM221 vector is from CSIRO plant industry, Australia) and then transformed into escherichia coli competent cell TOP10, after 10-12 hours, a single clone is picked for PCR positive detection, and after the positive clone is verified by sequencing, the nucleotide sequence is determined to be shown as SEQ ID NO: 1, the protein sequence coded by the gene is shown as SEQ ID NO: 2, respectively.
Example 2
Construction of overexpression vector of GhCBL2 gene
A. Construction of overexpression vectors
Cloning to pDONRTMGhCBL2 on 221 was recombined into a plant expression vector pk2gw7.0 using an LR reaction (Invitrogen) (wherein: the LR enzyme was purchased from Invitrogen, usa; the reaction was carried out at room temperature for 4 hours, the vector construction map is shown in fig. 2, and the reference numerals indicate that the vector backbone is pK2GW7, resistance in bacteria is spectinomycin, resistance in transgenic plants is kanamycin, the target gene GhCBL2 is driven by the constitutive promoter 35S; the vector pk2gw7.0 is from university of beth.) and escherichia coli competent cells TOP10 were transformed with the reaction product. After 10-12 hours, selecting a monoclonal antibody for PCR positive detection, wherein the forward primer is 35S-S: CTGACGTAAGGGATGACGC (SEQ ID NO.5), and the reverse primer is GhCBL 2-BP-A: GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGTATCATCAACTTGAG (SEQ ID NO. 4). The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; 30sec at 94 ℃, 30sec at 58 ℃, 1min at 72 ℃ and 28 cycles; extension at 72 ℃ for 5 min. And (3) carrying out positive monoclonal propagation and extracting a plasmid to obtain an overexpression plasmid pK2GW7.0-GhCBL2 for transformation.
B. Transformation of Agrobacterium with vectors
The constructed pK2GW7.0-GhCBL2 vector is transformed into an agrobacterium strain EHA105, a monoclonal colony is selected to be inoculated into an LB liquid culture medium containing 100mg/L spectinomycin and shaken at the speed of 150rpm and the temperature of 28 ℃ for 24h, the bacteria liquid is respectively subjected to positive detection by specific primers (the sequence of the primers is shown in example 2), and the positive bacteria liquid is stored at the temperature of-70 ℃ by using 20 percent of glycerol.
Example 3
Genetic transformation and screening identification of GhCBL2 gene
A. Agrobacterium-mediated genetic transformation
The test material was upland cotton strain (YZ1), full and consistent YZ1 seeds were selected, the seed coat was peeled off, sterilized with 0.1% mercuric chloride solution for 10-12min, shaken continuously during this period, the seeds were rinsed with sterile water for 3 times, and the seeds were placed on the surface of MS medium. Culturing at 30 deg.C for 1 day, culturing for 4-5 days.
Taking out a glycerol tube of an EHA105 strain which is preserved and contains a target gene (namely, the cloned GhCBL2 gene of the invention) from an ultra-low temperature refrigerator, melting the glycerol tube on ice, inoculating 10 mu L of the glycerol tube into 2ml of LB liquid containing 100mg/L spectinomycin, carrying out shaking culture at 28 ℃ for 1 day, inoculating 20 mu L of activated bacteria liquid into 15-20ml of fresh liquid LB containing 100mg/L spectinomycin, carrying out shaking culture at 28 ℃ for overnight, sucking 1ml of turbid bacteria liquid into 2ml of sterile centrifuge tube, centrifuging at 8000-10000rpm for 30s, collecting bacteria, resuspending the bacteria by using 20ml of MGL culture medium (the specific components are described later) containing 50mg/L Acetosyringone (AS), and carrying out shaking culture at 28 ℃ for 30-40min for infecting hypocotyl.
