LiSRM1 gene regulating the synthesis of volatile monoterpenoid compounds in lily and its application

Abstract

The present invention discloses a method for regulating the synthesis of lily monoterpene volatile compounds LiSRM1 The present invention further discloses the application of LiSRM1 in regulating the synthesis of volatile monoterpenoid compounds in lily. LiSRM1 Genes that enhance the synthesis and release of monoterpene volatiles in lily; overexpression LiSRM1 This gene inhibits the synthesis and release of monoterpene volatiles in lilies. This gene can be used to regulate the synthesis and release of floral monoterpenes in plants.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology, and in particular to a transcription factor LiSRM1 isolated and cloned from Lilium orientale 'Siberia' and its application in regulating monoterpene synthesis. Background Art

[0002] Lily (Lilium spp.), the general term for all species of the genus Lilium in the family Liliaceae (Liliaccae), is a perennial monocotyledonous herb. Lilies are native to my country, and there are currently approximately 115 species of Lilium worldwide, primarily distributed in temperate zones. After years of breeding, approximately 10,000 varieties with numerous excellent traits have been developed. However, the major lily varieties that dominate the large-scale commercial market are still the lily families of musk, Asiatic, and Oriental. Among them, Asian lilies have a variety of colors but are essentially fragranceless or have a light fragrance. Lilies of musk and Oriental lilies have large flowers but a strong fragrance that can easily cause discomfort in confined spaces. Therefore, enhancing the fragrance of light-scented lilies, reducing the fragrance of strong-scented lilies, and cultivating lilies with an appropriate and pleasant aroma are important goals in lily fragrance breeding.

[0003] Studies have shown that fragrance emission varies significantly between lily species and varieties. Volatile measurements of Asiatic, LA, Oriental, and musk lilies revealed significant differences in the types and amounts of fragrance components released by different lily types. Strong-fragrant lilies exhibited significantly higher amounts and total volatile release than light-fragrant lilies. Monoterpenes accounted for 80% of the total release in strong-fragrant lilies, while they were almost undetectable in light-fragrant lilies. This suggests that monoterpenoid volatiles are the primary contributor to lily fragrance variation. Therefore, screening for transcription factors upstream of monoterpene synthases is crucial for understanding the mechanisms regulating lily fragrance.

[0004] Transcriptional regulation research has been widely applied to explore the mechanisms underlying plant traits, but the complex and large genome severely limits research on lilies. Currently, most lily research focuses on the correlation between transcription factors and traits. Compared to other plants, research on the transcriptional regulation mechanisms underlying lily floral aroma synthesis is still relatively backward. Currently, there are no reports on the transcription factors that negatively regulate the synthesis of monoterpene volatiles in lilies. Summary of the Invention

[0005] Purpose of the invention: The main purpose of the present invention is to provide a LiSRM1 transcription factor capable of negatively regulating the synthesis of lily monoterpene volatile compounds, as well as its encoding gene and application.

[0006] In order to solve the above technical problems, the present invention discloses a LiSRM1 gene for regulating the synthesis of lily monoterpene volatile compounds, which has a coding region nucleotide sequence as shown in SEQ ID NO.13.

[0007] The present invention also discloses the protein encoded by the LiSRM1 gene. The amino acid sequence of the protein is shown in SEQ ID NO.14.

[0008] The present invention further provides a recombinant expression vector, which is a VIGS silencing vector or a transient overexpression vector, wherein the VIGS silencing vector comprises a LiSRM1-specific fragment and a pTRV2 vector, and the sequence of the LiSRM1-specific fragment is shown in SEQ ID NO.15; the transient overexpression vector comprises the LiSRM1 gene and the pSuper1300-GFP vector.

[0009] The present invention further proposes the use of any one of the above-mentioned LiSRM1 gene, the protein encoded by the LiSRM1 gene, or the recombinant expression vector in regulating the synthesis of lily monoterpene volatile compounds.

[0010] Specifically, silencing the LiSRM1 gene increased the synthesis and release of monoterpene volatiles in lily; overexpressing the LiSRM1 gene inhibited the synthesis and release of monoterpene volatiles in lily.

[0011] In a specific embodiment, the lily is Lilium 'Siberia'.

