CN117586369A - ScFT2 protein for delaying flowering or prolonging growth period, and encoding gene and application thereof - Google Patents
ScFT2 protein for delaying flowering or prolonging growth period, and encoding gene and application thereof Download PDFInfo
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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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Abstract
The present invention provides a method for delaying flowering or extending fertilityScFT2Protein and encoding gene and application thereof, belongs to the field of biotechnology, and particularly discloses sugarcaneScFT2Use of a gene for delaying flowering or for prolonging the fertility phase. The invention is realized by the method of the FT family of sugarcaneScFT2The gene is researched, so that the mechanism of sugarcane flower formation can be known, and the gene engineering technology and the like are utilized to induce plants to prolong the growth period and delay the flowering period, thereby regulatingThe mechanism research of the growth control period and the flowering period provides theoretical basis and reference.
Description
Technical Field
The invention belongs to the field of biotechnology, relates to plant transgenic biotechnology breeding, and in particular relates to a method for delaying flowering or prolonging the growth periodScFT2Protein and its coding gene and application.
Background
Sugarcane (sugar spp. Hybrid) is an important sugar crop, is widely planted in more than 90 countries worldwide, is the fifth largest economic crop worldwide, is the main source of 80% of sugar and 40% of bioethanol production raw materials worldwide, can reach 18.3 hundred million tons per year, and has a total annual yield value of up to 570 hundred million dollars (Zhang et al 2018). In recent years, the annual planting area of sugarcane in China is about 1800 mu, the annual yield of sugar is about 1000 ten thousand tons, but the import quantity is also continuously increased, and 2021 reaches 576 ten thousand tons. Researches show that the contribution rate of sugarcane variety improvement and new variety popularization to the sugarcane industry is more than 60%. However, on one hand, due to the problems of the lack of flowering period, the non-flowering of the sugarcane parents and the like, a plurality of excellent parents cannot be hybridized and paired, so that the genetic improvement of the sugarcane is hindered, and the utilization research of the hybridized parents and the progress of new variety creation are hindered; on the other hand, flowering of commercial sugarcane hybrids can have some adverse effect on sugar accumulation (Moore and Berding 2013). Therefore, the exploration of the flowering characteristics of the sugarcane, the flowering phase adjustment, the excavation of flowering genes and the flowering molecular mechanism have great significance for the hybrid seed production of the sugarcane.
The florigen FT is a key regulatory factor for transforming plants from vegetative growth to reproductive growth, the FT gene is induced to express in leaves under proper illumination, the FT synthesized in the leaves is transported to stem apical meristem through phloem, and the expression of the gene related to the flower meristem is activated, so that flowering is promoted (Corbesier et al 2007). FT genes belong to PEBP (phosphatidylethanolamine binding protein) family members, and PEBP gene families can be divided into3 major subfamilies: FT-like (Flowering Locus T-like), TFL1-like (Terminal flow 1-like) and MFT-like (Mother of FT and TFL-like) (Jin et al 2021). FT-like can be achieved by modulating plasma membrane H in addition to inducing flowering + ATPase activity to modulate stomatal opening and closing (Kinoshita et al 2011), involved in the growth and extension of lateral branches and roots and axillary buds (Hiraoka et al 2013; niwa et al 2013), involved in tuber formation by interaction with SWEET (Abeleneda et al 2019). The TFL1-like gene functions inversely to FT-like, inhibits the formation of floral primordia in shoot apical meristem, delays flowering, maintains infinite growth habit (Mimida et al 2001), and the MFT-like gene is expressed mainly in seeds and plays an important role in seed development and germination by regulating ABA and GA signaling pathways (Xi et al 2010).
