Sesame flowering regulation gene SiHd3a and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a sesame flowering regulation gene SiHd3a; the gene is located in a section of 13.54 Mb to 13.95 Mb on a No.11 chromosome of sesame, and is a heading data 3a of the sesame, and the nucleotide sequence of the gene is as shown in SEQ ID NO. 1; the amino acid sequence of the encoded protein is as shown in SEQ ID NO. 2; the invention also discloses application of the plant growth regulator in regulating the flowering time of plants. Connecting to form a cloning vector containing the SiHd3a gene by using a homologous recombination technology, and constructing an expression vector for plant expression; the invention further discloses a cloning vector containing the SiHd3a gene and application of an expression vector constructed by the cloning vector in regulating the flowering time of plants. The sequence of the sesame flowering regulation gene SiHd3a is disclosed for the first time, the problems of flowering asynchronism and introduction in plant early flowering breeding and cross breeding are solved by utilizing overexpression of the SiHd3a gene, plant flowering can be remarkably promoted, the growth period can be remarkably shortened, and gene resources are provided for plant early flowering breeding and introduction.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biotechnology, and particularly relates to a sesame flowering regulation gene SiHd3a, and also relates to the application of this gene in promoting early flowering breeding of plants, solving the problem of inconsistent flowering periods in cross-breeding, and introduction of plants. Background Art

[0002] Sesame (Sesamum indicum L.) belongs to the genus Sesamum of the Pedaliaceae family and is an ancient oil crop originating from Africa. Sesame is mainly planted in tropical and subtropical regions and has the advantages of being heat-loving, drought-tolerant, and barren-tolerant. Its seeds are rich in unsaturated fatty acids and the antioxidant component lignan, so it is known as the "queen of oils". Sesame is an important special high-quality oil crop and special agricultural product in China, with an annual demand of 1.8 million tons, but the domestic output is only 0.65 million tons, and the rest needs to rely on imports, with a relatively high degree of foreign dependence. Although the yield per unit area of sesame in China is 3 to 4 times the world average level, its planting area only ranks fifth in the world, behind Sudan, India, Myanmar, and Tanzania.

[0003] In 1935, Rhind first reported the phenomenon that short-day promotes sesame flowering while long-day delays flowering, and classified sesame as a short-day crop. Subsequent studies have also confirmed the differences in photoperiod sensitivity among different sesame germplasms. The flowering time of sesame germplasms ranges from 30 to 44 days. Zhang Tide et al. conducted a genetic analysis of the flowering time of sesame and revealed that the inheritance of sesame flowering time conforms to the additive-dominant-epistatic model of two major genes. The additive effects of these two major genes are similar and in the same direction, and the allele from photoperiod-insensitive is beneficial to shortening the flowering time.

[0004] Existing cultivated sesame is all domesticated from a wild species in the same origin center. Due to its repeated use in breeding work, the genetic basis of modern sesame varieties is extremely narrow. Some sesame germplasms in tropical and subtropical regions cannot flower at all in the long-day environment in temperate regions and have strong photoperiod sensitivity; while there are relatively few reports on the genetic research of photoperiod affecting sesame flowering, and its genetic mechanism is still unclear. During the plant growth and development process, flowering initiation is a key developmental stage. Plants perceive changes in the external environment and adjust their own developmental states, and select the best time to switch from vegetative growth to reproductive growth, so as to continue the life cycle. The early or late flowering time directly affects the growth period and environmental adaptability of plants. Therefore, in-depth study of the regulatory mechanism of photoperiod on sesame flowering is particularly important for cultivating new sesame varieties adapted to different environmental conditions.

