TW202111116A - Dna construct and method for silencing phytoene synthase gene expression in a specific plant tissues - Google Patents

Abstract

The invention discloses a DNA construct and method for silencing or reducing the expression level of phytoene synthase gene in a specific plant tissue. The DNA construct comprise a nucleotide sequence to control the mRNA expression of the phytoene synthase gene in a specific tissue. The invention is used to regulate the color, odor, or other plant physiological mechanisms which require carotenoid products.

Description

DNA construct and method for silencing phytoene synthase gene expression in specific plant tissues

The present invention relates to the silencing of phytoene synthase (PSY) gene expression to control the field of oncidium orchid color and the nucleotide fragment composition and method for regulating PSY gene expression, and more specifically It is about the production of RNA interference fragments in specific tissues by gene transfer of a DNA cassette, therefore down-regulating the expression of PSY gene in specific tissues of plants.

Oncidium is one of Taiwan’s main export cut flowers. 85% of Taiwan’s cut flowers are exported to other countries. Japan is the main export market for Taiwan’s cut flowers. Oncidium cut flowers are also sold in Taiwan. It is the first place in the total input volume of cut heart orchid flowers in Japan. However, the single variety is one of the serious difficulties in the current cut flower Oncidium industry. Therefore, the vision of the present invention is to create diversified and differentiated varieties, such as developing diversified patterns to meet the needs of different export countries and markets. , In order to open up a new vast market. The cut flowers of Taiwan Oncidium are mainly oncidium yellow flower. At present, the mainstream strains are all derived from Oncidesa Gower Ramsey through tissue culture. However, due to its origin from distant hybridization, the genome of the first hybrid obtained through distant mating has undergone major changes. The homologous chromosomes are unbalanced or even partially in the process of pairing. The chromosome deletion caused the Nanxi Oncidium to be unable to reproduce sexually. It requires tissue culture for seedling production, and it is also difficult to generate diversified new strains by traditional hybridization. In order to break through this phenomenon, the present invention uses RNA Interference technology uses gene transfer to develop different flower and color lines of Oncidium.

Color is one of the important characteristics of plant-based products. The flower color of higher plants is mainly produced by pigments such as betalains, flavonoids and carotenoids. The yellow lip part of the flower of the Nanxi strain Oncidium is composed of carotenoids, the main components are violaxanthin (violaxanthin), 9-cis-violaxanthin, neoxanthin (neoxanthin) and a small amount of lutein, and the upper part of the flower The reddish brown spots are composed of anthocyanins, the main components are cyanidin (cyanidin), delphinidin (delphinidin), and a small amount of peonidin (peonidin). Carotenoid biosynthesis mechanisms which plants, octahydro lycopene synthase (phytoene synthase, PSY) is a key enzyme determined rate, a two geranylgeranyl diphosphate to phytoene synthesis (octahydro lycopene), is the class The most upstream compound in the carotene biosynthesis pathway is followed by phytoene desaturase and ζ-carotene desaturase to form lycopene (lycopene), which is further catalyzed by downstream enzymes to form various products. Therefore, phytoene synthase is one of the important enzymes in the carotenoid production pathway. By inhibiting the expression of the phytoene synthase gene ( OgPSY ), the production of related carotenoids can be inhibited, thereby regulating flower color.

The principle of RNA interference or RNAi was first discovered by Fire et al. They injected double-stranded RNA (dsRNA) into the nematode ( Caenorhabidites elegan) and found that the expression of certain genes in the worm was blocked. In plants, the use of antisense RNA to change the pathway of flower color generation to create flowers of different colors, such as the use of antisense RNA to inhibit the gene expression of chalcone synthase ( PhCHS ) in Petunia hybrids to produce white petunia (Van der Krol et al., 1990); such as the use of antisense RNA to inhibit the expression of the chalcone synthase ( GCHS ) gene of Gerbera hybrida to create an orange gerbera. So far, the use of RNAi interference technology to adjust carotenoids to change flower color has only appeared in genetically transformed chrysanthemums. Knockdown CCD4 turns white chrysanthemums into bright yellow flower colors. In addition, since carotenoids are important elements for plant growth, if they are severely lacking, plants will face many growth and development problems, such as photosynthesis. Therefore, appropriate promoters should be selected to drive and regulate the catalysis in the production of carotenoid pigments. Enzyme genes, such as phytoene desaturase, Lycopene ε-cyclase, or Lycopene β-cyclase and other enzyme genes, enable specific genes to be specifically expressed in specific tissues, only to achieve the purpose of changing the color of the flowers, but still maintain the physiology of normal plant growth Function, this is an important key to technology.

