Lilium haichangyi gene and application thereof in regulating dormancy release of crops

Abstract

The application discloses a lily HAI1 Gene and application thereof in regulation of dormancy release of crops. It belongs to the technical field of genetic engineering. In the application, lily bulb of 'Longya red' is used as test material to observe morphological and physiological changes in the process of dormancy release. By using transcriptome sequencing technology, key regulation genes of the lily bulb of 'Longya red' for dormancy release are screened LbHAI1. The application explores the function of the gene in the process of dormancy release by cloning the key gene, heterologous overexpression of Arabidopsis thaliana and transient silencing of the key gene in lily bulb. The application provides a theoretical basis for lily dormancy regulation by using genetic engineering means.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a lily. HAI1 Genes and their application in regulating crop dormancy release. Background Technology

[0002] 'Longya Hong' lily is a new lily variety bred from a natural bud mutation of the Longya lily. It has an early maturity, strong disease resistance, high yield, and high value for food, medicine, and ornamental purposes. The cultivation, sale, ornamental tourism, and downstream product development of the 'Longyahong' lily provide significant support for rural development and revitalization.

[0003] After lilies enter dormancy in autumn, they need to be treated with low-temperature refrigeration for a period of time to break dormancy before they can germinate and grow normally. This means that 'Longya Hong' lilies, harvested in autumn, can only be replanted the following spring, making year-round production impossible and affecting their profitability.

[0004] Currently, there are many achievements in the physiological research on how low temperature breaks the dormancy of lily bulbs, but there is less research on the molecular mechanism of low temperature breaking the dormancy of lily bulbs, which needs to be explored in depth.

[0005] Therefore, screening the key regulatory genes for breaking dormancy in 'Longyahong' lily bulbs is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a lily HAI1 Genes and their application in regulating crop dormancy release.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A type of lily HAI1 The gene, whose nucleotide sequence is shown in SEQ ID No. 1.

[0009] The above-mentioned lilies HAI1 Application of genes in regulating crop dormancy release.

[0010] Furthermore, the regulation is negative regulation.

[0011] Furthermore, the crops mentioned are Arabidopsis thaliana and lily.

[0012] Furthermore, it is used to regulate the expression of dormancy-related genes.

[0013] Furthermore, the dormancy-related gene is AtGA2OX1 , AtFT , AtABI5 , AtSNRK2.8 .

[0014] Silent Lily HAI1 Application of genes in constructing a model to inhibit dormancy in lily bulbs.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention has the following beneficial effects: This invention uses 'Longyahong' lily bulbs as experimental material to observe the morphological and physiological changes during the dormancy-breaking process. Transcriptome sequencing technology is used to screen key regulatory genes for dormancy breaking in 'Longyahong' lily bulbs. LbHAI1 This invention investigates the function of a key gene during dormancy undosing by cloning a key gene, heterologously overexpressing it in Arabidopsis thaliana, and transiently silencing the key gene in lily bulbs. This invention provides a theoretical basis for regulating lily dormancy using genetic engineering techniques. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0017] Figure 1 In Embodiment 1 of the present invention LbHAI1 Gene amplification, where M: 2000 bp marker; 1: target band location.

[0018] Figure 2 This is a phylogenetic tree of LbHAI1 protein and Arabidopsis PP2C family proteins in Example 2 of the present invention.

[0019] Figure 3 This is a prediction of the secondary and tertiary structures of the LbHAI1 protein in Example 2 of the present invention, wherein A: prediction of the secondary structure of the LbHAI1 protein; B: prediction of the tertiary structure of the LbHAI1 protein.

[0020] Figure 4 This refers to the conserved domain of the LbHAI1 protein in Example 2 of this invention.

[0021] Figure 5 In Embodiment 3 of the present invention LbHAI1 Gene subcloning and pBI121 vector restriction enzyme digestion electrophoresis, where A: LbHAI1Gene subcloning; M: 2000 bp DNA Marker; 1: Amplified band of the target fragment; B: pBI121 vector digestion; M: 2000 bp DNA Marker; 1: Band after vector digestion; 2: pBI121 vector plasmid control.

[0022] Figure 6 pBI121- in Embodiment 3 of the present invention LbHAI1 Expression carrier map.

[0023] Figure 7 pBI121- in Embodiment 3 of the present invention LbHAI1 Expression vector PCR identification, where M: 2000 bp DNA Marker; 1-3: vector colony PCR amplification.

[0024] Figure 8 This is a subcellular localization map of the LbHAI1 protein in Example 4 of the present invention.

[0025] Figure 9 This document describes the dormancy breaking of 'Longya Hong' lily bulbs under 200 mg / L ABA treatment in Example 5 of this invention. A: Bud elongation of 'Longya Hong' lily under 200 mg / L ABA treatment; B: Statistical analysis of the ratio of bud length to bulb length of 'Longya Hong' lily under 200 mg / L ABA treatment. for p <0.05, the difference is significant; for p <0.01, the difference is extremely significant; the scale length is 1 cm.

[0026] Figure 10 In Example 5 of this invention, the 'Longyahong' lily plants treated with 200 mg / L ABA... LbHAI1 Expression levels of genes related to dormancy.