[0005] The invention patent "A molecular marker HSRC3911 closely linked to the major QTL locus for sesame flowering period and its application" with the application number CN202010177574.1 first located a major QTL locus qFT_LG08 controlling the flowering period in sesame, which can explain 41.50% - 57.55% of the phenotypic variation. At the same time, an SSR molecular marker HSR3911 closely linked to qFT_LG08 (genetic distance 0.8 cM) was obtained. Among them, the SSR marker HSR3911 is closely linked to the major QTL for sesame flowering period and is a co-dominant molecular marker. By detecting the amplification product of HSR3911 in breeding materials, the allelic state of qFT_LG08 in the materials can be determined; using this marker, the flowering period of sesame breeding materials can be accurately predicted and marker-assisted selection can be carried out in the early generations, which is not affected by the environment and significantly improves the breeding efficiency. Further research can be used to promote the genes controlling the flowering time of plants to ensure the meeting of flowering periods and shorten the growth period in crop cross-breeding. Summary of the Invention

[0006] To solve the deficiencies described in the above-mentioned prior art, the present invention further deeply studies the regulatory mechanism of photoperiod on sesame flowering based on the invention with the application number CN202010177574.1, and provides a sesame flowering regulatory gene SiHd3a and its application.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A sesame flowering regulatory gene SiHd3a, which is located in the 13.54 Mb - 13.95 Mb section on chromosome 11 of sesame and is the heading date 3a of sesame, and the nucleotide sequence of this gene is as shown in SEQ ID NO.1.

[0009] Furthermore, the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.2.

[0010] Furthermore, amino acid sequences with equivalent functions formed by different amino acid sites of this sequence.

[0011] An application of a sesame flowering regulatory gene SiHd3a in regulating the flowering time of plants.

[0012] Furthermore, using homologous recombination technology, a cloning vector containing the SiHd3a gene is ligated and an expression vector for plant expression is constructed.

[0013] Furthermore, an application of a cloning vector containing the SiHd3a gene and the expression vector constructed by it in regulating the flowering time of plants.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. The present invention discloses for the first time at home and abroad the sequence of the gene SiHd3a related to sesame flowering time. Overexpression of the SiHd3a gene in Yuzhi 4 and Bengalese small-seeded sesame can significantly promote plant flowering and shorten the growth period, providing gene resources for early flowering breeding of plants and introduction. Overexpression experiments in Arabidopsis thaliana showed that, compared with the control, in Arabidopsis thaliana overexpressing the SiHd3a gene under long-day conditions, the flowering period and growth period were significantly shortened, thus confirming the role of the SiHd3a gene in regulating the flowering period. Therefore, the present invention proposes to use overexpression of the SiHd3a gene to promote early flowering breeding of plants, solve the problem of non-coincidence of flowering periods in cross-breeding, and solve the problem of introduction, providing valuable gene resources for plant breeding.

[0016] 2. The SiHd3a gene of the present invention was cloned from the sesame varieties Yuzhi 4 and Bengalese small-seeded sesame. There are differences between the SiHd3a amino acid sequences of Yuzhi 4 and Bengalese small-seeded sesame, which are different at one amino acid site. The amino acid sequence encoded by this gene clusters together with FT in Arabidopsis thaliana, indicating their evolutionary correlation. The expression pattern of the SiHd3a gene in sesame shows that as the flowering process of Yuzhi 4 and Bengalese small-seeded sesame progresses, the expression level of this gene increases. In addition, the expression of the SiHd3a gene is hardly detectable in the roots, while the expression level in the leaves is relatively high, indicating that the SiHd3a gene plays an important role in the flowering process of sesame, especially the expression in the leaves may have special significance for flowering regulation.

[0017] 3. The present invention constructs the SiHd3a gene onto the expression vector C15 vector. Through the Agrobacterium-mediated genetic transformation technology, the SiHd3a gene carried by the C15 vector is transferred into Arabidopsis thaliana Col-0 and the ft-10 Arabidopsis mutant to obtain Arabidopsis thaliana overexpressing SiHd3a. The experimental results show that the SiHd3a gene has the function of promoting flowering and shortening the growth period. Description of the Drawings

[0018] Figure 1 Expression levels of the sesame flowering regulation gene SiHd3a in sesame leaves at different developmental stages of the early flowering sesame variety Yuzhi 4 (black) and the late flowering sesame variety Bengalese small-seeded sesame (gray) in the present invention;

[0019] 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 (pre-budding stage), S5: the 25th day after sowing (budding stage), S6: the 30th day after sowing (pre-flowering stage), S7: the 35th day after sowing (flowering stage), S8: the 40th day after sowing (initial capsule-setting stage).

