Genome site-directed editing for enhancing synthesis of 1,8-cineole by melaleuca alternifolia
阅读说明：本技术 基因组定点编辑增强互叶白千层合成1,8-桉叶素 (Genome site-directed editing for enhancing synthesis of 1,8-cineole by melaleuca alternifolia ) 是由 杨俊� 于 2020-07-14 设计创作，主要内容包括：本发明公开了基因组定点编辑强互叶白千层合成1,8-桉叶素,其特征是通过基因组定点编辑,改善互叶白千层单萜合成酶活性,提升了互叶白千层合成1,8-桉叶素的能力,其新鲜枝叶用水蒸馏提取,得到纯天然植物精油,即互叶白千层精油(茶树油)。该植株在广西成功繁育种植已超10年,其初蒸的互叶白千层精油中1,8-桉叶素含量超过75%,松油醇-4含量低于8%；该初蒸油再用分子蒸馏分离提纯,可获得1,8-桉叶素纯品,纯度超过95%,可开发出富含1,8-桉叶素的化妆品、日用品、药品等。本发明研究过程包括互叶白千层天然产物、1,8-桉叶素生物合成途径、酶组学,互叶白千层单萜合成酶基因组学和基因组定点编辑。(The invention discloses a method for synthesizing 1,8-cineole by editing a strong melaleuca alternifolia layer at a genome fixed point, which is characterized in that the activity of monoterpene synthetase of the melaleuca alternifolia layer is improved and the capability of synthesizing 1,8-cineole by the melaleuca alternifolia layer is improved by editing the genome fixed point, and fresh branches and leaves of the melaleuca alternifolia layer are distilled and extracted by water to obtain pure natural plant essential oil, namely melaleuca alternifolia essential oil (tea tree oil). The plant has been successfully bred and planted in Guangxi for more than 10 years, the content of 1,8-cineole in the primarily steamed melaleuca alternifolia essential oil exceeds 75 percent, and the content of terpineol-4 is lower than 8 percent; the primary distilled oil is separated and purified by molecular distillation to obtain 1,8-cineole pure product with purity over 95%, and can be used for developing cosmetics, daily necessities, medicines, etc. rich in 1, 8-cineole. The research process of the invention comprises the natural product of melaleuca alternifolia, 1,8-cineole biosynthesis pathway, enzymomics, melaleuca alternifolia monoterpene synthetase genomics and genome fixed-point editing.)
1. The method is characterized in that the activity of melaleuca alternifolia Monoterpene synthase (Monoterpene synthase) is improved through genome site-specific editing, the capability of melaleuca alternifolia in generating 1,8-Cineole (1, 8-Cineole) is improved, and new melaleuca alternifolia plants are cultivated.
2. The melaleuca alternifolia new plant of claim 1, wherein said new plant has been bred and planted in the Guangxi Zhuang autonomous region for more than 10 years, and its fresh branches and leaves are distilled and extracted with water to obtain pure natural plant essential oil, namely melaleuca alternifolia essential oil (tea tree oil ).
3. The melaleuca alternifolia essential oil of claim 2, wherein the blanched melaleuca alternifolia essential oil has a 1,8-cineole content of greater than 75% and a terpineol-4 content of less than 8%.
4. The primary distilled melaleuca alternifolia essential oil of claim 3, which is separated and purified by molecular distillation to obtain a pure 1,8-cineole product with a purity of over 95%.
5. The essential oil of cajeput of melaleuca alternifolia as claimed in claim 3 and the pure 1,8-cineole as claimed in claim 4, characterized in that the essential oil can be used for the development of cosmetics, daily necessities, drugs, etc. rich in 1, 8-cineole.
6. The method for synthesizing 1,8-cineole by editing melaleuca alternifolia at fixed points of a genome is characterized in that the research process comprises the following steps:
(1) analyzing natural products of melaleuca alternifolia and efficacy thereof;
(2) the study of the biological pathway of synthesizing 1,8-cineole from melaleuca alternifolia;
(3) an enzymomics for synthesizing 1,8-cineole from melaleuca alternifolia;
(4) genomics of melaleuca alternifolia monoterpene synthase;
(5) editing the genome of the melaleuca alternifolia monoterpene synthetase at a fixed point.
7. The melaleuca alternifolia monoterpene synthase of claim 6, which is a rate-limiting enzyme in the synthesis of 1,8-cineole by melaleuca alternifolia.