The specific steps of agrobacterium-mediated transformation of cotton hypocotyls are as follows:
(1) taking 30 aseptic seedlings in an ultraclean workbench, cutting hypocotyls into 0.5-0.8cm sections on aseptic filter paper, inoculating the sections into a 50ml aseptic conical flask, adding the sections into activated EHA105 agrobacterium liquid containing a target carrier pK2GW7.0-GhCBL2, infecting for 10min, and shaking for several times;
(2) pouring out the bacterial liquid, placing the hypocotyl on sterile filter paper, sucking to remove surface bacterial liquid, placing on an ultra-clean workbench, blowing for 10-15min, inoculating to 2,4-D induction culture medium (specific components are described later), and co-culturing at 19 deg.C in dark for 48-60 hr;
(3) after the co-culture is finished, the hypocotyl is cut into segments and inoculated into an induction culture medium (the specific components are described later) containing 2,4D of kanamycin (100mg/L) and cefamycin (100mg/L), and the mixture is cultured under the weak light at the temperature of 28 ℃;
(4) transferring to an induction culture medium (specific components are described later) containing antibiotic indolebutyric acid (IBA) for continuous subculture after 3 weeks until embryogenic callus appears;
(5) the embryonic callus is successively inoculated into an embryo differentiation culture medium (the specific components are described later) to be subcultured until the somatic embryo is mature, and the mature cotyledon embryo is inoculated into a rooting culture medium (the specific components are described later) to germinate until a complete plant is obtained.
The culture recipe used in this example:
MGL medium: tryptone 5g/L, NaCl 5g/L, MgSO4·7H2O 0.1g/L，KH2PO40.25g/L, mannitol 5g/L, glycine 1g/L, distilled water to make up to 1L.
2,4-D induction medium: MS is taken as a basic culture medium, 0.1mg/L of 2,4-D, 0.1mg/L of cytokinin (KT), 30g/L of glucose and 2.5g/L of Phytagel are added, and distilled water is used for supplementing to 1L. The pH was adjusted to 5.9.
IBA induction medium: MS is taken as a basic culture medium, 0.5mg/L IBA, 0.1mg/L KT, 30g/L glucose and 2.5g/L Phytagel are added, and distilled water is used for supplementing to 1L. The pH was adjusted to 5.9.
Embryo differentiation medium: MS is taken as a basic culture medium, and 1.9g/L KNO is added3KT 0.1mg/L, glucose 30g/L, Gln 1.0g/L, Asn 0.5g/L, Phytagel 2.5g/L, made up to 1L with distilled water. The pH was adjusted to 5.9.
Rooting culture medium: 1/2MS is used as a basic culture medium, 15g/L glucose and 2.5g/L Phytagel are added, and distilled water is used for supplementing to 1L. The pH was adjusted to 5.9.
The basic MS culture medium formula in the culture medium formula comprises macroelements (KNO)3 1.9g/L，NH4NO31.65g/L，KH2PO40.17g/L，MgSO4·7H2O 0.37g/L，CaCl2·2H2O0.44 g/L), trace elements (KI 0.83mg/L, H)3BO3 6.2mg/LMnSO4·4H2O 22.3mg/L，ZnSO4·7H2O 8.6mg/L，Na2MoO4·2H2O 0.25mg/L，CuSO4·5H2O 0.025mg/L，CoCl20.025mg/L), iron salt (Na)2·EDTA 37.3mg/L，FeSO4·7H2027.8 mg/L), organic components (inositol 100mg/L, Gly 2mg/L, VB)1 0.1mg/L，VB6 0.5mg/L，VB50.5mg/L)。
B. Identification of transgenic plants
(1) Positive detection and pure line detection of transgenic plant
Extracting genome DNA of young and tender leaves of a transgenic plant, wherein the DNA is extracted by adopting a plant genome DNA extraction kit of Tiangen Biochemical (Beijing) science and technology limited company (the specific operation steps are shown in the specification of the kit), and a forward primer is 35S-S: CTGACGTAAGGGATGACGC (SEQ ID NO.5), and the reverse primer is GhCBL 2-BP-A: GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGTATCATCAACTTGAG (SEQ ID NO.4) was subjected to PCR to determine whether there was a corresponding T-DNA insertion. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5 min; 30sec at 94 ℃, 30sec at 58 ℃, 1min at 72 ℃ and 28 cycles; extension at 72 ℃ for 5 min. The positive detection result of the GhCBL2 overexpression cotton T0 plant is shown as A in figure 1, and the reference signs indicate that: lane M shows the Marker electrophoresis results (300, 500, 800, 1000, 1500, 2000, 3000, 5000bp from top to bottom), WT shows the wild type receptor material, the transgenic material PCR can amplify the corresponding band, and the wild type has no band. The PCR amplification forward primer is 35S-S: CTGACGTAAGGGATGACGC (SEQ ID NO.5), reverse primer GhCBL 2-BP-A: GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGGTATCATCAACTTGAG (SEQ ID NO. 4). The results show that all the detected regeneration plants are transgenic positive plants.
