JP6866192B2 - Hydroponics method of Coptis Japonica and selection method of Coptis Japonica suitable for hydroponics - Google Patents
Hydroponics method of Coptis Japonica and selection method of Coptis Japonica suitable for hydroponics Download PDFInfo
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
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Description
本発明は、水耕栽培に適したコプティス・ジャポニカを用いた水耕栽培方法及び水耕栽培に適したコプティス・ジャポニカの選抜方法に関する。 The present invention relates to a hydroponic cultivation method using Coptis japonica suitable for hydroponics and a method for selecting Coptis japonica suitable for hydroponics.
黄連は、苦味健胃、整腸、止瀉等の作用を有し、胃炎、二日酔い、高血圧、神経症等に用いられる黄連解毒湯のほか、漢方処方の約１割に配合される重要生薬である。その主成分であるベルベリンは、抗菌、抗炎症に加え、最近、血中ＬＤＬコレステロール濃度低下や血糖値低下作用等が報告され、特に欧米ではメタボリックシンドロームに対する効果から注目されており、今後、需要の高まりが予想される。 Oren has the effects of bitterness, stomach regulation, antidiarrheal, etc. In addition to Oren-gedokuto, which is used for gastritis, hangover, hypertension, neuropathy, etc., it is important to be added to about 10% of Chinese herbal prescriptions. It is a crude drug. In addition to antibacterial and anti-inflammatory effects, berberine, which is the main component of berberine, has recently been reported to have a blood LDL cholesterol concentration lowering effect and a blood sugar level lowering effect. Expected to increase.
日本産黄連の品質は高く、かつては海外にも輸出されていた。しかし、日局規格（非特許文献１）を満たす生薬の生産には、畑栽培で５年、林床栽培では１５年以上の長い栽培期間を必要とする。黄連は、栽培農家の高齢化に加え、薬価の低下や安価な中国産黄連に押され、国内栽培は衰退した。 The quality of Japanese Coptis is high, and it was once exported overseas. However, the production of crude drugs that meet the Japanese Bureau standard (Non-Patent Document 1) requires a long cultivation period of 5 years for field cultivation and 15 years or more for forest floor cultivation. In addition to the aging of cultivated farmers, Coptis chinensis has declined due to the decline in drug prices and the cheap Chinese Coptis chinensis.
近年、高品質かつ安心・安全な黄連の効率的な生産技術の確立を目指し、黄連の原料の一つであるセリバオウレンの植物工場での水耕栽培が試みられ、１年間の水耕栽培によりベルベリン含量等の点で問題がなかったことが報告されている（非特許文献２）。 In recent years, with the aim of establishing high-quality, safe and secure efficient production technology for berberine, hydroponics has been attempted at a plant factory of Coptis chinensis, which is one of the raw materials for berberine, and hydroponics for one year. It has been reported that there was no problem in terms of berberine content and the like (Non-Patent Document 2).
しかしながら、セリバオウレンは水耕栽培であまり生育が良くない株が多く、どのよう株を選択すれば水耕栽培で生育が良いのかも不明であった。また、従来は水耕栽培の期間も長期であり、それが栽培コストを大きく引き上げ、実用化に至らない要因となっていた。 However, there are many strains of Seribaouren that do not grow very well in hydroponics, and it was unclear how to select the strains that grow well in hydroponics. In addition, conventionally, the period of hydroponics has been long, which has greatly increased the cultivation cost and has been a factor that has not been put into practical use.
本発明では、実用化に耐えうる、水耕栽培において格段に生育が良好なコプティス・ジャポニカを選抜する方法、その栽培方法を見出すことを、その課題とした。 An object of the present invention is to find a method for selecting Coptis japonica, which can withstand practical use and has remarkably good growth in hydroponics, and a method for cultivating the Coptis japonica.
本発明者らは、上記課題を解決するために鋭意研究した結果、特定の遺伝子の特定の型を有するコプティス・ジャポニカが水耕栽培での生育が格段に良好で、質量の増加も多いことを見出し、本発明を完成させた。 As a result of diligent research to solve the above problems, the present inventors have found that Coptis japonica, which has a specific type of a specific gene, grows remarkably well in hydroponics and has a large increase in mass. The heading has completed the present invention.
すなわち、本発明は、配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカを、水耕栽培することを特徴とするコプティス・ジャポニカの水耕栽培方法である。 That is, the present invention is a hydroponic cultivation method of Coptis japonica, which comprises hydroponically cultivating Coptis japonica having the Type I gene represented by SEQ ID NO: 1.