Although sugarcane is usually propagated through cuttage, sexual hybridization is an important means for cultivating new varieties of sugarcane, parent flowering is an important link of sexual hybridization, more genetic variation can be obtained through proper parent selection and pollen transmission, and more genetic diversity and excellent gene resources are provided for sugarcane breeding work. Flowering also has some effect on sugar accumulation for the developed sugarcane commercial hybrid (Moore and Berding 2013). When sugarcane plants enter the flowering stage, the growth and development center of gravity of the plants shifts to inflorescence formation and development, which results in changes in nutrient and energy distribution of the plants. Thus, the flowering process may lead to a decrease in sugar accumulation rate. In addition, the flowering process may also initiate nutrient loss from the plant. During the growth cycle of sugar cane, plants will accumulate nutrients in the stems for the juice of the cane. However, once flowering occurs, the plant will transfer a portion of the nutrients into the inflorescence and seeds, resulting in nutrient loss from the stems. This also has a negative effect on sugar accumulation. Thus, flowering of sugar cane can have some adverse effect on sugar accumulation. In order to keep sugar accumulation to the maximum extent and improve the sugar accumulation efficiency, the breeding of sugarcane non-flowering varieties is an important research direction in the field of sugarcane biological breeding. In addition, the transgenic technology is also one of important means for cultivating non-flowering varieties of sugarcane, and the growth and development process of the sugarcane can be changed by introducing exogenous genes or regulating and controlling the expression of endogenous genes (FT pathway related genes or plant hormone synthesis pathway related genes) through the transgenic technology, so that the non-flowering goal is realized.
Therefore, the research on the FT genes of the sugarcane has important theoretical and practical significance for improving the growth and development processes of the sugarcane.
Disclosure of Invention
In view of the above problems, the present invention provides a method for delaying flowering or prolonging fertilityScFT2Protein and its coding gene and application.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
for delaying flowering or extending the period of fertilityScFT2A protein, saidScFT2The amino acid sequence of the protein is shown in SEQ ID NO:1 is shown in the specification;
the CDS fragment region is 576bp long, 192 amino acids are encoded after translation, and the protein sequence comprises FT feature structural domains: PBP (PF 01161) domain;
ScFT2the protein sequence similarity of the gene and the orthologous gene SbFT2 of the sorghum reaches 90.63 percent,ScFT2the protein sequence similarity of the gene and the orthologous gene OsFTL8 of the rice is as high as 71.35%;
ScFT2proteins do not belong to the MFT-like and TFL1-like families, belong to the FT-like subfamily, and belong to the type II FT-like.
Encoding the above-mentioned compounds for delaying flowering or prolonging fertilityScFT2ProteinsScFT2Genes of the order ofScFT2The coding sequence of the gene is shown in SEQ ID NO: 2.
Further, the saidScFT2The cDNA sequence of the gene is shown in SEQ ID NO: 3.
Further, the saidScFT2The open reading frame of the gene is shown in SEQ ID NO:4.
comprises the aboveScFT2A plasmid for a gene, said plasmid comprisingScFT2The plasmid of the gene is usedScFT2The gene is obtained by connecting with a plasmid vector pBluescript II SK (+) which is cut by EcoRV single enzyme。
The method comprises the following steps ofScFT2A gene editing recombinant vector, saidScFT2The gene editing recombinant vector is prepared by using the above-mentioned vectorScFT2The plasmid of the gene is connected into a pBI121 plasmid vector after BamHI and SacI double enzyme digestion reaction, and the obtained plasmid is driven by a 35S promoterScFT2A kind of electronic deviceScFT2A recombinant vector for gene editing.
ComprisesScFT2Agrobacterium competence of a gene, said gene comprisingScFT2Agrobacterium competence of the genes is achieved by the aboveScFT2The gene editing recombinant vector was introduced into Agrobacterium GV 3101.
The method comprises the following steps ofScFT2Use of a gene for delaying flowering or for prolonging the fertility phase.
Further, the application is implemented by the method comprisingScFT2The agrobacterium of the gene competes to transform plants to obtain transformants to delay flowering of the transformants or to extend the growth period of the transformants.
Further, the plant is a gramineous plant or a cruciferous plant;
the application is to containScFT2The genetic agrobacterium competent transformed plant, the harvested T0 generation seed is sown and cultivated after disinfection, the T1 generation seed is harvested, sown and cultivated again, and the homozygous plant seed is harvested; planting the obtained homozygous strain seeds and verifying by phenotype identificationScFT2Function of the gene.
