Auxin response gene for regulating and controlling expansion of lily bulb and application of auxin response gene

Abstract

The invention discloses an auxin response gene LISAUR36 or LIIAA10 for regulating and controlling the expansion of a lily bulb. The nucleotide sequences of the auxin response gene LISAUR36 or LIIAA10 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2; an amino acid sequence coded by the LISAUR36 gene is as shown in SEQ ID NO: 3, and an amino acid sequence coded by the LIIAA10 gene is as shown in SEQ ID NO: 4. Complete coding segments of the two genes are cloned, an instantaneous silencing vector is constructed, and the regulation and control effect of the gene on bulb expansion is verified in the bulblet of the lily. According to the invention, the research on lily bulb expansion is promoted, and the specific application potential of the auxin in lily bulb breeding is explored.

Description

Technical Field

[0001] The invention relates to the field of molecular biology of garden plants, and in particular to auxin response genes LISAUR36 and LIIAA10 for regulating lily bulb swelling and applications thereof. Background Art

[0002] Lily (Lilium spp.) is a general term for perennial bulbous flowers of the genus Lilium in the family Liliaceae. It has a long history of cultivation, a wide distribution range, and rich germplasm resources. It is one of the bulbous flowers with important commercial value in the world. my country is the origin and distribution center of lily plants, and its wild resources are very rich. However, my country's lily bulb breeding system is backward, and the situation of relying on imported bulbs is gradually deepening. Therefore, conducting research on the expansion of lily bulbs and shortening the time for small bulbs to be cultivated into bulbs in the field is of great significance for the localization of lily bulbs and the creation of new varieties with independent advantages.

[0003] As endogenous metabolites of plants themselves, plant hormones play an important role in plant growth and development. Auxin can regulate the expansion of plant organs by promoting cell division and elongation. Currently, synthetic auxin analogs such as NAA and 2,4-D are widely used in agricultural production and have generated huge economic benefits. Therefore, exploring auxin-responsive genes that can regulate the expansion of lily bulbs through molecular biological methods can promote the application of plant growth regulators in the lily bulb industry and provide theoretical support for the localization of lily bulbs. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an auxin response gene for regulating the expansion of lily bulbs and an application thereof. The present invention explores an auxin response gene capable of regulating the expansion of lily bulbs, and performs transcriptome sequencing after treating Lilium plants with 100 mg / LIAA / NPA. Two auxin response genes, LISAUR36 and LIIAA10, are obtained through transcriptome analysis. Corresponding silencing vectors are constructed and transferred into lily bulblets to regulate the expansion of lily bulbs.

[0005] The invention discloses an auxin-responsive gene for regulating lily bulb expansion, wherein the gene is a LISAUR36 gene or a LIIAA10 gene, and the nucleotide sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0006] The expression protein of the auxin response gene for regulating lily bulb expansion of the present invention, the amino acid sequence encoded by the LISAUR36 gene is shown in SEQ ID NO:3, and the amino acid sequence encoded by the LIIAA10 gene is shown in SEQ ID NO:4.

[0007] The specific primer pair used to clone the auxin response gene that regulates lily bulb swelling described in the present invention, the specific primer pair of the LISAUR36 gene is LlSAUR36-F and LlSAUR36-R, and the nucleotide sequences thereof are shown in SEQ ID NO:5 and SEQ ID NO:6; the specific primer pair of the LIIAA10 gene is LIIAA10-F and LIIAA10-R, and the nucleotide sequences thereof are shown in SEQ ID NO:7 and SEQ ID NO:8.

[0008] The invention discloses an application of the auxin response gene for regulating lily bulb swelling in lily breeding.

[0009] The application described in the present invention, wherein the LISAUR36 gene or LIIAA10 gene can promote the enlargement of lily bulbs.