T to be collected1The seed coat of the generation is removed, the generation is sterilized with 0.1% mercuric chloride solution for 10-12min, shaking continuously during the sterilization, washing with sterile water for 3 times, and the seeds are placed on the surface of a cotton sterile seedling culture medium (containing 100mg/L kanamycin). Culturing at 30 deg.C in dark for 1 day, culturing in light room (illumination intensity of 3000Lux, 15h illumination/9 h dark), and observing for 5-6 days to determine whether there is resistance separation (if there is transgenic plant with long lateral root, it is positive transgenic plant). Then, each generation of single plants is reserved for selfing species for screening until resistance separation does not occur, namely, the transgenic pure line is used for next phenotypic analysis and functional identification.
(2) Detection of expression level of transgenic plant
The RNA is extracted from the main stem and the reverse leaf of the transgenic cotton plant by a conventional guanidinium isothiocyanate method. cDNA was synthesized by mixing 2. mu.g of total RNA as a template with 1. mu.L of 500. mu.g/ml oligo-dT (15) primer (available from Promega corporation), DEPC-water, in a total volume of 14. mu.L; then, denaturation is carried out at 70 ℃ for 5min, and ice quenching is carried out; then 10. mu.L of a mixture containing 5. mu.L of RT buffer, 1.25. mu.L of 10mM dNTP, 1.75. mu.L of DEPC-water, 1. mu.L
A mixture of ribonuclear Inhibitor (available from Promega, usa) and 1 μ L Superscript iii reverse transcriptase (available from Invitrogen, usa); the first chain is synthesized after being bathed for 1h at 42 ℃; after the reaction, Superscript III reverse transcriptase was inactivated by treatment at 70 ℃ for 15 min. Each cDNA was diluted to 200. mu.L and stored at-20 ℃ until use. Using the cDNA synthesized by the above reverse transcription as a template, specific PCR amplification was carried out using the primers in example 1, and the cotton GhUb7(GenBank accession number: DQ116441) gene was used as an internal control for relative quantitative analysis.
The results of the analysis are shown in FIGS. 1B and C, with the reference numbers indicating: b in figure 1 and C in figure 1 respectively represent RT-PCR semiquantitative and qRT-PCR quantitative expression detection of GhCBL2 overexpression cotton T0 plants. The results show that: most T0 transgenic plants have obviously higher expression level than the control plants, and strains with high expression level, such as CO7, CO13 and CO15, are selected in subsequent functional verification studies for further analysis.
(3) Copy number detection of transgenic plants:
extraction of cotton genomic DNA:
1) weighing 0.1-0.2g of young leaf of transgenic cotton plant, adding 200 μ L of DNA extraction buffer solution, grinding to 60s frequency 60Hz, adding 600 μ L of DNA extraction buffer solution, mixing, and centrifuging for 5min (11000 rpm at room temperature).
2) The supernatant was discarded, and 700. mu.L of GP1, a DNA extraction kit (a product of Tiangen Biochemical technology, Beijing, Ltd.), was added thereto and mixed well. The mixture was washed in a water bath at 65 ℃ for 40min, inverted and mixed every few minutes, and centrifuged for 8min (at room temperature, 11000 rpm).
3) The supernatant was added to a new centrifuge tube, 800. mu.L phenol chloroform was added, the tube was gently shaken for 20min, and the tube was centrifuged for 8min (12000 rpm at room temperature).
4) The supernatant was taken, 800. mu.L of chloroform was added thereto, and the mixture was shaken gently for 15min and centrifuged for 8min (room temperature, 12000 rpm).
5) Transferring the upper aqueous phase obtained in the last step into a new centrifuge tube, adding 700 mu L GP2, and fully mixing.
6) The mixed solution was transferred to an adsorption column CB3 (which was added in several portions), centrifuged at 12000rpm for 30sec, and the waste solution was discarded.