[0012] Specifically, the monoterpene volatiles are linalool and ocimene.

[0013] The present invention also proposes the use of any one of the above-mentioned LiSRM1 gene, the protein encoded by the LiSRM1 gene, or the recombinant expression vector in the directional cultivation of lily fragrance.

[0014] Beneficial Effects: The present invention discovered that LiSRM1 plays an important role in the biosynthesis and volatilization of lily monoterpenes. Based on this, addressing the gap in LiSRM1's ability to regulate lily monoterpenoid biosynthesis, the present invention provides the lily gene LiSRM1 and its application, which can provide a basis and direction for the targeted cultivation of lily floral fragrance. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the evolutionary tree of LiSRM1;

[0016] Figure 2 Expression analysis of LiSRM1 in different flower development stages (A) and different tissues (B) of the present invention; S7, bud stage; S8, initial opening stage; S9, full bloom stage; S10, withering stage. Root, root; Petal, petal; Stem, stem; Ovary, ovary; Filament, filament; Style, style; Anther, anther; Leaf, leaf;

[0017] Figure 3 for the subcellular localization of LiSRM1;

[0018] Figure 4 Figure 3 shows the transient silencing of the LiSRM1 gene in lily flowers, where (A) is the detection of LiSRM1 silencing efficiency, (B) is the detection of LiLiS and LiOcS expression levels, (C) is the detection of linalool and ocimene release, and (D) is the ion chromatogram of volatile composition measurements in silenced and control plants.

[0019] Figure 5 Figure 3 shows the transient overexpression of the LiSRM1 gene in lily flowers, where (A) is the detection of LiSRM1 overexpression efficiency, (B) is the detection of LiLiS and LiOcS expression levels, (C) is the detection of linalool and ocimene release, and (D) is the ion current diagram of volatile compound determination in silenced and control plants. DETAILED DESCRIPTION

[0020] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the above-mentioned and / or other advantages of the present invention will become more apparent. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Based on the previous transcriptome screening of differential transcription factors, the inventors preliminarily speculated that the LiSRM1 transcription factor is correlated with the synthesis and release of monoterpene volatiles in lily.

[0022] The materials involved in the following examples are:

[0023] Plant material: The material used in the experiment was Oriental lily 'Siberia'. The material used in the subcellular localization experiment was wild-type tobacco (Nicotiana benthamiana).

[0024] The test strains and vectors are shown in Table 1 below:

[0025] Table 1

[0026] Commonly used strains or plasmids Model or name Escherichia coli TOP10 Agrobacterium GV3101 cloning vector pClone-007 Overexpression / subcellular localization vector Super1300-GFP(C) VIGS vector pTRV1, pTRV2

[0027] The main kits used in the experiment were all manufactured by Beijing Quanshijin Biotechnology Co., Ltd.: RNA extraction: TransZolUp Plus RNA Kit; reverse transcription: Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR; DNA gel recovery: Quick Gel Extraction Kit.

[0028] The experimental methods designed in the experiment include:

[0029] (1) Vector construction

[0030] Based on the sequence information available in the lily transcriptome database, cloning primers were designed and PCR was performed using lily 'Siberia' cDNA as a template. The reaction system and reaction procedure are shown in Table 2 below:

[0031] Table 2

[0032]

[0033]

[0034] The obtained PCR products were run on agarose gel for verification and the gel recovery kit ( Quick Gel Extraction Kit). Then, the desired vector was double-digested, cutting 1 μg. The system is shown in Table 3 below:

[0035] Table 3

[0036]

[0037] The fragment enzyme digestion reaction system was denatured at 37°C for 30 min and 80°C for 20 min, and the liquid was recovered.

[0038] Next, use T4 ligase to ligate the digested product recovered from the previous step to the vector. Transform the ligated product into competent E. coli (TOP10) for transformation. Select the bacterial suspension that has been successfully transformed by sequencing and culture it overnight in a 50mL centrifuge tube. Then, use the Quanshijin Plasmid Mini Kit to extract the plasmid from the E. coli and store it for future use.

[0039] The primers, LiSRM1 gene sequence and the corresponding protein sequence encoded by it used in the experiment are shown in Table 4 below.