[0010] A method for gene silencing in lily bulblets by constructing a transient silencing vector comprises the following steps:

[0011] (1) Construction of transient silencing vector: Design PCR primers to amplify LISAUR36 gene and LIIAA10 gene, the primers are LlSAUR36-TRV2-F, LlSAUR36-TRV2-R, LlIAA10-TRV2-F and Ll1AA10-TRV2-R, and their nucleotide sequences are shown in SEQ ID NO:9 to SEQ ID NO:12; the amplified product is digested and connected to TRV2 vector, and transformed into Escherichia coli competent cells DH5α, and the colony PCR identification is positive, and the restriction site is EcoRI;

[0012] (2) TRV (Tobacco Rattle Virus)-mediated gene silencing method: The constructed expression vector was transformed into Agrobacterium competent EHA105 to obtain EHA105 / TRV2-LISAUR36 and EHA105 / TRV2-LIIAA10 for transformation of lily bulblets.

[0013] A specific primer pair for detecting the expression levels of LISAUR36 and LIIAA10 genes by real-time fluorescence quantitative PCR, wherein the specific primer pair for the LISAUR36 gene is LlSAUR36-qPCR-F and LlSAUR36-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO:13 and SEQ ID NO:14; the specific primer pair for the LIIAA10 gene is LIIAA10-qPCR-F and LIIAA10-qPCR-R, and their nucleotide sequences are shown in SEQ ID NO:15 and SEQ ID NO:16.

[0014] The auxin response gene for regulating lily bulb expansion of the present invention is different from the prior art in that:

[0015] The present invention discloses two auxin response genes capable of regulating the expansion of lily bulbs and specific applications, specifically disclosing auxin response genes LISAUR36 and LIIAA10 that positively regulate the expansion of lily bulbs and applications of these two genes. The lily LISAUR36 gene belongs to the auxin up-regulated small RNA (SAUR) gene family, and the lily LIIAA10 gene belongs to the auxin early response gene Aux / IAA family. The present invention cloned the complete coding segments of these two genes, constructed transient silencing vectors, and verified their regulatory effects on bulb expansion in lily bulblets. The present invention promotes the research on lily bulb expansion and explores the specific application potential of auxin in lily bulb breeding.

[0016] The present invention conducts research on lily bulb expansion, shortens the time for small bulbs to be cultivated into bulbs in the field, and is of great significance for the localization of lily bulbs and the creation of independent superior new varieties. The present invention mines auxin response genes that can regulate the expansion of lily bulbs through molecular biological methods, can promote the application of plant growth regulators in the lily bulb industry, and provide theoretical support for the realization of localization of lily bulbs.

[0017] The auxin response gene for regulating lily bulb expansion and its application of the present invention are further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The electrophoresis diagram of the clones of LISAUR36 and LIIAA10 genes of the present invention;

[0019] Figure 2 The bulb expansion of the two auxin response genes LISAUR36 and LIIAA10 silenced strains of the present invention;

[0020] Figure 3 It is the detection of viral COATPROTEIN protein of the two auxin response genes LISAUR36 and LIIAA10 silenced strains of the present invention;

[0021] Figure 4 This is a schematic diagram of the functions of the two auxin response genes LISAUR36 and LIIAA10 in regulating lily bulb expansion. DETAILED DESCRIPTION

[0022] The experimental methods and experimental materials involved in the embodiments of the present invention are conventional methods and conventional reagents unless otherwise specified.

[0023] The quantitative tests in the following examples were repeated three times, and the data were processed using SPSS11.5 statistical software, expressed as mean ± standard deviation, and tested using two-sided Student's t-test. P < 0.05 (*) indicated a significant difference, and P < 0.01 (**) indicated an extremely significant difference. The relative change in bulb size was determined by the difference between the circumference of the bulblets on the 0th day and the 14th day. The larger the difference, the greater the effect of the target gene on the development of the lily bulblets.