A method for delaying flowering or extending fertilityScFT2The protein and the encoding gene and application thereof have the beneficial effects that:
the invention is realized by the method of the FT family of sugarcaneScFT2The research of the genes is helpful for understanding the flowering mechanism of sugarcane, and the genetic engineering technology and the like are utilized to induce plants to prolong the growth period and delay the flowering period, so that theoretical basis and reference are provided for the mechanism research of regulating and controlling the growth period and the flowering period;
the invention extracts the total RNA and cDNA of the sugarcane from the sugarcane; and using qRT-PCR analysis result to show thatScFT2The expression quantity of the gene in the sugarcane stems is highest, especially the expression quantity in the mature stems and young leaves of the sugarcane is lower; and is verifiedScFT2Proteins not belonging to the MFT-like and TFL1-like families, belonging to FT-like subfamily and belong to the FT-like type II, which is characteristic of monocotyledonous plants, which has a guiding significance for guiding the relevant plants to delay flowering;
the invention is realized by constructingScFT2Gene editing recombinant vector and method for producing the sameScFT2Agrobacterium competence of the Gene, success will beScFT2Gene transfer into heterologous Arabidopsis thaliana, analysisScFT2Function of the gene in arabidopsis; the results show that in Arabidopsis thalianaScFT2After the gene is over-expressed, the plant platform has obvious functions of delaying plant platform (flowering) and prolonging the growth period; thus the invention disclosesScFT2The gene has important application value in improving the functions of deferring the plant to the platform (flowering) and prolonging the growth period of the original sugarcane and the homologous rice, sorghum and other gramineous plants, and can be applied to the heterologous arabidopsis and other cruciferous plants.
Drawings
FIG. 1 is a drawing of embodiment 1 of the present inventionScFT2Gel electrophoresis detection results and protein structural characteristics of CDS fragments of genes; wherein the left graph isScFT2Gel electrophoresis detection result of CDS fragment of gene, right image isScFT2Protein structural features of CDS fragments of genes;
FIG. 2 is a schematic diagram of example 1 of the present inventionScFT2And (3) carrying out alignment analysis on homologous protein sequences of sorghum and rice, wherein,ScFT2representing the sugarcane of the inventionScFT2The sequence of the gene,SbFT2representing the homologous gene sequence of sorghum,OsFTL8representing homologous gene sequences of rice, and Consensus representing sequences consistent with the three genes;
FIG. 3 is a schematic diagram of example 1 of the present inventionScFT2Phylogenetic analysis with other FT; wherein At represents arabidopsis thaliana, st represents potato, bd represents brachypodium distachyon, sc represents sugarcane (i.e., the present invention)ScFT2Protein), OS represents rice, sb represents sorghum;
FIG. 4 is a graph of sugarcane in example 1 of the present inventionScFT2The expression level of the gene in the mature stems, young stems, mature leaves and young leaves; wherein, the A diagram is of sugarcaneScFT2Expression abundance of genes in mature stems, young stems, mature leaves and young leaves, panel B is sugarcaneScFT2Gene maturationRelative expression levels in stems, young stems, mature leaves, and young leaves;
FIG. 5 shows the structure and cleavage verification result of the plant expression vector pBI121-ScFT2 in example 1 of the present invention; wherein, the left graph shows the structure of the plant expression vector pBI121-ScFT2, and the right graph shows the result of enzyme digestion verification of the plant expression vector pBI121-ScFT 2;
FIG. 6 is a diagram of example 2 of the present inventionScFT2Positive seedling screening and expression quantity detection results of transgenic plants; wherein the left side isScFT2Positive seedling screening results of transgenic plants are shown on the right by semi-quantitative PCRScFT2The expression level of the transgenic plants is detected;
FIG. 7 in example 2 of the present inventionScFT2Phenotype result diagram of transgenic plant with over-expressed gene;
FIG. 8 in example 2 of the present inventionScFT2Bolting time statistics results of transgenic plants with over-expressed genes;
FIG. 9 in example 2 of the present inventionScFT2Counting the number of rosette leaves of the transgenic plant with the over-expressed gene;
FIG. 10 in example 2 of the present inventionScFT2Statistical results of the growth period time of transgenic plants over-expressed in genes.
Detailed Description
The following description of the technical solution in the embodiments of the present invention is clear and complete. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways other than those described herein, and persons skilled in the art will readily appreciate that the present invention is not limited to the specific embodiments disclosed below. The present invention is described in further detail below in conjunction with specific embodiments for understanding by those skilled in the art.
