Gene SiFTL related to flowering of sesame and application of gene SiFTL

Abstract

The invention belongs to the technical field of molecular biology, particularly relates to a gene SiFTL related to sesame flowering, and also relates to application of the gene to promotion of flowering asynchronism and introduction in plant early flowering breeding and cross breeding. The gene is located in a 13.54 Mb-13.95 Mb segment on a No.11 chromosome of sesame and is a floating locus T-like of the sesame, the nucleotide sequence of the gene is shown as SEQ ID NO: 1, the amino acid sequence of the gene is shown as SEQ ID NO: 2, and silencing of the sequence can cause flowering delay or no flowering. The sesame flowering related gene SiFTL is applied to overexpression of Arabidopsis Col-0 and ft-10 Arabidopsis mutants, it is shown that the gene can promote flowering of Arabidopsis, and the gene has potential application value for flowering asynchronism and introduction problems in crop early flowering breeding and cross breeding.

Description

Technical Field

[0001] The invention belongs to the field of molecular biotechnology, and specifically relates to a gene SiFTL related to sesame flowering, and also relates to the application of the gene in promoting early flowering breeding of plants, flowering delay in hybrid breeding and introduction problems. Background Art

[0002] Sesame (Sesamum indicum L.) belongs to the genus Sesamum in the family Sesamum in the subclass Lamiaceae of the class Rosaceae. It is a traditional high-quality oil crop in my country and one of the six most widely grown oil crops in the world. In 2020, the planting area of ​​sesame in my country was 2.78×105 hm2, with a total output of 4.55×105 t, and its yield per unit area was 1636.34 kg / hm2, ranking first in the world.

[0003] The existing cultivated sesame in my country is domesticated from the same wild species. Due to the repeated use of backbone parents in breeding work, the genetic basis of modern sesame varieties is extremely narrow, which greatly limits the further improvement of my country's sesame breeding level. Strengthening the collection and utilization of sesame germplasm resources at home and abroad is conducive to improving existing sesame varieties and enriching the genetic diversity of sesame. Some sesame germplasms from tropical and subtropical regions cannot bloom under the long-day environment of temperate regions, and it is difficult to successfully introduce them between regions. They are often ignored in early breeding work. Although short-day treatment can obtain hybrid offspring with local germplasm, it will also affect the growth period and regional adaptability of new sesame varieties after multiple generations of breeding, which seriously limits its promotion and planting area in my country. Therefore, analyzing the regulatory mechanism of photoperiod on sesame flowering is particularly critical for breeding new sesame varieties that adapt to different environmental conditions.

[0004] The invention patent with application number CN202010177574.1, "A molecular marker HSRC3911 tightly linked to the main effect QTL site of sesame flowering period and its application", located a main effect QTL site qFT_LG08 controlling the flowering period in sesame for the first time, which can explain 41.50% to 57.55% of the phenotypic variation, and obtained the SSR molecular marker HSR3911 tightly linked to qFT_LG08 (genetic distance 0.8cM). Among them, the SSR marker HSR3911 is tightly linked to the main effect QTL of sesame flowering period and is a co-dominant molecular marker. By detecting the amplification product of HSR3911 in the breeding material, the allele state of qFT_LG08 in the material can be determined; the marker can be used to accurately predict the flowering period of sesame breeding materials and marker-assisted selection in the early generation, which is not affected by the environment and significantly improves the breeding efficiency. In this way, further research can be conducted on genes that can be used to promote the flowering time of plants to ensure the encounter of flowering periods and shorten the growth period in crop hybrid breeding. Summary of the invention

[0005] To address the deficiencies described in the above-mentioned prior art, the present invention further studies genes that can be used to promote the flowering time of plants to ensure the encounter of flowering periods and shorten the growth period in crop hybrid breeding, and provides a sesame flowering-related gene SiFTL and its application.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is: A gene SiFTL related to sesame flowering is located in the 13.54 Mb-13.95 Mb segment on chromosome 11 of sesame and is the flowering locus T-like of sesame. The nucleotide sequence of the gene is shown in SEQ ID NO.1. Silencing of the sequence will cause late flowering and no flowering under long daylight. The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.2.

