Anthurium andraeanum aahds gene and application thereof in regulating aroma

Abstract

The application discloses an AaHDS gene of ananas comosus and application of the AaHDS gene in regulating aroma, and belongs to the field of genetic engineering of plants. The AaHDS gene of the ananas comosus is obtained by PCR amplification and cloning, the DNA sequence of the gene in the ananas comosus with aroma and the ananas comosus without aroma has obvious difference, and the expression amount of the gene is the highest in the inflorescence. The VIGS technology is used to silence the gene, the expression amount of the gene is reduced by 49.1 %, and the contents of alpha-pinene, beta-pinene and eucalyptol are significantly reduced. The AaHDS gene cloned by the application is beneficial to genetic improvement of plant aroma characteristics, and can be used for cultivating more new varieties of the ananas comosus with pleasant aroma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of genetic engineering and plant breeding technology. More specifically, it relates to Anthurium andraeanum AaHDS gene and its application in regulating aroma. BACKGROUND

[0002] Anthurium andraeanum is a perennial evergreen herb of Araceae Anthurium, which is famous for its unique shape of spadix, bright and diverse flower color, and long ornamental period. It is a famous cut flower and pot flower plant in the world. Flower fragrance is an important breeding target trait of ornamental plants. There are few anthurium varieties with fragrance on the current market, and more fragrant anthuriums are bred as the target of anthurium fragrance breeding. Therefore, breeding more new anthurium varieties with different fragrances has important significance for further improving the competitiveness and benefits of China's anthurium industry and promoting the high-quality development of the anthurium industry.

[0003] Flower scent is an important feature of ornamental plants, not only to attract customers and improve the commercial potential of ornamental plants, but also to attract pollinators and improve disease resistance. Flower scent 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 / benzenoids, and fatty acid derivatives. Among them, terpenes are the largest class of plant volatile organic compounds, which are synthesized by cytoplasmic mevalonate (MVA) pathway and plastidial methylerythritol phosphate (MEP) pathway. Scented anthurium plants release fragrance and volatile compounds during the pistil stage of flower development, and the main released compounds include 1,8-cineole, α, β-pinene, sabinene, myrcene and limonene, as well as some benzene compounds. Due to the great difference in flower scent of anthurium varieties, some of them release more volatile compounds. In 1999, the artificial smelling method was used to classify the types of scent released by scented anthurium and its hybrid F1 generation, mainly sweet, spicy, pungent, fruity, orange, pine and mint, etc. (Kuanprasert, 1998). Croatian people observed that the smell of several native female anthuriums ranged from pleasant to unpleasant. A more extensive investigation of 147 anthurium species and hybrids collected by the University of Hawaii and the Missouri Botanical Garden germplasm resources showed that 76% of the plants released a smell from flower scent to fishy or foul odor, from very weak to very strong (Kuanprasert, 1998). The scent of most anthuriums is relatively strong, pungent, not very pleasant, and long-term smelling can cause dizziness. For example, anthurium A. 'Armeniense' (with strong sweet smell) and A. 'Mystral' (with strong sweet smell). Therefore, it is particularly important to adjust the intensity of the scent of scented anthuriums. Existing researches take adjusting the intensity of the scent of scented anthuriums as a new breeding goal, which is of great significance for cultivating more scented anthuriums with suitable, delicate and pleasant smell.

[0004] So far, there are few studies on the flower fragrance components and formation mechanism of Anthurium andraeanum. Through the analysis of the main aroma components of Anthurium andraeanum, the expression level of the aroma synthesis genes of the fragrant varieties is significantly higher than that of the non-fragrant varieties, including AaDXS, AaDXR, AaMDS, AaHDS, AaTPS, AaDAHPS, AaADT2, AaPAL1 and AaPAL2. At present, a plurality of Anthurium andraeanum aroma synthesis genes have been obtained, but their functions have not been verified, and whether they can be used for flower fragrance regulation of the fragrance type Anthurium andraeanum and Anthurium andraeanum flower fragrance breeding is unknown. Therefore, carrying out and excavating more cloning and function researches of Anthurium andraeanum aroma regulation genes has very important significance for promoting the development of Anthurium andraeanum transgenic breeding, breaking through the 'neck' technology of Anthurium andraeanum aroma breeding, continuously cultivating new flower fragrance Anthurium andraeanum varieties with independent intellectual property rights and market competitiveness, quickly narrowing the gap between China's Anthurium andraeanum breeding and foreign countries, and promoting the efficient and sustainable development of Anthurium andraeanum industry.