[0020] Figure 2 The expression profiles of sesame flowering regulation gene SiHd3a in roots (R), old leaves (OL), young leaves (NL), shoot apices (SAM), and small flower buds (B) of Yuzhi 4 and Bengalese small-seeded sesame in this invention, where the black columns represent Yuzhi 4 and the gray columns represent Bengalese small-seeded sesame.

[0021] Figure 3 Comparison diagram of the flowering status of T2 generation plants overexpressing SiHd3a Arabidopsis thaliana under long-day conditions (16 h light: 8 h dark);

[0022] Among them, Col-0: Arabidopsis thaliana Col-0; EV: Arabidopsis thaliana Col-0 overexpressing empty vector; OE-SiHd3a: Arabidopsis thaliana Col-0 overexpressing SiHd3a; Photos were taken 37 days after sowing, and the scale bar is 1 cm.

[0023] Figure 4 Comparison diagram of the flowering time of T2 generation plants overexpressing SiHd3a Arabidopsis thaliana under long-day conditions (16 h light: 8 h dark);

[0024] Among them, Col-0: Arabidopsis thaliana Col-0; EV: Arabidopsis thaliana Col-0 overexpressing empty vector; OE-SiHd3a: Arabidopsis thaliana Col-0 overexpressing SiHd3a.

[0025] Figure 5 Comparison diagram of the flowering status of T2 generation plants of the Arabidopsis thaliana mutant overexpressing SiHd3a ft-10 under long-day conditions (16 h light: 8 h dark);

[0026] Among them, ft-10: Arabidopsis thaliana mutant ft-10; EV: Arabidopsis thaliana mutant ft-10 overexpressing empty vector C15; OE-SiHd3a: Arabidopsis thaliana mutant ft-10 overexpressing SiHd3a.

[0027] Figure 6 Comparison diagram of the flowering time of T2 generation plants of the Arabidopsis thaliana mutant overexpressing SiHd3a ft-10 under long-day conditions (16 h light: 8 h dark);

[0028] Among them, ft-10: Arabidopsis thaliana mutant ft-10; EV: Arabidopsis thaliana mutant ft-10 overexpressing empty vector C15; OE-SiHd3a: Arabidopsis thaliana mutant ft-10 overexpressing SiHd3a. Detailed implementation manners

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Specifically, the DNA fragment containing the complete coding region of the SiHd3a gene of the present invention is cloned, the expression pattern of the SiHd3a gene, and the method for verifying the function of the SiHd3a gene are described.

[0030] The method for cloning the flowering-related gene SiHd3a of the present invention is a commonly used method in the art. The total RNA extraction method refers to the instruction manual of the RNA extraction kit (Omega). Cloning genes, constructing vectors, floral dipping, etc. are also commonly used techniques in the art. The reagents, competent cells, antibiotics, etc. involved can all be obtained commercially. Among them, the C15 plasmid as an expression vector has the cauliflower mosaic virus CaMV35S promoter and the yellow fluorescent reporter protein YFP, and can achieve constitutive and overexpression of genes. The specific introduction is from the following paper materials:

[0031] 1. Jie Liu, Fengli Zhao, Ye Guo, Xiucai Fan, Yuejin Wang, Yingqiang Wen. The ABA receptor-like gene VyPYL9 from drought-resistance wild grapevine confers drought tolerance and ABA hypersensitivity in Arabidopsis. Plant Cell, Tissue and Organ Culture, 2019, 138: 543–558.

[0032] 2. Hu Y, Cheng Y, Yu X, Liu J, Yang L, Gao Y, Ke G, Zhou M, Mu B, Xiao S, Wang Y, Wen YQ. Overexpression of two CDPKs from wild Chinese grapevine enhances powdery mildew resistance in Vitis vinifera and Arabidopsis. New Phytol. 2021 Jun; 230(5): 2029-2046. doi: 10.1111 / nph.17285. Epub 2021 Mar 26. PMID: 33595857.