[0027] Figure 11 In Embodiment 6 of the present invention LbHAI1 Gene PCR amplification and pCambia1305 vector restriction enzyme digestion electrophoresis, where A: LbHAI1 Gene amplification; M: 2000 bp DNA Marker; 1: Electrophoresis of target fragment amplification; B: Restriction digestion of pCambia1305 vector; M: 2000 bp DNA Marker; 1: Restriction digestion of pCambia1305 vector; 2: pCambia1305 vector plasmid control.

[0028] Figure 12 pCambia1305- in Embodiment 6 of the present invention LbHAI1 Expression carrier map.

[0029] Figure 13 pCambia1305- in Embodiment 6 of the present invention LbHAI1 Expression vector colony PCR identification, where M: 2000 bp DNA Marker; 1-3: single colony PCR amplification; y: negative control.

[0030] Figure 14 pCambia1305- in Embodiment 7 of the present invention LbHAI1 PCR identification of Arabidopsis thaliana plants infected with the expression vector, where M: 2000 bp Marker; WT: wild-type Arabidopsis thaliana plants; 1: transgenic plant identification band; Y: positive control; y: negative control.

[0031] Figure 15 pCambia1305- in Embodiment 7 of the present invention LbHAI1 RT-qPCR identification of Arabidopsis thaliana plants infected with the expression vector, among which, for p <0.01, the difference is extremely significant.

[0032] Figure 16 In Embodiment 7 of the present invention LbHAI1 The germination of overexpressing Arabidopsis thaliana seeds on MS plates was analyzed, including: A: Germination of transgenic Arabidopsis thaliana seeds on MS plates on day 3; B: Germination rate of transgenic Arabidopsis thaliana seeds on MS plates; C: Cotyledon unfolding of transgenic Arabidopsis thaliana seeds on MS plates on day 4; D: Cotyledon unfolding rate of transgenic Arabidopsis thaliana seeds on MS plates on day 4. for p <0.01, the difference is extremely significant; the scale length is 1 cm.

[0033] Figure 17 In Embodiment 7 of the present invention LbHAI1 Germination of overexpressing Arabidopsis thaliana seeds on MS +0.8 µM ABA plates: A: Germination of transgenic Arabidopsis thaliana seeds on MS +0.8 µM ABA plates on day 5; B: Germination rate of transgenic Arabidopsis thaliana seeds on MS +0.8 µM ABA plates; C: Cotyledon unfolding of transgenic Arabidopsis thaliana seeds on MS +0.8 µM ABA plates on day 8; D: Cotyledon unfolding rate of transgenic Arabidopsis thaliana seeds on MS +0.8 µM ABA plates on day 8. for p <0.01, the difference is extremely significant; the scale length is 1 cm.

[0034] Figure 18 In Embodiment 7 of the present invention LbHAI1 Overexpression of dormancy-related genes in Arabidopsis thaliana, among which, for p <0.05, the difference is significant; for p <0.01, the difference is extremely significant.

[0035] Figure 19 In Embodiment 8 of the present invention LbHAI1 Gene fragment amplification and pTRV2 vector restriction enzyme digestion electrophoresis, where A: LbHAI1 Partial gene fragment amplification; M: 2000 bp DNA Marker; 1: Electrophoresis of target fragment amplification; B: pTRV2 vector digestion; M: 2000 bp DNA Marker; 1: pTRV2 vector digestion; 2: pTRV2 vector plasmid control.

[0036] Figure 20 pTRV2- in Embodiment 8 of the present invention LbHAI1 Expression carrier map.

[0037] Figure 21 pTRV2- in Embodiment 8 of the present invention LbHAI1 Colony PCR identification of expression vectors, where M: 2000 bp DNA Marker; 1-3: single colony PCR amplification; 4: pTRV2 empty vector PCR amplification; y: negative control.

[0038] Figure 22 In the case of transient silence in Embodiment 9 of the present invention LbHAI1 The dormancy release status of the 'Dragon Tooth Red' lily gene, including A: transient silencing. LbHAI1 Post-Longya Red lily bud elongation; B: Instantaneous silence LbHAI1 Statistics on the ratio of bud length to bulb length of 'Longyahong' lily; for p <0.05, the difference is significant; the scale length is 1 cm.

[0039] Figure 23 In Example 9 of this invention, the 'Dragon Tooth Red' lily is momentarily silenced. LbHAI1 Gene identification and expression levels of dormancy-related genes. Detailed Implementation

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1 Target gene cloning (1) RNA extraction and cDNA synthesis of 'Longyahong' lily Approximately 100 mg of stem tips from the 'Longyahong' lily, refrigerated at 4°C for 60 days, was rapidly frozen and ground in liquid nitrogen. RNA was extracted from the 'Longyahong' lily according to the instructions of the RNA extraction kit. RNA concentration was determined after extraction, and RNA quality was detected by gel electrophoresis. Using 'Lilium Longyahong' RNA as a template, cDNA was synthesized according to the cDNA synthesis kit instructions.

[0042] (2) LbHAI1 Gene fragment amplification and ligation into pCE2 TA cloning vector Design specific amplification primers (KL-) for the target gene using SnapGene software. LbHAI1 -F: ATGGCTGAGATCTGCTG, SEQ ID No. 2; KL- LbHAI1 -R: CTAGTACCTCCTCAGATCAACC (SEQ ID No. 3), using 'Lilium Longyahong' cDNA as a template, the target gene was amplified. Gel electrophoresis was used to check if the length of the amplified fragment was correct, yielding a band of approximately 1200 bp. Figure 1 (Refer to the instructions for the gel recovery kit) LbHAI1 The gene amplification product was purified and stored at -20°C. Referring to the TA cloning kit instructions, the target gene fragment was ligated into the pCE2TA cloning vector (Nanjing Novizan Biotechnology Co., Ltd., catalog number 100001_2005205005_C601).