7) To the adsorption column CB3, 500. mu.L of buffer GD (DNA extraction kit, available from Tiangen Biochemical technology Co., Ltd.) was added, and the mixture was centrifuged at 12000rpm for 30sec to discard the waste liquid.
8) To the adsorption column CB3, 600. mu.L of PW (absolute ethanol was added before use) was added, and the mixture was centrifuged at 12000rpm for 30sec to discard the waste liquid. And repeating the steps again. Then carrying out air centrifugation for 2 min; the adsorption column was left to air dry at room temperature.
9) The adsorption column CB3 is transferred to a new centrifuge tube with 1.5mL, 60 mu L of eluent TE is added to the middle part of the adsorption film, the mixture is placed at room temperature for 2-5min, and is centrifuged at 12000rpm for 2min, and DNA is collected in the centrifuge tube.
DNA enzyme digestion and electrophoresis separation of DNA
1) Mu.g of DNA sample, 80U of restriction enzyme (HindIII-HF), 8. mu.L of the corresponding CutSmart Buffer were added to a 200. mu.L microcentrifuge tube, mixed on a vortex apparatus and centrifuged slightly and digested at 37 ℃ for 72 h.
2) 0.8% 0.5 XTBE agarose gel was prepared in DYY-III 34A type electrophoresis chamber; adding 2 mu L of loading buffer solution into each sample, mixing uniformly, slightly centrifuging, and then carrying out spotting; performing 250V high-pressure electrophoresis in 0.5 xTBE electrophoresis buffer solution for 10 minutes, and then performing 40V electrophoresis for 12-14 hours.
DNA denaturation and membrane transfer
1) Cutting the glue: stopping electrophoresis, taking out the rubber plate, cutting the spot hole at the upper end, cutting the spot hole at the two sides about 0.5cm away from the spot hole, cutting the lower end along the edge of the bromophenol blue, and cutting the upper left corner to show the direction.
2) Denaturation: acid denaturation for 15min, alkali denaturation for 20min, and gentle shaking of the gel mass from time to time during denaturation.
3) Carrying out salt bridging: pouring alkali transfer liquid into a cleaned 20X 30cm porcelain plate, transversely placing a clean glass plate on the plate, wetting the glass plate by the alkali transfer liquid after balancing, flatly laying filter paper with a salt bridge on the glass plate, enabling two ends of the paper to naturally droop into the plate, driving off an air pocket between the glass plate and the paper by a glass rod, laying a second layer of filter paper according to the same method, placing the front side of the glue upwards in the center of the filter paper, driving off air bubbles, and separating the wide part of the glue, which is about 0.5cm wide, from the filter paper by an X-ray film strip, so that the alkali transfer liquid must enter the water absorption paper through gel to ensure that DNA in the glue is fully transferred to a nylon membrane. Putting a nylon membrane with the size equal to that of the rubber block on the rubber accurately, removing air bubbles, putting two pieces of filter paper with the size equal to that of the nylon membrane, putting absorbent paper with the thickness of about 10cm, putting a glass plate and a weight of about 500g, leveling, and imprinting for 18-24 h.
4) Soaking the transferred nylon membrane in 2 XSSC for 15min, repeating the soaking for one time, taking out, sucking water with filter paper, wrapping with clean filter paper, drying at 80 deg.C for 2 hr, wrapping with preservative film, and storing at-20 deg.C.
Southern hybridization
1) Pre-hybridization: soaking the pre-hybridized nylon membrane in 2 XSSC for 15-30min, taking out the nylon membrane, filling the nylon membrane into a hybridization tube, removing air bubbles, adding 25ml of pre-hybridization solution into the hybridization tube, pre-hybridizing at 42 ℃, rotating at a low speed, and checking whether the solution leaks or not after a few minutes. 320 μ L/403 μ L salmon sperm was added without leakage.
2) And (3) hybridization: pipette 500. mu.L of hybridization solution from the hybridization tube into a new centrifuge tube, add probe, denature at 98 ℃ for 5 minutes, immediately place on ice for 3 min. And adding the denatured probe into a hybridization tube, fully and uniformly mixing, and hybridizing for 10-12 h at 42 ℃.