[0024] 1. Cloning of two auxin-responsive genes, LISAUR36 and LIIAA10

[0025] Two auxin response genes, LISAUR36 and LIIAA10, were obtained by transcriptome sequencing after treating Lilium lancifolium plants with 100 mg / L IAA / NPA. Total RNA from Lilium lancifolium scales was extracted using an RNA extraction kit (Vazyme, R401), and 1 μg RNA was synthesized into the first strand of cDNA using a reverse transcription kit (Vazyme, R312) to obtain Lilium lancifolium scale cDNA. Specific cloning primers for open reading frames (ORFs) were designed using Premier 5 software, and the specific primer sequences are shown in Table 1. PCR amplification was performed using the cDNA of Lilium lancifolium scales as a template, with a total amplification system of 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), cultured in LB solid medium (containing 50 mg / L Kana) at 37°C for 12-18 hours, and single clones were randomly picked and subjected to colony PCR detection. 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 LISAUR36 and LIIAA10 genes, the full-length genes of 528 bp and 516 bp were obtained, respectively, as shown in Table 4. Figure 1 shown.

[0026] Table 1 Gene cloning primer names and sequences

[0027]

[0028] Table 2 High-fidelity enzyme PCR reaction system

[0029]

[0030] Table 3 High-fidelity enzyme PCR reaction program

[0031]

[0032] Table 4 Colony PCR reaction system

[0033]

[0034] Table 5 Colony PCR reaction procedure

[0035]

[0036] LISAUR36 gene sequence:

[0037] ATGCTTCCTATCAGCACATGCATTGTCCCAAATCCCATTAGGGAAGAAACTCCACTTGTCTCTTGTATT

[0038] TCCCGGCTCCATATATGTACCTCGTCCCTCCATTACAGCACAACACCATTGCCTATCAATTCCTCTCTCT

[0039] TACAGCCGACCATGCTGAACACAAAAAGGATTCTGGAGAGAGCCAGGAAATCTTCTGCCTTCAGGAG

[0040] AAGGGTTTCCCTCTCTTCACCAAAAAGATGGAATTCATCAGCTAATGATGTTATATGCACACACAGTTC

[0041] CACCGAAATTGCCAGCAAAGGACATCTATTCGTCTACACAATCGACGGGGAGCGCTTCATGATACCAA

[0042] TTGAATACCTCAACACCAACATCATCCGAGAGCTCCTCAAATCTCTGAAGAAGAATTCAGATTACAGC

[0043] ATGAAGGTCCTATCATGCTCCATTGTGATGCCCCGTGCATCCAATACATCATCTCTTTGCTTCGATCTAG

[0044] GGTTTCCAGAGAAGTTGAAAGGGCTCTGCTTACATAAATCGCTGTGA

[0045] Protein sequence encoded by the LISAUR36 gene:

[0046] MLPISTCIVPNPIREETPLVSCISRLHICTSSLHYSTTPLPINSSLLQPTMLNTKRILERARKSSAFRRRVSLSSP

[0047] KRWNSSANDVICTHSSTEIASKGHLFVYTIDGERFMIPIEYLNTNIIRELLKSLKKNSDYSMKVLSCSIVMP

[0048] RASNTSSLCFDLGFPEKLKGLCLHKSL

[0049] LIIAA10 gene sequence:

[0050] ATGGGCAGCCCTACAACGGAGAAGAGGGATAATTACAGCCTCGGCTTCGAGGCCACCGAGCTGCGCC

[0051] TCGGCTTGCCCGGAGATGGTGAGGCTGCGAGGGTTTCAGGGAAGAGGGGTTTTTCTGAGACGATAGA

[0052] CTTGAAATTGAAGCTTCAGACCACGGATTCCGAGGATGTGAAGGGAGAGATAGAGAAGATGAAGAG

[0053] AACTCCTAGTGTGAAGAATCTTGTGTCGGTTAATGATGTAGATCCAGCAAAGCCTCCGGCTCCAAAGG

[0054] CACAAGTGGTGGGTTGGCCACCGGTTCGATCATACAGGAAGAACGCCTTATCGGTTCGACATGATAAG

[0055] GTAGTGAAAGAGGAGAAGCATAGCAACAACACATCGTTTGTGAAGGTTAGCATGGATGGCGCACCTT

[0056] ACCTACGTAAGGTGGACTTGAAGATGTACAAAAGCTATCAAGAACTCTCGAAAGCCTTGGATGCGAT

[0057] GTTCAACTCCTTCACTATATCATATTCACAAACACAATTGTACTAA

[0058] Protein sequence encoded by LIIAA10 gene:

[0059] MGSPTTEKRDNYSLGFEATELRLGLPGDGEAARVSGKRGFSETIDLKLKLQTTDSEDVKGEIEKMKRTPS

[0060] VKNLVSVNDVDPAKPPAPKAQVVGWPPVRSYRKNALSVRHDKVVKEEKHSNNTSFVKVSMDGAPYLR

[0061] KVDLKMYKSYQELSKALDAMFNSFTISYSQTQLY

[0062] 2. Application of two auxin-responsive genes, LISAUR36 and LIIAA10

[0063] 1. Construction of gene silencing vector

[0064] (1) Total RNA extraction and cDNA synthesis from Lilium scales

[0065] Total RNA from Lilium scales was extracted using an RNA extraction kit (Vazyme, R401), and 1 μg of RNA was synthesized into the first-strand cDNA using a reverse transcription kit (Vazyme, R312) to obtain Lilium scale cDNA.

[0066] (2) Ligation and transformation of target fragment and vector

[0067] According to the sequence alignment of LISAUR36 and LIIAA10 transcriptomes in bulbil formation treated with IAA / NPA, PCR primers were designed to amplify LISAUR36 and LIIAA10 genes using lily scale cDNA as template. The specific primer sequences are shown in Table 6. The amplified products were digested and connected to the TRV2 vector, and transformed into Escherichia coli competent cells DH5α, spread on the corresponding resistance screening medium, and inverted in a 37°C constant temperature incubator for 12-16h. After the colony PCR identification was positive, sequencing was performed (the reaction system and procedure were the same as Table 4 and Table 5), and the sequencing analysis was completed by Shanghai Shenggong Biotechnology Service Co., Ltd.

[0068] Table 6 Primers for transient silencing target genes

[0069]

[0070] 2. Gene function verification

[0071] The middle and outer scales of lily bulbs that were free of pests and diseases and uniform in size were selected as test materials. They were disinfected by soaking them in carbendazim for 30 minutes. After the scales were washed and dried, they were inserted into 7g / L water agar until small bulbs were produced at the base of the mother scales, which were used as plant materials for transient silencing verification.

[0072] The recombinant plasmids TRV2-LISAUR36 and TRV2-LIIAA10, which were correctly recombined according to sequencing results, as well as the empty plasmids TRV1 and TRV2, were transferred into EHA105 Agrobacterium competent cells. The Agrobacterium strains were streaked on LB solid medium containing antibiotics (50 mg / L Kana and 25 mg / L Rif), inverted and cultured for two days, and then each Agrobacterium strain was placed in 100 ml of LB liquid containing Kana and Rif antibiotics and shaken overnight. The cells were collected in a 50 mL centrifuge tube at 5000 rpm for 10 min and resuspended with a resuspension solution (10 mM MgCl 2 , 10 mM MES, 200 μM AS; pH 5.8) to resuspend the bacteria at the bottom of the centrifuge tube and use a spectrophotometer to adjust the concentration of the Agrobacterium solution OD 600 =0.8-1.0. TRV1 was mixed with TRV2 and recombinant TRV2 at an equal volume ratio of 1:1 and allowed to stand for 3 hours in the dark.

