Anthurium andraeanum AaHDS gene and application thereof in regulating aroma

Abstract

The invention discloses an anthurium andraeanum AaHDS gene and application of the anthurium andraeanum AaHDS gene in aroma regulation. The anthurium andraeanum AaHDS gene is obtained through PCR amplification cloning, the DNA sequences of the gene in fragrant anthurium andraeanum and non-fragrant anthurium andraeanum have obvious differences, and the expression quantity of the gene in meat spike inflorescence is the highest; the gene is silenced by adopting a VIGS technology, the expression quantity of the gene is reduced by 49.1%, and the contents of alpha-pinene, beta-pinene and eudesmol are remarkably reduced. The AaHDS gene cloned by the invention is beneficial to genetic improvement of plant aroma traits, and can be used for cultivating more fragrant anthurium andraeanum new varieties with pleasant aroma.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering and plant breeding, and more specifically, relates to anthurium andraeanum AaHDS gene and its application in regulating aroma. Background Art

[0002] Anthurium andraeanum is a perennial evergreen herb in the genus Anthurium of the family Araceae. It is famous for the unique shape of its spathe, bright and diverse flower colors, and a long ornamental period. It is a world-famous cut flower and potted flower plant. Flower fragrance is an important breeding target trait for ornamental plants. There are currently few fragrant anthurium varieties on the market, and the goal of anthurium aroma breeding is to cultivate more fragrant anthuriums. Therefore, cultivating more new varieties of anthurium with different fragrances is of great significance to further improve the competitiveness and benefits of my country's anthurium industry and promote the high-quality development of the anthurium industry.

[0003] Flower fragrance is an important feature of ornamental plants. It not only attracts customers and improves the commercial potential of ornamental plants, but also attracts pollinators and improves disease resistance. Flower fragrance is composed of a series of low molecular weight volatile organic compounds (VOCs), which can be divided into three categories according to their biosynthetic sources: terpenes, phenylpropanoids / benzenes, and fatty acid derivatives. Among them, terpenoids are the largest category of plant volatile organic compounds, synthesized through the cytoplasmic mevalonic acid (MVA) pathway and the plastid methylerythritol phosphate (MEP) pathway. Fragrant anthurium plants release fragrance and volatile compounds during the pistil stage of flower development. The main compounds released include 1,8-cineole, α, β-pinene, sabinene, myrcene and limonene, as well as some benzene compounds. Because the flower fragrance of anthurium varieties varies greatly, some fragrant anthuriums release more volatile compounds. In 1999, the method of artificial olfactory smelling was used to classify the types of aroma substances released by fragrant anthuriums and their hybrid F1 generations, mainly sweet, spicy, smelly, fruity, orange, pine and minty (Kuanprasert, 1998). Croatians observed that the odors emitted by several native female anthuriums ranged from pleasant to unpleasant. A more extensive survey of the presence of floral fragrance in 147 species and hybrids of anthuriums collected by the University of Hawaii and Missouri Botanical Garden germplasm resources showed that 76% of the plants emitted odors ranging from floral to fishy or foul, from very weak to very strong (Kuanprasert, 1998). The aroma of most anthuriums is relatively strong, pungent, and not very pleasant. If you smell the fragrance for a long time, you will feel dizzy, such as the fragrant varieties of anthurium A.'Armeniense' (with a strong sweet taste) and A.'Mystral' (with a strong fragrance). Therefore, it is particularly important to adjust the intensity of the aroma of fragrant anthuriums. Existing research has set regulating the intensity of the fragrance of fragrant anthurium as a new breeding goal, which is of great significance for breeding more fragrant anthuriums with appropriate fragrance, elegant and fresh smell, and pleasant odor.

[0004] So far, there are few studies on the composition and formation mechanism of the flower fragrance of Anthurium. Existing studies have analyzed the main aroma components of Anthurium and found that the expression level of aroma synthesis genes in fragrant varieties is significantly higher than that in non-fragrant varieties, including AaDXS, AaDXR, AaMDS, AaHDS, AaTPS, AaDAHPS, AaADT2, AaPAL1 and AaPAL2. At present, multiple aroma synthesis genes of Anthurium have been obtained, but their functions have not been verified, and it is unknown whether they can be used for the regulation of the flower fragrance of fragrant Anthurium and the breeding of Anthurium flower fragrance. Therefore, it is of great significance to carry out and explore the cloning and functional research of more Anthurium aroma regulation genes to promote the development of transgenic breeding of Anthurium, break through the "bottleneck" technology of Anthurium aroma breeding, continuously cultivate new varieties of Anthurium with independent intellectual property rights and market competitiveness, narrow the gap between my country's Anthurium breeding and foreign countries as soon as possible, and promote the efficient and sustainable development of the Anthurium industry.