The purpose of the present invention is to use a specific promoter to generate interfering RNA fragments to attenuate or inhibit the expression of the phytoene synthase gene in Oncidium flowers, thereby reducing the production of carotenoids, thereby achieving the purpose of changing the flower color. These applications of interfering RNA technology on ornamental plants are completely different from the aforementioned cases for regulating different genes and plant species.

The main purpose of the preferred embodiments of the present invention is to provide a method that can silence or reduce the expression of Oncidium phytoene synthase gene, and provide a DNA card that can silence Oncidium phytoene synthase gene The DNA cassette is a nucleotide sequence, and the RNA fragments transcribed from the nucleotide sequence can be used to regulate the content of message RNA (mRNA) of the phytoene synthase gene in Oncidium, So it can control the color, smell, Or other plant physiological mechanisms that require carotenoid products.

In order to achieve the above-mentioned purpose, the mRNA produced by the transcription of the nucleotide sequence of the present invention uses phytoene synthase mRNA as the target of complementary binding, because it binds to phytoene synthase mRNA into a double-stranded form. After RNA, the mRNA that interferes with phytoene synthase is further translated into protease, so the biosynthetic reaction of carotenoids is affected. The nucleotide sequence is a promoter and a DNA cassette capable of transcribing interfering RNA fragments. The promoter includes the nucleotide sequence of SEQ ID NO:1.

A preferred embodiment of the present invention provides a DNA cassette, which contains a promoter (P), and then a DNA fragment of SEQ ID NO: 3 is connected to the 3'end, and then an intron fragment containing the GUS gene is connected (intronGUS nucleotide sequence), and a DNA fragment of SEQ ID NO: 3 inverted, and finally connected with a terminating fragment of the NOS gene (T-NOS nucleotide sequence). The above-mentioned recombinant plastids or vectors include, but are not limited to, specific promoters, introns, or terminator fragments, or other recombinant plastids or vectors applicable to the present invention. Among them, a nucleotide sequence is placed after the promoter Pchrc . This nucleotide sequence includes two short nucleotide fragments of SEQ ID NO: 3 of about 150 nucleotides each, and the two are reversed. Arranged with a GUS-intron nucleotide fragment in the middle, this nucleotide sequence fragment can be transcribed to produce a hairpin RNA molecule under the drive of the promoter Pchrc. This RNA fragment has the target mRNA, such as the mRNA of the phytoene synthase gene, and forms a double-stranded RNA structure due to the nucleotide sequence complementation.

Encoding SEQ ID NO: 3 is a nucleotide sequence encoding SEQ ID NO: 2 with a length of 150 nucleotides from the 51st nucleotide.

A preferred embodiment of the present invention provides a nucleotide sequence, which includes encoding SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3; and SEQ ID NO: 1, SEQ ID NO: 2. A nucleotide sequence complementary to the sequence of SEQ ID NO: 3.

The nucleotide sequence of the preferred embodiment of the present invention is provided in a vector or a shuttle vector.

The nucleotide sequence of the preferred embodiment of the present invention is provided in a pseudo-protosphere

The vector in a preferred embodiment of the present invention can be introduced into a plant cell or a protosphere (PLB, Protocorm-like body) by transfection.

The nucleotide sequence of a preferred embodiment of the present invention is provided in a plant tissue.

In order to achieve the above objective, the method for down-regulating and silencing the Oncidium phytoene synthase gene of the preferred embodiment of the present invention includes: providing at least one DNA cassette, the nucleotide sequence of the DNA cassette includes a specific A promoter and a specific nucleotide fragment that can be transcribed into a hairpin interfering RNA fragment; introducing the nucleotide sequence into a plant cell (or PLB) to obtain a transgenic plant cell (or PLB); and using The transformed plant cell regenerates and develops into a plant, and achieves the purpose of changing the flower color of the plant.