[0073] Add the resuspension solution to cover the scales, use a vacuum pump to suck, maintain at an atmospheric pressure of 0.8MPa for 15min, then slowly release the air to restore the vacuum pump to standard atmospheric pressure, and dry the excess Agrobacterium liquid on the scales. The scales were cut on 7g / L water agar, 20 scales were placed in each culture dish, and cultured in a dark environment for 14d for observation, statistical phenotype and sampling. The gene expression of the LISAUR36 and LIIAA10 silenced strains was detected by real-time fluorescence quantitative PCR. The reaction procedures and primers are shown in Tables 7 and 8, respectively. The silencing gene effect was determined by detecting the viral COAT PROTEIN protein. The reaction procedure is shown in Table 9. The relative change in bulb size was determined by the difference in the circumference of the bulblets on the 0th day and the 14th day after infection. 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 bulblets.

[0074] Table 7 Real-time fluorescence quantitative PCR reaction system

[0075]

[0076] Table 8 Primer names and sequences used in real-time fluorescence quantitative PCR

[0077]

[0078] Table 9 Virus COAT PROTEIN protein reverse transcription system

[0079]

[0080] The present invention silences LISAUR36 and LIIAA10 genes in Lilium lily bulblets, selects 0d and 14d after infection as sampling time to statistically sample Lilium lily bulblets, and detects gene expression and viral COAT PROTEIN protein to indicate gene silencing effect, such as Figure 2 and Figure 3 The results showed that the expression levels of LISAUR36 and LIIAA10 genes decreased significantly in the silenced strain, causing the circumference of the bulblets of Lilium lily to decrease by about 50% compared with the control group. This indicates that LISAUR36 and LIIAA10 genes play a positive regulatory role in the expansion of the bulblets of Lilium lily.

[0081] 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. An auxin response gene for regulating lily bulb expansion, characterized in that: The gene is the LISAUR36 gene or the LIIAA10 gene, and the nucleotide sequences thereof are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

2. The expression protein of the auxin response gene for regulating lily bulb expansion according to claim 1, wherein the amino acid sequence encoded by the LISAUR36 gene is shown in SEQ ID NO: 3, and the amino acid sequence encoded by the LIIAA10 gene is shown in SEQ ID NO:

4.

3. A specific primer pair for cloning the auxin response gene that regulates lily bulb swelling as described in claim 1, wherein the specific primer pair for the LISAUR36 gene is LlSAUR36-F and LlSAUR36-R, and their nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6; the specific primer pair for the LIIAA10 gene is LIIAA10-F and LIIAA10-R, and their nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:

8.

4. Use of the auxin response gene for regulating lily bulb expansion as claimed in claim 1 in lily breeding.

5. The use according to claim 4, characterized in that: The LISAUR36 gene or LIIAA10 gene can promote the enlargement of lily bulbs.

6. A method for gene silencing in lily bulblets by constructing a transient silencing vector, characterized in that: The steps include: (1) Construction of transient silencing vector: Design PCR primers to amplify LISAUR36 gene and LIIAA10 gene, the primers are LlSAUR36-TRV2-F, LlSAUR36-TRV2-R, LlIAA10-TRV2-F and Ll1AA10-TRV2-R, and their nucleotide sequences are shown in SEQ ID NO:9 to SEQ ID NO:12; the amplified product is digested and connected to TRV2 vector, and transformed into Escherichia coli competent cells DH5α, and the colony PCR identification is positive, and the restriction site is EcoRI; (2) TRV-mediated gene silencing method: The constructed expression vector was transformed into Agrobacterium competent EHA105 to obtain EHA105 / TRV2-LISAUR36 and EHA105 / TRV2-LIIAA10 for transformation of lily bulblets.

7. A specific primer pair for detecting the expression levels of LISAUR36 and LIIAA10 genes by real-time fluorescence quantitative PCR, the specific primer pair for the LISAUR36 gene being LlSAUR36-qPCR-F and LlSAUR36-qPCR-R, and the nucleotide sequences thereof being shown in SEQ ID NO:13 and SEQ ID NO:14; the specific primer pair for the LIIAA10 gene being LIIAA10-qPCR-F and LIIAA10-qPCR-R, and the nucleotide sequences thereof being shown in SEQ ID NO:15 and SEQ ID NO:16.

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