In addition, the specific techniques or conditions are not specified in the specific examples disclosed below, and are carried out according to techniques or conditions described in the literature in the art (for example, refer to the "molecular cloning laboratory Manual", third edition, scientific Press ", et al, compiled by sambrook et al, huang Peitang, et al) or according to the product specifications. The reagents used were not manufacturer-identified and were all conventional commercially available products.
Example 1 sugarcaneScFT2Cloning of genes and construction of plant expression vectors
The extraction material of the total RNA of the sugarcane is selected from noble species Badila.
The wild type Arabidopsis thaliana (Col-0) and the transgenic Arabidopsis thaliana lines required for the experiment were both grown in a 16h/8h (light/dark) incubator at 22 ℃.
The kit used in the experiment comprises a plant total RNA extraction kit OMEGA, TIANGEN plasmid extraction kit, a TAKARA reverse transcription kit and a TIANGEN gel recovery kit, and E.coli competence (DH 5 a) of Shanghai Weidi Biotechnology Co.
SugarcaneScFT2The specific process of cloning the gene and constructing the plant expression vector is as follows:
1) SugarcaneScFT2Cloning of genes
SugarcaneScFT2The gene is obtained by cloning from sugarcane (noble species Badila) by RT-PCR amplification technology, and is specifically as follows:
11 RNA extraction and reverse transcription
Grinding leaves of Saccharum sinensis Roxb (Badila) in liquid nitrogen into powder, extracting total RNA of Saccharum sinensis Roxb with OMEGA kit, reverse transcribing RNA into cDNA with TAKARA reverse transcription kit, and storing the obtained total RNA and cDNA in-80deg.C refrigeratorScFT2The cDNA sequence of the gene is shown in SEQ ID NO: 3.
12 Cloning of genes
Cloning by RT-PCR amplification using sugarcane cDNA as templateScFT2The expression frame of the gene, and the pair primer adopted in RT-PCR amplification is ScFT-F2:5'-ATGTCGATGACGTCAAGGGACAG-3', scFT2-R:5'-CTAGTTTAGGACGCCGGCGCCCGGC-3'. The RT-PCR amplification procedure was: 94 ℃ (pre-denaturation) for 5min;94 ℃ (denaturation), 10s;55 ℃ (annealing), 15s;72 ℃ (extension), 2min;35cycle;72 ℃ (final extension), 10min.
Recovering and purifying amplified products by using a radices root gel recovery kit after RT-PCR amplification is completed, and detecting the products by gel electrophoresis to obtainScFT2PCR purification of the gene.
To be obtainedScFT2The PCR purified product of the gene was ligated with EcoRV single digested plasmid vector pBluescript II SK (+) (pBSK) to transform E.coli DH5a to obtain plasmid pB-ScFT2 (i.e., comprisingScFT2Plasmids of genes) and sequencing the cloned genes.
Wherein, pBSK connects the reaction system and is: 1. Mu.L of T4 DNA Ligase buffer, 2. Mu.L of vector, 6. Mu.LScFT21. Mu.L of T4 DNA enzyme; the pBSK ligation system was placed in a water bath at 16℃for more than 10h (in this example, in a water bath at 16℃for 10 h).
The transformation method of the escherichia coli DH5a comprises the following steps: the ligation product or plasmid (plasmid pB-ScFT2 in this example) was added to 50. Mu.L of DH5a E.coli competent cells, gently mixed, and ice-bathed for 25min; immediately carrying out ice bath for 3-5 min after heat shock for 90s in a water bath at 42 ℃, and adding 800 mu L of LB liquid culture medium to perform preculture for 40-65 min at 37 ℃; then, the cells were collected by centrifugation at 7500rpm for 3min, the supernatant was discarded, and the cells were suspended in the remaining about 200. Mu.L of the culture solution and uniformly spread on an ampicillin-added plate, and 5. Mu.L of IPTG and 30. Mu.L of X-gal (20 mg/mL) were added before spreading; the plates were incubated overnight at 37 ℃ with single colonies picked the next day for subsequent PCR and sequencing verification.
Proper sequencingScFT2The coding sequence of the gene is shown in SEQ ID NO: as shown in figure 2, the number of the parts is two,ScFT2the open reading frame of the gene is shown in SEQ ID NO:4.