[0040] Table 4

[0041]

[0042]

[0043]

[0044]

[0045] (2) VIGS infection method for lily petals

[0046] In order to construct the VIGS silencing vector, a 334bp specific fragment of the LiSRM1 gene was constructed into the pTRV2 vector as shown in SEQ ID NO.15 and named TRV2-LiSRM1. 1 μg TRV2-LiSRM1, TRV2 empty load and TRV1 were respectively transferred into the Agrobacterium competent state (GV3101), pipetted and mixed, and then ice bathed for 5 minutes, liquid nitrogen for 5 minutes, 37°C for 5 minutes, ice bathed for 5 minutes, and then 500 μL of anti-liquid LB was added and shaken at 28°C and 200rpm for 3 hours. After the culture was completed, 100 μL of each was spread on LB solid culture medium containing 50 μg / mL and 25 μg / mL, and inverted in a 28°C incubator for 48 hours. Subsequently, a single clone was picked and inoculated into 500 μL LB liquid culture medium (containing Kan+Rif), shaken and cultured at 28°C for 16 hours, and then PCR was performed to detect positive clones. Transfer the small shaken bacterial solution that has been successfully detected by PCR into a 50mL centrifuge tube (5-10mL of resistance LB) and shake culture at 28℃ for 8h. Then transfer the 2mL medium shaken bacterial solution into 200mL resistance LB and shake culture at 28℃ for 12h. Centrifuge the cultured large shaken bacterial solution for bacteria collection, centrifuge at 5,000rpm for 10min, and discard the supernatant. Resuspend the bacteria separately with infection solution (containing 200mM acetosyringone, 10mM magnesium chloride, 10mM MES) and adjust the OD 600 To 1.0. After resuspending the infection solution, TRV2 and the TRV2 recombinant vector were mixed with TRV1 in equal proportions and kept in the dark for 3 hours. Then, lily buds with good and consistent growth were selected for infection. Using a 1mL syringe, the infection solution was injected into the lily petals along the petal veins until all the petals were soaked with the infection solution. After 24 hours of darkness, the lily petals were cultured under normal light for 5-7 days. After the petals reached the blooming stage, samples were collected and the silencing effect was detected by qRT-PCR and the floral fragrance was detected by GC-MS. The primers used were TRV2-LiSRM1-F and TRV2-LiSRM1-R.

[0047] (3) Lily petal transient overexpression infection method

[0048] Construction of lily transient overexpression vector The full-length LiSRM1 was constructed into the pSuper1300-GFP vector and named pSuper1300-LiSRM1. The successfully constructed recombinant plasmid, pSuper-GFP empty vector, and p19 were respectively transformed into GV3101 Agrobacterium, and the culture conditions were the same as above. The pSuper-GFP recombinant plasmid and empty vector were respectively adjusted to OD 600 To 1.5, p19 adjust OD 600Adjust the OD600 to 1.0. Mix the recombinant plasmid and the empty vector with equal volumes of p19. Cultivate the cells in the same manner. After approximately three days of cultivation, when the cells reach full bloom, sample the cells for overexpression efficiency testing and floral scent collection. Primers 1300-LiSRM1-F and 1300-LiSRM1-R were used.

[0049] (4) Collection and determination of lily volatiles

[0050] This experiment used headspace and solid phase microextraction techniques to collect volatile compounds from lily flowers, and used gas chromatography-mass spectrometry-mass spectrometry (GC-MS) to perform quantitative and qualitative analysis of the volatile compounds. The specific procedures are as follows:

[0051] Lilium 'Siberia' petals (approximately 1 g) were collected in full bloom and immediately placed in a 15 mL vial. 10 μL of the internal standard trioctanol was added and equilibrated at 40°C for 5 minutes. Extraction was then performed using a solid-phase microextraction (SPME) needle for 40 minutes, followed by chromatography on a GC. The extraction needle was placed in the GC inlet for 3 minutes, using a DB-5MS column (30 m × 0.25 mm × 0.25 μm) with He as the carrier gas. The GC temperature program was as follows: hold at 40°C for 2 minutes, then increase the temperature to 180°C at a rate of 4°C / min, then to 270°C at a rate of 15°C / min, and hold for 3 minutes. The MS ionization mode was set to EI, with an electron source energy of 70 eV and an atomic scan range of 29-600 amu. The interface and ion source temperatures were 250°C and 230°C, respectively. The NIST 2008 database was used for retrieval, and the release of other volatile compounds was calculated based on the release of the standard trioctanol.