また、本発明は、コプティス・ジャポニカから、配列番号１で示されるType I遺伝子を検出することを特徴とする水耕栽培に適したコプティス・ジャポニカの選抜方法である。 Further, the present invention is a method for selecting Coptis Japonica suitable for hydroponics, which comprises detecting the Type I gene represented by SEQ ID NO: 1 from Coptis Japonica.
更に、本発明は、配列番号１で示されることを特徴とするType I遺伝子である。 Furthermore, the present invention is a Type I gene, which is represented by SEQ ID NO: 1.
また更に、本発明は、配列番号２または３に記載のオリゴヌクレオチドからなることを特徴とするType I遺伝子増幅用プライマーである。 Furthermore, the present invention is a Primer for Type I gene amplification, which comprises the oligonucleotide according to SEQ ID NO: 2 or 3.
本発明のコプティス・ジャポニカの水耕栽培方法は、水耕栽培に適したコプティス・ジャポニカを栽培するため、短期間で効率良く栽培することができる。 Since the hydroponic cultivation method of Coptis japonica of the present invention cultivates Coptis japonica suitable for hydroponic cultivation, it can be efficiently cultivated in a short period of time.
また、本発明の水耕栽培に適したコプティス・ジャポニカの選抜方法は、水耕栽培に適したコプティス・ジャポニカを高い精度で選抜できる。 In addition, the method for selecting Coptis japonica suitable for hydroponics of the present invention can select Coptis japonica suitable for hydroponics with high accuracy.
更に、上記選抜方法で選抜されたコプティス・ジャポニカは、水耕栽培に適したものであり、これを水耕栽培することにより、従来のものに比べて有害元素含量が低い、より安全性が高いものであると共に、ベルベリン含量も安定し、品質が安定する。 Furthermore, Coptis japonica selected by the above selection method is suitable for hydroponics, and by hydroponics, the content of harmful elements is lower than that of the conventional one, and the safety is higher. In addition to being a product, the berberine content is stable and the quality is stable.
本発明のコプティス・ジャポニカの水耕栽培方法（以下、「本発明栽培方法」という）は、配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカを、水耕栽培するものである。なお、配列番号１で示されるType I遺伝子は、ベルベリンの生合成経路鍵酵素の一つと考えられているS-adenosyl-L-methionine : 3’-hydroxy-N-methylcoclaurine 4’-O-methyltrans ferase （Cj4'OMT）をコードする遺伝子のゲノムＤＮＡの多型の一つである。 The hydroponic cultivation method of Coptis japonica of the present invention (hereinafter referred to as "the cultivation method of the present invention") is to hydroponicly cultivate Coptis japonica having the Type I gene represented by SEQ ID NO: 1. The Type I gene shown in SEQ ID NO: 1 is S-adenosyl-L-methionine: 3'-hydroxy-N-methylcoclaurine 4'-O-methyltrans ferase, which is considered to be one of the key enzymes for biosynthetic pathway of velverin. It is one of the genomic DNA polymorphisms of the gene encoding (Cj4'OMT).
本発明栽培方法に用いられるコプティス・ジャポニカは、配列番号１で示されるType I遺伝子を有するものである。コプティス・ジャポニカとしては、キクバオウレン（Coptis japonica Makino var. japonica Satake）、セリバオウレン（Coptis japonica Makino var. dissecta Nakai）、コセリバオウレン（Coptis japonica Makino var. major Satake）が挙げられる。これらの中でもセリバオウレンが好ましい。また、これらの由来は特に限定されないが、不定胚培養由来のものが好ましい。 The Coptis japonica used in the cultivation method of the present invention has the Type I gene shown in SEQ ID NO: 1. Examples of Coptis japonica include Coptis japonica Makino var. Japonica Satake, Coptis japonica Makino var. Dissecta Nakai, and Coptis japonica Makino var. Major Satake. Of these, seribaouren is preferable. The origin of these is not particularly limited, but those derived from adventitious embryo culture are preferable.