ScFT2translation of the nucleic acid sequence of the Gene intoScFT2The protein is used for preparing the protein,ScFT2the amino acid sequence of the protein is shown in SEQ ID NO: 1.
Cloning from cDNA of sugarcane (noble species Badila)ScFT2The gel electrophoresis detection result of CDS fragment of gene is shown as A diagram in FIG. 1, the left side of A diagram in FIG. 1 is Marker gel electrophoresis detection result, the middle and right side are two groupsScFT2The result of gel electrophoresis of CDS fragments of the gene is consistent with the expected size, and the sequencing result shows that the CDS fragments are 576bp in length, 192 amino acids are encoded after translation, and the protein sequence comprises FT feature domains: the PBP (PF 01161) domain is as in FIG. 1, thusScFT2Proteins belong to the FT family.
As shown in the figure 2 of the drawings,SbFT2andOsFTL8respectively areScFT2Gene orthologous genes in sorghum and rice, and Consensus represents the sequence of the three genes in agreement, it can be seen thatScFT2Gene ortholog of gene and sorghumSbFT2The protein sequence similarity of the (a) is as high as 90.63%,ScFT2gene and orthologous gene of riceOsFTL8The protein sequence similarity of (2) is as high as 71.35%.
13）ScFT2Gene and geneScFT2Phylogenetic analysis of proteins and other FT
The isolated genes were further confirmed to belong to the PEBP family by analysis of the encoded protein domains using the on-line website NCBI (https:// www.ncbi.nlm.nih.gov /). Pairs by online tools ExPASy (https:// www.expasy.org /), SOPMA (http:// npsa-pbil. Ibcp. Fr/cgi-bin/npsa_automation. Plpage=npsa_sopma. Html) and MEME (https:// memesulite. Org/MEME/index. Html)ScFT2The physicochemical properties, secondary, tertiary structure and motif of the proteins were analyzed and predicted, and PEBP homologous protein sequences were retrieved by on-line website Phytozome (https:// Phytozome-next. Jgi. Doe. Gov /) and NCBI (https:// www.ncbi.nlm.nih.gov), and multiple sequence alignment and phylogenetic tree construction were performed using MEGA (maximum likelihood method).
Will beScFT2The result of phylogenetic tree construction of FT protein sequences of Sorghum (Sb), paddy (Oryza sativa OS), brevibacterium (Brachypodium distachyon Bd), potato (Solanum tuberosum St) and Arabidopsis thaliana (Arabidopsis thaliana At) for protein (Sc) shows that the result is shown in FIG. 3ScFT2Proteins do not belong to the MFT-like and TFL1-like families, belong to the FT-like subfamily, and belong to the FT-like of type II, which is characteristic of monocotyledonous plants. The method has a certain guiding significance for guiding related plants to delay flowering.
14）ScFT2Tissue expression specific analysis of genes
To exploreScFT2The expression conditions of the genes in different organs of the sugarcane are analyzedMature stems, young stems, mature leaves and young She Zhuailu groupScFT2Simultaneously uses cDNA of mature stems, young stems, mature leaves and young leaves of sugarcane as templates, uses qRT-PCR to performScFT2The expression quantity of the gene in different organs of sugarcane is analyzed, the internal reference gene is ScGAPDH, and the specific analysis process is as follows:
respectively taking mature stems, young stems, mature leaves and young leaves of sugarcane, obtaining RNA by the method in the step 11), performing reverse transcription to obtain cDNA, using the corresponding cDNA obtained by reverse transcription as a template cDNA, and detecting by using a Berle CFX real-time quantitative PCR instrumentScFT2The relative expression level of the genes,ScFT2the quantitative PCR primer of the gene is qFT2-F (namely a front primer): GCGAGGTGATTTGCTACGAGA, qFT2-R (i.e.post primer): GGCTGGCAGGTGAAGAAGG the internal reference primer is qGAPDH-F: CACGGCCACTGGAAGCA, qGAPDH-R: TCCTCAGGGTTCCTGATGCC.