[0043] (3) Escherichia coli transformation Following the instructions from Weidi Biotechnology's DH5α E. coli competent cells, the ligation product was transformed into E. coli. Successfully transformed DH5α E. coli were screened using LB agar plates containing 50 mg / L Kan. Single colonies were picked for PCR amplification using KL- LbHAI1-F(ATGGCTGAGATCTGCTG, SEQ ID No. 2) and KL- LbHAI1 -R (CTAGTACCTCCTCAGATCAACC, SEQ ID No. 3), gel electrophoresis detection LbHAI1 Whether the gene was successfully ligated to the cloning vector Use a pipette tip to pick up a single positive colony and add it to 5 mL of LB liquid medium (containing 50 mg / L Kan), then incubate at 37°C with shaking at 200 rpm for 12 h. 300 µL of different positive monoclonal bacterial cultures were pipetted into a 1.5 mL centrifuge tube and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Comparison of the sequencing results indicated successful cloning. LbHAI1 The gene has a sequence length of 1221 bp. Take 600 µL of successfully sequenced bacterial culture and mix it 1:1 with 50% glycerol solution, then store at -80 ℃.

[0044] LbHAI1 The nucleotide sequence of the gene is as follows:

[0045] The amino acid sequence of LbHAI1 protein is as follows: MAEICCGAVTGATAAAAAVSEISPRAARLRRMQIRRFVSDETEISEKRKKPRFETSVSSPPSTPPSAGETVAGDGKERSTVSKSTIPSGAGETAAGDGKERSTVSKSILSAGDSFSSSAPAPAPPVPADLSRYGITSVCGRRRDMEDAVAVHRSFAAGFDYYGVFDGHGCSHVAVTCKDRMHHIVAEELGYGPLSTEADWTL IMGRSFNRMDTEAIVLGGAPPPIGNCKCELQMPKCDHVGSTAVVAVVDAERVVVANCGDSRAVLCRDGIAVPLSVDHKPDRPDELQRIEDAGGRVIFWDGARVLGVLAMSRAIGDGYLKPFVIADPEVTVTERSGGDECLIVASDGLWDVVSPEMACDVVRTCLRSSSVEAACTDATVLLTKLALARQSSDNVSVVVVDLRRY , SEQ ID No.4.

[0046] (4) Extraction of recombinant vector plasmids Take 4 mL of successfully sequenced bacterial culture and extract the recombinant vector plasmid according to the instructions of Novizan Biosciences' plasmid extraction kit. After determining the plasmid concentration, store it at -20 ℃.

[0047] Example 2 LbHAI1 Gene bioinformatics analysis (1) Classification of LbHAI1 protein The amino acid sequence of LbHAI1 and the amino acid sequences of Arabidopsis PP2C family members were imported into TBtools to construct a phylogenetic tree, and the phylogenetic tree was visualized using the ITOL website.

[0048] The results are as follows Figure 2 As shown, LbHAI1 is most closely related to AtPP2CA, and also to 10 Arabidopsis species. PP2C Family members all belong to sub-tribe A.

[0049] (2) Physicochemical properties of LbHAI1 protein The physicochemical properties of LbHAI1 protein were analyzed using the ProtParam and Protscale tools in Expasy. The chemical formula of LbHAI1 protein is C. 1846 H 2983 N 543 O 590 S 23 With a molecular weight of 42961.66 Da, an aliphatic index of 81.18, and an instability coefficient of 58.04, combined with the analysis of the hydrophilicity of LbHAI1 protein, it is predicted that LbHAI1 protein is an unstable hydrophilic protein.

[0050] (3) Prediction of the secondary and tertiary structures of LbHAI1 protein The secondary structure of the LbHAI1 protein was predicted using the SOPMA website, such as... Figure 3 In structure A, the secondary structure consists of 34.73% alpha helix, 12.32% extended strand, and 52.96% random coil, with the random coil being the most prevalent.

[0051] The tertiary structure of the LbHAI1 protein was predicted using the SWISS-MODEL tool on the Expasy website, such as... Figure 3 As shown in Figure B, the tertiary structure of the LbHAI1 protein contains a large proportion of irregular coils and α-helices, consistent with the predicted results of the secondary structure.

[0052] (4) Conserved domain analysis of LbHAI1 protein The conserved domains of the LbHAI1 protein from 'Lilium Longyahong' were analyzed using the NCBI website. like Figure 4 Within the amino acid sequence of the LbHAI1 protein, from 133 to 390, there is a PP2Cc domain containing multiple active sites. This domain is a catalytic domain, and its catalytic mechanism is related to... PP1 , PP2A , PP2B Proteins in the family are similar, but the LbHAI1 protein does not share sequence similarity with proteins in these families. This catalytic domain endows the LbHAI1 protein with enzymatic activity that dephosphorylates SnRK2 kinase, enabling it to interact with ABA receptors and thus placing the LbHAI1 protein in a negative regulatory role in the ABA signaling pathway.