[0005] References: Kuanprasert et al, ARKuehnle, CSTang, Floral fragrance compounds of some anthurium (ARACEAE) species and hybrids, Phytochemistry, Volume 49, Issue 2, 1998. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the defect of pungent aroma of existing fragrant anthurium and the shortage of existing anthurium aroma breeding gene resources, and to provide anthurium AaHDS gene and its application in regulating aroma.

[0007] The first object of the present invention is to provide anthurium andraeanum aroma gene AaHDS.

[0008] The second object of the present invention is to provide the coding protein of the anthurium aroma gene AaHDS.

[0009] The third object of the present invention is to provide a recombinant expression vector.

[0010] The fourth object of the present invention is to provide a genetically engineered bacterium.

[0011] The fifth object of the present invention is to provide an application of regulating the aroma gene of anthurium andraeanum.

[0012] The sixth object of the present invention is to provide the use of a preparation for silencing or inhibiting the expression of anthurium aroma gene.

[0013] A seventh object of the present invention is to provide a product for regulating the aroma of anthurium.

[0014] The eighth object of the present invention is to provide a method for regulating the aroma of anthurium andraeanum.

[0015] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0016] The present invention clones the anthurium AaHDS gene in the anthurium genome, the full length of the gene DNA sequence is 17264bp, encoding 745 amino acids, the ORF nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. Studies have shown that the gene is a key gene for controlling aroma, and the highest expression level is in the anthurium spadix, which is 15 times that in the root, followed by the spathe, with an expression level of 3.8 times that in the root. Expression analysis in the fragrant anthurium through gene silencing technology shows that silencing the AaHDS gene will reduce its expression level, and at the same time inhibit the synthesis of anthurium monoterpenoid compounds, and reduce the content of α-pinene, β-pinene and eucalyptol; silencing the AaHDS gene significantly reduces the content of anthurium aroma components, indicating that the gene is a key gene for regulating the synthesis of anthurium aroma compounds, and can be used to cultivate more new varieties of fragrant anthurium with pleasant aroma, which is beneficial to the genetic improvement of plant aroma traits.

[0017] Therefore, the present invention provides the Anthurium andraeanum AaHDS gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0018] The present invention provides a protein encoded by the AaHDS gene of anthurium andraeanum, and the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0019] The invention provides a recombinant expression vector containing the anthurium andraeanum AaHDS gene.

[0020] The present invention provides a genetically engineered bacterium comprising the above-mentioned recombinant expression vector.

[0021] The present invention provides application of anthurium andraeanum AaHDS gene in regulating the synthesis of anthurium andraeanum aroma or aroma compounds.

[0022] Furthermore, the aroma compound is a monoterpene compound, and silencing the AaHDS gene of anthurium andraeanum can reduce the content of eucalyptol, β-pinene and α-pinene in anthurium andraeanum.

[0023] The invention provides an application of a preparation for silencing or inhibiting the expression of anthurium andraeanum AaHDS gene in reducing the aroma of anthurium andraeanum or reducing the synthesis of anthurium andraeanum aroma compounds.

[0024] The present invention provides application of a preparation for silencing or inhibiting the expression of anthurium AaHDS gene in preparing a product for regulating the aroma of anthurium.

[0025] The present invention provides a product for regulating the aroma of anthurium, comprising a preparation for silencing or inhibiting the expression of anthurium AaHDS gene, or a silencing vector or engineered bacteria containing anthurium aroma gene.

[0026] The present invention provides a method for regulating the aroma of anthurium or a method for cultivating a light-fragrant anthurium plant, silencing or inhibiting the expression of anthurium AaHDS gene in anthurium, or using the anthurium aroma regulating product to treat anthurium.

[0027] The method further specifically comprises the following steps:

[0028] S1. Construction of silencing vector: Using pCaBS-γ as a vector, the silencing vector pCaBS-AaHDS-CHS was constructed by homologous recombination and transformed into Agrobacterium;

[0029] S2. Obtaining gene silenced plants: When the spathe of the Anthurium plant has not yet opened, inject the Agrobacterium solution containing the silencing vector into the peduncle and culture until the spathe opens;

[0030] S3. Obtaining aromatic plants: When the spathe is unfolded, observe the color of the spathe. Plants whose color becomes lighter are silent plants, that is, plants with silent anthurium aroma genes.