3) Washing the membrane: 2 XSSC + 0.1% SDS at room temperature for 2 washes, 15min each; washing with 0.1 XSSC + 0.1% SDS at 68 deg.C for 3 times, the first 2 times for 15min, the 3 rd time for 10 min; washing for 1 time for 2-3 min by Washing Buffer; washing with maleic acid buffer solution for 1 time and 2-3 min; diluting 10 Xblocking Solution into 1 Xblocking Solution by maleic acid buffer Solution, taking 80ml of the Solution to block the background, and discarding the Solution after shaking gently for 1h at normal temperature; tube No.4 (Anti-AP) in the kit is centrifuged for 5min at 12000rmp, 2 mu L of the Solution is taken and added with 20ml of 1 × Blocking Solution, the prepared Blocking Solution is added into a hybridization tube, the hybridization furnace is shaken for 40min at 37 ℃, then a nylon membrane is taken out, and the nylon membrane is washed for 3 times by 500ml of Washing Buffer in a porcelain plate, and each time is 15 min.
Lamination and development
1) And rinsing the nylon membrane by using a detection buffer solution for 3-5 minutes at room temperature.
2) Film pressing: cutting a big enough self-sealing bag, spreading the self-sealing bag on a table, sucking 800 mu L CSPD to uniformly drip on a plastic bag, taking out a nylon membrane to put the DNA surface downwards, placing the self-sealing bag on the self-sealing bag, pressing the membrane, and sealing the self-sealing bag by a sealing machine to prevent bubbles. After incubation at room temperature for 10min, the membrane was incubated at 37 ℃ for 5-10min to enhance the chemiluminescent reaction.
3) Pressing a phosphor screen: and (3) placing the DNA surface of the membrane in a phosphor screen in an upward mode, placing 1 piece of X-ray film, covering the phosphor screen, and exposing for 10-20 min.
4) And (3) developing: immersing the X-ray film in developing solution, repeating for several times, washing with water, immersing in fixing solution for 5min, and washing.
The results of the copy number identification of the transgenic lines are shown in FIG. 3. Description of reference numerals: lane M shows the results of Marker electrophoresis (2027, 2322, 4361, 6557, 9416, 23130bp from top to bottom), in which the CO7, CO12 and CO13 transgenic lines are all single copies.
Example 4
Functional verification of GhCBL2 gene by using transgenic cotton
The method comprises the following specific steps:
determination of soluble sugar content in GhCBL2 overexpression lines
The transgenic pure line materials of CO7, CO13 and CO15 and the materials of four lines of negative control (Null) and wild type material (WT) are planted in a transgenic test field, 20 plants of each line are planted, when the plants grow to the flowering period, the leaves at the same part of the oriented light side are respectively selected, 4 biological replicates of each line are performed, and the samples are quickly put into liquid nitrogen for freezing storage after being taken. Grinding the sample with liquid nitrogen under laboratory conditions, weighing 0.1g per sample, adding 1ml distilled water, placing in water bath kettle, extracting at 80 deg.C in water bath for 30min, and mixing for several times.