[0053] Example 3 pBI121- LbHAI1 Construction of expression carrier pBI121- was constructed using homologous recombination. LbHAI1 expression carrier The pBI121 vector with a GFP label was selected as the overexpression vector. The empty vector plasmid was linearized by single digestion with BamHI restriction endonuclease. An undigested plasmid was used as a control. Gel electrophoresis was used to detect whether the digestion was successful. Figure 5 B is the single enzyme digestion product of the pBI121 vector with BamHI restriction endonuclease. Its band position differs significantly from that of the undigested pBI121 vector plasmid, indicating successful digestion of the pBI121 vector. It can be used for subsequent vector construction. The successfully digested linearized vector is then purified using a gel extraction method.

[0054] The CDS sequence of the target gene was imported into SnapGene software and the stop codon was removed. Subcloning primers were designed (pBI12-LbHAI1-F: CACGGGGGACTCTAGAGGATCCATGGCTGAGATCTGCTGC, SEQ ID No. 5; pBI121-LbHAI1-R: GGGACTGACCACCCGGGGATCCGTACCTCCTCAGATCAAC, SEQ ID No. 6). The primers contained anterior and posterior homologous arms and BamHI restriction sites, using pCE2- LbHAI1 Using the vector plasmid as a template, the target fragment was obtained by PCR amplification. Gel electrophoresis is used to determine if the target fragment is of the correct length. Figure 5 A is pCE2- LbHAI1 The PCR amplification product of the vector plasmid had a band length of approximately 1200 bp, consistent with the predicted target gene band length. The detected target fragment was then purified using a gel extraction method.

[0055] The purified products of the linearized vector and the target fragment were ligated using homologous recombinase. The ligation product was then transformed into DH5α competent cells. Successfully transformed DH5α *E. coli* were screened using LB agar plates containing 50 mg / L Kan. Sequencing alignment results were correct, indicating that pBI121- LbHAI1 The expression carrier was successfully constructed. Figure 6 pBI121- LbHAI1 Expression vector map

[0056] pBI121- sequenced successfully LbHAI1The expression vector and the empty pBI121 plasmid were transformed into GV3101 competent cells. Successfully transformed GV3101 Agrobacterium cells were selected using LB agar plates containing 50 mg / L Kan, and colony PCR was performed for identification. The results are as follows: Figure 7 This indicates that pBI121- LbHAI1 The expression vector was successfully transformed into Agrobacterium GV3101. Select a single positive colony and shake it to incubate. Mix the bacterial culture with a 1:1 ratio of 50% glycerol solution and store at -80°C for later use.

[0057] Example 4 Subcellular localization analysis Sow tobacco seeds evenly on the surface of nutrient soil, water, cover with a plastic bag to keep moist, and place in a refrigerator at 4 ℃ for 2 days. After that, remove them and place them in a light incubator (16 hours of light, 8 hours of darkness, 21 ℃) for about 2 weeks. Then, transplant the tobacco seedlings into black square pots containing nutrient soil and place them in a light incubator (16 hours of light, 8 hours of darkness, 21 ℃) until they have 6-8 true leaves, at which point they can be inoculated.

[0058] Will contain pBI121- LbHAI1 The GV3101 Agrobacterium bacterial suspension containing the expression vector and pBI121 empty plasmid was removed from the -80 ℃ freezer, and the bacterial suspension was streaked onto LB agar plates containing 50 mg / L Kan using a pipette tip. The plates were then incubated at 28 ℃ for 48 h. Use a pipette tip to pick up a single colony and inoculate it into 3 mL of LB liquid medium (containing 50 mg / L Kan). Incubate at 28°C with shaking for approximately 36 h until the bacterial culture becomes turbid. Using a pipette tip, inoculate 400 µL of bacterial culture into an Erlenmeyer flask containing 40 mL of LB liquid medium (containing 50 mg / L Kan). Incubate overnight at 28 °C with shaking until the OD600 reaches approximately 1.2. Centrifuge the culture at 5500 rpm for 10 min, discard the supernatant, and resuspend the culture in resuspending buffer (10 mM MES + 10 mM MgCl2 + 0.2 mM AS, pH = 5.6) until the OD600 reaches 1.0. Incubate in the dark for 2 h.

[0059] Using the needle of a 1 mL syringe, gently make a small incision on the underside of a tobacco leaf. Inject the bacterial solution into the entire leaf through the incision. Inject two leaves per tobacco plant. The injected tobacco was cultured in the dark for 24 h, then placed in a light incubator (16 h light, 8 h dark, 21 ℃) for 24 h. Fluorescence was observed under a laser confocal microscope.

[0060] The results showed that pBI121- LbHAI1 The emitted green fluorescence coincides with the red fluorescence of the cell nuclear marker, indicating that the LbHAI1 protein is localized in the cell nucleus. Figure 8 )

[0061] Example 5 Effects of exogenous ABA treatment on dormancy breaking in 'Longyahong' lily bulbs Sixty 'Longya Hong' lily bulbs of uniform size and free from pests and diseases, which were in a dormant state, were selected. Thirty bulbs were soaked in a 200 mg / L ABA solution for 1 hour, while the other 30 bulbs served as a control and were soaked in plain water for 1 hour. After drying the surface moisture of the bulbs, they were stored in a moist substrate and placed in a cold storage at 4 ℃. Sixty days after treatment, lily bulbs were dissected to observe bud elongation, and samples were taken from the buds, flash-frozen in liquid nitrogen, and stored at -80 ℃.