8. The melaleuca alternifolia monoterpene synthase gene group site-specific editing method of claim 6, which is characterized in that after a specific part of a gene is cut by using a CRISPR/Cas9 technology, an exogenous gene (melaleuca alternifolia monoterpene synthase gene and specific promoter introduction) is introduced into the corresponding gene to obtain a recombinant melaleuca alternifolia monoterpene synthase gene.
9. The recombinant melaleuca alternifolia monoterpene synthase gene of claim 8, which is introduced into melaleuca alternifolia cells by transgenic technology to achieve gene expression and enhance the ability of melaleuca alternifolia to synthesize 1, 8-cineole.
10. The cell of claim 9, wherein said recombinant melaleuca alternifolia monoterpene synthase gene is introduced, and wherein said new melaleuca alternifolia plant is grown by tissue culture.
Technical Field
The invention belongs to the field of biomedicine, and relates to a method for synthesizing 1,8-Cineole (1, 8-Cineole) by enhancing Melaleuca alternifolia through genome fixed-point editing, namely, the activity of Monoterpene synthetase (Monoterpene synthase) of Melaleuca alternifolia is improved through genome fixed-point editing, the capability of the Melaleuca alternifolia for generating 1,8-Cineole (1, 8-Cineole) is improved, and fresh branches and leaves are extracted by water distillation to obtain pure natural plant essential oil, namely Melaleuca alternifolia essential oil (also called tea tree oil, tea tree essential oil and tea tree oil). Fresh branches and leaves of a new melaleuca alternifolia plant improved by the technology, the content of 1,8-Cineole (1, 8-Cineole) in the primarily steamed melaleuca alternifolia essential oil (tea tree oil ) is more than 75 percent, and the content of terpineol-4 (Terpinen-4-o 1) is less than 8 percent; the primarily steamed cajeput essential oil (tea tree oil ) is separated and purified by molecular distillation to obtain a pure 1,8-Cineole (1, 8-Cineole) product with purity of over 95 percent, and the product can be used for developing cosmetics, daily necessities, medicines and the like rich in 1,8-Cineole (1, 8-Cineole).
Background
In 1770, when the couk captain in the uk landed in australia, it was found that local hair users boiled Tea with leaves of a strong-smelling plant, and therefore called "Tea Tree" (Tea Tree) "; meanwhile, it is found that when the hair is injured in the field, the tea leaves can be immediately picked off and applied to the affected part after being mashed, and the wound can be healed and the congestion and swelling can be quickly relieved. Curiously, the couk captain collected "tea leaves" in australia, bringing it back to the uk and to chemist analysis, which was the beginning of the scientific study of "tea tree oil" by human beings. In the twentieth of the last century, a scientific research booming of Tea tree oil (Tea tree oil) was started. In fact, this modern name for "tea plant" is "Melaleuca alternifolia".
Melaleuca alternifolia (Melaleuca alternifolia) is a shrub of the genus Melaleuca of the family Myrtaceae, native to Australia, and is produced primarily in the northern coastal region of New southern Wales, Australia. In the nineties of the last century, the wide introduction of seeds has been carried out in the places of Guangdong, Guangxi, Hainan, Fujian and the like in China, and fresh branches and leaves are distilled and extracted by water to obtain pure natural plant essential oil, namely melaleuca alternifolia essential oil, which is also called Tea tree oil or Tea tree essential oil (Tea tree oil). The melaleuca alternifolia essential oil (tea tree oil) is colorless to light yellow liquid, has characteristic fragrance, can effectively inhibit pathogenic bacteria and mould, has the functions of bacteriostasis, anti-inflammation, insect expelling and mite killing, and has strong permeability, high safety, no pollution and no corrosiveness.
The use of Melaleuca alternifolia (Melaleuca alternifolia) by indigenous residents in Australia has been known for nearly a thousand years; during the second war, each Australian soldier and Sailor was provided with Melaleuca alternifolia essential oil (tea tree oil ) for the treatment of tropical infections and traumatic injuries. All studies show that the melaleuca alternifolia essential Oil (Tea Tree Oil) has the effects of refreshing, disinfecting, resisting bacteria, diminishing inflammation and promoting wound healing. Melaleuca alternifolia essential oil (tea tree oil ) is used as a broad-spectrum antibacterial agent and is widely applied to industries such as daily chemicals, pharmacy, food and the like, such as agricultural fungicides, sanitizers, preservatives, air fresheners, air-conditioning bactericides, anti-acne (acne) cleaners, automobile cleaners, carpet deodorizers, tableware cleaners, refreshing moisturizers, deodorants, bath cleaners, human cleaners, hygienic products for pets and the like. Melaleuca alternifolia essential oil (tea tree oil) is listed in the purchase list of the food and drug administration of the United states, in European pharmacopoeia 2001, and in the fourth bulletin 2003 of the Ministry of health of China, melaleuca alternifolia essential oil (tea tree oil) is also listed in the list of food flavors. Although Melaleuca alternifolia (Melaleuca alternifolia) is not listed in the Chinese pharmacopoeia, the application of Melaleuca alternifolia essential oil (tea tree oil ) shows that the Melaleuca alternifolia essential oil has the characteristics of bacteriostasis, anti-inflammation and high safety.