[0005] Reference: Kuanprasert et al, A.R. Kuehnle, C.S. Tang, Floral fragrance compounds of some anthurium (ARACEAE) species and hybrids, Phytochemistry, Volume 49, Issue 2, 1998. SUMMARY

[0006] The technical problem to be solved by the present application is to overcome the defects of the existing fragrance type Anthurium andraeanum that the aroma is pungent, and the lack of existing Anthurium andraeanum aroma breeding gene resources, and to provide an Anthurium andraeanum AaHDS gene and its application in aroma regulation.

[0007] The first object of the present application is to provide an Anthurium andraeanum aroma gene AaHDS.

[0008] The second object of the present application is to provide a coding protein of the Anthurium andraeanum aroma gene AaHDS.

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

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

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

[0012] The sixth object of the present application is to provide an application of a preparation for silencing or inhibiting the expression of the Anthurium andraeanum aroma gene.

[0013] The seventh object of the present application is to provide a product for regulating the aroma of Anthurium andraeanum.

[0014] An eighth object of the present application is to provide a method for regulating the aroma of an anthurium.

[0015] The above objects of the present application are achieved by the following technical solutions.

[0016] The present application clones an anthurium AaHDS gene from an anthurium genome, the full-length DNA sequence of the gene is 17264 bp, encoding 745 amino acids, the ORF nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. Research shows that the gene is a key gene for controlling aroma, and the expression amount in an anthurium inflorescence is the highest, being 15 times that in roots, and the expression amount in a spathe is 3.8 times that in roots. Through gene silencing technology, expression analysis in an aroma anthurium shows that the expression amount is reduced after silencing the AaHDS gene, and the synthesis of monoterpenes in the anthurium is also inhibited, reducing the contents of alpha-pinene, beta-pinene and eucalyptol; the content of aroma components in the anthurium is significantly reduced after silencing the AaHDS gene, indicating that the gene is a key gene for regulating the synthesis of aroma compounds in the anthurium, and can be used for cultivating more new varieties of aroma anthurium with pleasant aroma, and is beneficial to the genetic improvement of plant aroma traits.

[0017] Therefore, the present application provides an anthurium AaHDS gene, and the nucleotide sequence thereof is shown as SEQ ID NO. 1.

[0018] The present application provides a coding protein of an anthurium AaHDS gene, and the amino acid sequence thereof is shown as SEQ ID NO. 2.

[0019] The present application provides a recombinant expression vector containing the above-mentioned anthurium AaHDS gene.

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

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

[0022] Further, the aroma compound is a monoterpene compound, and the contents of eucalyptol, beta-pinene and alpha-pinene in the anthurium are reduced after silencing the anthurium AaHDS gene.

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

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

[0025] The application provides a product for regulating the aroma of an anthurium, which contains a preparation for silencing or inhibiting the expression of an anthurium AaHDS gene or a silencing vector containing an anthurium aroma gene or an engineering bacterium.

[0026] The application provides a method for regulating the aroma of an anthurium or a method for cultivating an anthurium plant with light aroma, which comprises the following steps: silencing or inhibiting the expression of an anthurium AaHDS gene or treating an anthurium with the product for regulating the aroma of an anthurium.

[0027] Further, the method specifically comprises the following steps:

[0028] S1. Constructing a silencing vector: a pCaBS-gamma vector is used to construct a silencing vector pCaBS-AaHDS-CHS through a homologous recombination method, and the silencing vector is transferred into an agrobacterium;

[0029] S2. Obtaining a gene-silenced plant: when a spathe of the anthurium plant has not been unfolded, an agrobacterium liquid containing the silencing vector is injected into a flower stalk, and the anthurium is cultured until the spathe is unfolded;

[0030] S3. Obtaining an aroma plant: when the spathe is unfolded, the color of the spathe is observed, and the plant with light color is a silenced plant, i.e., a plant with a silenced anthurium aroma gene.