コプティス・ジャポニカが、配列番号１で示されるType I遺伝子を有しているかどうかを判断する方法は、特に限定されず、公知の遺伝子の検出方法を用いることができる。具体的には、コプティス・ジャポニカのゲノムDNA、コプティス・ジャポニカのCj4'OMTをコードする遺伝子のゲノムDNA等の塩基配列を公知の方法で解析して配列番号１で示されるType I遺伝子があるかどうかを判断する方法、配列番号２および３に記載のオリゴヌクレオチドからなるType I遺伝子増幅用プライマーを用い、公知の方法でType I遺伝子が増幅されるかどうかで判断する方法等が挙げられる。これらの方法の中でも配列番号２および３に記載のオリゴヌクレオチドからなるType I遺伝子増幅用プライマーを用いてType I遺伝子が増幅されるかどうかで判断する方法が好ましい。 The method for determining whether or not Coptis Japonica has the Type I gene shown in SEQ ID NO: 1 is not particularly limited, and a known gene detection method can be used. Specifically, is there a Type I gene shown by SEQ ID NO: 1 by analyzing the base sequences of the genomic DNA of Coptis japonica, the genomic DNA of the gene encoding Cj4'OMT of Coptis japonica, etc. by a known method? Examples thereof include a method for determining whether or not the type I gene is amplified by a known method using a Type I gene amplification primer consisting of the oligonucleotides shown in SEQ ID NOs: 2 and 3. Among these methods, a method of determining whether or not the Type I gene is amplified by using the Type I gene amplification primer consisting of the oligonucleotides shown in SEQ ID NOs: 2 and 3 is preferable.
配列番号２および３に記載のオリゴヌクレオチドからなるType I遺伝子増幅用プライマーを用いてType I遺伝子を増幅する方法は特に限定されないが、例えば、PCR、LAMP等が挙げられる。これらの方法の中でもPCRが好ましい。PCRの条件は特に限定されないが、例えば、公知の方法で調製したコプティス・ジャポニカの遺伝子サンプル（ゲノムDNA）に対し、配列番号２および３に記載のオリゴヌクレオチドからなるType I遺伝子増幅用プライマーを使用し、94℃〜98℃で、DNAを一本鎖に解離させ、好ましくは、50℃〜65℃、特に好ましくは54℃〜56℃でプライマーを結合させ、68℃〜72℃でDNAを増幅させることが好ましい。 また、遺伝子増幅用プライマーの濃度は、0.4μＭ〜0.5μＭ、更に0.42μＭが好ましい。 The method for amplifying the Type I gene using the Primer for amplifying the Type I gene consisting of the oligonucleotides shown in SEQ ID NOs: 2 and 3 is not particularly limited, and examples thereof include PCR and LAMP. Among these methods, PCR is preferable. The PCR conditions are not particularly limited, but for example, a Primer for Type I gene amplification consisting of the oligonucleotides shown in SEQ ID NOs: 2 and 3 is used for a gene sample (genomic DNA) of Coptis japonica prepared by a known method. Then, the DNA is dissociated into a single strand at 94 ° C to 98 ° C, the primer is bound at preferably 50 ° C to 65 ° C, particularly preferably 54 ° C to 56 ° C, and the DNA is amplified at 68 ° C to 72 ° C. It is preferable to let it. The concentration of the gene amplification primer is preferably 0.4 μM to 0.5 μM, more preferably 0.42 μM.
また、このType I遺伝子の増幅の際には別途、PCRの鋳型として用いるゲノムDNAの品質そのものに問題がないことを示すために、ITS領域（約0.7kbp）の増幅も確認することが好ましい。 In addition, when amplifying this Type I gene, it is preferable to separately confirm the amplification of the ITS region (about 0.7 kbp) in order to show that there is no problem in the quality of the genomic DNA used as a PCR template.
