Lilium lancifolium LlELP1 gene and application thereof

Abstract

The invention discloses a lilium lancifolium LlELP1 gene, the nucleotide sequence of the lilium lancifolium LlELP1 gene is as shown in SEQ ID NO: 1, and the amino acid sequence of expression protein of the lilium lancifolium LlELP1 gene is as shown in SEQ ID NO: 2; according to a specific primer pair of the lilium lancifolium LlELP1 gene, the nucleotide sequences of the LlELP1-F and the LlELP1-R are as shown in SEQ ID NO: 3 and SEQ ID NO: 4. The invention also discloses an application of the lilium lancifolium LlELP1 gene in lilium lancifolium breeding. The lilium lancifolium LlELP1 gene can promote the generation process of bulbils of lilium lancifolium and increase the number of bulbils. The chitinase related gene capable of regulating and controlling the bulbil generation number is excavated for the first time, directional selection of the bulbil generation number is facilitated, and reference is provided for molecular breeding.

Description

Technical Field

[0001] The invention relates to the field of molecular biology of garden plants, and in particular to a Lilium lily LlELP1 gene and an application thereof. Background Art

[0002] Lily (Lilim spp.) is a perennial bulbous plant of the genus Lilium in the family Liliaceae. It is one of the important ornamental flowers in my country and is widely used in cut flowers, potted flowers, and flower borders. Lily has both edible and medicinal value. Its scales have the effects of moistening the lungs, relieving coughs, and clearing away heat. Its flower fragrance has the effects of calming the nerves and soothing the mind. The annual trade volume is as high as more than 2 billion US dollars. However, the domestic lily bulb breeding is often accompanied by the risk of native diseases. The bulb degeneration and low reproduction efficiency caused by native diseases are one of the main bottlenecks restricting the development of my country's lily industry and one of the important reasons why my country relies on imported bulbs. In addition to bulb breeding, a new breeding method, bulbil breeding, is considered to have great breeding potential, mainly manifested in its low virus carrying rate, high reproduction rate, and retention of maternal advantages. It can be seen that the research on lily bulbil breeding has become an important starting point for optimizing the important technology of lily variety breeding in my country and enhancing the competitiveness of my country's lily industry. It has important development significance for promoting the advantageous breeding of lilies.

[0003] With the development of molecular biotechnology, molecular breeding has become one of the important means of horticultural plant breeding. Bulbs are considered to have great breeding potential. Regulating the number of bulbils is an important factor in determining the yield of bulbils. It is crucial to discover the important genes that regulate the number of lily bulbils and explore their molecular regulatory mechanisms. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Lilium lily LlELP1 gene and its application. By analyzing the expression pattern of the gene in the process of bulbilogenesis, it is confirmed that the gene is involved in the process of bulbilogenesis, and the complete coding segment of the gene is cloned, a transient silencing vector is constructed, and its regulatory effect on the number of bulbils is verified in the axil of lily leaves.

[0005] The nucleotide sequence of the Lilium lily LlELP1 gene of the present invention is shown in SEQ ID NO:1.

[0006] The amino acid sequence of the expressed protein of the Lilium lily LlELP1 gene of the present invention is shown in SEQ ID NO:2.

[0007] The specific primer pair for the Lilium lily LlELP1 gene of the present invention, LlELP1-F and LlELP1-R, have nucleotide sequences as shown in SEQ ID NO:3 and SEQ ID NO:4.

[0008] The invention discloses an application of the Lilium lily LlELP1 gene in Lilium lily breeding.

[0009] In the application of the present invention, the Lilium lily LlELP1 gene can promote the occurrence process of Lilium lily bulbils and increase the number of bulbils.

[0010] The method for analyzing the expression pattern of the Lilium lily L1ELP1 gene of the present invention comprises the following steps:

[0011] (1) Samples were collected from the leaf axils of lilies during bulbilogenesis: dormant bulbs were planted in a solar greenhouse and the leaf axils on the upper part of the stems were used as test materials. The sample collection period was the bulbilogenesis stage (S0), the small white spot stage (S1), the green bulb stage (S2), and the purple bulb stage (S3).

[0012] (2) Total RNA extraction and cDNA synthesis from lily leaf axils during bulbilogenesis:

[0013] (3) Real-time fluorescence quantitative PCR quantitative detection of gene expression: Real-time fluorescence quantitative PCR primers were designed according to the candidate gene CDS, cDNA was amplified, LlFP was used as the internal reference gene, and each sample was repeated three times;

[0014] (4) Analysis of the expression patterns of candidate genes during bulbilogenesis: significant differences were analyzed based on the results of fluorescence quantitative PCR.