[0033] 3. Wang W, Devoto A, Turner JG, Xiao S. 2007. Expression of the

[0034] membrane-associated resistance protein RPW8 enhances basal defense against

[0035] biotrophic pathogens. Molecular Plant–Microbe Interactions 20, 966 - 976.

[0036] Example 1

[0037] The acquisition of a sesame flowering regulation gene SiHd3a was obtained by screening from the major QTL locus qFT_LG08 for sesame flowering period in the invention patent "A molecular marker HSRC3911 closely linked to the major QTL locus for sesame flowering period and its application" with the application number CN202010177574.1. The specific acquisition method is as follows:

[0038] Using the annotation information of the SiHd3a gene in Yuzhi 4, the position of this gene on the Yuzhi 4 genome was obtained, its DNA and CDS sequences were searched, and primers for amplifying its complete CDS sequence were designed based on the CDS sequence of the SiHd3a gene. Total RNA was extracted from the young leaves of newly budding Yuzhi 4 and Bengalese small-seeded sesame (purchased from the total RNA extraction kit of Omega company), and the operation was carried out according to the instructions. The reverse transcriptase provided by TaKaRa company was used to reverse-transcribe the extracted total RNA into cDNA, and the operation steps followed the instructions of the TaKaRa company reverse transcriptase kit.

[0039] Using the obtained cDNA as a template, PCR amplification was carried out with the following primers:

[0040] SiHd3a-F: 5’-ATGGCAAGACAAATTAGGGACCCTC-3’,

[0041] SiHd3a-R: 5'-TCGCTTTCACTAGTTAACAGATCTTCGG-3'. Using the above primers, a cDNA fragment encoding the complete coding region of the SiHd3a gene was amplified to obtain the complete SiHd3a gene sequence, as shown in SEQ ID NO.1. The specific SiHd3a nucleotide sequence (Sin05627) is as follows: atggcaagacaaattagggaccctctggaagttggtcgtatcatcggagatgttgtggactcttttaggtcgtctgttgatcttcgggtttattacggaggcagaattgtgattaatggctgtgatctgaggcctccccaagttgctaatcagcccagagttgaagttgggggtgatgactttcgaactttctatactcttgttttggttgatcccgatgcaccaagtcctagtaacccacacctacgggaatatctgcactggttggttactgacattccaggaagcactggcgcaagttttggaactgaagtggtgtgctacgaaagccctcggccgacgatgggaatccaccgccttgttttcgtgctgttccgtcagctggggcggcagacggtgtatgctccagggtggcgtcagaacttcataaccagagactttgctgaactctacaaccttggctcgccggtcgcagctgtgtactacaactgccaaagagaaagtggcaccggaggccgaagatctgttaactag

[0042] The amino acid sequence of the protein encoded by it is shown in SEQ ID No.2. The specific SiHd3a amino acid sequence is as follows:

[0043] marqirdplevgriigdvvdsfrssvdlrvyyggrivingcdlrppqvanqprvevggddfrtfytlvlvdpdapspsnphlreylhwlvtdipgstgasfgtevvcyesprptmgihrlvfvlfrqlgrqtvyapgwrqnfitrdfaelynlgspvaavyyncqresgtggrrsvn*

[0044] There is a difference of one amino acid in the amino acid sequences encoded by the sesame flowering regulation gene SiHd3a in Yuzhi 4 and Bengalese small-seeded sesame, as shown below, but it does not affect its function of promoting flowering.

[0045]

[0046]

[0047] Example 2

[0048] Regarding the expression level of the endogenous SiHd3a gene in sesame, the following experiments were conducted:

[0049] The early-flowering sesame variety Yuzhi 4 and the late-flowering sesame variety Bengalese small-seeded sesame were selected for analyzing the expression level of the SiHd3a gene. At different growth stages of Yuzhi 4, young leaves were collected every 5 days from sowing to after flowering; for Bengalese small-seeded sesame, young leaves in the corresponding growth states as Yuzhi 4 were taken, and after flowering, root, old leaf, young leaf, shoot tip and small flower bud samples of Yuzhi 4 and Bengalese small-seeded sesame were collected respectively. Total RNA of these samples was extracted and sent to the company for transcriptome sequencing analysis.