[0031] Preferably, the primers for constructing the silencing vector are upstream primer: 5ˊ-caggtcgactctagaggatccATGGCTACTGGCACAGTCCCG-3ˊ, and downstream primer: 5ˊ-cgatcggggaaattcgagctcTTCTTCTGTTGGTGGATCAACCC-3ˊ.

[0032] The present invention has the following beneficial effects:

[0033] The present invention clones a 17264bp AaHDS gene in the anthurium genome. Studies have shown that the gene is a key gene for controlling aroma, with the highest expression level in the anthurium spadix, which is 15 times that in the root, followed by the spathe, with an expression level of 3.8 times that in the root. Expression analysis was performed in the fragrant anthurium through gene silencing technology, showing that the expression level of the AaHDS gene would be reduced after silencing, while also inhibiting the synthesis of anthurium monoterpenoid compounds, and reducing the content of α-pinene, β-pinene and eucalyptol; it was shown that silencing the AaHDS gene significantly reduced the content of anthurium aroma components, regulated the intensity of the aroma of the fragrant anthurium, and could be used to cultivate more new varieties of fragrant anthurium with pleasant aroma, which is beneficial to the genetic improvement of plant aroma traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The nucleotide sequence alignment of the AaHDS gene of fragrant and non-fragrant anthurium.

[0035] Figure 2 This is a sequence alignment of the amino acids of fragrant and non-fragrant anthurium.

[0036] Figure 3 This is a diagram showing the differences in the AaHDS gene structure between fragrant and non-fragrant anthuriums (Note: red arrows indicate intron fragments with different bases).

[0037] Figure 4 This is a graph showing the expression levels of the AaHDS gene in different tissues of Anthurium andraeanum.

[0038] Figure 5 The AaHDS gene silencing vector of Anthurium andraeanum and its identification results (a. Schematic diagram of the structure of the AaHDS gene silencing vector; b. PCR identification results of the AaHDS gene silencing vector).

[0039] Figure 6 To silence Anthurium andraeanum plants and spathes (a. injected with Agrobacterium containing an empty vector control; b. injected with Agrobacterium containing the pCaBS-AaHDS-CHS vector).

[0040] Figure 7 is the expression level of AaHDS gene in the spadix of control and silenced plants (Note: * indicates p value < 0.05, the relative expression level between control plants and silenced plants is significantly different).

[0041] Figure 8 The components and contents of aroma substances in the spadix of the control and AaHDS silenced plants (Note: * indicates p value < 0.05, ** indicates p value < 0.01, and the relative contents between the control plants and the silenced plants are significantly different). DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0043] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0044] The anthurium plants used in this example are the fragrant variety A.'Mystral' and the non-fragrant variety A.'Alabama', both of which are provided by Guangzhou Flower Research Center.

[0045] Example 1 Cloning and Analysis of Anthurium andraeanum AaHDS Gene

[0046] 1. DNA extraction

[0047] Tender leaves of the fragrant variety Anthurium A.'Mystral' and the non-fragrant variety Anthurium A.'Alabama' were taken and fully ground in liquid nitrogen, and DNA was extracted using the modified CTAB method for later use.

[0048] 2. DNA sequence acquisition

[0049] According to the genome data of Anthurium, several pairs of gene cloning primers were designed using Primer 5.0, as shown in Table 1, and the full length of DNA was cloned in segments. PCR amplification was performed using DNA extracted from Anthurium A.'Mystral' and A.'Alabama' as templates, respectively. The PCR reaction system and reaction procedure are shown in Tables 2 and 3.

[0050] Table 1 Primer sequences used for gene cloning

[0051]

[0052] Table 2 PCR reaction system

[0053]

[0054] Table 3 PCR reaction program

[0055]

[0056] The amplified products were then subjected to 1% agarose gel electrophoresis using Novozymes GelDNAExtraction Mini Kit was used to recover DNA, and the recovered fragments were ligated with pTOPO001 (Jinsha Biotechnology) cloning vector. The ligation products were transformed into competent Escherichia coli DH5α, DH5α was cultured, and single positive colonies were selected. After expansion and culture, they were sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. DNASTAR software was used to splice the sequencing results.