[0062] The results are as follows Figure 9 As shown, the bulb bud length of 'Longyahong' lily treated with 200 mg / L ABA was significantly shorter than that of the water control. Figure 9 A) The average shoot length / bulb length ratio in the water control group was 0.884, while the average shoot length / bulb length ratio in the 200 mg / L ABA treatment group was 0.311, which was significantly lower than that in the water control group. Figure 9 B) indicates that soaking 'Longyahong' lily bulbs in 200 mg / L exogenous ABA inhibits dormancy release.

[0063] The expression levels of dormancy-related genes in the bulb buds of 'Longyahong' lily treated with 200 mg / L ABA were measured using RT-qPCR. The results are as follows: Figure 10 After treatment with 200 mg / L ABA, the buds of 'Longyahong' lily bulbs showed... LbHAI1 , LbSNRK2.8 , LbFT and LbGA2OX1 The expression level of was significantly increased. LbABI5 The expression level was significantly reduced.

[0064] Example 6 pCambia1305- LbHAI1 Construction of expression carrier pCambia1305- was constructed using homologous recombination. LbHAI1 expression carrier pCambia1305 with a 35S promoter was selected as the overexpression vector. The empty vector plasmid was linearized by single digestion with BamHI restriction endonuclease. An undigested plasmid was used as a control. Gel electrophoresis was used to detect whether the digestion was successful. Figure 11 B is the single-enzyme digestion product of the pCambia1305 vector with BamHI restriction endonuclease. Its band position differs significantly from that of the undigested pCambia1305 vector plasmid, indicating successful digestion and suitability for subsequent vector construction. The successfully digested linearized vector was then purified using a gel extraction method.

[0065] The CDS sequence of the target gene was imported into SnapGene software and the stop codon was removed. Subcloning primers were designed (1305-LbHAI1-F: AGAGACACGGGGGACGGATCCGCCACCATGGCTGAGATCTGCTGC, SEQ ID No. 7; 1305-LbHAI1-R: CATGGTCTTTGTAGTCGGATCCGTACCTCCTCAGATCAAC, SEQ ID No. 8). The primers contained anterior and posterior homologous arms and BamHI restriction sites, using pCE2- LbHAI1 Using the vector plasmid as a template, the target fragment was obtained by PCR amplification. Gel electrophoresis is used to determine if the target fragment is of the correct length. Figure 11 A is pCE2- LbHAI1 The PCR amplification product of the vector plasmid had a band length of approximately 1200 bp, consistent with the predicted target gene band length. The detected target fragment was then purified using a gel extraction method.

[0066] The purified products of the linearized vector and the target fragment were ligated using homologous recombinase. The ligation product was then transformed into DH5α competent cells. Successfully transformed DH5α *E. coli* were screened using LB agar plates containing 50 mg / L Kan. Sequencing alignment results were correct, indicating that pCambia1305- LbHAI1 The expression carrier was successfully constructed. Figure 12 pCambia1305- LbHAI1 Expression vector map

[0067] pCambia1305- was successfully sequenced LbHAI1 The expression vector and the empty pCambia1305 plasmid were transformed into GV3101 competent cells. Successfully transformed GV3101 Agrobacterium cells were selected using LB agar plates containing 50 mg / L Kan, and colony PCR was performed for identification. The results are as follows: Figure 13This indicates that pCambia1305- LbHAI1 The expression vector was successfully transformed into Agrobacterium GV3101. Select a single positive colony and shake it to incubate. Mix the bacterial culture with a 1:1 ratio of 50% glycerol solution and store at -80°C for later use.

[0068] Example 7 Obtaining transgenic Arabidopsis plants and seed germination experiments (1) Cultivation of wild-type Arabidopsis thaliana Wild-type Arabidopsis thaliana was sterilized and sown on MS plates, then placed in a 4 ℃ refrigerator for 2 days. Afterward, it was placed in a light incubator (16 h light, 8 h dark, 21 ℃) for approximately 2 weeks. The Arabidopsis seedlings from the plates were then planted in black square pots containing nutrient soil and placed in a light incubator (16 h light, 8 h dark, 21 ℃) for approximately 30 days. After this period, the Arabidopsis began to flower and could be used for genetic transformation experiments.

[0069] (2) Activation of bacterial culture Will have pCambia1305- LbHAI1 The GV3101 Agrobacterium expression vector was removed from the -80 °C freezer, and the bacterial culture was streaked onto LB agar plates containing 50 mg / L Kan using a pipette tip. The plates were then incubated at 28 °C for 48 h. Use a pipette tip to pick up a single colony and inoculate it into 3 mL of LB liquid medium (containing 50 mg / L Kan). Incubate at 28 °C with shaking for about 36 h until the bacterial culture becomes turbid. Using a pipette tip, inoculate 400 µL of bacterial culture into an Erlenmeyer flask containing 40 mL of LB liquid medium (containing 50 mg / L Kan). Incubate overnight at 28 °C with shaking until the OD600 reaches approximately 1.2. Centrifuge the culture at 5500 rpm for 10 minutes, discard the supernatant, and resuspend the cells in 5% sucrose solution (containing 0.02% Silwet L-77) until the OD600 reaches 1.0.