[0007] Furthermore, the cloning method of the gene SiFTL includes: extracting sesame RNA, reverse transcribing it into cDNA, using cDNA as a template and the following sequences as primers to obtain the SiFTL gene sequence by RT-PCR, wherein the primer sequences are: SiFTL-F: 5'-ATGGATATAAACTCACGAGACCCTTTGG-3' and SiFTL-R: 5'-AGATCTTCGGCCTCCAGTCCC-3'.

[0008] Furthermore, homologous recombination technology is used to connect and form a cloning vector containing the SiFTL gene, and an expression vector for plant expression is constructed.

[0009] Furthermore, the cloned SiFTL gene, and the vector or host cell containing the SiFTL gene are used in promoting plant flowering.

[0010] The beneficial effects of the present invention are: 1. The present invention is the first to report the flowering-related sesame gene SiFTL sequence at home and abroad, and its overexpression can significantly promote plant flowering and shorten the growth period.

[0011] 2. The overexpression test in Arabidopsis showed that the flowering period and growth period of Arabidopsis plants overexpressing SiFTL gene were significantly shortened compared with the control (Arabidopsis Col-0 and ft-10 Arabidopsis mutants). Therefore, the present invention proposes to use the overexpression of SiFTL gene to promote early flowering breeding of plants, flowering period problems in hybrid breeding and introduction problems, and provide valuable gene resources for plant breeding.

[0012] 3. The SiFTL gene of the present invention was cloned from the sesame variety Yuzhi No. 4. The amino acid sequence encoded by the gene is clustered with the Arabidopsis FT gene, indicating that they are evolutionarily related. The expression level of SiFTL increases with the flowering process of Yuzhi No. 4, especially in old leaves, with the highest expression value, while there is no significant difference in the expression level in young leaves, stem tips and small flower buds. In addition, no expression value of the SiFTL gene was detected in all developmental stages and different tissues of Bengal small-seed sesame, which indicates that the silencing of the SiFTL gene in Bengal small-seed sesame may cause the growth period of this variety to be delayed, and it cannot bloom normally under long-day conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The expression levels of the sesame flowering-related SiFTL genes in the present invention in sesame leaves at different developmental stages of the early-flowering sesame variety Yuzhi No. 4 (black column) and the late-flowering sesame variety Bengal small-seed sesame (not detected); Among them, S1: the 5th day after sowing, S2: the 10th day after sowing, S3: the 15th day after sowing, S4: the 20th day after sowing (early budding period), S5: the 25th day after sowing (budding period), S6: the 30th day after sowing (early flowering period), S7: the 35th day after sowing (flowering period), S8: the 40th day after sowing (initial capsule formation period).

[0014] Figure 2 The expression of SiFTL gene in the present invention in roots (R), old leaves (OL), young leaves (NL), stem tips (SAM) and small flower buds (B) of Yuzhi No. 4 and Bangladesh small-seed sesame, among which Yuzhi No. 4 is black column and no value was detected in Bangladesh small-seed sesame.

[0015] Figure 3 This is a comparison of the flowering status of the T2 generation of Arabidopsis thaliana overexpressing the SiFTL gene under long-day conditions (16h light: 8h dark); Among them, Col-0: Arabidopsis Col-0; EV: Arabidopsis Col-0 overexpressing empty vector C15; OE-SiFTL: Arabidopsis Col-0 overexpressing SiFTL gene; photos were taken 37 days after sowing, and the scale is 1 cm.

[0016] Figure 4 This is a comparison of flowering time of Arabidopsis T2 generation overexpressing SiFTL gene under long-day (16h light: 8h dark); Among them, Col-0: Arabidopsis Col-0; EV: Arabidopsis Col-0 overexpressing empty vector C15; OE-SiFTL: Arabidopsis Col-0 overexpressing SiFTL gene.