Melaleuca alternifolia essential oil (tea tree oil ) the main natural products include Terpineol-4 (Terpinen-4-o 1), 1,8-Cineole (1, 8-Cineole), gamma-Terpinene (gamma-Terpinene), alpha-Terpinene (alpha-Terpinene), alpha-Terpineol (alpha-Terpineol) and p-cymene (p-cymene: (p-cymene))p-Cymene). Wherein terpineol-4 (Terphin-4-o 1) and 1,8-Cineole (1, 8-Cineole) are most important, both have molecular weight of 154.25 and molecular formula of C10H18O (FIG. 1).
Foreign standards such as Australian standard (2782-. 20
TABLE 1 Standard of tea Tree oil Main Components established by domestic and foreign standards
Melaleuca alternifolia is divided into three types of terpineol-4, 1,8-cineole and mixed type, and is mainly planted at home and abroad. From the criteria of Table 1, it can be seen that the content of terpineol-4 (Terphin-4-o 1) in the freshly distilled oil of Melaleuca alternifolia (Melaleuca alternifolia), i.e. the essential oil of the freshly distilled Melaleuca alternifolia (tea tree oil ), is more than 30% and the content of 1,8-Cineole (1, 8-Cineole) is not more than 15% (ISO 4730-1996). The research literature reports that the same is true for melaleuca alternifolia (Melaleuca alternifolia) introduced domestically (see the university of forestry, southwest, proceedings 2019; 39(6): 1-8) (FIG. 2).
The research of the inventor shows that the functions of terpineol-4 (Terphin-4-o 1) and 1,8-Cineole (1, 8-Cineole) in melaleuca alternifolia essential oil (tea tree oil ) are different, namely 1,8-Cineole (1, 8-Cineole) in melaleuca alternifolia essential oil (tea tree oil ) mainly plays the effects of sterilization and bacteriostasis and wound healing promotion, and terpineol-4 (Terphin-4-o 1) mainly plays the effects of skin penetration, inflammation resistance and pain relief. Therefore, the cultivation of the new melaleuca alternifolia plant for efficiently synthesizing the 1,8-Cineole (1, 8-Cineole) is the key for obtaining the high-
Disclosure of Invention
The invention discloses that genome site-directed editing enhances the synthesis of 1,8-Cineole (1, 8-Cineole) by Melaleuca alternifolia, namely, the activity of Monoterpene Synthase (MS) of Melaleuca alternifolia is improved by genome site-directed editing, the capability of the Melaleuca alternifolia for generating 1,8-Cineole (1, 8-Cineole) is improved, and fresh branches and leaves are extracted by water distillation to obtain pure natural plant essential oil, namely, Melaleuca alternifolia essential oil (tea tree oil ). The technology improves Melaleuca alternifolia (Melaleuca alternifolia), new Melaleuca alternifolia plants are obtained, the plants are successfully planted in Yulin city of Guangxi Zhuang province for more than 10 years, the content of 1,8-Cineole (1, 8-Cineole) in Melaleuca alternifolia essential oil (tea tree oil ) of which fresh branches and leaves are steamed for the first time exceeds 75%, and the content of terpineol-4 (Terphin-4-o 1) is lower than 8%; the primarily steamed melaleuca alternifolia essential oil (tea tree oil ) is separated and purified by molecular distillation to obtain pure 1,8-Cineole (1, 8-Cineole) with purity of over 95%.
The efficacy of Melaleuca alternifolia (Melaleuca alternifolia) as main natural product
The inventor researches show that 1,8-Cineole (1, 8-Cineole) and terpineol-4 (Terphin-4-o 1) have different efficacies. Research on common pathogenic bacteria (such as staphylococcus aureus, staphylococcus epidermidis, beta hemolytic streptococcus, escherichia coli, pseudomonas aeruginosa, candida albicans, spore anaerobe, clostridium botulinum, salmonella and vibrio parahemolyticus) shows that 1,8-Cineole (1, 8-Cineole) has the effects of sterilizing and inhibiting bacteria and promoting wound healing which are far stronger than terpineol-4 (Terphin-4-o 1). It is demonstrated that 1,8-Cineole (1, 8-Cineole) in melaleuca alternifolia essential oil (tea tree oil ) mainly plays a role in sterilization, bacteriostasis and wound healing promotion, and terpineol-4 (Terpinen-4-o 1) mainly plays a role in transdermal, anti-inflammatory and analgesic effects.
