Gibberellin related gene and application thereof

Abstract

The invention discloses a gibberellin related gene, which is an L1GA20ox2 gene, an L1GID1C gene or an L1CIGR2 gene, and the nucleotide sequences of the genes are respectively shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; according to the expression protein of the gibberellin related gene, an amino acid sequence coded by the LlGA20ox2 gene is shown as SEQ ID NO: 4, an amino acid sequence coded by the LlGID1C gene is shown as SEQ ID NO: 5, and an amino acid sequence coded by the LlCIGR2 gene is shown as SEQ ID NO: 6. The gibberellin related gene is identified and verified to participate in lily bulb development, is beneficial to the high-quality breeding process of lily bulbs, and is of great significance to localization of the lily bulbs in China.

Description

Technical Field

[0001] The invention relates to the field of molecular biology of garden plants, in particular to three gibberellin-related genes and applications thereof. Background Art

[0002] Lily (Lilium spp.) is a plant that integrates multiple uses such as food, medicine, health care, ornamental, and greening. It is one of the important bulbous flowers in the world. The market for lily deep-processing products is vast, and its demand increases by 25% every year. At present, the development of my country's lily industry is faced with prominent problems such as bulb diseases, serious obstacles to continuous cropping, backward deep processing technology, and serious dependence on imported bulbs, which seriously restrict the further development of the lily industry. Lily bulb propagation has the advantages of high reproduction coefficient and short reproduction cycle, and is currently the main reproduction method in my country's lily industry. As an important material in lily asexual reproduction, the development process of small bulbs is regulated by hormones such as gibberellins. Therefore, in-depth exploration of key genes that promote the reproduction of lily bulbs, shortening the reproduction cycle of lily bulbs, and cultivating high-quality and high-yield lily varieties with high reproduction coefficients are the key to achieving quality improvement and efficiency growth in my country's lily industry. Summary of the invention

[0003] The invention discloses the role and application of a positive regulatory gene LlGA20ox2 related to a gibberellin synthesis pathway, and positive regulatory genes LlGID1C and LlCIGR2 related to gibberellin signal transduction in the development of lily bulbs.

[0004] The present invention uses the small bulb induced by the outer scales in the lily bulb as a material, and after the expression of LlGA20ox2, LlGID1C and LlCIGR2 genes is inhibited by using the virus-induced gene silencing (VIGS) technology, the length and width of the lily small bulb are reduced, and the development of the lily small bulb is inhibited. The present invention uses the small bulb induced by the outer scales in the lily bulb as a material, and LlGA20ox2 is overexpressed by using a transient overexpression system, so as to promote the development of the small bulb. Compared with the control group, the length of the bulb increases by 25%, and the development of the lily small bulb is promoted. After the present invention silences and overexpresses the LlGA20ox2 gene respectively, the phenotypic changes of the small bulb are observed and it is found that the length and width of the small bulb are changed, and the gene regulates the development process of the lily small bulb.

[0005] The present invention discloses a gibberellin-related gene, which is LlGA20ox2 gene, LlGID1C gene or LlCIGR2 gene, and the nucleotide sequences thereof are shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively.

[0006] The expression protein of the gibberellin-related gene of the present invention, the amino acid sequence encoded by the LlGA20ox2 gene is shown in SEQ ID NO:4, the amino acid sequence encoded by the LlGID1C gene is shown in SEQ ID NO:5, and the amino acid sequence encoded by the LlCIGR2 gene is shown in SEQ ID NO:6.

[0007] The specific primer pairs used to clone the gibberellin-related genes of the present invention are: the specific primer pair for the LlGA20ox2 gene is LlGA20ox2-F and LlGA20ox2-R, and their nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:8; the specific primer pair for the LlGID1C gene is LlGID1C-F and LlGID1C-R, and their nucleotide sequences are shown in SEQ ID NO:9 and SEQ ID NO:10; the specific primer pair for the LlCIGR2 gene is LlCIGR2-F and LlCIGR2-R, and their nucleotide sequences are shown in SEQ ID NO:11 and SEQ ID NO:12.