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

[0032] The application has the following beneficial effects:

[0033] The application clones an AaHDS gene with a length of 17264bp from an anthurium genome. Research shows that the gene is a key gene for controlling aroma, and the expression amount thereof in a spadix inflorescence is the highest, 15 times that in roots, and the expression amount thereof in a spathe is 3.8 times that in roots. Through a gene silencing technology, expression analysis is performed on an aroma anthurium, and it is shown that the AaHDS gene silencing can reduce the expression amount thereof, and also inhibit the synthesis of monoterpenes in the anthurium, and reduce the content of alpha-pinene, beta-pinene and eucalyptol; it is shown that the AaHDS gene silencing can significantly reduce the content of aroma components in the anthurium, and regulate the light and dark degree of the aroma of the aroma anthurium, and the application can be used for cultivating more new varieties of aroma anthurium with pleasant aroma, and is beneficial to the genetic improvement of the aroma trait of a plant. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 Nucleotide sequence alignment of AaHDS genes of anthuriums with aroma and without aroma.

[0035] Figure 2 Sequence alignment of amino acids of AaHDS from fragrant and non-fragrant A. belotti.

[0036] Figure 3 Figure 4 is a diagram showing the differences in AaHDS gene structure between fragrant and non-fragrant A. belotti (Note: the red arrow indicates the intron fragment with base difference).

[0037] Figure 4 Figure 5 is a diagram showing the expression amount of AaHDS gene in different tissues of A. belotti.

[0038] Figure 5 Figure 6 is a diagram showing the AaHDS gene silencing vector of A. belotti and the identification results (a. AaHDS gene silencing vector structure diagram; b. AaHDS gene silencing vector PCR identification results).

[0039] Figure 6 Figure 7 is a diagram showing the A. belotti plants and spathes after silencing (a. injection of Agrobacterium containing empty control; b. injection of Agrobacterium containing pCaBS-AaHDS-CHS vector).

[0040] Figure 7 Figure 8 is a diagram showing the expression amount of AaHDS gene in the inflorescence of control and silenced plants (Note: * indicates that the p value is less than 0.05, and the relative expression amount of the control plant and the silenced plant is significantly different).

[0041] Figure 8 Figure 9 is a diagram showing the aroma substance composition and content in the inflorescence of control and silenced AaHDS plants (Note: * indicates that the p value is less than 0.05, and ** indicates that the p value is less than 0.01, and the relative content of the control plant and the silenced plant is significantly different). DETAILED DESCRIPTION

[0042] The present application will be further described in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

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

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

[0045] Example 1 Cloning and analysis of AaHDS gene of A. belotti

[0046] 1. DNA extraction

[0047] Fresh leaves of A. 'Mystral' and A. 'Alabama' were collected and ground in liquid nitrogen. DNA was extracted by modified CTAB method and stored at -20℃.

[0048] 2. DNA sequence acquisition

[0049] According to the genome data of A. hybridus, a number of pairs of primers were designed by Primer 5.0 for cloning the full-length DNA of the gene, as shown in Table 1. The extracted DNA of A. 'Mystral' and A. 'Alabama' was used as the template for PCR amplification, and the PCR reaction system and reaction procedure are shown in Table 2 and Table 3.

[0050] Table 1 Primer sequences used for gene cloning

[0051]

[0052] Table 2 PCR reaction system

[0053]

[0054] Table 3 PCR reaction procedure

[0055]

[0056] Subsequently, the amplification products were subjected to 1% agarose gel electrophoresis, and the DNA was recovered by using the QIAGEN Gel DNA Extraction Mini Kit. The recovered fragments were ligated with the pTOPO001 cloning vector, and the ligation products were transformed into competent E. coli DH5a. The DH5a was cultured, and single positive colonies were selected and expanded for sequencing by Shengwu Bioengineering (Shanghai) Co., Ltd. The sequencing results were subjected to sequence splicing by using the DNASTAR software. After cloning and sequencing, a 17264 bp gene was amplified from the DNA of the fragrant variety A. 'Mystral', which was named AaHDS gene. The ORF nucleotide sequence of the gene is shown in SEQ ID NO. 1. The gene encodes 745 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2, with a relative molecular mass of 82877.04 Da.

[0057] Further comparison of the sequences of the fragrant and non-fragrant varieties of A. hybridus showed that the AaHDS gene of the fragrant variety A. 'Mystral' had a 1 bp insertion at the 163rd nucleotide, which resulted in a 1 aa insertion at the 54th aa of the AaHDS protein.