Cineole (1, 8-Cineole) biosynthetic pathway
Through researches on the chemistry, physiology and the like of natural products of Melaleuca alternifolia (Melaleuca alternifolia) 1,8-Cineole (1, 8-Cineole), the inventor determines the synthetic pathway of 1,8-Cineole (1, 8-Cineole) in Melaleuca alternifolia (Melaleuca alternifolia) (figure 3), and defines the key point of 1,8-Cineole (1, 8-Cineole) synthesized by Melaleuca alternifolia (figure 4).
Enzymology for synthesizing 1,8-Cineole (1, 8-Cineole) from Melaleuca alternifolia
The inventor researches the composition, structure and action rule of related enzymes in the synthesis of 1,8-Cineole (1, 8-Cineole) by Melaleuca alternifolia, and finds that the Melaleuca alternifolia monoterpene synthase (Monoterenythase) is a rate-limiting enzyme for the synthesis of 1,8-Cineole (1, 8-Cineole) by Melaleuca alternifolia.
Analysis of Antheprot 5.0 software shows that the molecular formula of melaleuca alternifolia Monoterpene synthetase (Monoterpene synthase) is C2947H4635N814O871S20The molecular weight is 66042.3Da, the theoretical isoelectric point is 5.623, the number of positive charge amino acid residues is 67, the number of negative charge amino acid residues is 81, and the protein instability coefficient is 51.68, and belongs to unstable proteins; the amino acid sequence of the enzyme has 12 signal peptide cleavage sites (16, 21, 141, 182, 269, 272, 310, 316, 340, 443 and 449 aa).
Software analysis of Swiss Model Server and VMD1.8.5 shows that after being folded for many times, the enzyme forms a three-dimensional structure which takes alpha-helix as a main part and has 2 beta-sheet structure areas; meanwhile, after several substrate binding sites, such as Arg297, Trp305, Asp333, Arg474 and the like, are folded, a "pocket-type" substrate binding region is formed, and substrate inclusion can be catalyzed (FIG. 5).
Genomics of melaleuca alternifolia Monoterpene synthase (Monoterpene synthase)
The present inventors studied the genomic composition, the structure of each gene in a group and the interrelation of melaleuca alternifolia Monoterpene synthase (monterepen synthase). Selecting young leaves of Melaleuca alternifolia (Melaleuca alternifolia), extracting total RNA, and reverse transcribing into cDNA, 5 '-RACE cDNA and 3' -RACE cDNA.
And (3) comparing Monoterpene synthetase (Monoterpene synthase) sequences in an NCBI database, and designing a primer. Taking the cDNA of the tender leaves as a template, carrying out PCR amplification, and recycling, cloning and sequencing products. As a result of sequencing of 5 '-RACE and 3' -RACE fragments, the sequence portion of the vector was removed, and the fragments were spliced with the partial fragment of the conserved region to obtain the full-length cDNA of melaleuca alternifolia Monoterpene synthase (Monoterpene synthase) (FIG. 6).
The Vector NTI 11.0 software translates the Open reading frame (Open reading frame) of the melaleuca alternifolia Monoterpene synthase (Monoterpene synthase) gene into a protein sequence, and finds that the protein sequence has higher homology with Monoterpene synthase (Monoterpene synthase) from different sources, the homology of the N end and the C end of the amino acid sequence of the Monoterpene synthase (Monoterpene synthase) is lack of obvious homology, but the middle sequence is relatively conserved, wherein, the positions of Arg297, Trp305, Asp333, Asp337, Arg474, Leu475, Asn477, Asp478, Glu480, Tyr548 and the like form a bag-shaped substrate binding region after being spatially folded by the protein, thereby forming the catalytic function of the enzyme (figure 7).
Point-of-use editing of melaleuca alternifolia Monoterpene synthase (Monoterpene synthase) genome
By using a CRISPR/Cas9 technology, after cutting a specific part of a gene, introducing an exogenous gene (a Monterpen synthsase gene and a specific promoter) into the corresponding gene to obtain a recombinant Monterpen synthsase gene.