[0015] In the method of the present invention, the real-time fluorescence quantitative PCR primers used in step (3) are LlELP1-qRT-F and LlELP1-qRT-R, and their nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:6.

[0016] The method for constructing a transient silencing vector of the Lilium lily L1ELP1 gene of the present invention comprises the following steps:

[0017] (1) Obtain the target fragment, design specific primers according to the gene sequence, add restriction sites at both ends of the primers, use the cloning vector as a template, perform PCR amplification, and obtain the LlELP1 coding region amplification product with restriction sites, i.e., the target fragment; the restriction sites are EcoRⅠ and XhoⅠ; the primer sequences are Primer F and Primer R, and their nucleotide sequences are shown in SEQ ID NO:7 and SEQ ID NO:8;

[0018] (2) Recovering the target fragment, digesting the LlELP1 gene PCR product fragment with restriction sites and the vector pTRV2 in step (1), and recovering the restriction products respectively, and then connecting the recovered LlELP1 gene PCR product fragment and the vector pTRV2;

[0019] (3) connecting the target fragment digested and recovered in step (2) to the pTRV2 vector digested and recovered;

[0020] (4) The ligation product of step (3) was transformed into Escherichia coli competent cells DH5α, and positive recombinants were identified by colony PCR;

[0021] (5) The expression vector constructed in step (4) was transformed into Agrobacterium competent EHA105, and the obtained engineered bacteria EHA105 / pTRV2-LlELP1 was used to transform lily

[0022] The present invention is different from the prior art in that:

[0023] The invention discloses an application method for regulating the number of bulbils based on the LlELP1 gene of Lilium lily, which regulates the synthesis of chitinase protein. The LlELP1 gene is 717 bp in length, including a 717 bp open reading frame (coding sequence), encoding 238 amino acids, and predicting that the LlELP1 protein has no transmembrane domain; the invention confirms that the candidate gene is involved in regulating the occurrence process of lily bulbils by analyzing the expression pattern of the candidate gene during the bulbilogenesis process. After the invention uses the virus-induced gene silencing technology to inhibit the expression of the LlELP1 gene, the number of bulbils of Lilium lily is significantly reduced, and it is determined that the LlELP1 gene is related to the number of bulbils. The research of the invention is of great significance to the occurrence of lateral tissues. The invention first excavates chitinase-related genes that can regulate the number of bulbils, which is conducive to the directional selection of the number of bulbils, and provides a reference for molecular breeding.

[0024] The Lilium lily LlELP1 gene of the present invention and its application are further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of bacterial liquid PCR for cloning the full-length sequence of the LlELP1 gene in the present invention;

[0026] Figure 2 The expression pattern of the LlELP1 gene in two varieties (the variety with bulbils 'Hotel California' and the variety without bulbils 'Brasilia') in the present invention;

[0027] Figure 3 is the expression pattern of the LlELP1 gene during bulbilogenesis in the present invention;

[0028] Figure 4 This is the bulbil occurrence of the LlELP1 silenced strain in the present invention;

[0029] Figure 5 This is a schematic diagram of the function of the LlELP1 gene in regulating the number of bulbils produced. DETAILED DESCRIPTION

[0030] In order to explore the key genes that can regulate the number of lily bulbils, the present invention samples the upper leaf axils of Lilium stem segments during the bulbilogenesis of lily, and performs KEGG enrichment analysis on the transcriptome data of lily bulbilogenesis, and finds that the differentially expressed genes are most enriched in the "carbohydrate metabolism" pathway. The differential genes in the carbohydrate metabolism pathway are screened to obtain the candidate gene LlELP1 related to bulbilogenesis. By analyzing its expression pattern during the bulbilogenesis, it is determined that it is involved in the bulbilogenesis process, and a corresponding silencing vector is constructed. The LlELP1 silencing vector is transferred into the plant to adjust the number of bulbils.

[0031] The experimental methods in the examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are repeated three times and the results are averaged.

[0032] Example 1: L1ELP1 gene cloning

[0033] The LlELP1 gene was obtained by screening the transcriptome data of bulbil development in Lilium lancifolium, and its transcript sequence was put into ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to obtain its open reading frame (ORF) sequence. The longest ORF sequence obtained was compared in NCBI protein BLAST (https: / / blast.ncbi.nlm.nih.gov / -Blast.cgi), and the comparison was successful if it matched the transcriptome annotation result. The ORF sequence was obtained in the previous step. Specific cloning primers were designed using Premier 5 software, as shown in Table 1, and PCR amplification was performed using Lilium lancifolium bulbil cDNA as a template. The total amplification system was 50 μL, the specific reaction system was shown in Table 2, and the reaction procedure was 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 Escherichia coli competent DH5α (Qingke, Beijing), cultured in LB solid medium (containing 50 mg / L kanamycin) 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.