[0008] The real-time fluorescence quantitative primers used in the gene expression analysis of gibberellin-related genes of the present invention, wherein the nucleotide sequences of the real-time fluorescence quantitative primers used to detect the expression of the LlGA20ox2 gene are shown in SEQ ID NO:13 and SEQ ID NO:14; the nucleotide sequences of the real-time fluorescence quantitative primers used to detect the expression of the LlGID1C gene are shown in SEQ ID NO:15 and SEQ ID NO:16; the nucleotide sequences of the real-time fluorescence quantitative primers used to detect the expression of the LlCIGR2 gene are shown in SEQ ID NO:17 and SEQ ID NO:18.

[0009] The invention discloses an application of the gibberellin-related gene in lily breeding.

[0010] In the application of the present invention, the LlGA20ox2 gene, LlGID1C gene or LlCIGR2 gene can promote the development of lily bulblets.

[0011] A method for gene silencing in lily bulblets by constructing a transient silencing vector comprises the following steps:

[0012] (1) The specific fragments of LlGA20ox2, LlGID1C, and LlCIGR2 were respectively connected to the pTRV2 vector, and the primer information was as follows: LlGA20ox2-TRV2-F and LlGA20ox2-TRV2-R, whose nucleotide sequences are shown in SEQ ID NO: 19 and SEQ ID NO: 20; LlGID1C-TRV2-F and LlGID1C-TRV2-R, whose nucleotide sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22; LlCIGR2-TRV2-F and LlCIGR2-TRV2-R, whose nucleotide sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24;

[0013] (2) The empty plasmids pTRV1 and pTRV2 and the recombinant plasmids pTRV2-LlGA20ox2, pTRV2-LlGID1C, and pTRV2-LlCIGR2 were transformed into Agrobacterium EHA105, and LlGA20ox2, LlGID1C, and LlCIGR2 were transiently silenced in lily bulblets. The middle and outer scales of disease-free lily bulbs were selected and cultured in complete darkness on 7 g / L water agar for 20 days. Then, disease-free bulblets of uniform size induced from the base of the mother scales were selected and transformed with the pTRV2 silencing vector of the above genes. The cells were vacuum infected twice at 0.07 MPa for 5 min and cultured in complete darkness at 22±2°C.

[0014] A method for constructing a transient overexpression vector of the LlGA20ox2 gene comprises the following steps:

[0015] (1) Designing PCR upstream and downstream primers to amplify LlGA20ox2, the primers are LlGA20ox2-eGFP-F and LlGA20ox2-eGFP-R, and the nucleotide sequences thereof are shown in SEQ ID NO:25 and SEQ ID NO:26; the amplified product is digested and ligated into the 35S:eGFP / pCAMBIA2300 vector, and transformed into Escherichia coli competent cells DH5α, and the colony PCR identification is positive, and the restriction site is EcoRI;

[0016] (2) The constructed expression vector was transformed into Agrobacterium competent EHA105 to transiently overexpress the target gene in lily bulblets; the transformed bacterial solution was cultured to OD 600 is 1.0, resuspended in buffer; select disease-free small bulbs of uniform size induced from the base of the mother scale for infection; vacuum infect twice, 0.07MPa, 5min, culture in complete darkness on 7g / L water agar at 23±2℃, and repeat the infection every 4 days.

[0017] The present invention is different from the prior art in that: the present invention discloses the role and application of the positive regulatory gene LlGA20ox2 related to the gibberellin synthesis pathway, and the positive regulatory genes LlGID1C and LlCIGR2 related to gibberellin signal transduction in the development of lily bulbs. The present invention clones the coding segments of the above genes, verifies the role of these genes in the development of lily bulbs through transient silencing and overexpression systems of lily bulblets, and observes and statistics the size changes of bulblets. The present invention determines that the genes LlGA20ox2, LlGID1C and LlCIGR2 regulate the length and width of the bulblets and participate in the development process of lily bulbs. The present invention preliminarily explores the regulatory mechanism of gibberellin-related genes in the development of lily bulbs, provides a solid theoretical basis for studying the molecular mechanism of lily bulb development, and accelerates the development of my country's lily bulb high-quality breeding industry.

[0018] The present invention obtains genes related to lily bulb development through transcriptome analysis, clones the coding region of the target gene, constructs transient silencing and overexpression vectors, and verifies them in lily bulblets, verifying that these candidate genes are involved in regulating lily bulb development. The present invention identifies genes related to gibberellins and verifies that they are involved in lily bulb development, which is beneficial to the high-quality breeding process of lily bulbs and has important significance for the localization of lily bulbs in my country.