By utilizing a transgenic technology (such as an agrobacterium-mediated method), the gene of the recombinant melaleuca alternifolia monoterpene synthase (Monoterenythase) is introduced into melaleuca alternifolia cells to realize excellent gene expression and enhance the ability of melaleuca alternifolia (Melaleuca alternifolia) to synthesize 1,8-Cineole (1, 8-Cineole). The cell is subjected to tissue culture to obtain a new melaleuca alternifolia plant.
Distilling fresh branches and leaves of a new melaleuca alternifolia plant with water to obtain melaleuca alternifolia essential oil (tea tree oil ); detecting the content of 1,8-Cineole (1, 8-Cineole) and screening out a new melaleuca alternifolia plant for efficiently synthesizing 1,8-Cineole (1, 8-Cineole).
Detection of Melaleuca alternifolia essential oil (tea tree oil )
The new melaleuca alternifolia plant cultivated by the technology of the inventor is bred and planted in Yulin city of autonomous region of Guangxi province for 10 years, fresh branches and leaves of the new melaleuca alternifolia plant are subjected to water distillation to obtain primarily steamed melaleuca alternifolia essential oil (tea tree oil ), and the content of 1,8-Cineole (1, 8-Cineole) is higher than 75% and the content of terpineol-4 (Terphin-4-o 1) is lower than 8% by detection of an Agilent gas chromatography analyzer (model: Intuvo 9000) (figure 8).
As a result, the inventors of the present invention have confirmed that there was also a problem in the Stadium of university of southern China, professor of Stadium university (Nature science edition) 2012, 51, 5, 7-13.
Development of 1,8-Cineole (1, 8-Cineole) product
The invention provides a technology for synthesizing 1,8-Cineole (1, 8-Cineole) by 'genome fixed-point editing of strong Melaleuca alternifolia', namely, the activity of Monoterpene synthetase (Monoterpene synthase) of Melaleuca alternifolia is improved by genome fixed-point editing, the capability of the Melaleuca alternifolia for generating 1,8-Cineole (1, 8-Cineole) is improved, and a new Melaleuca alternifolia plant is obtained. The new plant is bred and planted in Guangxi Yulin city for more than 10 years, fresh branches and leaves of the new plant can be cut for 1 time in the first year and cut for 2 times every year later, and the oil yield of the fresh branches and leaves is 2-3%.
Distilling fresh branches and leaves of a new melaleuca alternifolia plant with water to obtain a primary steamed melaleuca alternifolia essential oil (tea tree oil ), wherein the content of 1,8-Cineole (1, 8-Cineole) is more than 75%, and the content of terpineol-4 (Terpinen-4-o 1) is less than 8%; and separating and purifying the primarily steamed melaleuca alternifolia essential oil (tea tree oil) by molecular distillation to obtain a pure 1,8-Cineole (1, 8-Cineole) product with purity of over 95%.
The 1,8-Cineole (1, 8-Cineole) pure product has broad-spectrum bactericidal and bacteriostatic effects and effects of promoting wound healing, is non-toxic and high in safety, and can be developed into a new medicine. In addition, the primarily steamed cajeput essential oil (tea tree oil ) can be used for developing cosmetics rich in 1,8-Cineole (1, 8-Cineole), such as 100% tea tree oil, 5% tea tree oil emulsion, tea tree oil ointment, tea tree oil shampoo, etc.; the 1,8-Cineole has strong mosquito repellent performance and moistens skin, is safe and nontoxic, and can be used for developing daily necessities rich in 1,8-Cineole (1, 8-Cineole) (figure 9).
In conclusion, the innovation point of the invention is that the route of synthesizing 1,8-Cineole (1, 8-Cineole) by melaleuca alternifolia is successfully improved for the first time in the world, namely, the rate-limiting enzyme for synthesizing 1,8-Cineole (1, 8-Cineole) is determined by analyzing natural products and biosynthetic route of melaleuca alternifolia (1, 8-Cineole), the research of enzymomics and genomics is carried out, finally, genome fixed-point editing is adopted, new melaleuca alternifolia plants for efficiently synthesizing 1,8-Cineole (1, 8-Cineole) are cultivated, and fresh branches and leaves of the new melaleuca alternifolia plants are distilled and extracted by water to obtain melaleuca alternifolia essential oil (tea tree oil ). The content of 1,8-Cineole (1, 8-Cineole) in the primarily steamed melaleuca alternifolia essential oil (tea tree oil ) is more than 75 percent, the content of terpineol-4 (Terpinen-4-o 1) is less than 8 percent, and a series of products rich in 1,8-Cineole (1, 8-Cineole) can be developed, wherein the products comprise raw materials of melaleuca alternifolia essential oil (tea tree oil ), cosmetics, daily necessities, medicines and the like (figure 10).
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