上記した配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカを水耕栽培する方法は特に限定されないが、例えば、配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカを、栽培ベッド部、養液タンク部、養液供給部、酸素（空気）供給ユニット（エアポンプ、ドリップ装置等）、光照射部及びそれらのコントロールユニット等を備えた水耕栽培装置で水耕栽培を行えばよい。水耕栽培の栽培ベッド部に設置する培地は、保水性と通気性を有する素材であれば特に限定されない。水耕栽培に用いる養液は、特に限定されないが、例えば、マツザキ１号及び２号（マツザキアグリビジネス製）、ウオーターファーム（清和肥料製）、ＯＡＴハウス肥料（ＯＡＴアグリオ製）等の水耕栽培用の肥料を、地下水、井戸水、水道水等の常水に溶解させたもの等を用いればよい。栽培条件は、特に限定されず、例えば、温度１０℃〜２５℃、湿度３０％〜７０％等のあまり高温多湿にならない環境下で直射日光に当てない様な条件で６か月以上である。 The method of hydroponically cultivating Coptis japonica having the Type I gene represented by SEQ ID NO: 1 described above is not particularly limited. For example, Coptis japonica having the Type I gene represented by SEQ ID NO: 1 can be cultivated in the cultivation bed section. Hydroponics may be carried out in a hydroponic cultivation device equipped with a nutrient solution tank unit, a nutrient solution supply unit, an oxygen (air) supply unit (air pump, drip device, etc.), a light irradiation unit, and a control unit thereof. The medium installed in the cultivation bed for hydroponics is not particularly limited as long as it is a material having water retention and breathability. The nutrient solution used for hydroponic cultivation is not particularly limited, but for example, hydroponic cultivation of Matsuzaki Nos. 1 and 2 (manufactured by Matsuzaki Agribusiness), Water Farm (manufactured by Seiwa Fertilizer), OAT House Fertilizer (manufactured by OAT Agrio), etc. The fertilizer for this purpose may be dissolved in normal water such as groundwater, well water, tap water, or the like. The cultivation conditions are not particularly limited, and are, for example, 6 months or more under conditions such as a temperature of 10 ° C. to 25 ° C. and a humidity of 30% to 70% so as not to be exposed to direct sunlight in an environment where the temperature and humidity are not so high and humid.
なお、水耕栽培に用いる配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカの形態は特に限定されないが、上記したような方法でType I遺伝子を有することが確認されたコプティス・ジャポニカから誘導された培養クローンを用いることが好ましい。 The morphology of Coptis japonica having the Type I gene shown in SEQ ID NO: 1 used for hydroponics is not particularly limited, but it is derived from Coptis Japonica confirmed to have the Type I gene by the above method. It is preferable to use the cultured clone.
上記した水耕栽培装置としては、例えば、フィールド水耕栽培装置（エスペックミック製）等の市販の装置を利用することもできる。このフィールド水耕栽培装置を利用する場合、栽培ベッド部に設置する培地としては、ココピート、パミス、ハイドロボール、ピートモス、パーライト、バーミキュライト、ゼオライト等が好ましい。養液としては、ＯＡＴハウス１号とＯＡＴハウス２号（いずれも、ＯＡＴアグリオ製）を組合せたものを常水に溶解させたものを用いることが好ましい。栽培条件は温度１８℃〜２２℃、湿度４５％〜６０％、太陽光＋補助光源で１２時間〜１６時間明期で６か月以上である。 As the hydroponic cultivation device described above, for example, a commercially available device such as a field hydroponic cultivation device (manufactured by Especmic) can be used. When this field hydroponic cultivation apparatus is used, as the medium to be installed in the cultivation bed portion, coco pumice, pumice, hydroball, peat moss, perlite, vermiculite, zeolite and the like are preferable. As the nutrient solution, it is preferable to use a combination of OAT House No. 1 and OAT House No. 2 (both manufactured by OAT Agrio) dissolved in normal water. Cultivation conditions are temperature 18 ° C to 22 ° C, humidity 45% to 60%, sunlight + auxiliary light source for 12 hours to 16 hours, and 6 months or more in the light period.
以上のようにして水耕栽培された配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカは、同じ期間、土耕栽培された場合と比較して、根の生育が早く且つ大きくなる。また、Type I遺伝子を有していないコプティス・ジャポニカと比較して、水耕栽培を行った際の生存率が高い。そのため、配列番号１で示されるType I遺伝子を有するコプティス・ジャポニカは、水耕栽培に適していると判断できる。 The roots of Coptis japonica having the Type I gene shown in SEQ ID NO: 1 hydroponically cultivated as described above grow faster and grow larger than those cultivated in soil for the same period. In addition, the survival rate when hydroponically grown is higher than that of Coptis Japonica, which does not have the Type I gene. Therefore, it can be determined that Coptis japonica having the Type I gene shown in SEQ ID NO: 1 is suitable for hydroponics.
本発明の水耕栽培に適したコプティス・ジャポニカの選抜方法（以下、「本発明選抜方法」という）は、コプティス・ジャポニカから、配列番号１で示されるType I遺伝子を検出するものである。 The method for selecting Coptis japonica suitable for hydroponics of the present invention (hereinafter referred to as "the method for selecting the present invention") is to detect the Type I gene represented by SEQ ID NO: 1 from Coptis japonica.