The qRT-PCR reaction system comprises: 10.0. Mu.L of SYBR Premix Ex Taq (2X), 0.5. Mu.L of pre-primer (10. Mu.M), 0.5. Mu.L of post-primer (10. Mu.M), 2.0. Mu.L of template cDNA and 7.0. Mu.L of ddH 2 O；
qRT-PCR reaction procedure: (1) pre-denaturation at 95℃for 5min; (2) denaturation at 95℃for 10s; (3) annealing at 56 ℃ for 20s; (4) real-time fluorescence data acquisition; (5) returning to the second step, 40 cycles; (6) dissolution profile program setting: 95 ℃ for 10s,60 ℃ for 20s, and 60 ℃ for 1s; (8) 8 ℃ for preservation.
After the detection is finished, 2- ΔΔ The CT method calculates the final relative expression, wherein CT: cycle number when fluorescence intensity reaches a threshold value.
With specific results in fig. 4, it can be seen that,ScFT2the highest expression level of the gene in the sugarcane stems, especially the mature stems and young leaves of the sugarcane, shows thatScFT2The gene may play an important role in the process of the flowering regulation and the nutritional growth to reproductive growth of sugarcane.
2) Construction of plant expression vector
The plasmid pB-ScFT2 with correct sequence was ligated into pBI121 (kanamycin resistance) plasmid vector after BamHI and SacI double cleavage reaction to obtain a plasmid vector driven by 35S promoterScFT2Plant expression vector of (a)The volume pBI121-ScFT2 (i.eScFT2Gene editing recombinant vector), specifically as follows:
mu.L of plasmid pB-ScFT2, 2. Mu.L of 10 Xbuffer, 1. Mu.L of BamHI (TaKaRa), 1. Mu.L of SacI (TaKaRa) and 10. Mu.L of ddH were taken 2 O is mixed, the obtained double enzyme digestion reaction system is incubated for 2 to 3 hours at 37 ℃, and then enzyme digestion products are separated by 1.0 percent agarose gel electrophoresis, thus obtainingScFT2After the target band was recovered, 2. Mu.L of pBI121 plasmid vector, 1. Mu.L of T4 DNA Ligase buffer, 6. Mu.L ofScFT2Mixing the target band with 1 μL of T4 DNA Ligase, standing the obtained mixture in water bath at 16deg.C for more than 10 hr for pBI121 ligation reaction to obtain a DNA sequence driven by 35S promoterScFT2The specific structure of the plant expression vector pBI121-ScFT2 is shown in the left panel of FIG. 5.
The correctness of the cloning was verified by BamHI and SacI cleavage of the plant expression vector pBI121-ScFT2, i.e., 6. Mu.L of the plant expression vector pBI121-ScFT2, 2. Mu.L of 10 Xbuffer, 1. Mu.L of BamHI, 1. Mu.L of SacI and 10. Mu.L of ddH 2 O is evenly mixed and then is incubated for 2 to 3 hours at 37 ℃, then the enzyme digestion products are separated by 1.0 percent agarose gel electrophoresis, the 3 to 5cI enzyme digestion is utilized to verify the correctness (3 to 5c, d) of cloning, the result is shown in the right graph of FIG. 5, and it can be seen that the plant expression vector pBI121-ScFT2 containsScFT2And (3) a gene.
3) Plant expression vector pBI121-ScFT2 transformed with Agrobacterium GV3101 competent (i.e., comprisingScFT2Construction of Agrobacterium competence of Gene
The correct plant expression vector pBI121-ScFT2 is introduced into agrobacterium GV3101 to obtain the competent agrobacterium GV3101 transformed by plant expression vector pBI121-ScFT2 (i.e. comprisingScFT2Agrobacterium competence of the gene), the specific process is as follows:
taking 100 mu L of agrobacterium GV3101 competent to be completely melted on ice, adding 1-10 mu L of plant expression vector pBI121-ScFT2 into the solution by using a pipette, gently mixing the solution, and carrying out ice bath for 30min; then quick-freezing for 3-5 min by utilizing liquid nitrogen, and then carrying out warm bath for 5min at the temperature of 37 ℃; then 1000 mu L of LB liquid medium is added, shake culture is carried out for 5 hours at 28 ℃, centrifugation is carried out at 7500rpm for 1.5 minutes, supernatant is discarded, and then the mixture is coated on LB solid culture containing 50 mu g/mL of rifamycin (Rif for short) and 50 mu g/mL of kanamycin (Kan for short),inverted culturing at 28deg.C for 3 days; finally, picking single colony by a pipette into an Ep tube filled with LB liquid medium (the LB liquid medium contains 50 mug/mL kanamycin), shake culturing and expanding culture at 28 ℃ for 24 hours to obtain the competent agrobacterium GV3101 transformed by the plant expression vector pBI121-ScFT2 (namely, the strain comprisesScFT2Agrobacterium competence of the gene).