[0058] Figure 1 ​As shown, the DNA sequences of fragrant and unfragrant Anthuriums differ: the ORF sequences of both the fragrant variety A.'Mystral' and the unfragrant variety A.'Alabama are 17264 bp in length, consisting of 19 exons and 18 introns. The exon sequences are both 2238 bp in length, with 9 base differences, leading to differences in 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 fourteenth intron. Figure 3 ).

[0059] 3. Gene expression analysis

[0060] The expression of the cloned AaHDS gene was analyzed. Roots, stems, leaves, spathes, and spadixes of the fragrant variety A.'Mystral' and the non-fragrant variety A.'Alabama were used as materials. Total RNA was extracted using the OmniPlant RNA Kit (DNase I) column method. The RNA was then reversed into cDNA using the Vazyme HiScript III 1st Strand cDNA Synthesis Kit. Expression levels were detected by qPCR. The reaction procedures and systems are shown in Tables 4 and 5 below. Relative gene expression levels were analyzed using a 2-1 -ΔΔCt We conducted an analysis to investigate the expression level of this gene in different tissues of Anthurium.

[0061] Table 4 qPCR reaction procedures

[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 higher than that in the root. The expression level is also 3.8 times higher in the spathe. 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 the Anthurium gene and its effects

[0067] 1. Silencing vector construction

[0068] The AaHDS gene cloned in Example 1 was silenced by VIGS technology, and the primers for silencing vector construction were designed as shown in Table 6. A silencing vector pCaBS-AaHDS-CHS was constructed by homologous recombination technology, wherein CHS is chalcone synthase, which is used as a marker gene, and fading indicates the success of the system, which can detect floral fragrance components. The structure of the silencing vector pCaBS-AaHDS-CHS is shown in Figure 5 a, and then the constructed vector was transformed into Agrobacterium (for details, see: Yuan C, 2018, Application of barley mosaic virus-induced gene silencing in plant gene function analysis). The colonies were picked and identified by PCR, and the bacterial liquid with correct fragment size was sent for sequencing as shown in Figure 5 b. The sequencing results showed that the inserted fragment was consistent with the inserted sequence, and the vector construction was successful.

[0069] Table 6 Primer sequences in silencing vector construction

[0070]

[0071] 2. Obtaining A. 'Mystral' after gene silencing

[0072] A. 'Mystral' plants were used, and when the spathe had not yet unfolded, bacterial liquid containing the silencing vector was injected into the inflorescence peduncle, and Agrobacterium containing empty vector (pCaBS-γ) was injected as a control group. The injection was performed continuously for 5 days, 1 mL per day, and the plants were cultured in a 20°C incubator after injection. The color of the spathe was observed during the spathe unfolding period.

[0073] The results are shown in Figure 6 , which shows that VIGS silencing of AaHDS gene and CHS causes the spathe to fade.

[0074] Subsequently, the spathe color of the plant was taken, and gene expression analysis was performed on the inflorescence. The total RNA was extracted from the inflorescence of A. 'Mystral', and the cDNA was reverse transcribed. qPCR detection was performed, and the reaction program and reaction system are shown in Tables 4 and 5. The relative expression of the gene was analyzed by 2 -ΔΔCt The expression of AaHDS gene in the inflorescence of the silenced plant was analyzed.

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

[0076] 3. Changes in fragrance of A. 'Mystral' after gene silencing

[0077] The GC-MS method is used, the GC model is Agilent 7890A, the MS model is Agilent 5975C, the chromatographic column model is Agilent DB-5ms (30m*0.25mm), and the specific detection method is referred to: Wang Yue. Preliminary study on the molecular genetic mechanism of the aroma characteristics of anthurium[D]. Guangzhou: South China Agricultural University, 2021. The aroma components of the inflorescence of the silenced plant and the unloaded plant are determined, and the contents of the aroma compounds, such as eucalyptol, β-pinene and α-pinene, are determined.