[0019] The gibberellin-related gene of the present invention and its application are further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The electrophoresis results of the gibberellin-related genes LlGA20ox2, LlGID1C and LlCIGR2 gene cloning in the present invention;

[0021] Figure 2 The development of bulblets in which LlGA20ox2 is transiently silenced in the bulblets of Lilium lily in the present invention;

[0022] Figure 3 The development of bulblets in which LlGA20ox2 is overexpressed in Litsea tangerine bulblets in the present invention;

[0023] Figure 4 The development of bulblets in which LlGID1C and LlCIGR2 are transiently silenced in the bulblets of Lilium lily in the present invention;

[0024] Figure 5 This is a schematic diagram of the functions of several gibberellin-related genes in the present invention in regulating lily bulb development. DETAILED DESCRIPTION

[0025] 1. Cloning of three gibberellin-related genes LlGA20ox2, LlGID1C and LlCIGR2 that regulate lily bulb development

[0026] The invention obtains candidate genes LlGA20ox2, LlGID1C and LlCIGR2 related to the development of lily bulbs by analyzing the transcriptome of lily bulbs at different developmental stages. During the development of lily bulbs, disease-free dormant bulbs with a diameter of 3.5 to 5.0 cm are collected, and the bulb samples are immediately frozen in liquid nitrogen and stored at -80°C. The total RNA of Lilium lancifolium bulbs is extracted using an RNA extraction kit (Vazyme, R401), and 1 μg of RNA is synthesized into the first chain of cDNA using a reverse transcription kit (Vazyme, R312) to obtain the cDNA of Lilium lancifolium bulbs. The specific cloning primers of open reading frames (ORF) are designed using Premier 5 software, and the specific primer sequences are shown in Table 1. PCR amplification is performed using the cDNA of Lilium lancifolium scales as a template, and the total amplification system is 50 μL. The specific reaction system is shown in Table 2, and the reaction procedure is shown in Table 3. The PCR amplification product was detected by 1.2% agarose gel electrophoresis, and the target fragment was recovered using a DNA recovery kit (Novozyme, DC301); the target fragment was connected to the TA / Blunt-Zero intermediate vector (Novozyme, Nanjing), and transformed into the competent Escherichia coli DH5α (Qingke, Beijing), and cultured at 37°C for 12-18h in LB solid medium (containing 50mg / L Kana). Single clones were randomly picked and colony PCR was performed. The colony PCR reaction system is shown in Table 4, and the reaction procedure is shown in Table 5. Sequencing analysis was completed by Beijing Qingke Biotechnology Co., Ltd., and the sequencing results were compared in DNAMAN software. By cloning the genes of LlGA20ox2, LlGID1C and LlCIGR2, the full-length genes of 1134bp, 1011bp and 1647bp were obtained, respectively, as shown in Table 4. Figure 1 shown.

[0027] Table 1 Gene cloning primer names and sequences

[0028]

[0029] Table 2 High-fidelity enzyme PCR reaction system

[0030]

[0031] Table 3 High-fidelity enzyme PCR reaction program

[0032]

[0033] Table 4 Colony PCR reaction system

[0034]

[0035] Table 5 Colony PCR reaction program

[0036]

[0037]

[0038] LlGA20ox2 gene sequence:

[0039] ATGAATTCAAGCCCTACTTCGGTCCTCCTCCTCCCGACTCTAGACCATCTCAAGAGGGTCGACGCCAA

[0040] TGGCGGTGGCTGCATGGTCTTCGACTCCTCCCACCTCCGCAAACAAGCCCGCATACCCACCCAATTCA

[0041] TCTGGCCTCGCAATGACCGCCTCACCGCCATTGACGAACTTGAAGCCCCCATCATCGACCTCCAAGGC

[0042] TTCCTCGACGGAGACGAAGCATCCACCCTTGACGCCGCCAAACTCGTCGCCTCGGCTTGCACCCGCC

[0043] ACGGCTTCTTCCAGGTGACAAACCACGGCGTCGACACCTCCTTAGTCCGAGAGGCTTTTGCGCGCTT

[0044] GGATGAATTCTTCTTGCTCCCACTCAATGAGAAGCTGAGGGCACGACGGAAGCCTGGCAGCATGTGG

[0045] GGCTACGCCGGCGCCCACACTGACCGCTTCTCCTCGAAACTCCCATGGAAGGAAACGCTCTCCTTCG

[0046] GCTTTGATGACGCCGGCGACAAAGCTGTCGTGGCCGACTACATGAAATCTCAGCTAGGGAACGAGTT

[0047] TGAACAAATGGGGCTGATATACCAAAAATACTGCGAGGCAATGAGAAAGTTATCGGCGGCAATCGTC

[0048] GACCTCCTCGCCATCAGCATGGGAGTGGATCCATCATATTTCCGTGACTTCTTTGACGAGAGCAGCTC

[0049] GATCATGCGTTGCAACTACTACCCGTCCTGCCAGGAGCCGGAGTTGGCTCTCGGCACAGGGCCGCAC

[0050] ACCGACCCAACTTCCCTGACGATCCTACAGCAGGATCAGGTGGGCGGGCTCGAGGTATTCACTGACG

[0051] GCGAGTGGAAAATGATCAGCCCCGTCCGCGACGCGCTCGTGATCAACATCGGCGATACATTCATGGCT

[0052] TTGTCTAATGGTATGTACAAGAGCTGCCTGCACCGGGCTGTCGTAAACAGCCACTGTGAGCGGCGGTC

[0053] CCTGGCCTTCTTCCTCTGTCCGAGGAGCGACAAGACGGTGCGACCGCCGGTGGAGCTCTGCAACACC

[0054] GATAGGCAGTTTCCGAACTTCACCTGGGCACAGCTGTTGCAGTTCACGCAGCAGCACTACCGTGCCG

[0055] ATGTGAGAACACTACAAGCTTTCACCCAGTGGCTCGGCAGCAACCCTATTTGCTAALlGA20ox2 amino acid sequence encoded by the gene:

[0056] MNSSPTSVLLLPTLDHLKRVDANGGGCMVFDSSHLRKQARIPTQFIWPRNDRLTAIDELEAPIIDLQGFLD

[0057] GDEASTLDAAKLVASACTRHGFFQVTNHGVDTSLVREAFARLDEFFLLPLNEKLRARRKPGSMWGYAGA

[0058] HTDRFSSKLPWKETLSFGFDDAGDKAVVADYMKSQLGNEFEQMGLIYQKYCEAMRKLSAAIVDLLAISM

[0059] GVDPSYFRDFFDESSSIMRCNYYPSCQEPELALGTGPHTDPTSLTILQQDQVGGLEVFTDGEWKMISPVRD

[0060] ALVINIGDTFMALSNGMYKSCLHRAVVNSHCERRSLAFFLCPRSDKTVRPPVELCNTDRQFPNFTWAQLL

[0061] QFTQQHYRADVRTLQAFTQWLGSNPIC

[0062] LlGID1C gene sequence:

[0063] ATGGCTGTGCCACTCAACACATGGGTGCTGATCTCCAACTTCAAGGTGGCCTACAACATGCTACGCCG

[0064] CCCTGACGGTACCTTCGACCGCCATCTCGCTGAGTTTCTGGACCGGAAGGTGCCTGCGAATGCCGCC

[0065] GCTGTCAACGGTGTCCGCTCCTTCGACCTCATCATCGACCGCCCCACCAGCCTCCTCGCCCGCATCTA

[0066] CCGCCCCGCCGACGATGCCCAGGCCCCGCTCTCCGCCCCCGCCCTCCTCGCCGAGCTCCGCCGCCCC

[0067] CCGTCGCAGGACCCCTTCCCAGTCATTATCTTCTTCCACGGCGGAAGCTTCGCCCACTCCTCCGCAAA

[0068] CAGCGCCATCTACGACTCCCTCTGCCGCCGCCTCGTCACGCTCTGTAACGGCGCTGTCGTCGTCTCCG

[0069] TAAACTACCGCCGATCCCCGGAGCACCGCTTCCCCTCCGCCTACGAGGACGGGTGGGCTGCGCTTAG

[0070] GTGGGCAGCCAACCAGCCCTGGCTGCGCAGCGGCAGCGACGACAAGCTCCGCATCTTCCTCTCCGGC

[0071] GACAGCTCCGGCGGGAACATCGCCCACCATGTCGCCCTCCGCGCAGTCGAGTCCGGCATCGACATTG

[0072] CCGGCAACATTCTCCTCAACCCGATGTTCGGTGGCAACGATCGCACGGACTCCGAGAACCGGCTTGA

[0073] TGGGAAGTACTTCGTCACCGTCCAGGACAGGGACTGGTACTGGAAGGCATTCCTTCCGGAGGAAGCC

[0074] GACCGTGACCATGAAGCATGCAATCCGTTCGGTCCAAACGCCACTAAACTCGACGGTCTCCCCTTCCC

[0075] AAAGAGCCTCGTAGTGGTGGCGGGACTGGACCTGATTTCCGACTGGCAGCTTGCTTATGCCGAGGGG

[0076] CTCGAGAAAGCCGGACATGATGTGAAGCTGATCTACCGTGAGCAGGCCACGATCGGGTTCTACCTACT

[0077] GCCCAACACCAGCCATTTCCATGAAGTGATGGAGGAGATCAGTACCTTTGTGAGCACTAATTGCTAGLlGID1C gene-encoded amino acid sequence:

[0078] MAVPLNTWVLISNFKVAYNMLRRPDGTFDRHLAEFLDRKVPANAAAVNGVRSFDLIIDRPTSLLARIYRPA

[0079] DDAQAPLSAPALLAELRRPPSQDPFPVIIFFHGGSFAHSSANSAIYDSLCRRLVTLCNGAVVVSVNYRRSPE

[0080] HRFPSAYEDGWAALRWAANQPWLRSGSDDKLRIFLSGDSSGGNIAHHVALRAVESGIDIAGNILLNPMFG

[0081] GNDRTDSENRLDGKYFVTVQDRDWYWKAFLPEEADRDHEACNPFGPNATKLDGLPFPKSLVVVAGLDLI

[0082] SDWQLAYAEGLEKAGHDVKLIYREQATIGFYLLPNTSHFHEVMEEISTFVSTNCLlCIGR2 gene sequence:

[0083] ATGGTTGACATACCAGTTTCTTTTGCAAGCTACTCTCTAGACAACTCGATTATTAAAGCTTCTAAAGGT

[0084] CAGGCTTCACATTTACAGCAAACTTCGATGCCTGAGATGGAAGGCGATTCTCATTTTCTTCCTCCGAAA

[0085] TACCAATCTGCTAACCATTTTTACCCTGAAGACGGATCACAGTTAAGAAATAACACCCATCAGACAGC

[0086] CCATGGCCAATACTGCACTCTTGAGTCCTCAATGACAGCCGGTGGTTACGCAGTTCACAGCTCCCCGT

[0087] CCAGTCTCAGTTTCTCCCCTGCCACCGGGAGCCCCATATCCCCGCAAGAATGCCAGTCAGATAACACT

[0088] TACGGGTCACCAGTAAGCGTGTCATGTGTAACTGAAGACCCGAATGAACTGAAACACAAGCTAAGGG

[0089] AATTAGAGACTGCGATGCTCGGACCAGACTTGGATATTGTCAACAGCTTCGAGAGCACGTTCCACAGT

[0090] CATCTCATGCTGGAGCCGGAGAAGTGGAAGCAAGTGATCGAGGTTCCTCGAGATCTAAAACAGGTTC

[0091] TTATAGCATGCGCAAGGGCAGTCGCGGATAATGACTTTTACATGGTGGAATGGTTGGTTACCGAACTG

[0092] AGGCAGATGGTGTCTGTTTCCGGGGAACCAATCGAACGTTTGGGCGCCTACTTGTTGGAAGGCCTCAT

[0093] CGCGAGGCTAGCTTCATCAGGCAGTTCGATCTACAAGGCGTTGAGGTGCAAAGAACCTGCGAGTTCC

[0094] GACCTCCTCTCATACATGCATGTTCTTTACGAGGTCTGCCCGTTCTTCAAATTCGGCTACATGTCTGCA

[0095] AATGGTGCCATTGCGGAGGCTATGAAGGGTGAGGACATGGTTCACATTATCGATTTCCAGATTGCACA

[0096] AGGAAGCCAGTGGGTGTCACTGATTCAGGCGCTTGCAGCGAGACCAGGCGGGCCGCCACGGGTGAA

[0097] GATAACCGGAATTGATGACTCTGTGTCTGCTTATGCTCGAGGTGGCGGTTTACAAATCGTGGGCAAGA

[0098] GACTATCTCAGCTGGCCGCATCCTGTAACGTGCTGTTCGAGTTTCATGCTGCGGCCATGTCTGGTACCG

[0099] ACGTAGAGCTTGAGCATCTCGGTGTCAAGCCTGGGGAGGCATTGGCCGTCAACTTCGCCTTCCAGCT

[0100] GCATCACATGCCGGATGAGAGTGTCAGTACTAAGAATCACCGTGACAGGATTCTGAGGATGGTGAAG

[0101] AGCCTCTCGCCAAAGGTGGTCACTCTTGTTGAGCAGGAGTCGAATACGAACACCGCTCCGTTCTTTCC

[0102] GCGGTTCTTGGAGACTCTCGACTATTACAACGCCATTTTTGAATCAATTGATGTTAGCCTCTCGAGGGA

[0103] GAACAAGCAACGGATTAATGTGGAGCAGCACTGCCTAGCGAGGGATATAGTCAATATCATCGCTTGTG

[0104] AGGGAGCGGAGAGAGTCGAAAGGCACGAACTTCTGGGAAAATGGAAGGCGAGGTTCAGAATGGCG

[0105] GGGTTTACTCCGTACCCGCTAAGTTCATTGGTGAATGTGACGATCAAATCCCTGTTAGGGAACTACTGT

[0106] GAGCACTACTGGCTTGAGGAGAGGGATGGAATTCTTTATCTTGGATGGAAAAATAGGGTTTTGGTTGT

[0107] CTCCTGCGCATGGCGATGA

[0108] Amino acid sequence encoded by the LlCIGR2 gene:

[0109] MVDIPVSFASYSLDNSIIKASKGQASHLQQTSMPEMEGDSHFLPPKYQSANHFYPEDGSQLRNNTHQTAH

[0110] GQYCTLESSMTAGGYAVHSSPSSLSFSPATGSPISPQECQSDNTYGSPVSVSCVTEDPNELKHKLRELETAM

[0111] LGPDLDIVNSFESTFHSHLMLEPEKWKQVIEVPRDLKQVLIACARAVADNDFYMVEWLVTELRQMVSVS

[0112] GEPIERLGAYLLEGLIARLASSGSSIYKALRCKEPASSDLLSYMHVLYEVCPFFKFGYMSANGAIAEAMKG

[0113] EDMVHIIDFQIAQGSQWVSLIQALAARPGGPPRVKITGIDDSVSAYARGGGLQIVGKRLSQLAASCNVLFE

[0114] FHAAAMSGTDVELEHLGVKPGEALAVNFAFQLHHMPDESVSTKNHRDRILRMVKSLSPKVVTLVEQESN

[0115] TNTAPFFPRFLETLDYYNAIFESIDVSLSRENKQRINVEQHCLARDIVNIIACEGAERVERHELLGKWKARF

[0116] RMAGFTPYPLSSLVNVTIKSLLGNYCEHYWLEERDGILYLGWKNRVLVVSCAWR

[0117] 2. Construction of gene silencing vector and transformation of lily bulblets

[0118] The specific steps for constructing the gene silencing vector are as follows:

[0119] Specific fragments were selected for silencing in the non-conserved domains of the LlGA20ox2, LlGID1C and LlCIGR2 sequences, and PCR upstream and downstream primers were designed to amplify LlGA20ox2, LlGID1C and LlCIGR2, and restriction sites EcoRI were added to both ends of the primers. The specific primer sequences are shown in Table 6; the amplified products were digested and connected to the TRV2 vector, and transformed into the competent Escherichia coli cells DH5α, and the colony PCR identification was positive; the specific reaction system and reaction procedure are the same as Tables 2 and 3. The constructed expression vector was transformed into the competent Agrobacterium EHA105, and the bacterial solution with positive bacterial solution PCR detection was bidirectionally sequenced. The colony PCR reaction system and reaction procedure are the same as Tables 4 and 5, and the sequencing results were compared with the constructed vector sequence. If the alignment was correct, EHA105 / TRV2-LlGA20ox2, EHA105 / TRV2-LlGID1C and EHA105 / TRV2-LlCIGR2 were obtained and used to transform lily bulblets.