Example 2 selection, phenotypic identification and statistics of transformed Arabidopsis plants
To further determine the sugarcane FT-associated genesScFT2Role in plant extraction and flowering first, agrobacterium GV3101 transformed with plant expression vector pBI121-ScFT2 competes with flower dipping to transform Arabidopsis thaliana, the specific procedure is as follows:
after the competence of agrobacterium GV3101 transformed by a plant expression vector pBI121-ScFT2 is verified to be accurate by PCR, wild arabidopsis thaliana (Col-0) is transformed by a flower dipping method, the harvested T0 generation seeds are sterilized and sown on a 1/2 MS solid medium flat plate containing 50mg/L kanamycin for screening, the obtained plants with resistance are transferred into a plug tray for continuous culture in a greenhouse at 22 ℃ for 16h/8h (light/dark), DNA detection is carried out on the resistant plants, positive seedlings are reserved for continuous culture, T1 generation seeds are harvested, after continuous culture according to a T0 generation seed culture method, positive seedlings are reserved, and T2 generation seeds are harvested, namely homozygous plant seeds.
Simultaneously planting homozygous strain seeds (namely a T2 generation high-expression strain Col-0) in a greenhouse with the speed of 16h/8h (light/dark), and parallelly planting three groups of seeds marked as OE-1, OE-2 and OE-3 respectively, simultaneously planting wild arabidopsis thaliana (Col-0) as a blank control group marked as WT, and recording the pumping time, the growing period and the rosette number in the planting process; and planting seeds of the homozygous strain by semi-quantitative PCRScFT2The transgenic plants are subjected to expression analysis, and actin2 is taken as an internal reference gene to screenScFT2The identification results of the lines with high transcription level are shown in fig. 6 to 8.
The left panel in FIG. 6 shows seed planting of homozygous linesScFT2As can be seen from the screening result of positive seedlings of transgenic plants, the seedlings obtained by planting seeds of homozygous linesScFT2Transgenic plants(also resistant strains) can be grown on MS medium containing kanamycin; the right panel in FIG. 6 shows the use of semi-quantitative PCR pairsScFT2The expression level detection result diagram of the transgenic plants can be used for finding out three groups of transgenic plantsScFT2The gene expression levels were significantly increased and could be used for subsequent phenotypic analysis (i.e. for the time of pumping, growth period and rosette number analysis).
FIG. 7 is a diagram ofScFT2Phenotypic outcome of transgenic plants overexpressed in the three groups of transgenic plants (i.e., three groups of overexpressing lines)ScFT2The genes all obviously lead arabidopsis to delay the flowering of the extraction platform. The average pumping period for plants grown in the placebo group was 33 days, while the pumping periods for over-expressed strains OE-1, OE-2 and OE-3 were 56, 55 and 54 days, respectively, all differenced to a significant level compared to the placebo group (as shown in FIG. 8). When the table was drawn, the number of rosette leaves of the three over-expressed lines was also greater, 22, 24 and 23, respectively, and the difference was significant compared to rosette leaf number 13 in the blank group (as shown in fig. 9). At the same time, a significant extension of the whole growth period occurred, with the growth periods of the blank and three over-expressed lines being 63, 105, 106 and 105 days, respectively (as shown in fig. 10).
These above experiments and results are well documented,ScFT2the gene has the functions of delaying plant's top-hat and prolonging the growth period.