コプティス・ジャポニカから、配列番号１で示されるType I遺伝子を検出する方法は、上記した公知の遺伝子の検出方法を用いることができるが、好ましくは配列番号１で示されるType I遺伝子の検出にあたり、配列番号２および３に記載のオリゴヌクレオチドからなるType I遺伝子増幅用プライマーを用いる方法である。配列番号１で示されるType I遺伝子の検出にあたり、配列番号２および３に記載のオリゴヌクレオチドからなるType I遺伝子増幅プライマーを用いれば、他のType IIa、Type IIb、Type IIIa、Type IIIb、Type IIIc、Type IIId等の遺伝子を有するコプティス・ジャポニカと鑑別できる。 As a method for detecting the Type I gene represented by SEQ ID NO: 1 from Coptis japonica, the above-mentioned known method for detecting a gene can be used, but preferably, in detecting the Type I gene represented by SEQ ID NO: 1. This is a method using a Primer for Type I gene amplification consisting of the oligonucleotides shown in SEQ ID NOs: 2 and 3. In detecting the Type I gene shown in SEQ ID NO: 1, if the Type I gene amplification primer consisting of the oligonucleotides shown in SEQ ID NOs: 2 and 3 is used, other Type IIa, Type IIb, Type IIIa, Type IIIb, and Type IIIc , Type IIId, etc., can be distinguished from Coptis japonica.
以下、本発明を実施例を挙げて本発明を説明するが、本発明はこれら実施例に何ら限定されるものではない。 Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.
参 考 例 １
セリバオウレンの不定胚培養物の塩基配列解析：
セリバオウレンのベルベリンの生合成経路における鍵酵素の一つであるCj4'OMTをコードする遺伝子のゲノムＤＮＡ（Cj4'OMTg）の種間多型を、以下のようにして調べた。
Reference example 1
Nucleotide sequence analysis of adventitious embryo culture of Seribaouren:
The interspecific polymorphism of the genomic DNA (Cj4'OMTg) of the gene encoding Cj4'OMT, which is one of the key enzymes in the berberine biosynthetic pathway of seribaouren, was investigated as follows.
＜サンプル＞
セリバオウレンの由来の全て異なる不定胚培養物４クローン（クローン１、２、３、４）をサンプルとした。
<Sample>
Four clones of adventitious embryo cultures (
＜方法＞
セリバオウレンの4’OMT遺伝子間で保存されている領域について設計したプライマー、Cj4’OMT-668S（センスプライマー、sense primer）及びCj4’OMT-801A（アンチセンスプライマー、antisense primer）のセット（配列番号４および５）を用い、ゲノムDNAを鋳型に、Cj4’OMTg遺伝子の増幅を行った。PCR条件は表１に示す。各プライマーの最終濃度は、1 μMとした。
<Method>
A set of primers, Cj4'OMT-668S (sense primer) and Cj4'OMT-801A (antisense primer) designed for the region conserved between the 4'OMT genes of seribaouren (SEQ ID NO: 4). And 5) were used to amplify the Cj4'OMTg gene using genomic DNA as a template. The PCR conditions are shown in Table 1. The final concentration of each primer was 1 μM.
（PCR条件）
得られた増幅産物は末端A付加ののち、アガロースゲル電気泳動及びWizard（登録商標）SV Gel and PCR Cleanup System (Promega)を用いたゲル精製により未反応のプライマー等を除去し、T-vectorにクローニングした。大腸菌DH5aを形質転換し、アンピシリン耐性の各植物由来8クローンについてコロニーダイレクトPCRを行い、解析領域を増幅し、得られた増幅産物を鋳型としてF-primer（フォワードプライマー、forward primer）またはR-primer（リバースプライマー、reverse primer）を用いたダイレクトシーケンシングに供した。
塩基配列の解析にはBigDye Terminator v3.1 Cycle Sequencing Kit (ABI)を用いたサイクルシーケンシング法を適用し、 ABI PRISM 3130-Avant DNA sequencer、80 cm キャピラリー、POP-7ポリマー(ABI)により解析を行った。得られた塩基配列データは、DNASIS V3.7 (Hitachi software)、FinchTV (Geospiza Inc.)、BioEdit ver. 7.1.11を用い解析し、分子系統樹解析にはTreeView (1.6.6)を使用した。
After addition of terminal A, the obtained amplification product was subjected to agarose gel electrophoresis and gel purification using Wizard® SV Gel and PCR Cleanup System (Promega) to remove unreacted primers and the like to form a T-vector. It was cloned. Escherichia coli DH5a was transformed, colony direct PCR was performed on 8 clones derived from each plant resistant to ampicillin, the analysis region was amplified, and the obtained amplification product was used as a template for F-primer (forward primer) or R-primer. It was subjected to direct sequencing using (reverse primer).