Taking out the extracted sample, cooling to room temperature, centrifuging, taking the supernatant, diluting the supernatant by 10 times, preparing a gradient standard solution by glucose, taking 100 mu L of each of the standard solution and the sample diluent, adding 200 mu L of anthrone sulfate solution (0.2 anthrone is added into 100ml of 98% sulfuric acid solution), uniformly mixing, placing the sample in 90 ℃ water bath for 10min, taking 200 mu L of the sample after the water bath is finished, adding the sample into an ELISA plate, detecting the light absorption value under 630nm by using an ELISA reader, converting the content of soluble sugar according to a standard curve, performing statistical analysis and difference significance detection on data results by using EXCEL software, and marking and explaining the result in a figure 4: (a and B in fig. 4) shows soluble total sugar (a in fig. 4) and glucose (B in fig. 4) content analysis in GhCBL2 overexpressed cotton, error bars indicate standard deviation of 6 biological replicates,. P <0.05,. P <0.01, T-test. The results show that: compared with wild type and Null, the over-expression strains CO7, CO13 and CO15 greatly promote the content of soluble sugar in the plant.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
Application of <120> over-expression GhCBL2 gene in promotion of accumulation of soluble sugar in cotton leaves
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 681
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgttgcagt gcatagacgg attgaagcat ttatttgctt cagtgctgca atgctgtgat 60
attgatttgt acaagcaatc aagaggtctt gttgatccag aacttcttgc aagagagacc 120
gtttttagtg taagtgaaat agaagcactt tatgagcttt tcaagaagat aagcagtgct 180
gttatagatg atgggctgat caacaaggag gagttccaat tggcgttatt caaaacaaac 240
aaaaaggaga gtttgtttgc tgatcgggta tttgacttgt ttgatacaaa gcataatgga 300
attctaggtt tcgaagagtt tgctcgtgct ctctctgtct tccatccaaa tgcgcccatt 360
gacgataaga ttgatttctc ttttcaacta tacgatctca agcagcaagg ttttattgag 420
aggcaggagg tgaagcaaat ggtagtggct accctagctg aatctggcat gaacctttca 480
gatgatgtta tagaaagtat aattgacaag acctttgagg aagctgatac aaaacatgac 540
gggaggatcg acaaggaaga gtggagaagc cttgttttgc gacatccatc ccttctgaaa 600
aatatgaccc tgcaatacct taaggacatc accacaacgt tcccaagctt tgttttccac 660
tctcaagttg atgatacctg a 681
<210> 2
<211> 226
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Leu Gln Cys Ile Asp Gly Leu Lys His Leu Phe Ala Ser Val Leu
1 5 10 15
Gln Cys Cys Asp Ile Asp Leu Tyr Lys Gln Ser Arg Gly Leu Val Asp
20 25 30
Pro Glu Leu Leu Ala Arg Glu Thr Val Phe Ser Val Ser Glu Ile Glu
35 40 45
Ala Leu Tyr Glu Leu Phe Lys Lys Ile Ser Ser Ala Val Ile Asp Asp
50 55 60
Gly Leu Ile Asn Lys Glu Glu Phe Gln Leu Ala Leu Phe Lys Thr Asn
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Lys Lys Glu Ser Leu Phe Ala Asp Arg Val Phe Asp Leu Phe Asp Thr
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Lys His Asn Gly Ile Leu Gly Phe Glu Glu Phe Ala Arg Ala Leu Ser
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Val Phe His Pro Asn Ala Pro Ile Asp Asp Lys Ile Asp Phe Ser Phe
115 120 125
Gln Leu Tyr Asp Leu Lys Gln Gln Gly Phe Ile Glu Arg Gln Glu Val
130 135 140
Lys Gln Met Val Val Ala Thr Leu Ala Glu Ser Gly Met Asn Leu Ser
145 150 155 160
Asp Asp Val Ile Glu Ser Ile Ile Asp Lys Thr Phe Glu Glu Ala Asp
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Thr Lys His Asp Gly Arg Ile Asp Lys Glu Glu Trp Arg Ser Leu Val
180 185 190
Leu Arg His Pro Ser Leu Leu Lys Asn Met Thr Leu Gln Tyr Leu Lys
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ggggacaagt ttgtacaaaa aagcaggctc catgttgcag tgcatagacg g 51
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggggaccact ttgtacaaga aagctgggtc tcaggtatca tcaacttgag 50
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
ctgacgtaag ggatgacgc 19
Claims (4)
1. The over-expression nucleotide sequence is shown as SEQ ID NO.1GhCBL2Application of GhCBL2 protein with gene or amino acid sequence shown in SEQ ID NO.2 in promoting accumulation of soluble sugar in cotton leaf.
2. The over-expression nucleotide sequence is shown as SEQ ID NO.1GhCBL2The application of GhCBL2 protein with gene or amino acid sequence shown in SEQ ID NO.2 in culturing cotton with high soluble sugar accumulation in leaves.
3. The over-expression nucleotide sequence is shown as SEQ ID NO.1GhCBL2Gene pKKWFFS7.0 vector for promoting cotton leavesAnd accumulation of soluble sugars.
4. The over-expression nucleotide sequence is shown as SEQ ID NO.1GhCBL2Application of the gene agrobacterium in promoting accumulation of soluble sugar in cotton leaves.
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