[0034] Table 1 Names and sequences of primers for cloning L1ELP1 gene

[0035]

[0036] Table 2 High-fidelity enzyme PCR reaction system

[0037]

[0038] Table 3 High-fidelity enzyme PCR reaction program

[0039]

[0040] Table 4 Colony PCR reaction system

[0041]

[0042] Table 5 Colony PCR reaction procedure

[0043]

[0044] Figure 1The present invention obtained the LlELP1 gene by screening the transcriptome data of bulbil development of Lilium lily, put its transcript sequence into ORF Finder (https: / / www.ncbi.nlm.nih.gov / orffinder / ) to obtain its open reading frame (ORF) sequence, and BLAST to a 717bp CDS series through the NCBIORF Finder online website. Specific cloning primers were designed using Premier 5 software, and PCR amplification was performed using the cDNA of mature bulbils of Lilium lily as a template. After the PCR amplification product was recovered by gel, the recovered product was connected to the TA / Blunt-Zero intermediate vector and transformed into the competent Escherichia coli DH5α, cultured at 37°C for 12-18h in LB solid medium (containing 50mg / L kana), and single clones were picked and colony PCR detection was performed.

[0045] LlELP1 gene sequence:

[0046] ATGGCTTCTCTTCCCCTCTCTCTCCTCCTCCTCGGCCTCCTCTCTGCTTCACTTCCATCCACCTCCGCAGCCCTTTTTCGTGAATACATCGGCGCCGAGTTCAACGGCGTCAGGTTTTCCCAAGTCCCCATCAACCCCGGCGTTGATTTCCATTTCATCCTCTCCTTCGCCATTGACTACACCACCAGCTCCAATCCCACCCCTACCAACGGCCACTTCTCTCCCTTCTGGGACACAGACAGCCTCTCCGCCTCCCAAGTCGGCGCCATCAAACGCGCTCATGGCAACGTGAAGGTGGCAGTCAGCCTCGGCGGCGACAGCGTGGGCGGGAAGAGTGCCTTCTTCAGCCCTACCTCAGTCTCGTCGTGGGTCCGTAATGCCGTCTCGTCTCTGACCGGGATCATCAAGCAGTATGGTCTTGATGGCATTGACATCGATTATGAGCATTTCAATGGGGACCCTAATACGTTTGCTGAATGTATCGGGCAGCTGGTGACGACGCTGAAGAAGAACGGCGTGGTATCGTTTGCGTCCATTGCCCCGTTCGATGATGATCAGGTGCAGAGTCACTACTTGGCGCTATGGAAGAAGTACGGACAGCAGATAGACTATGTGAACTTCCAGTTCTATGCTTACTCTGCAGGGACGACGGTGTCACAGTTTCTCAATTACTTTCAGACCCAGAGCTCTAATTACGCAGGAGGGAAGAACTCTTAA

[0057] Amino acid sequence encoded by the LlELP1 gene:

[0058] MASLPLSLLLLGLLSASLPSTSAALFREYIGAEFNGVRFSQVPINPGVDFHFILSFAIDYTTSSNPTPTNGHFSPFWDTDSLSASQVGAIKRAHGNVKVAVSLGGDSVGGKSAFFSPTS VSSWVRNAVSSLTGIIKQYGLDGIDIDYEHFNGDPNTFAECIGQLVTTLKKNGVVSFASIAPFDDDQVQSHYLALWKKYGQQIDYVNFQFYAYSAGTTVSQFLNYFQTQSSNYAGGKNS

[0062] Example 2: Analysis of candidate gene expression patterns during bulbilogenesis

[0063] 1. Take materials from the axils of lily leaves during the bulbilization process

[0064] The lily bulbs were domestically bred Lilium lily bulbs, which were planted in a solar greenhouse after dormancy was released. Total RNA was extracted from the upper leaf axils of the stem segments at four stages: S0 (bulb buds not yet formed), S1 (small white dot stage), S2 (green bulb stage), and S3 (purple bulb stage), and candidate gene fluorescence quantitative PCR was performed.