[0017] Figure 5This is a comparison of the flowering status of the T2 generation of the Arabidopsis mutant ft-10 overexpressing the SiFTL gene under long-day conditions (16h light: 8h dark); Among them, ft-10: ft-10 Arabidopsis mutant; EV: ft-10 Arabidopsis mutant overexpressing empty vector; OE-SiFTL: Arabidopsis ft-10 overexpressing SiFTL gene; photos were taken 51 days after sowing, and the scale is 1 cm.

[0018] Figure 6 This is a comparison of the flowering time of the T2 generation of the Arabidopsis mutant ft-10 overexpressing the SiFTL gene under long-day conditions (16 h light: 8 h dark); Among them, ft-10: ft-10 Arabidopsis mutant; EV: ft-10 Arabidopsis mutant overexpressing empty vector; OE-SiFTL: ft-10 Arabidopsis overexpressing SiFTL gene. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below, specifically describing the method of cloning a DNA fragment containing the complete coding region of the SiFTL gene, the expression pattern of the SiFTL gene, and verifying the function of the SiFTL gene.

[0020] The method of cloning the flowering-related gene SiFTL described in the present invention is a commonly used method in the art. The total RNA extraction method is carried out with reference to the instructions of the RNA extraction kit (Omega). Cloning genes, constructing vectors, inflorescence infection, etc. are also commonly used techniques in the art. The reagents, competent cells, antibiotics, etc. involved can all be obtained from commercial channels. Among them, the C15 plasmid involved as an expression vector has a cauliflower mosaic virus CaMV35S promoter and a yellow fluorescent reporter protein YFP, which can achieve constitutive and overexpression of genes. The specific introduction comes from the following paper materials: 1. Jie Liu, Fengli Zhao, Ye Guo, Xiucai Fan, Yuejin Wang, YingqiangWen. The ABA receptor-like gene VyPYL9 from drought-resistance wildgrapevineconfers drought tolerance and ABA hypersensitivity in Arabidopsis. Plant Cell, Tissue and Organ Culture, 2019, 138: 543–558. 2. Hu Y, Cheng Y, Yu 10.1111 / nph.17285. Epub 2021 Mar26.PMID: 33595857. 3. Wang W, Devoto A, Turner JG, Xiao S. 2007. Expression of the membrane-associated resistance protein RPW8 enhances basal defenseagainst biotrophic pathogens. Molecular Plant–Microbe Interactions 20, 966-976. Example 1 A gene SiFTL related to sesame flowering is obtained by the following method: Using the annotation information of the SiFTL gene in Yuzhi No. 4, the location of the gene on the genome of Yuzhi No. 4 was obtained, and its DNA and CDS sequences were searched accordingly. Based on the CDS sequence of the SiFTL gene, primers that can amplify its complete CDS sequence were designed. Total RNA was extracted from the newly budding young leaves of Yuzhi No. 4 (purchased from the total RNA extraction kit of Omega Company) according to the instructions. The extracted total RNA was reverse transcribed into cDNA using the reverse transcriptase provided by TaKaRa Company. The operation steps followed the instructions of the reverse transcriptase kit of TaKaRa Company.

[0021] The obtained cDNA was used as a template and PCR amplification was performed using the following primers: SiFTL-F: 5'-ATGGATATAAACTCACGAGACCCTTTGG-3', SiFTL-R: 5'-AGATCTTCGGCCTCCAGTCCC-3', The above primers were used to amplify the cDNA fragment of the complete coding region of the SiFTL gene to obtain the complete SiFTL gene sequence, as shown in SEQ ID NO. 1. The specific SiFTL nucleotide sequence (Sin05626) is as follows: atggatataaactcacgagaccctttggaagttagtcgtgtcgtcggagatgttgtagacccttttacatcgtccactagtctacggatttcctatggtggcaggacagtggctaatggctgtgatctgaga actccccatgttatcagccagcccagttgagatagggggtgatgactttcgaactttctatactctcttgttttggtggaccctgatgcgccaagccctagcaacccacacctaagagaatacttgcattgg ttggtgacagacatcccaggaagcactggcacaagttttggaactgaagttgtgtgttacgagagccctcagccgacgataggaatccaccgcttggttttcttgttgttccgccaactggggcggcagacg gtgtatgctccggggtggcgccagaacttcataactagagacttcgctgaagtgtacaatcttggctcgccggttgccgctgtgtactacaactgccaaagggaaaacgggactggaggccgaagatcttag The amino acid sequence of the protein encoded by the SiFTL gene is shown in SEQ ID No. 2. The specific SiFTL amino acid sequence (Sin05626) is as follows: mdinsrdplevsrvvgdvvdpftsstslrisyggrtvangcdlrtphvisqprveiggddfrtfytlvlvdpdapspsnphlreylhwlvtdipgstgtsfgtevvcyespqptigihrlvfllfrqlgrqtvyapgwrqnfitrdfaevynlgspvaavyyncqrengtggrrs The SiFTL gene of the present invention is cloned from the sesame variety Yuzhi No. 4. The amino acid sequence encoded by the gene is clustered with the Arabidopsis FT gene, indicating that they are evolutionarily related.