The nucleotide sequence of the preferred embodiment of the present invention is obtained through gene cloning.

In a preferred embodiment of the present invention, the transformed plant cell is used to change the expression level of phytoene synthase in a plant.

The preferred embodiment of the present invention utilizes the transformed plant cells to regenerate and produce a transformed plant.

In a preferred embodiment of the present invention, the plant cell is selected from an orchid plant cell.

Figure 1. Schematic diagram of DNA cassette construction for silencing phytoene synthase gene Figure.

Figure 2. The DNA cassette construction diagram of silencing the phytoene synthase gene. LB/RB: left/right border of T-DNA, P: promoter

Figure 3. Flower photos, from left to right show the flowers of wild-type (WF) Oncidesa Honey Angel, the transformed plant Oncidesa MF-1, and the transformed plant Oncidesa MF-5.

Figure 4. The carotenoid content in flower tissue. The black icon shows the wild type (WF) Oncidesa Honey Angel, the gray icon shows the transformed plant Oncidesa MF-1, and the white one shows the transformed plant Oncidesa MF- 5. The four types of carotene shown on the horizontal axis are 9-cis-violaxanthin, neoxanthin, violaxanthin and β-carotene ).

Figure 5, using semi-quantitative real-time PCR analysis to analyze wild-type (WF) Oncidesa Honey Angel (a), the transformed plant Oncidesa MF-1 (b), and the transformed plant The expression level of the phytoene polymerase regulatory gene (OgPSY ) in each part of the plant Oncidesa MF-5(c). The abbreviations are: flowers (F), HPTII (hygromycin phosphotransferase II gene).

Figure 6, Northern blot analysis was used to analyze the expression levels of small RNA molecules (SiRNA) in various plant tissues. From left to right are the flower tissues and the transformed plants of Oncidesa MF-5. Flower tissue of the plant Oncidesa MF-1, the leaf tissue of the transgenic plant Oncidesa MF-5, and the flower tissue of the wild species (WF) Oncidesa Honey Angel.

In order to fully understand the present invention, preferred embodiments are exemplified below and described in detail with the accompanying drawings, and they are not intended to limit the present invention.

The Oncidium phytoene synthase gene of the preferred embodiment of the present invention is suitable for providing its gene product or its encoded protein, but it is not intended to limit the scope of the present invention. Furthermore, the Oncidium phytoene synthase gene and its regulation method according to the preferred embodiment of the present invention are suitable for gene transformation in various plant cells or pseudo-protospheres by various genetic engineering, but its It is not intended to limit the scope of the present invention.

For example, the oncidium used in the preferred embodiment of the present invention is a yellow-flowered variety (such as lemon green oncidium; Oncidesa Honey Angel), but the present invention is applicable to various other oncidium varieties, which is not intended to limit the present invention. The scope of the invention.

A preferred embodiment of the present invention is a technique for silencing the gene expression of Oncidium phytoene synthase, which is a DNA cassette that produces interfering RNA. The schematic diagram of the structure is shown in Fig. 2 to construct a transcribable DNA cassette. Combine the transfer DNA (T-DNA) cassette of the hairpin RNA, and then combine it in the vector. The DNA cassette contains a promoter and is connected to the 3'end of the nucleoside containing SEQ ID NO: 3 in the forward direction. The acid sequence, and the intronGUS intron fragment (intronGUS nucleotide sequence), followed by a nucleotide sequence inverted in the direction of SEQ ID NO: 3, and then followed by the T-NOS nucleotide sequence of the terminating fragment of the NOS gene. The aforementioned recombinant plastids or vectors include, but are not limited to, specific promoters or intron fragments, or other recombinant plastids or vectors applicable to the present invention.