[0050] The analysis results showed that, as Figure 1 shown, with the flowering process of Yuzhi 4 and Bengalese small-seeded sesame, the expression level of the SiHd3a gene gradually increased, indicating that the SiHd3a gene plays an important role in regulating the flowering time of sesame; as Figure 2 shown, in these two cultivated varieties, the SiHd3a gene had a relatively high expression level in leaves, and the expression levels were significantly reduced in shoot tips and small flower buds. As for roots, no expression of SiHd3a was detected. These expression patterns further confirmed the importance of the SiHd3a gene in the sesame flowering process and revealed that its expression in leaves may have special significance for flowering regulation.

[0051] Example 3

[0052] An application of a sesame flowering regulation gene SiHd3a in promoting plant flowering is as follows:

[0053] 1) Using homologous recombination technology for the above-mentioned sesame flowering regulation gene SiHd3a, the SiHd3a gene was ligated to the C15 plasmid to construct a vector for plant expression, named C15-SiHd3a. The specific operation is as follows:

[0054] First, a linearized vector was obtained by using the single enzyme digestion (BamHI, Takara) method, and a high-purity linearized vector was purified through agarose gel electrophoresis and a gel recovery kit (Tiangen Biochemical Technology Co., Ltd.).

[0055] Add the target fragment DNA and the linearized vector to a 200-μL PCR tube at a molar ratio of 3:1 for recombination reaction. After mixing evenly, place it in a PCR instrument at 50 °C for about 15 min. Then add 20 μL of DH5α competent cells (removed from the -80 °C refrigerator and thawed on ice for 5 min), gently mix with a pipette, incubate on ice for another 20 min, perform heat shock in a 42 °C water bath for 45 seconds, and then quickly cool on ice for 2 min.

[0056] In a clean bench, spread the incubated competent cells on an LB solid medium containing Kan resistance, and culture it upside down in an incubator at 37 °C for 16 - 24 h.

[0057] Pick several clones transformed by the recombination reaction for colony PCR identification. Pick the positive single colonies and culture them overnight in a liquid LB medium containing Kan antibiotic in an incubator at 37 °C and 200 rpm. Extract the plasmids, and directly sequence the corresponding bacterial solutions to identify the accuracy of the vector. Through sequence alignment, the sequencing results are completely matched with the full-length CDS sequence of the SiHd3a gene, confirming that the constructed plant expression vector is accurate.

[0058] 2) Transfer the vector C15-SiHd3a prepared in step 1) into Agrobacterium tumefaciens GV3101 (Shanghai Weidi Biotechnology Co., Ltd.), and then introduce it into Arabidopsis thaliana plants. The specific operations are as follows:

[0059] (1) Transfer the recombinant vector into Agrobacterium tumefaciens GV3101:

[0060] ① Add 200 ng of plasmid DNA to every 20 μL of Agrobacterium tumefaciens GV3101 competent cells, gently tap the bottom of the tube to mix evenly, and let it stand 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 in sequence.

[0061] ② Spread the incubated competent cells evenly on an LB solid medium containing Kan and Rif, culture it upside down in an incubator at 28 °C for 2 days. Pick several positive clones, and colony PCR with SiHd3a-F / R primers can amplify the target fragment, indicating that the vector C15-SiHd3a has been transferred into Agrobacterium tumefaciens GV3101.

[0062] (2) Genetic transformation of Arabidopsis thaliana:

[0063] ① Transform the Agrobacterium tumefaciens containing the target vector into competent cells, and resuspend the competent cells with 5% sucrose and 0.02% Silwet-L-77.

[0064] ② Immerse the Arabidopsis thaliana inflorescence into the competent cells and gently shake for 20 s.

[0065] ③ After 12 hours of darkness, cultivate under normal conditions and transform again after one week.