[0022] Example 2 Regarding the expression level of the endogenous SiFTL gene in sesame, the experiment was as follows: The early-flowering sesame variety Yuzhi No. 4 and the late-flowering sesame variety Bengal small-seed sesame were selected to analyze the expression level of SiFTL genes. At different growth stages of Yuzhi No. 4, young leaves were collected every 5 days from sowing to flowering; for Bengal small-seed sesame, young leaves of the corresponding growth state of Yuzhi No. 4 were taken, and after flowering, roots, old leaves, young leaves, stem tips and small flower buds of Yuzhi No. 4 and Bengal small-seed sesame were collected, and the total RNA of these samples was extracted and sent to the company for transcriptome sequencing analysis.

[0023] The analysis results show that: Figure 1 As shown in Figure 2, the expression level of SiFTL gene increased with the flowering process of Yuzhi 4 sesame; Figure 2 As shown in Figure 2, in Yuzhi 4, the expression of SiFTL gene was not detected in the root, but the expression level was highest in old leaves, and there was no significant difference in the expression levels in young leaves, stem tips and small flower buds. Figure 1 and Figure 2 As shown, no expression values ​​of SiFTL gene were detected in all developmental stages and different tissues of Bengal small-seed sesame, indicating that the silencing of SiFTL gene in Bengal small-seed sesame may be the reason for its delayed flowering period. These findings reveal the important role of SiFTL gene in regulating the flowering process of sesame.

[0024] Example 3 An application of a gene SiFTL related to sesame flowering in promoting plant flowering is as follows: 1) The sesame flowering-related gene SiFTL was connected to the C15 plasmid using homologous recombination technology to construct a vector for plant expression, named C15-SiFTL. The specific operations are as follows: First, the linearized vector was obtained by single enzyme digestion (BamHI, Takara) method, and then purified by agarose gel electrophoresis and gel recovery kit (Tiangen Biochemical Technology Co., Ltd.) to obtain a high-purity linearized vector.

[0025] Add the target fragment DNA and linearized vector to a 200-μl PCR tube at a molar ratio of 3:1 for recombination reaction. After mixing, place in a PCR instrument at 50°C for about 15 min, add 20 μL DH5a competent cells (taken out of a -80°C refrigerator and thawed on ice for 5 min), mix gently with a pipette, incubate on ice for 20 min, heat shock in a 42°C water bath for 45 seconds, and quickly cool on ice for 2 min.

[0026] In a clean bench, the competent cells after incubation were spread on LB solid medium containing Kan resistance and incubated upside down in a 37°C incubator for 16-24 hours.

[0027] Several clones transformed by the recombination reaction were selected for colony PCR identification, and then the single colonies identified as positive were selected and cultured overnight in a liquid LB medium containing Kan antibiotics at 37°C and 200 rpm in an incubator, plasmids were extracted, and the corresponding bacterial liquid was directly sequenced to identify the accuracy of the vector. Through sequence comparison, the sequencing results completely matched the full-length CDS sequence of the SiFTL gene, indicating that the constructed plant expression vector was accurate.