[0078] The results are shown in Figure 8 , indicating that in the inflorescence of the plant injected with pCaBS-γ bacterial solution, the content of monoterpenes eucalyptol is 2.20 μg·gFW -1 h -1 , the content of β-pinene is 0.171 μg·gFW -1 h -1 , and the content of α-pinene is 0.078 μg·gFW -1 h -1 ; while in the inflorescence of the plant injected with pCaBS-AaHDS-CHS bacterial solution, the content of eucalyptol is 1.26 μg·gFW -1 h -1 , the content of β-pinene is 0.136 μg·gFW -1 h -1 , and the content of α-pinene is 0.045 μg·gFW -1 h -1 ; the aroma of anthurium becomes weak, indicating that AaHDS gene is a key gene for controlling aroma, silencing the gene significantly reduces the content of aroma components of anthurium, and adjusting the aroma intensity of the aroma type anthurium can be used to cultivate more aroma suitable, delicate and fresh aroma type anthurium, which is beneficial to the genetic improvement of plant aroma characteristics.

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

[0080] S1. Construct pCaBS-AaHDS-CHS silencing vector: use pCaBS-γ as vector, construct pCaBS-AaHDS-CHS silencing vector by homologous recombination, and transfer into Agrobacterium;

[0081] S2. Obtain gene silenced plant: inject Agrobacterium containing silencing vector into inflorescence stem when spathaceous bract of anthurium plant has not yet unfolded, continuously inject for 5 days, and place the injected plant in incubator at 22℃ until the spathaceous bract unfolds; observe the color of the spathaceous bract when the spathaceous bract unfolds, and the color becomes weak, the aroma becomes weak, the aroma is suitable, and the aroma is delicate and fresh, which changes the plant aroma characteristics.

[0082] In conclusion, the application clones a DNA full length of 17264bp AaHDS gene from the genome of the fragrant Anthurium andraeanum, the gene encodes 745 amino acids, the nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence is shown as SEQ ID NO. 2. Research shows that the gene is a key gene for controlling the fragrance of Anthurium andraeanum, the expression amount of the gene in the Anthurium andraeanum inflorescence is the highest, which is 15 times of that in the root, and the expression amount of the gene in the spathe is 3.8 times of that in the root. Through gene silencing, expression analysis in the fragrant Anthurium andraeanum shows that the gene silencing can reduce the expression, and also inhibit the synthesis of monoterpenes in Anthurium andraeanum, reduce the content of alpha-pinene, beta-pinene and eucalyptol, and significantly reduce the content of the fragrance components of Anthurium andraeanum by silencing the AaHDS gene, so as to adjust the fragrance intensity of the fragrant Anthurium andraeanum, and cultivate more new varieties of fragrant Anthurium andraeanum with delicate and fresh fragrance, which is beneficial to the genetic improvement of the plant fragrance trait.

[0083] The above embodiment is a preferred embodiment of the application, but the embodiments of the application are not limited by the above embodiment, and any change, modification, replacement, combination, simplification made without departing from the spirit and principle of the application should be an equivalent replacement mode, and all are included in the protection scope of the application.

Claims

1. Zantedeschia aethiopica 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 of claim 1 ， characterized in that its amino acid sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector, characterized in that, comprising the gene of claim 1.

4. A genetically engineered bacterium, characterized by, comprising the recombinant expression vector of claim 3.

5. Use of the gene of claim 1 in regulating the synthesis of aroma or aroma compounds of an anthurium, characterized in that, said regulation is silencing or inhibiting the expression of the gene of claim 1.

6. Use of the gene of claim 1 in breeding of an anthurium fragrant plant, characterized in that, silencing or inhibiting the expression of the gene of claim 1.

7. Use of an agent that silences or inhibits the expression of the gene of claim 1 in reducing the aroma of an Anthurium or in reducing the synthesis of an aroma compound of an Anthurium.

8. Use of a preparation that silences or suppresses the expression of the gene according to claim 1 in the manufacture of a product for regulating the aroma of an anthurium, characterized in that, said regulation is silencing or inhibiting the expression of the gene of claim 1; and said agent is a silencing vector of the gene of claim 1.

9. A product for regulating the aroma of Anthurium andraeanum, characterized by, an agent that silences or inhibits the gene of claim 1, said agent comprising a silencing vector of the gene of claim 1.

10. A method for regulating the aroma of an Anthurium or a method for cultivating a low-scent type Anthurium plant, characterized by, silencing or inhibiting the expression of the gene of claim 1 in an Anthurium, or treating an Anthurium with the product of claim 9.

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