[0052] (5) Extraction of total RNA from lily and qRT-PCR

[0053] The total RNA of lily was extracted using TransZol Up Plus RNA Kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.). After the RNA was tested and qualified, it was used for the preparation of cDNA. Uni All-in-One First-Strand cDNA Synthesis SuperMix reverse transcription kit was used to reverse cDNA according to the instructions of the reverse transcription kit. Real-time fluorescence quantitative PCR (qRT-PCR) was performed using the KAPA SYBR FAST qPCR KIT kit and ABI's PRISM Step ONE Plus Real-time PCR System (Applied Biosystems, Foster City, CA, USA) instrument to perform qRT-PCR analysis and calculate the relative expression of each gene. Each experiment included three biological sample replicates and three technical replicates. Among them, the primers used to detect LiSRM1, LiLiS, LiOcS and Actin (internal reference) are shown in Table 4, where the suffix -F is the forward primer and the suffix -R is the reverse primer.

[0054] (6) Subcellular localization of LiSRM1 protein

[0055] pSuper-LiSRM1 plasmid, pSuper-GFP empty vector, NF-YA4-mCherry plasmid (Zhang et al., 2019) and P19 were transformed into Agrobacterium GV3101. Culture conditions and OD 600 The values are the same as those under overexpression conditions. The recombinant plasmid and the empty vector were mixed with equal volumes of p19 and NF-YA4-mCherry, respectively, and incubated in the dark for 3 hours before being injected into tobacco plants containing four true leaves. After 3 days of culture under normal conditions, fluorescence confocal microscopy (Olympus FluoView FV1000) was used for observation.

[0056] Example 1 Screening of LiSRM1 gene.

[0057] Preliminary selection of transcription factors for floral development in Lilium 'Siberia':

[0058] The inventors conducted a comparative analysis of the transcriptome of 'Siberia' lily flower development, performed a correlation analysis with the release of monoterpene volatiles, and finally screened out a negatively correlated transcription factor. Through gene structure analysis, it was found that it was a MYB-type transcription factor. The gene coding region sequence was obtained from the lily transcriptome database, and PCR amplification was performed using the 'Siberia' lily petal cDNA as a template (amplification primers were LiSRM1_F and LiSRM1_R). Sequencing results showed that the gene coding region was 888bp in length (see SEQ ID NO.13) and encoded 296 amino acids (see SEQ ID NO.14). Analysis using NCBI software showed that the sequence had a relatively conserved structural domain as a MYB transcription factor. The NCBI database was selected for comparison, and the results showed that it had a high homology with SRM1 of Argentine mesquite (Prosopis alba), so it was named LiSRM1 ( Figure 1 ).

[0059] Example 2 Analysis of tissue-specific expression of LiSRM1 in lily.

[0060] To clarify the role of LiSRM1 in the synthesis and release of monoterpenes in lily, the expression characteristics of LiSRM1 in different flower development stages and different tissues of lily were analyzed. The expression trend of LiSRM1 during flowering is that it first increases and reaches the highest level in the early opening stage, then decreases in the full bloom stage, and then increases again in the decay stage ( Figure 2 A). LiSRM1 is highly expressed in ovary, filament, style, stem and leaves, but is lowly expressed in petals, the main tissue for monoterpene release ( Figure 2 B). Combined with its expression during floral development, it is speculated that it may negatively regulate the synthesis and release of monoterpenes.

[0061] Example 3 Subcellular localization of transcription factor LiSRM1.

[0062] To determine the subcellular localization of LiSRM1, the full-length LiSRM1 construct was inserted into the pSuper1300-GFP vector and infected with tobacco. It was observed that the green fluorescence of LiSRM1 overlapped with the red fluorescence signal of the nuclear marker to form yellow fluorescence, while the negative control empty vector showed green fluorescence in both the cell membrane and the cell nucleus. Therefore, it was concluded that LiSRM1 was localized in the cell nucleus ( Figure 3 ).