[0057] After cloning and sequencing, a 17264bp gene was amplified from the fragrant variety A.'Mystral' DNA and named AaHDS gene. The ORF nucleotide sequence of the gene is shown in SEQ ID NO.1; the gene encodes 745 amino acids, and its amino acid sequence is shown in SEQ ID NO.2, with a relative molecular mass of 82877.04Da.

[0058] Further comparison of the sequences of fragrant and non-fragrant Anthurium varieties revealed the following results: Figure 1As shown, the gene DNA sequences of fragrant and non-fragrant anthuriums are different: the gene ORF sequences of the fragrant variety A.'Mystral' and the non-fragrant variety A.'Alabama' are both 17264bp long, and are composed of 19 exons and 18 introns. The exon sequence length is 2238bp, and there are 9 base differences, which lead to different amino acids at positions 4, 127 and 556 ( Figure 2 ). The differences in the introns mainly include: 15 base differences in the first intron; 10 base differences in the second intron, 5 base differences in the third intron, 1 base difference in the fourth intron, 2 base differences in the seventh intron, 4 base differences in the eighth intron, and 2 base differences in the 14th intron ( Figure 3 ).

[0059] 3. Gene expression analysis

[0060] The expression of the AaHDS gene cloned above was analyzed. The roots, stems, leaves, spathes and spadixes of the fragrant variety A.'Mystral' and the non-fragrant variety A.'Alabama' were used as materials. The total RNA was extracted using the column-based universal plant RNA extraction kit OmniPlant RNAKit (DNase I), and then converted into cDNA using Vazyme's HiScript III 1stStrand cDNASynthesis Kit. The expression level was detected by qPCR. The reaction procedure and reaction system are shown in Tables 4 and 5 below. The relative expression of the gene was measured using 2 -ΔΔCt Analysis was conducted to explore the expression level of this gene in different tissues of Anthurium.

[0061] Table 4 qPCR reaction procedure

[0062]

[0063] Table 5 qPCR reaction system

[0064]

[0065] The results are as follows Figure 4 As shown, this gene is a key gene controlling aroma. Its expression level is highest in the spadix of Anthurium, which is 15 times that in the root, followed by the spathe, which is 3.8 times that in the root. The main release site of Anthurium aroma is the spadix. The high expression of this gene in the spadix may be related to the formation of aroma substances.

[0066] Example 2 Silencing of Anthurium andraeanum genes and their effects

[0067] 1. Construction of silencing vector

[0068] The AaHDS gene cloned in Example 1 was silenced by VIGS technology, and primers for constructing the silencing vector were designed, as shown in Table 6. Homologous recombination technology was used to construct the silencing vector pCaBS-AaHDS-CHS, wherein CHS is chalcone synthase, which is used as a marker gene. Fading indicates that the system is successful, and floral fragrance components can be detected. The schematic diagram of the silencing vector pCaBS-AaHDS-CHS structure is shown in Figure 5 As shown in a, the constructed vector was then transferred into Agrobacterium (for specific methods, refer to: Yuan Cheng, 2018, Application of gene silencing induced by barley stripe mosaic virus in plant gene function analysis). Colonies were selected for PCR identification, and bacterial liquid with the correct fragment size was selected and sent to the company for sequencing. Figure 5 As shown in b. The sequencing results showed that the inserted fragment was consistent with the inserted sequence, confirming that the vector was successfully constructed.

[0069] Table 6 Primer sequences in silencing vector construction

[0070]

[0071] 2. Anthurium obtained after gene silencing

[0072] A.'Mystral' plants were used. When the spathe had not yet unfolded, the bacterial solution containing the silencing vector was injected into the inflorescence peduncle. At the same time, the control group was injected with Agrobacterium containing an empty vector (pCaBS-γ). The injections were continued for 5 days, 1 mL per day. After injection, the plants were cultured in an incubator at 20°C, and the color of the spathe was observed during the spathe unfolding period.

[0073] The results are as follows Figure 6 Shown are VIGS silencing of the AaHDS gene and CHS-induced spathe fading.

[0074] Then, the spadix of the plants with lighter spathe color was taken for gene expression analysis. Total RNA was extracted from the spadix of Anthurium andraeanum and reverse transcribed into cDNA. qPCR detection was performed. The reaction procedure and reaction system are shown in Table 4 and Table 5. The relative expression of genes was measured by 2 -ΔΔCt The expression level of AaHDS gene in the spadix of silenced plants was analyzed.

[0075] qPCR test results Figure 7 As shown, the expression level of AaHDS gene in the spadix of silenced A.'Mystral' was reduced by 49.1% compared with the material infected with pCaBS-γ.