[0070] (3) Inflorescence infection Cut off the pods from the Arabidopsis inflorescence with scissors, immerse the inflorescence in the bacterial solution for 30 seconds, and after infection, blot the bacterial solution off the leaves and stems with paper towels. Place the Arabidopsis in a plastic bag to maintain humidity and incubate in the dark for 24 hours. Afterward, remove the Arabidopsis from the plastic bag and place it in a light incubator (16 hours light, 8 hours dark, 21°C) for further cultivation. When the pods turn yellow and split open, the seeds are mature. Harvest the Arabidopsis seeds and store them in a refrigerator at 4 ℃ to dry.

[0071] (4) Screening of transgenic plants T0 generation Arabidopsis thaliana seeds were placed in sterile 2 mL centrifuge tubes and sterilized in a laminar flow hood. 1 mL of 4% NaClO solution was added to the centrifuge tubes, and the tubes were shaken to ensure thorough contact between the seeds and the NaClO solution. The NaClO solution was then quickly removed, and the seeds were rinsed 5-6 times with sterile water to completely remove the NaClO solution. The seeds were then inoculated onto MS plates containing 50 mg / L Hyg and 200 mg / L Cef and placed in a 4 ℃ refrigerator for 2 days. Afterward, they were placed in a light incubator (16 h light, 8 h dark, 21 ℃) for approximately 2 weeks. The phenotype of the Arabidopsis thaliana seedlings was observed. Transgenic seedlings grew normally on the resistance medium plates, while non-transgenic seedlings did not grow normally and gradually yellowed and died.

[0072] (5) Identification of transgenic plants The selected transgenic Arabidopsis seedlings were planted in black square pots containing nutrient soil and placed in a light incubator (16 h light, 8 h dark, 21 ℃). After 2 weeks, leaves from both transgenic and wild-type Arabidopsis were harvested, and Arabidopsis DNA was extracted according to the DNA extraction kit instructions. The extracted DNA concentration was measured and stored at -20 ℃ for later use. PCR amplification was performed using extracted Arabidopsis DNA as a template, with pCambia1305- LbHAI1 The expression vector plasmid served as a positive control, and wild-type Arabidopsis DNA served as a negative control. The selection of transgenic plants was determined using PCR primers 35S-F (TCAACAAAGGGTAATATCCGG, SEQ ID No. 9) and 1305-. LbHAI1 -R (SEQ ID No. 8)

[0073] The results showed that the Arabidopsis DNA obtained through screening could be amplified into bands, and the positions were similar to those of pCambia1305- LbHAI1 The bands amplified by the expression vector plasmid in the positive control were consistent with those in the negative control and the wild-type Arabidopsis control, indicating that the Arabidopsis plants obtained through screening were transgenic plants. Figure 14 )

[0074] RT-qPCR was used to detect whether transgenic Arabidopsis thaliana overexpressed the gene. LbHAI1 The result is as follows Figure 15 As shown, in transgenic Arabidopsis thaliana LbHAI1 The expression level of [specific gene] was significantly higher than that of wild-type Arabidopsis, indicating that transgenic Arabidopsis overexpressed [specific gene]. LbHAI1

[0075] (6) Screening of T3 generation transgenic Arabidopsis seeds Seeds of the successfully identified T1 generation transgenic Arabidopsis thaliana were collected and stored in a refrigerator at 4 ℃ for drying. T1 generation Arabidopsis seeds were screened using MS plates containing 50 mg / L Hyg and 200 mg / L Cef to obtain T2 generation transgenic Arabidopsis. T2 generation seeds were screened again to obtain T3 generation transgenic plants. T3 generation Arabidopsis seeds were harvested and stored at 4°C in a dry place.

[0076] (7) Observation and statistics of germination phenology of transgenic Arabidopsis thaliana seeds Twenty-six transgenic Arabidopsis thaliana T3 seeds and wild-type Arabidopsis thaliana seeds were sterilized and sown on MS medium plates and MS medium plates containing 0.8 µM ABA. The experiment was repeated three times. Seed germination was observed and recorded. Data analysis and graphing were performed using GraphPad Prism software.

[0077] MS medium plates: Both overexpressing and wild-type Arabidopsis seeds began to germinate 2 days after sowing, and most seeds completed germination after 3 days, but the overexpressing seeds... LbHAI1 The germination rate of Arabidopsis thaliana seeds was significantly higher than that of wild-type Arabidopsis thaliana seeds 2 days after sowing. The difference in germination rates decreased after 3 days, but overexpression... LbHAI1 The germination rate of Arabidopsis thaliana seeds is still higher than that of wild-type Arabidopsis thaliana seeds. Figure 16 A and Figure 16 B) The cotyledon unfolding rate of Arabidopsis seeds 4 days after sowing was statistically analyzed. It was found that the cotyledons of both overexpressed and wild-type Arabidopsis seeds unfolded normally, but the overexpressed... LbHAI1 The cotyledon unfolding rate of Arabidopsis thaliana seeds was significantly higher than that of wild-type Arabidopsis thaliana seeds. Figure 16 C and Figure 16 D) The experimental results show that LbHAI1 It can promote the breaking of dormancy in Arabidopsis seeds.