[0065] 2. Total RNA extraction and cDNA synthesis from lily leaf axils during bulbilogenesis

[0066] Total RNA from the axils of lily leaves was extracted using an RNA extraction kit (Tiangen, DP441), and 1.5 μg RNA was synthesized into the first strand of cDNA using a reverse transcription kit (Vazyme, R323) to obtain cDNA from the axils of lily leaves at S0 (bulb buds not formed), S1 (small white dot stage), S2 (green bulb stage), and S3 (purple bulb stage). The reverse transcription system is as follows:

[0067] Table 6 Reverse transcription system

[0068]

[0069] 3. Real-time fluorescence quantitative PCR (qPCR) quantitative detection of gene expression

[0070] According to the candidate gene CDS, the real-time fluorescence quantitative PCR primers were designed. The specific primers are shown in Table 7. The cDNA was amplified using a real-time fluorescence quantitative PCR kit (Vazyme, Q111). The fluorescence quantitative reaction system and reaction procedures are shown in Tables 8 and 9. LlFP was used as the internal reference gene, and the measurement instrument was an ABI 7500 quantitative PCR instrument (ABI 7500 Real-Time PCR System, ABI, USA). Each sample was repeated three times.

[0071] Table 7 Fluorescence quantitative PCR primer list

[0072]

[0073] Table 8 Fluorescence quantitative PCR system

[0074]

[0075] Table 9 Fluorescence quantitative PCR reaction program

[0076]

[0077] 4. Analysis of the expression pattern of candidate genes during bulbilogenesis

[0078] According to the above fluorescence quantitative PCR results, GraphPad Prism8 and DPS were used for significant difference analysis. The asterisk indicates the significant difference analysis by ANOVA (P<0.05). The experimental results showed that the expression of LlELP1, the gene controlling the number of bulbils, was highly upregulated during the occurrence of bulbils, and then showed a downward trend, indicating that LlELP1 is a key gene regulating the occurrence of bulbils.

[0079] Figure 2-3 The expression of LlELP1 in the upper leaf axils of the bulbil varieties 'Hotel California' and the non-bulil varieties 'Brasilia' and the leaf axils of Lilium lily S0-S3 was detected by qRT-PCR. The results showed that the expression of LlELP1 was higher in the bulbil varieties, and the expression of S1 and S2 in the bulbils of Lilium lily was higher and showed an up-regulation trend, which suggested that LlELP1 may play a promoting role in the bulbilogenesis process.

[0080] Example 3: Application of the bulbil number gene L1ELP1

[0081] 1. Construction of gene silencing vector

[0082] (1) Cloning of target fragment and purification and recovery of product. The PCR reaction system is as follows: The primers in the following table are primers for cloning the full length of the LlELP1 coding region (ORF). The reaction system is as follows:

[0083]

[0084] Note: The cDNA template is a mixture of cDNA from the leaf axils of lilies S1, S2, S3, and S4 in Experimental Example 1 at a ratio of 1:1:1:1; Primer F and Primer R are primer sequences for cloning the full length with restriction sites. The primer sequences are as follows:

[0085]

[0086] The PCR reaction system was placed in a PCR amplifier (Bio-Rad) to start the PCR reaction. The reaction procedure is shown in Table 3. After the reaction is completed, 1.0%-2.0% agarose gel electrophoresis is used to detect whether the amplified band is the target band. The correct band is then cut and recovered. The PCR product was purified using a gel recovery purification kit (provided by Nanjing Novozyme). For specific operations, refer to the kit instructions.

[0087] (2) Enzyme digestion and purification and recovery of target fragment and cloning vector: The purified and recovered target fragment and cloning vector are digested using the following enzyme digestion system:

[0088]

[0089] After mixing, incubate at 37°C for 15 min and at 80°C for 20 min. Purify and recover the product after enzyme digestion using a purification kit (provided by Nanjing Novozymes). For specific operations, refer to the kit instructions.

[0090] (3) Connect the target fragment to the vector. Use the T4 ligation method to connect the target gene to the cloning vector pTRV2. The reaction system is as follows:

[0091]

[0092] After mixing, connect at 50°C for 20 min.

[0093] (4) Transformation of ligation products: The ligation products were transformed into competent E. coli DH5a, and then placed on ice for 30 min, 42°C for 90 s, and on ice for 2 min. Then, they were mixed with 700 μL of LB solution without antibodies, shaken at 37°C at 200 rpm for 1 h, and centrifuged at 6000 rpm for 1 min. The supernatant was removed, the remaining liquid was mixed by pipetting, and then spread on the corresponding resistance screening medium, and incubated in a constant temperature incubator at 37°C for 12-16 h. Single clones that were positive by colony PCR were selected for sequencing. Various sequence sequencing analyses were completed by Shanghai Shenggong Biotechnology Service Co., Ltd.