[0120] The specific fragments of the target genes LlGA20ox2, LlGID1C and LlCIGR2 were cloned into the pTRV2 vector, respectively. The specific fragments of pTRV1, pTRV2 and pTRV2 were transformed into Agrobacterium EHA105. The middle and outer scales of disease-free lily bulbs were selected, and after culturing in complete darkness on 7g / L water agar for 20 days, small bulbs of uniform size induced at the base of the mother scales were selected and infected with the transformed EHA105 bacterial solution. Vacuum infiltration was performed twice (0.07MPa, 5min), and culture was carried out in complete darkness at 22±2℃. The relative change in bulb size was determined by the difference in length and width between the 0th day and the 14th day, and the significance was determined using the t-test (*p<0.05; **p<0.01). The larger the difference, the greater the effect of the target gene on the development of the small bulbs. Figure 2 and Figure 4 shown.

[0121] The present invention silenced the LlGA20ox2, LlGID1C and LlCIGR2 genes in the bulblets of Lilium lily. The 0th and 14th days after infection were selected as sampling times for sampling the bulblets of Lilium lily. The gene silencing effect was indicated by detecting the gene expression amount. The primer sequences are shown in Table 6. The results are shown in Table 6. Figure 2 and Figure 4 The results showed that the expression levels of LlGA20ox2, LlGID1C and LlCIGR2 genes decreased significantly in the silenced strains, causing the length and width of the bulblets of Lilium lily to decrease by about 20% to 50% compared with the control group. This indicates that LlGA20ox2, LlGID1C and LlCIGR2 genes play a positive regulatory role in the expansion of the bulblets of Lilium lily.

[0122] Table 6 Primer names and sequences used for gene silencing

[0123]

[0124]

[0125] 3. Construction of transient overexpression vector and transformation of lily bulblets

[0126] The specific steps of transient overexpression vector construction are as follows: design upstream primer LlGA20ox2-eGFP-F and downstream primer LlGA20ox2-eGFP-R to amplify the LlGA20ox2 gene with a fragment length of 1134bp, and add restriction sites at both ends of the primers. The specific primer sequences are shown in Table 7, the specific reaction system is the same as Table 2, and the reaction procedure is the same as Table 3. The amplified product is digested and connected to the 35S:eGFP / pCAMBIA2300 vector, and transformed into Escherichia coli competent cells DH5α, and the colony PCR identification is positive. The specific reaction system and procedure are the same as Tables 4 and 5, and the restriction site is EcoRI. The constructed expression vector LlGA20ox2-eGFP is transformed into Agrobacterium competent EHA105 to obtain EHA105 / eGFP-LlGA20ox2 for transformation of lily bulblets.

[0127] The specific steps for verifying the function of transiently overexpressed genes are as follows: transiently overexpress the LlGA20ox2 gene in the small bulbs induced by the mother scales as described above. Cultivate the EHA105 / eGFP-LlGA20ox2 bacterial solution to OD 600 The pH value was 1.0 and resuspended in a buffer solution (10 mM MgCl2, 10 mM MES, pH 5.8, 200 μM acetosyringone). Lily bulblets of uniform size were selected and inoculated twice with the resuspension by vacuum (0.07 MPa, 5 min), and then cultured in complete darkness on water agar (7 g / L) at a temperature of 23 ± 2 °C. The infection was repeated every 4 days. The relative size change was calculated as the difference between the length and width of the bulblets on the 0th day and the 14th day. The significance was determined using a t-test (*p<0.05; **p<0.01). The larger the difference, the greater the effect of the target gene on the development of the bulblets, such as Figure 3 shown.

[0128] The present invention transiently overexpresses the LlGA20ox2 gene in the bulblets of Lilium lily, selects the 0th and 14th day after infection as sampling time to statistically sample the bulblets of Lilium lily, and detects the gene expression amount to indicate the gene overexpression effect. The primer sequences are shown in Table 7. The results are as follows: Figure 3 The results showed that the expression level of LlGA20ox2 gene was significantly increased in the overexpression strain, and the length of the bulblet of Lilium lily increased by about 20% compared with the control group. This indicated that LlGA20ox2 gene played a positive regulatory role in the expansion of the bulblet of Lilium lily.