[0078] MS + 0.8 µM ABA medium plates: overexpression LbHAI1 Arabidopsis seeds began to germinate 2 days after sowing, while wild-type Arabidopsis seeds were inhibited by ABA, with a germination rate of 0% at 2 days and germination only beginning 3 days after sowing. After overexpression LbHAI1Arabidopsis seeds germinated rapidly, with an average germination rate of 97.436% at 5 days after sowing, compared to an average germination rate of 61.539% for wild Arabidopsis seeds at the same time, significantly lower than that of overexpressed Arabidopsis seeds. LbHAI1 Arabidopsis seeds, and in subsequent statistics, the germination rate of wild Arabidopsis seeds was still lower than that of overexpressed seeds. LbHAI1 Arabidopsis seeds ( Figure 17 A and Figure 17 B) The cotyledon unfolding rate of Arabidopsis seeds 8 days after sowing was statistically analyzed. In wild Arabidopsis seeds, cotyledon unfolding was inhibited by ABA, resulting in fewer seeds with unfolded cotyledons and no greening of the cotyledons. However, overexpression of ABA... LbHAI1 Arabidopsis seeds and cotyledons can unfold normally and grow green. Figure 17 C), overexpression LbHAI1 The cotyledon unfolding rate of Arabidopsis thaliana seeds was significantly higher than that of wild-type Arabidopsis thaliana seeds. Figure 17 D) The test results show that LbHAI1 Dormancy breaking in Arabidopsis seeds can be promoted by inhibiting ABA regulation.

[0079] (8) RT-qPCR detection of gene expression levels in T3 generation transgenic Arabidopsis thaliana RNA was extracted from the basal leaves of T3 generation transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana plants before they produced their scapes. Following the instructions of the cDNA synthesis kit, synthesize cDNA for RT-qPCR. Using transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana cDNA as templates, AtUBQ The gene is an internal reference gene, used to measure important genes related to dormancy regulation. AtGA2OX1 , AtSnRK2.8 , AtABI5 , AtFT , AtSVP Gene expression levels, and the relative expression levels of each gene are expressed in 2π. -ΔΔCt The data was obtained through calculation and then imported into GraphPad Prism software for analysis and plotting.

[0080] The results are as follows Figure 18 As shown, overexpression LbHAI1 Arabidopsis thaliana AtFT The expression level was significantly higher in Arabidopsis thaliana than in wild-type Arabidopsis thaliana, and overexpression LbHAI1 Arabidopsis thaliana AtGA2OX1 The expression level was significantly lower than that in wild-type Arabidopsis thaliana. AtABI5 , AtSNRK2.8 The expression level was significantly lower than that of wild-type Arabidopsis thaliana, while AtSVP The expression level was not significantly different from that of wild-type Arabidopsis. Experiments show LbHAI1 It may be by affecting dormancy-related genes in Arabidopsis thaliana. AtGA2OX1 , AtFT , AtABI5 , AtSNRK2.8 The expression of [something] promotes the breaking of dormancy in Arabidopsis seeds.

[0081] Example 8 pTRV2- LbHAI1 Construction of expression carrier pTRV2- was constructed using homologous recombination. LbHAI1 expression carrier The pTRV2 plasmid was linearized by single-enzyme digestion with EcoRI restriction endonuclease, with an undigested plasmid serving as a control. Gel electrophoresis was used to determine if digestion was successful. Figure 19 B is the single enzyme digestion product of the pTRV2 vector with EcoRI restriction endonuclease. Its band position differs significantly from that of the undigested pTRV2 vector plasmid, indicating successful digestion of the pTRV2 vector and its suitability for subsequent vector construction. The successfully digested linearized vector was then purified using a gel extraction method.

[0082] A 300 bp sequence fragment was selected from the non-conserved domain of the target gene's CDS sequence. Subcloning primers (pTRV2-LbHAI1-F: TGTGAGTAAGGTTACCGAATTCCGGATTGAGGATGCTGGC, SEQ ID No. 10; pTRV2-LbHAI1-R: ATGGAGGCCTTCTAGAGAATTCCAACGCCAGCTTCGTCAG, SEQ ID No. 11) were designed using SnapGene software. The primers contained anterior and posterior homologous arms and EcoRI restriction sites, with pCE2- LbHAI1 Using the vector plasmid as a template, the target fragment was obtained by PCR amplification. Gel electrophoresis is used to determine if the target fragment is of the correct length. Figure 19 A is pCE2- LbHAI1 The PCR amplification product of the vector plasmid had a band length of approximately 300 bp, consistent with the predicted target gene band length. The target fragment was then purified using a gel extraction method.

[0083] The purified products of the linearized vector and the target fragment were ligated using homologous recombinase. The ligation product was then transformed into DH5α competent cells. Successfully transformed DH5α *E. coli* were screened using LB agar plates containing 50 mg / L Kan. Sequencing alignment results were correct, indicating that pTRV2- LbHAI1 The expression carrier was successfully constructed. Figure 20 pTRV2- LbHAI1Expression vector map

[0084] pTRV2- sequenced successfully LbHAI1 Expression vectors and empty plasmids pTRV2 and pTRV1 were transformed into GV3101 competent cells. Successfully transformed GV3101 Agrobacterium cells were screened using LB agar plates containing 50 mg / L Kan, and colony PCR was performed for identification. The results are as follows: Figure 21 This indicates that pTRV2- LbHAI1 The expression vector was successfully transformed into Agrobacterium GV3101. Afterwards, select positive single colonies and shake them to incubate. Mix the bacterial suspension with 50% glycerol solution at a 1:1 ratio and store at -80 ℃ for later use.