[0028] 2) The vector C15-SiFTL prepared in step 1) was transferred into Agrobacterium tumefaciens GV3101 (Shanghai Weidi Biotechnology Co., Ltd.), and then introduced into Arabidopsis plants. The specific operation is as follows: (1) Transformation of the recombinant vector into Agrobacterium GV3101: ① Add 200 ng of plasmid DNA to every 20 μL of GV3101 Agrobacterium competent cells, gently stir the bottom of the tube to mix, and then place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0029] ② Spread the competent cells evenly on LB solid medium containing Kan and Rif, and culture them upside down in a 28°C incubator for 2 days. Pick several positive clones and use SiFTL-F / R primers to perform colony PCR to detect the target fragment, indicating that the vector C15-SiFTL has been transferred into Agrobacterium tumefaciens GV3101.

[0030] (2) Genetic transformation of Arabidopsis thaliana: ① Transform the Agrobacterium containing the target vector into competent cells and resuspend the competent cells in a medium containing 5% sucrose and 0.02% Silwet-L-77.

[0031] ② Immerse the Arabidopsis inflorescence into the competent cells and shake gently for 20 s.

[0032] ③ After 12 hours of darkness, culture normally and transform again after one week.

[0033] 3) Screening positive plants.

[0034] Seeds harvested from the T0 generation of Arabidopsis were planted and sown on the substrate. When the seedlings grew to the two true leaves stage, they were sprayed with 2500 times 10% basta. One week later, plants that remained green and grew normally were selected, and the positive plants were transplanted into the soil for further cultivation. Genomic DNA was extracted from the leaves of the positive plants, and the SiFTL gene was identified by PCR, and the target gene in the transgenic strain was molecularly verified. In addition, the expression of the YFP reporter gene of the SiFTL gene in the positive strain was observed using a fluorescence microscope to confirm that the SiFTL gene had been successfully transferred into the T1 generation positive plants. By the same method, the T2 generation positive strain was screened and obtained.

[0035] 4) Sesame flowering gene SiFTL promotes flowering in Arabidopsis The T2 generation overexpression Arabidopsis positive lines obtained from Example 3 were cultured together with the control group (Arabidopsis Col-0, ft-10 Arabidopsis mutants) in a light incubator (6 h light: 8 h dark) until the transgenic plants bloomed. Figure 3 and Figure 4 The sesame flowering gene SiFTL overexpression showed that the flowering time of Arabidopsis was 13 days earlier than that of the control group, indicating that the sesame SiFTL gene has the activity of promoting flowering. Figure 5 and Figure 6 The results showed that the flowering gene SiFTL overexpressed in the sesame ft-10 Arabidopsis mutant flowered 28 days earlier than the control group, further confirming that the SiFTL gene has the function of promoting plant flowering.

Claims

1. A gene SiFTL related to sesame flowering, characterized in that: The gene is located in the 13.54 Mb-13.95 Mb segment on chromosome 11 of sesame and is the flowering locus T-like of sesame. The nucleotide sequence of the gene is shown in SEQ ID NO.

1. Silencing of the sequence will cause late flowering and no flowering under long daylight.

2. The gene SiFTL related to sesame flowering according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. The gene SiFTL related to sesame flowering according to claim 1, characterized in that: The cloning method includes: extracting sesame RNA, reverse transcribing it into cDNA, using cDNA as a template and the following sequences as primers to obtain SiFTL gene sequence through RT-PCR, wherein the primer sequences are: SiFTL-F: 5'-ATGGATATAAACTCACGAGACCCTTTGG-3' and SiFTL-R: 5'-AGATCTTCGGCCTCCAGTCCC-3'.

4. The gene SiFTL related to sesame flowering according to claim 1, characterized in that: Using homologous recombination technology, a cloning vector containing the SiFTL gene is connected and an expression vector for plant expression is constructed.

5. The gene SiFTL related to sesame flowering according to claim 3 or 4, characterized in that: The cloned SiFTL gene, and the vector or host cell containing the SiFTL gene are used in promoting plant flowering.

Images