[0129] Table 7 Primer names and sequences used for transient overexpression

[0130]

[0131] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A gibberellin-related gene, characterized in that: The gene is LlGA20ox2 gene, LlGID1C gene or LlCIGR2 gene, and the nucleotide sequences thereof are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

2. The expression protein of the gibberellin-related gene according to claim 1, wherein the amino acid sequence encoded by the LlGA20ox2 gene is shown in SEQ ID NO:4, the amino acid sequence encoded by the LlGID1C gene is shown in SEQ ID NO:5, and the amino acid sequence encoded by the LlCIGR2 gene is shown in SEQ ID NO:

6.

3. A specific primer pair for cloning the gibberellin-related gene according to claim 1, wherein the specific primer pair for the LlGA20ox2 gene is LlGA20ox2-F and LlGA20ox2-R, and their nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:8; the specific primer pair for the LlGID1C gene is LlGID1C-F and LlGID1C-R, and their nucleotide sequences are shown in SEQ ID NO:9 and SEQ ID NO:10; the specific primer pair for the LlCIGR2 gene is LlCIGR2-F and LlCIGR2-R, and their nucleotide sequences are shown in SEQ ID NO:11 and SEQ ID NO:

12.

4. The real-time fluorescence quantitative primer used in the gene expression analysis of the gibberellin-related gene according to claim 1, characterized in that: The nucleotide sequences of the real-time fluorescence quantitative primers for detecting the expression of the LlGA20ox2 gene are shown in SEQ ID NO: 13 and SEQ ID NO: 14; the nucleotide sequences of the real-time fluorescence quantitative primers for detecting the expression of the LlGID1C gene are shown in SEQ ID NO: 15 and SEQ ID NO: 16; the nucleotide sequences of the real-time fluorescence quantitative primers for detecting the expression of the LlCIGR2 gene are shown in SEQ ID NO: 17 and SEQ ID NO:

18.

5. Use of the gibberellin-related gene according to claim 1 in lily breeding.

6. The use according to claim 5, characterized in that: The LlGA20ox2 gene, LlGID1C gene or LlCIGR2 gene can promote the development of lily bulblets.

7. A method for gene silencing in lily bulblets by constructing a transient silencing vector, characterized in that: The steps include: (1) The specific fragments of LlGA20ox2, LlGID1C, and LlCIGR2 were respectively connected to the pTRV2 vector, and the primer information was as follows: LlGA20ox2-TRV2-F and LlGA20ox2-TRV2-R, whose nucleotide sequences are shown in SEQ ID NO: 19 and SEQ ID NO: 20; LlGID1C-TRV2-F and LlGID1C-TRV2-R, whose nucleotide sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22; LlCIGR2-TRV2-F and LlCIGR2-TRV2-R, whose nucleotide sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24; (2) The empty plasmids pTRV1 and pTRV2 and the recombinant plasmids pTRV2-LlGA20ox2, pTRV2-LlGID1C, and pTRV2-LlCIGR2 were transformed into Agrobacterium EHA105, and LlGA20ox2, LlGID1C, and LlCIGR2 were transiently silenced in lily bulblets. The middle and outer scales of disease-free lily bulbs were selected and cultured in complete darkness on 7 g / L water agar for 20 days. Then, disease-free bulblets of uniform size induced from the base of the mother scales were selected and transformed with the pTRV2 silencing vector of the above genes. The cells were vacuum infected twice at 0.07 MPa for 5 min and cultured in complete darkness at 22±2°C.

8. A method for constructing a transient overexpression vector of LlGA20ox2 gene, characterized in that: The steps include: (1) Designing PCR upstream and downstream primers to amplify LlGA20ox2, the primers are LlGA20ox2-eGFP-F and LlGA20ox2-eGFP-R, and the nucleotide sequences thereof are shown in SEQ ID NO:25 and SEQ ID NO:26; the amplified product is digested and ligated into the 35S:eGFP / pCAMBIA2300 vector, and transformed into Escherichia coli competent cells DH5α, and the colony PCR identification is positive, and the restriction site is EcoRI; (2) The constructed expression vector was transformed into Agrobacterium competent EHA105 to transiently overexpress the target gene in lily bulblets; the transformed bacterial solution was cultured to OD 600 is 1.0, resuspended in buffer; Select disease-free small bulbs of uniform size induced from the base of the mother scales for infection; vacuum infect twice, 0.07MPa, 5min, culture in complete darkness on 7g / L water agar at 23±2℃, and repeat the infection every 4 days.

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