[0085] Example 9 'Dragon Tooth Red' lily LbHAI1 Gene silencing (1) Preparation of bacterial culture Will have pTRV2- LbHAI1 The expression vectors, pTRV2, and pTRV1 Agrobacterium tumefaciens GV3101 bacterial suspensions were removed from the -80℃ freezer, and the bacterial suspensions were streaked onto LB agar plates containing 50 mg / L Kan using a pipette tip. The plates were then incubated at 28℃ for 48 h. Use a pipette tip to pick up a single colony and inoculate it into 3 mL of LB liquid medium (containing 50 mg / L Kan). Incubate at 28 °C with shaking for about 36 h until the bacterial culture becomes turbid. Using a pipette tip, inoculate 1.5 mL of the bacterial culture into an Erlenmeyer flask containing 150 mL of LB liquid medium (containing 50 mg / L Kan). Incubate overnight at 28 °C with shaking until the OD600 reaches approximately 1.2. Centrifuge the culture at 5500 rpm for 10 minutes, discard the supernatant, and resuspend the culture in resuspending buffer (10 mM MES + 10 mM MgCl2 + 0.2 mM AS, pH = 5.6) until the OD600 reaches 1.0. Separately, inoculate pTRV1 with pTRV2... LbHAI1 Mix with pTRV2 at a 1:1 volume ratio and let stand in the dark for 3 hours.

[0086] (2) Vacuum inoculation method for infecting 'Longyahong' lily bulbs Ninety uniformly sized, disease- and pest-free dormant 'Longya Hong' lily bulbs were selected and divided into three groups: a silent group, a control group, and a wild group. Holes were made in the base scales and bulb disc of the bulbs in the silent and control groups using a needle. The bulbs from the silent group were then placed in pTRV2- LbHAI1The control group was placed in a mixture of pTRV2 and pTRV1 bacterial culture. Both the silent group and the control group were subjected to vacuum treatment at 0.09 MPa for 10 min, repeated twice. After infection, the bulbs were removed from the bacterial solution and washed with clean water. They were then placed in mesh bags and kept in a dark, room-temperature environment for 3 days to dry. The silent group, control group, and wild group were then stored in a slightly moist substrate and allowed to stand in a cold storage at 4 ℃.

[0087] (3) Observation and statistics of phenotypic characteristics of infected bulbs After 60 days of cold storage, the infected bulbs were removed, dissected, and the bud length and bulb length were measured. The data were analyzed and plotted using GraphPad Prism software. Sampling of bulb buds from each experimental group

[0088] The results are as follows Figure 22 As shown, momentary silence LbHAI1 The bulb buds of the gene were significantly shorter than those of wild-type lily bulbs and the empty control group. Figure 22 A) Instantaneous silence LbHAI1 The average bud length / bulb length ratio of the gene-mutated lily bulbs was 0.454, while the average bud length / bulb length ratio of the empty vector control group was 0.652, significantly higher than that of the transiently silenced lily bulbs. LbHAI1 Genetically modified lily bulbs ( Figure 22 B) Experiments show that silence LbHAI1 Genes inhibited the dormancy release of 'Dragon Tooth Red' lily bulbs.

[0089] (4) Detection of dormancy-related gene expression levels in infected bulbs Infected with pTRV2- LbHAI1 Using pTRV2 bulb bud cDNA as a template, the assay was performed. LbHAI1 Genes and LbFT , LbSNRK2.8 , LbABI5 and LbGA2ox1 The expression levels were analyzed and plotted using GraphPad Prism software.

[0090] The results are as follows Figure 23 Compared with the empty control group, the infection of pTRV2- LbHAI1 'Dragon Tooth Red' lily bulb buds LbHAI1 The expression level of [a substance] decreased significantly, indicating that in 'Lilium Longyahong'... LbHAI1 Gene silencing successful Located in the abscisic acid signaling pathway LbHAI1downstream of genes LbSNRK2.8 and LbABI5 The expression level of [the substance] was significantly increased compared to the empty vector control group. LbFT Gene expression levels were significantly lower compared to the empty vector control group. Experimental results show that the silent 'Dragon Tooth Red' lily bulbs LbHAI1 Genes can induce positively regulatory genes in the abscisic acid signaling pathway, such as LbSNRK2.8 and LbABI5 The expression level of these genes was upregulated, and they induced dormancy-related genes. LbFT The expression level of [something] is downregulated, thereby inhibiting [something]. LbHAI1 'Dragon Tooth Red' lily bulbs have broken dormancy.

[0091] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0092] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of lily HAI1 Genes, characterized by, Its nucleotide sequence is shown in SEQ ID No.

1.

2. The lily as described in claim 1 HAI1 Application of genes in regulating crop dormancy release.

3. The application as described in claim 2, characterized in that, The regulation mentioned is negative regulation.

4. The application as described in claim 2, characterized in that, The crops mentioned are Arabidopsis thaliana and lily.

5. The application as described in claim 2, characterized in that, Used to regulate the expression of dormancy-related genes.

6. The application as described in claim 5, characterized in that, The dormancy-related gene is AtGA2OX1 , AtFT , AtABI5 , AtSNRK2.8 .

7. Silent Lily HAI1 Application of genes in constructing a model to inhibit dormancy in lily bulbs.

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