1. Fishing the promoter from Oncidesa Honey Angel (Oncidesa Honey Angel).

The method is to use Oligotex mRNA Kit (Qiagen) to purify the total RNA of Lime Green Oncidium to obtain Poly(A)+RNA. According to the operating method provided by the Clontech manufacturer, using Rapid Amplification of cDNA Ends (RACE), a full-length DNA was amplified from the 5'end and 3'end of the cDNA to obtain Lime Green Oncidium chromoplast specific carotenoid The full-length cDNA nucleotide sequence of the associated protein ( CHRC) gene. The promoter selection method used in the present invention is to use molecular biology gene walking technology (GenomeWalker Universal Kit, Clontech) to amplify the promoter fragment Pchrc of this gene from CHRC cDNA, with a total of 1703 bases (Figure 1 ).

2. RNA interference target selection.

The homology analysis of the phytoene synthase nucleotide sequence of four monocot species (rice, wheat, corn, phalaenopsis) in the NCBI database was conducted, and a 150bp phytoene synthase regulatory gene was found The cDNA region (+51bp~+200bp of the open reading frame) contains a 22-bp conservative sequence with 100% homology. In the corresponding complementary DNA (cDNA) sequence, it is selected as the target nucleus for RNA interference Nucleotide sequence.

3. Gene transfer and cultivation methods.

The DNA cassette containing the present invention is constructed into a vector, and the Agrobacterium carrying the vector is used to infect the Protocorm-like body (PLB) of Onc. Honey Angel, and screened with a medium containing an appropriate concentration of antibiotics The transformed protospheres are then induced into complete plants with shooting and rooting media, and the plants are planted in pots for two years to observe their flower color. The above-mentioned transfer methods include but are not limited to: Agrobacterium vector method, genetic recombinant virus infection method, jumper vector transfer method, gene gun transfer method, electroporation, microinjection method, pollen tube method, liposome vector The method of colonization, the method of colonization with ultrasonic media, the method of colonization with silicon carbide fiber, etc. are familiar to those skilled in the art to which the present invention belongs; the method of orchid tissue culture is also familiar to those skilled in the art to which the present invention belongs, so it will not be repeated.

"Example 1: Oncidium orchid color control"

As shown in Figure 3, from left to right, the flowers of wild type (WF) Oncidesa Honey Angel, the transgenic strain Oncidesa MF-1 implanted with the nucleotide sequence of the DNA cassette of the present invention, and the implanted The transgenic plant Oncidesa MF-5 of the DNA cassette nucleotide sequence of the present invention can be seen that MF-1 has a pale white flower color, and MF-5 has a snow white flower color.

In order to maintain the phenotypic stability of the product, this example uses Murashige & Skoog (MS) medium containing a half concentration (1/2) of 30 ppm hygromycin (hygromycin) to cultivate the transgenic plants Oncidesa MF-1 and Oncidesa MF- 5 Tiny meristem buds at P _{0 stage.} After protospheroid differentiation and plant tiller growth, the two lines each produced 2,000 P ₁ seedlings after 2 years of cultivation and were planted in the greenhouse. The seedling growth temperature is 30°C/25°C (day/night), and the light intensity is controlled in an environment where the photosynthetic photon flux density (PPFD) is about 300 μmolm ^-2 s ^-1 .

"Example 2: Carotenoid content in Oncidium flowers"

As shown in Figure 4, the carotenoids of the transformed plants Oncidesa MF-1 and Oncidesa MF-5 are greatly reduced, and the 9-cis-violaxanthin content of the aforementioned two plants is 40% of that of the wild type, while Oncidesa MF-1 Neoxanthin, violaxanthin and β-carotene are all 30% of the wild type; in Oncidesa MF-5, which has a pure white flower color, the content of these three pigments are all It is only 1-3% of the wild type. This result shows that the DNA cassette nucleotide sequence of the present invention can effectively control the production of carotenoids to achieve the effect of controlling flower color.

"Example 3: Oncidium tissue gene expression level"

Figure 5 shows the expression level of the phytoene synthase gene (OgPSY ). It can be seen that the OgPSY expression level of the wild-type Oncidium is high, and the transgenic plants Oncidesa MF-1 and Oncidesa MF-5 do not see bands, indicating that it is The expression level is significantly reduced, while the wild type still expresses OgPSY . Figure 6 can also show that the siRNA (small interfering RNA) of the transgenic plants Oncidesa MF-1 and Oncidesa MF-5 performed strongly, and the results can be judged from the gene expression. This also shows that the present invention can successfully interfere with the performance of OgPSY and achieve the purpose of controlling pigments and flower colors.