Because of the PEBP gene family, it plays an important role in plant floral transformation, tuber formation, seed development and germination. At present, numerous PEBP genes have been isolated from a variety of plants such as arabidopsis, rice, maize, sorghum, and soybean. Plant PEBP family genes are divided into 3 subfamilies, namely FT-like, TFL1-like and MFT-like. The invention relates toScFT2The gene belongs to the type II FT-like gene, which is characteristic of monocotyledonous plants and whose function is generally to promote or have no effect on the table. For example, sbFT4 (Sobic.003G295300) of sorghum was studied to promote the extraction of table (Wolabu et al 2016), whereas SbFT14 (Sobic.010G164200) had no effect on the extraction (Wolabu et al 2016); rice OsFTL8 (LOC_Os01g10590) was studied to find out that no matter in short sunshineNeither over-expression nor post knockout of the soybean in long sunlight conditions was altered (Zhang et al 2020). In contrast, the flowering of Zhuzhu has been an important area of research in the cultivation of new varieties of sugarcane, and some FT genes, such as ScFT3, have been studied by cloning to promote flowering of transgenic lines after heterologous expression in Arabidopsis (Kinoshita et al 2011). However, the FT gene which delays flowering has not been cloned. In the present invention, the expression is carried out by overexpression in Arabidopsis thalianaScFT2The gene can obviously prolong the vegetative growth, delay the transformation from the vegetative growth to the reproductive growth, increase rosette leaves and prolong the growth period, and further proves thatScFT2The gene can regulate and control the flowering phase.
Due toScFT2The gene can realize over-expression in heterologous arabidopsis thaliana and then prolong the growth period, thereby regulating and controlling the flowering period, and can also realize over-expression on the original sugarcane or the homologous rice, sorghum and other gramineous plants, thereby prolonging the growth period and regulating and controlling the flowering period. And the sugarcane blooming can produce a desugaring phenomenon, so that the sucrose content is reduced. Delay flowering can play a role of 'open source', prolong the vegetative growth period of sugarcane and accumulate more sugar on one hand; meanwhile, the sugar-removing effect of flowering is reduced, so that the sugar content of the sugarcane is improved.
In summary, the sugar caneScFT2The gene belongs to type II FT-like gene for regulating and controlling flower formation transformation, and is over-expressed in heterologous ArabidopsisScFT2The results of (2) confirm that it does have the functions of delaying flowering and extending the period of fertility.
Other parts not described in detail are prior art. Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, those skilled in the art may, in light of the present disclosure, obtain additional embodiments without undue experimentation, and are within the scope of the invention.
Claims (9)
1. For delaying flowering or extending the period of fertilityScFT2A protein, characterized in that theScFT2The amino acid sequence of the protein is shown in SEQ ID NO: 1.
2. A method of delaying flowering or prolonging the period of fertility according to claim 1ScFT2ProteinsScFT2A gene characterized in that the gene comprisesScFT2The coding sequence of the gene is shown in SEQ ID NO: 2.
3. According to claim 2ScFT2A gene characterized in that the gene comprisesScFT2The cDNA sequence of the gene is shown in SEQ ID NO: 3.
4. A method according to claim 2 or 3ScFT2A gene characterized in that the gene comprisesScFT2The open reading frame of the gene is shown in SEQ ID NO:4.
5. comprisesScFT2A plasmid containing a gene, characterized in that the plasmid containsScFT2A plasmid of a gene according to any one of claims 2 to 4ScFT2The gene was ligated with EcoRV single digested plasmid vector pBluescript II SK (+).
6. The method comprises the following steps ofScFT2A recombinant vector for gene editing, characterized in that it comprisesScFT2A recombinant vector for gene editing comprising the vector according to claim 5ScFT2The plasmid of the gene is connected into a pBI121 plasmid vector after BamHI and SacI double enzyme digestion reaction, and the obtained plasmid is driven by a 35S promoterScFT2A kind of electronic deviceScFT2A recombinant vector for gene editing.
7. ComprisesScFT2The Agrobacterium competence of the gene, characterized in that the gene comprisesScFT2Genetic Agrobacterium competence using the method of claim 6ScFT2The gene editing recombinant vector is introduced into agrobacterium to obtain.
8. The method comprises the following steps ofScFT2Use of a gene for delaying flowering or for prolonging the fertility phase.
9. The application of claim 8, wherein the application is a use of a composition comprisingScFT2The agrobacterium competence of the gene is used for transforming arabidopsis thaliana, sugarcane, rice or sorghum to obtain a transformant so as to delay the flowering of the transformant or prolong the growth period of the transformant.
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