The cycle sequencing method using the Big Dye Terminator v3.1 Cycle Sequencing Kit (ABI) was applied to the base sequence analysis, and the analysis was performed using the ABI PRISM 3130-Avant DNA sequencer, 80 cm capillary, and POP-7 polymer (ABI). went. The obtained nucleotide sequence data was analyzed using DNASIS V3.7 (Hitachi software), FinchTV (Geospiza Inc.), BioEdit ver. 7.1.11, and TreeView (1.6.6) was used for molecular phylogenetic tree analysis. ..
＜結果＞
全てのセリバオウレン不定胚培養物でCj4'OMTgが増幅された。Cj4'OMTg塩基配列解析の結果、Cj4'OMTgの多型はType I、Type IIa、Type IIb、Type IIIa、Type IIIb、Type IIIc、Type IIIdの7種であった。これらの出現頻度はクローン１でType I : Type IIIb (5 : 2)、クローン２では、Type IIa : Type IIIa : Type IIIc : Type IIId (3 : 3 : 1 : 1)、クローン３では、Type IIIa : Type IIIc (6 : 1)、クローン４では、Type IIa : Type IIb : Type IIIb (3 : 2 : 3)であった。
<Result>
Cj4'OMTg was amplified in all seribaouren adventitious embryo cultures. As a result of Cj4'OMTg nucleotide sequence analysis, there were seven types of Cj4'OMTg polymorphisms: Type I, Type IIa, Type IIb, Type IIIa, Type IIIb, Type IIIc, and Type IIId. The frequency of these occurrences is Type I: Type IIIb (5: 2) for
実 施 例 １
セリバオウレンの水耕栽培：
由来の異なるセリバオウレン（Ａ株：１４株、Ｂ株：６株、Ｃ株：１６株）の苗（２〜５ｇ程度）を定植し、７か月間、水耕栽培した。水耕栽培の条件は以下の通りである。
Example 1
Hydroponics of Seribaouren:
Seedlings (about 2 to 5 g) of Seribaouren (A strain: 14 strains, B strain: 6 strains, C strain: 16 strains) of different origins were planted and hydroponically cultivated for 7 months. The conditions for hydroponics are as follows.
＜水耕栽培条件＞
栽培条件：非閉鎖温室 設定温度20℃、16時間明期、湿度60％
栽培装置：底面潅水方式水耕栽培装置
栽培支持体：パミス 特小粒 粒度2.4〜5.0 mm（大江化学工業製）
養液：ＯＡＴハウス1号（ＯＡＴアグリオ製）0.1875g/l＋ＯＡＴハウス２号（ＯＡＴアグリオ製）0.125/l、pH5.7、EC 0.37mS/cm
<Hydroponic cultivation conditions>
Cultivation conditions: Non-closed greenhouse Set temperature 20 ℃, 16 hours light period, humidity 60%
Cultivation equipment: Bottom irrigation method Hydroponic cultivation equipment Cultivation support: Pumice Extra small grain size 2.4-5.0 mm (manufactured by Oe Chemical Industry Co., Ltd.)
Nutrient solution: OAT House No. 1 (manufactured by OAT Agrio) 0.1875 g / l + OAT House No. 2 (manufactured by OAT Agrio) 0.125 / l, pH5.7, EC 0.37 mS / cm
水耕栽培後、Ａ株、Ｂ株、Ｃ株の収穫時の地上部生存率（平均値）、栽培開始時に対する質量増加率（平均値）を表２に示した。 Table 2 shows the above-ground survival rate (average value) at the time of harvesting of A, B, and C strains after hydroponics, and the mass increase rate (average value) at the start of cultivation.