[0094] 2. Construction of gene silencing vector

[0095] The recombinant plasmid pTRV2-LlELP1, pTRV2 empty plasmid and pTRV1 empty plasmid with correct sequencing results were transferred into Agrobacterium EHA105, and the bacterial solution was resuspended to OD600 = 0.8 using a resuspension solution (10mM MgCl2, 10mM MES pH5.8, 200μM Acetosyringone / AS). The Agrobacterium containing the recombinant plasmid and the Agrobacterium containing the pTRV1 plasmid (pTRV2: pTRV1 = 1:1) were mixed in equal volumes, and empty pTRV2 + empty pTRV1 was used as a control. The lily bulbs were infecting after standing in a 28°C incubator in the dark for 3 hours. The infected bulbs were planted in a solar greenhouse, and the occurrence of bulbils in the gene-silenced strains was observed after 6 weeks. The number of bulbils was used as an indicator. The results showed that compared with the control (empty pTRV2 + empty pTRV1), the expression of the LlELP1 gene in the Lilium plants of pTRV2-LlELP1 was downregulated by 61.31% (p = 0.0001) compared with the control, and the number of bulbils was downregulated by 89.74% (p < 0.0001) compared with the control, indicating that LlELP1 plays a positive role in regulating the number of bulbils.

[0096] Figure 4 The present invention silenced the gene in the bulbils of Lilium lily, and selected 45 days as the sampling time to count and sample the number of bulbils of Lilium lily. The results showed that the expression of the LlELP1 gene was down-regulated by 61.31% in the silenced strain compared with the control. After silencing LlELP1 in the bulbils for 45 days, the number of bulbils of Lilium lily was down-regulated by 89.74% compared with the control. This shows that LlELP1 has a positive regulatory effect on the occurrence of bulbils of Lilium lily.

[0097] 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 Lilium lily L1ELP1 gene, Features: Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The expressed protein of the Lilium lily L1ELP1 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO:

2.

3. A specific primer pair for cloning the Lilium lily LlELP1 gene of claim 1, LlELP1-F and LlELP1-R, whose nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO:

4.

4. Use of the Lilium lily L1ELP1 gene according to claim 1 in Lilium lily breeding.

5. The use according to claim 4, Features: The Lilium lily LlELP1 gene can promote the occurrence process of Lilium lily bulbils and increase the number of bulbils.

6. The method for analyzing the expression pattern of the Lilium lily L1ELP1 gene according to claim 1, Features: The steps include: (1) Samples were collected from the leaf axils of lilies during bulbilogenesis: dormant bulbs were planted in a solar greenhouse and the leaf axils on the upper part of the stems were used as test materials. The sample collection period was the bulbilogenesis stage (S0), the small white spot stage (S1), the green bulb stage (S2), and the purple bulb stage (S3). (2) Total RNA extraction and cDNA synthesis from lily leaf axils during bulbilogenesis: (3) Real-time fluorescence quantitative PCR quantitative detection of gene expression: Real-time fluorescence quantitative PCR primers were designed according to the candidate gene CDS, cDNA was amplified, LlFP was used as the internal reference gene, and each sample was repeated three times; (4) Analysis of the expression patterns of candidate genes during bulbilogenesis: significant differences were analyzed based on the results of fluorescence quantitative PCR.

7. The method according to claim 6, It is characterized in that The real-time fluorescence quantitative PCR primers used in step (3) are LlELP1-qRT-F and LlELP1-qRT-R, and their nucleotide sequences are shown in SEQ ID NO:5 and SEQ ID NO:

6.

8. The method for constructing the transient silencing vector of the Lilium lily L1ELP1 gene according to claim 1, Features: The steps include: (1) Obtain the target fragment, design specific primers according to the gene sequence, add restriction sites at both ends of the primers, use the cloning vector as a template, perform PCR amplification, and obtain the LlELP1 coding region amplification product with restriction sites, i.e., the target fragment; the restriction sites are EcoRⅠ and XhoⅠ; the primer sequences are Primer F and Primer R, and their nucleotide sequences are shown in SEQ ID NO: 7 and SEQ ID NO: 8; (2) Recovering the target fragment, digesting the LlELP1 gene PCR product fragment with restriction sites and the vector pTRV2 in step (1), and recovering the restriction products respectively, and then connecting the recovered LlELP1 gene PCR product fragment and the vector pTRV2; (3) ligating the target fragment digested and recovered in step (2) to the pTRV2 vector digested and recovered; (4) The ligation product of step (3) was transformed into Escherichia coli competent cells DH5α, and positive recombinants were identified by colony PCR; (5) The expression vector constructed in step (4) was transformed into Agrobacterium competent cell EHA105, and the obtained engineered bacteria EHA105 / pTRV2-LlELP1 was used to transform lily.

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