[0066] 3) Screen positive plants.

[0067] Sow the seeds harvested from the T0 generation of Arabidopsis thaliana onto the substrate. When the seedlings grow to the two true leaf stage, spray them with 2500-fold 10% basta. After one week, select the plants that remain green and grow normally, and transplant the positive plants into the soil for continued cultivation. Extract genomic DNA from the leaves of the positive plants and identify the SiHd3a gene using the PCR method to conduct molecular verification of the target gene in the transgenic plants. Conduct molecular verification of the target gene in the transgenic lines. In addition, use a fluorescence microscope to observe the expression of the YFP reporter gene of the SiHd3a gene in the positive lines to confirm that the SiHd3a gene has been successfully transferred into the T1 generation positive plants. Through the same method, screen and obtain the T2 generation positive lines.

[0068] 4) Sesame flowering gene SiHd3a promotes Arabidopsis thaliana flowering

[0069] Cultivate the T2 generation positive Arabidopsis thaliana transformed plants obtained from Example 3 together with the controls (Arabidopsis thaliana Col-0, ft-10 Arabidopsis thaliana mutant) in a light incubator (6 h light: 8 h dark) until the transgenic plants flower.

[0070] Figure 3 It shows the flowering state of the T2 generation plants of Arabidopsis thaliana overexpressing SiHd3a under long-day conditions (16 h light: 8 h dark). Among them, Col-0: Arabidopsis thaliana Col-0; EV: Arabidopsis thaliana Col-0 overexpressing the empty vector; OE-SiHd3a: Arabidopsis thaliana Col-0 overexpressing SiHd3a; Photos were taken 37 days after sowing, and the scale bar is 1 cm. Figure 4 In the overexpression of the SiHd3a gene, the flowering time of Arabidopsis thaliana is 16 days earlier than that of the control group, indicating that the sesame SiFTL gene has the activity of promoting flowering.

[0071] Figure 5 It shows the flowering state of the T2 generation plants of the ft-10 Arabidopsis thaliana mutant overexpressing SiHd3a under long-day conditions (16 h light: 8 h dark). Among them, ft-10: ft-10 Arabidopsis thaliana mutant; EV: ft-10 Arabidopsis thaliana mutant overexpressing the empty vector C15; OE-SiHd3a: ft-10 Arabidopsis thaliana mutant overexpressing SiHd3a; Photos were taken 51 days after sowing, and the scale bar is 1 cm. Figure 6 In the overexpression of the SiHd3a gene in the ft-10 Arabidopsis thaliana mutant, the flowering time is 27 days earlier than that of the control group, further confirming that the sesame SiHd3a gene can not only promote flowering but also shorten the growth period of plants.

Claims

1. A sesame flowering regulatory gene SiHd3a, characterized in that: The gene is located in the 13.54Mb-13.95Mb segment on chromosome 11 of sesame, which is heading date 3a of sesame. The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The sesame flowering regulatory gene SiHd3a according to claim 1, characterized in that: The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

3. The sesame flowering regulatory gene SiHd3a according to claim 2, characterized in that: The amino acid sequences encoded by it are different in different types of sesame, and different amino acid sites can also form amino acid sequences with the same function.

4. The sesame flowering regulating gene SiHd3a according to claim 3, characterized in that: There is one amino acid difference between the amino acid sequence encoded in Yuzhi No. 4 and the amino acid sequence encoded in Bangladesh small-seed sesame, but it does not affect its function of promoting flowering.

5. The sesame flowering regulatory gene SiHd3a according to claim 1, characterized in that: Application in regulating the flowering time of sesame.

6. The sesame flowering regulating gene SiHd3a according to claim 1 or 5, characterized in that: Using homologous recombination technology, a cloning vector containing the SiHd3a gene was formed by connection, and a vector for plant expression was constructed.

7. The sesame flowering regulatory gene SiHd3a according to claim 6, characterized in that: Application of a cloning vector containing SiHd3a gene and an expression vector constructed therefrom in regulating the flowering time of plants.

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