[0076] 3. Changes in the aroma of Anthurium after gene silencing

[0077] The GC-MS method was used, the GC model was Agilent 7890A, the MS model was Agilent 5975C, and the chromatographic column model was Agilent DB-5ms (30m×0.25mm). The specific detection method is referenced to: Wang Yue. Preliminary study on the molecular genetic mechanism of aroma traits of Anthurium [D]. Guangzhou: South China Agricultural University, 2021. The aroma components of the spadix of silent plants and the spadix of empty plants were determined, and the contents of aroma compounds monoterpenoids such as eucalyptol, β-pinene, and α-pinene were determined.

[0078] The results are as follows Figure 8 As shown, the content of monoterpenoid compound eucalyptol in the spadix injected with pCaBS-γ bacterial solution was 2.20 μg·gFW -1 h -1 , β-pinene content is 0.171μg·gFW -1 h -1 , α-pinene content is 0.078μg·gFW -1 h -1 ; In the spadix injected with pCaBS-AaHDS-CHS solution, the content of eucalyptol was 1.26 μg·gFW -1 h -1 , β-pinene content is 0.136μg·gFW -1 h -1 , α-pinene content is 0.045μg·gFW -1 h -1 The fragrance of anthurium becomes lighter, indicating that the AaHDS gene is the key gene controlling the fragrance. Silencing this gene significantly reduces the content of aroma components in anthurium and regulates the intensity of the fragrance of the fragrant anthurium. It can be used to cultivate more anthuriums with suitable, elegant and fresh fragrance, which is beneficial to the genetic improvement of plant aroma traits.

[0079] Example 3 A method for regulating the aroma of anthurium

[0080] S1. Construction of pCaBS-AaHDS-CHS silencing vector: Using pCaBS-γ as a vector, the pCaBS-AaHDS-CHS silencing vector was constructed by homologous recombination and transferred into Agrobacterium;

[0081] S2. Obtaining gene-silenced plants: Before the spathe of the anthurium plant unfolds, inject the Agrobacterium solution containing the silencing vector into the inflorescence peduncle, and continue the injection for 5 days. Place the injected plants in an incubator and culture them at 22°C until the spathe unfolds. When the spathe unfolds, observe the color of the spathe. If the color becomes lighter, it is a silenced plant. Its fragrance becomes lighter and has a suitable fragrance, with a light and fresh fragrance, which changes the aroma traits of the plant.

[0082] In summary, the present invention cloned an AaHDS gene with a DNA full length of 17264bp in the genome of the fragrant anthurium, which encodes 745 amino acids, and its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. Studies have shown that this gene is a key gene controlling the aroma of anthurium, and the gene is expressed in the spadix of anthurium, which is 15 times that in the root, followed by the spathe, and the expression is 3.8 times that in the root. After gene silencing, expression analysis in the fragrant anthurium showed that gene silencing would reduce expression, and at the same time inhibit the synthesis of monoterpenoid compounds in anthurium, and reduce the content of α-pinene, β-pinene and eucalyptol. Silencing the AaHDS gene significantly reduces the content of anthurium aroma components, regulates the intensity of the fragrance of the fragrant anthurium, and can be used to cultivate more elegant, fresh, and pleasant new varieties of fragrant anthurium, which is beneficial to the genetic improvement of plant aroma traits.

[0083] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Anthurium andraeanum AaHDS gene, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

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

2.

3. A recombinant expression vector, characterized in that: Contains the gene according to claim 1.

4. A genetically engineered bacterium, characterized in that: Contains the recombinant expression vector according to claim 3.

5. Use of the gene according to claim 1 in regulating the aroma or aroma compound synthesis of Anthurium.

6. Use of the gene according to claim 1 in breeding of anthurium andraeanum aroma plants.

7. Use of an agent for silencing or inhibiting the expression of the gene according to claim 1 in reducing the aroma of anthurium or reducing the synthesis of aroma compounds of anthurium.

8. Use of a preparation for silencing or inhibiting the expression of the gene according to claim 1 in the preparation of a product for regulating the aroma of anthurium.

9. A product for regulating the aroma of anthurium, characterized in that: A preparation containing the gene silenced or inhibited according to claim 1, or a silencing vector or engineered bacteria containing the gene according to claim 1.

10. A method for regulating the aroma of anthurium or a method for cultivating anthurium plants with a light aroma, characterized in that: Silencing or inhibiting the expression of the gene of claim 1 in anthurium or using the product of claim 9 to treat anthurium.

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