<110> Ye Kaiwen

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The DNA cassette construction diagram of silencing the phytoene synthase gene. A preferred embodiment of the present invention is a DNA cassette that exhibits interfering RNA fragments in specific plant tissues to silence the phytoene synthase gene. The nucleotide fragments contained in this DNA cassette can be used in specific plant tissues. It is transcribed into an interfering RNA fragment. The schematic diagram of the structure is shown in Figure 2. Construct a DNA cassette vector, including a promoter (P), and then add a DNA fragment of SEQ ID NO: 3 at the 3'end, and then Followed by an intron fragment containing the GUS gene (intronGUS nucleotide sequence), followed by a DNA fragment of SEQ ID NO: 3 with an inverted direction, and finally a terminator fragment of the NOS gene (T-NOS nucleotide sequence) ). The aforementioned recombinant plastids or vectors include, but are not limited to, specific promoters, introns, or terminator fragments, or other recombinant plastids or vectors applicable to the present invention.

Claims (10)

A DNA cassette construct for regulating the color of oncidium orchids, comprising: a combination of several nucleotide sequences, the DNA cassette can interfere with the mRNA of the phytoene synthase gene in specific plant tissues For the purpose of color regulation, the DNA cassette contains a promoter and a DNA nucleotide sequence that can produce hairpin-type interfering RNA. The DNA cassette described in item 1 of the scope of patent application, wherein the promoter comprises the nucleotide sequence of SEQ ID NO:1, and the promoter can make specific genes be specifically expressed in specific tissues, and only achieves the purpose of changing flower color Purpose, but still maintain the physiological function of normal plant growth. In the DNA cassette described in item 1 of the scope of patent application, one of the DNA nucleotide sequences that can produce hairpin-type interfering RNA can be driven by the promoter described in item 2 of the scope of patent application. A hairpin interfering RNA sequence is produced by transcription in a specific tissue to interfere with the mRNA of the phytoene synthase gene, so that it cannot be further translated into protease, and is composed of the following nucleotide sequence: PSY nucleus placed in the forward direction Nucleotide sequence, intronGUS nucleotide sequence, inverted PSY nucleotide sequence, and termination fragment of NOS gene (T-NOS nucleotide sequence), wherein the PSY nucleotide sequence comprises SEQ ID NO: 3 nucleoside Acid sequence. The DNA cassette described in claim 1, wherein the nucleotide sequence is provided in a vector or a shuttle vector or a pseudo-protosphere, wherein the vector can be introduced into a plant cell or a pseudo-protosphere by transgenic The original sphere. The nucleotide sequence of SEQ ID NO: 3 as described in the scope of patent application 3 is a nucleotide sequence of 150 nucleotides in length from the 51st nucleotide in the nucleotide sequence of SEQ ID NO: 2. A DNA cassette construction method for regulating oncidium orchid color, comprising: providing at least one DNA cassette, the nucleotide sequence of the DNA cassette includes a specific promoter and a segment that can be transcribed into a hairpin type Interfering with the specific nucleotide fragment of the RNA fragment, introducing (or translocating) the nucleotide sequence into a plant cell to obtain a transgenic plant cell, and using the transgenic plant cell to regenerate and develop into a plant, and achieve The purpose of changing the color of the plant. According to item 6 of the scope of patent application, a method for constructing a DNA cassette for regulating oncidium orchid color, wherein the nucleotide sequence is obtained by gene cloning. According to item 6 of the scope of patent application, a DNA cassette construction method for regulating oncidium orchid color is used, wherein the transformed plant cell is used to change the expression of a protein that controls carotenoid pigments in a plant. According to the method for constructing a DNA cassette for regulating the color of oncidium orchids described in item 6 of the scope of patent application, the transformed orchid cells are used to regenerate a transformed plant. A preferred embodiment of the present invention provides a nucleotide sequence comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. The sequence is complementary to the nucleotide sequence.

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