[0063] Obtaining LiSRM1 transiently silenced lily plants and phenotypic identification of their monoterpene volatiles

[0064] To determine whether LiSRM1 plays a role in linalool biosynthesis in Lilium 'Siberia', we used VIGS to verify its transient silencing function. First, we constructed a TRV2-LiSRM1 recombinant vector and infected Lilium 'Siberia' petals with Agrobacterium-mediated transfection. qRT-PCR was used to select silenced plants. The expression of LiSRM1 transcripts was significantly downregulated ( Figure 4 A), the expression of LiLiS and LiOcS was detected under the background of LiSRM1 silencing, and it was found that the expression of LiLiS and LiOcS was significantly increased ( Figure 4 B). The release of linalool and ocimene was also significantly induced to increase ( Figure 4 C), while the abundance of linalool and ocimene was significantly higher than that of the control ( Figure 4 D). These results indicate that LiSRM1 negatively regulates the expression of LiLiS, thereby affecting the synthesis of linalool and ocimene.

[0065] Example 4 Obtaining lily LiSRM1 transiently overexpressing plants and phenotypic identification of their monoterpene volatiles.

[0066] To further clarify the role of LiSRM1 in linalool biosynthesis in 'Siberia' lily, a pSuper1300-LiSRM1 recombinant vector was constructed and infected with lily petals using Agrobacterium tumefaciens. qRT-PCR was used to detect endogenous expression of LiSRM1, and effective overexpression plants were selected for subsequent analysis. The results showed that LiSRM1 was successfully overexpressed ( Figure 5 A). The expression of LiLiS and LiOcS and the release of linalool and ocimene were then detected, and the results showed that the expression of LiLiS and LiOcS was significantly inhibited ( Figure 5 B), the release of linalool and ocimene was significantly reduced compared with the control group ( Figure 5 C). However, the ion flow analysis of overexpressed volatiles revealed that the abundance of linalool and ocimene in overexpressed plants was significantly lower than that in the control ( Figure 5 D).

[0067] Floral fragrance is the main characteristic trait of lily, and its research mechanism has always lagged behind other traits. At the same time, its rich fragrance has also troubled many consumers, and there are currently no reports on its negative regulation. The inventors obtained a negatively correlated transcription factor LiSRM1 through transcriptome data screening. After transient silencing of LiSRM1, the synthesis and release of lily monoterpene volatiles were significantly improved, while after transient overexpression of LiSRM1, the synthesis and release of monoterpenes were significantly inhibited. The above results show that overexpression of LiSRM1 can inhibit the expression of LiLiS and LiOcS, thereby reducing the synthesis of linalool and ocimene. The results show that LiSRM1 plays a negative regulatory role in the synthesis of 'Siberian' linalool.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for regulating the synthesis of volatile monoterpenoid compounds from lily LiSRM1 A gene characterized by It has a coding region nucleotide sequence as shown in SEQ ID NO.

13.

2. The protein encoded by the LiSRM1 gene according to claim 1, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.

14.

3. Containing the LiSRM1 Recombinant gene expression vector.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is a VIGS silencing vector or a transient overexpression vector, wherein the VIGS silencing vector comprises LiSRM1 Specific fragment and pTRV2 vector, the sequence of the LiSRM1 specific fragment is shown in SEQ ID NO. 15; the transient overexpression vector includes LiSRM1 gene and pSuper1300-GFP vector.

5. The method according to claim 1 LiSRM1 Use of any one of the gene, the protein encoded by the LiSRM1 gene according to claim 2, or the recombinant expression vector according to claim 3 in regulating the synthesis of lily monoterpene volatile compounds.

6. The use according to claim 5, characterized in that silence LiSRM1 Genes that enhance the synthesis and release of monoterpene volatiles in lily; overexpression LiSRM1 Genetic inhibition of the synthesis and release of monoterpene volatiles in lily.

7. The use according to claim 5, characterized in that The lily is Lilium 'Siberia'.

8. The use according to claim 5, characterized in that The monoterpene volatiles are linalool and ocimene.

9. The method according to claim 1 LiSRM1 Use of any one of the gene, the protein encoded by the LiSRM1 gene according to claim 2, or the recombinant expression vector according to claim 3 in the directed cultivation of lily fragrance.

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