以上の結果から、水耕栽培において、A株が特に生育が良好であることが判明した。A株のCj4'OMTgの遺伝子型を実施例2により調べたところ、Type I遺伝子が検出された。その他のB株、C株においてはType Iの遺伝子は検出されなかった。オウレン株のCj4'OMTgの遺伝子型を調べ、Type Iが検出されるものを選別すれば、水耕栽培に適した株を選抜できることが判明した。 From the above results, it was found that the A-share grows particularly well in hydroponics. When the genotype of Cj4'OMTg of the A strain was examined by Example 2, the Type I gene was detected. No Type I gene was detected in the other strains B and C. By examining the genotype of Cj4'OMTg of Coptis chinensis strain and selecting those in which Type I is detected, it was found that a strain suitable for hydroponics can be selected.
実 施 例 ２
Type I遺伝子用プライマーの設計：
実施例１の結果から、Type I遺伝子を有するセリバオウレンが、水耕栽培に適していることが判明した。このType I遺伝子を有するセリバオウレンを鑑別できるように、Type I遺伝子に特有のプライマーを設計した（配列番号２および３）。これらのプライマーと、GoTaq Green Master Mix（Promega製）を用い、各サンプルのType I遺伝子の増幅を行った。PCR条件は表３に示す。各プライマーの最終濃度は、0.42 μMとした。結果を図１に示した。
Example 2
Primer design for Type I genes:
From the results of Example 1, it was found that seribaouren having a Type I gene is suitable for hydroponics. Primers specific to the Type I gene were designed so that seribaourene having this Type I gene could be differentiated (SEQ ID NOS: 2 and 3). Using these primers and GoTaq Green Master Mix (manufactured by Promega), the Type I gene of each sample was amplified. The PCR conditions are shown in Table 3. The final concentration of each primer was 0.42 μM. The results are shown in FIG.
＜サンプル＞
Type I遺伝子を有さないセリバオウレン２種、Type I遺伝子を有さないセリバオウレン２種、Type I遺伝子を有さないコセリバオウレン、Type I遺伝子を有さないキクバオウレン、Type I遺伝子を有さないC.chinensis、Type I遺伝子を有するセリバオウレンを用いた。
<Sample>
Two types of Seribaouren without the Type I gene, two types of Seribaouren without the Type I gene, Koselibaouren without the Type I gene, Kikubaouren without the Type I gene, and C. chinensis without the Type I gene , Seribaouren having a Type I gene was used.
（PCR条件）
Type I遺伝子用プライマーを用いることによりType I遺伝子の増幅産物以外の非特異的増幅産物の出現が抑制されていた。 The appearance of non-specific amplification products other than the amplification products of the Type I gene was suppressed by using the primers for the Type I gene.
なお、PCRの鋳型として用いるゲノムDNAの品質そのものに問題がないことを示すために、各サンプルのITS領域（約0.7kbp）が増幅されることも確認した。 It was also confirmed that the ITS region (about 0.7 kbp) of each sample was amplified in order to show that there was no problem in the quality of the genomic DNA used as a PCR template.
実 施 例 ３
Type I遺伝子を有するセリバオウレンの栽培：
参考例１においてType I遺伝子を有するオウレン株の葉柄から誘導した不定胚培養由来のType I遺伝子を有するセリバオウレンの苗（平均生質量１．９３ｇ）を、以下の条件で土耕栽培または水耕栽培し、栽培189日後に生薬の原料となる根茎の質量を凍結乾燥機で乾燥後、測定した。その結果を表４に示した。また、土耕栽培189日後のセリバオウレンの外観を図２に、水耕栽培189日後のセリバオウレンの外観を図３に示した。
Actual example 3
Cultivation of Seribaouren carrying the Type I gene:
In Reference Example 1, Coptis chinensis seedlings (average crude mass 1.93 g) having a Type I gene derived from adventitious embryo culture derived from the leaf stalk of a Coptis chinensis strain having a Type I gene are cultivated in soil or hydroponics under the following conditions. Then, after 189 days of cultivation, the mass of rhizomes, which are the raw materials for crude drugs, was measured after drying with a freeze-dryer. The results are shown in Table 4. The appearance of Seribaouren after 189 days of soil cultivation is shown in FIG. 2, and the appearance of Seribaouren after 189 days of hydroponics is shown in FIG.
＜土耕栽培条件＞
3寸鉢：赤玉土：培養土（クレハ製）：堆肥＝3：1：1
肥料：ハイポネックス6-10-5（ハイポネックスジャパン製） 500倍液を週１回散布
<Soil cultivation conditions>
3 inch pot: Akadama soil: Potting soil (made by Kureha): Compost = 3: 1: 1
Fertilizer: Hyponex 6-10-5 (manufactured by Hyponex Japan) 500 times solution is sprayed once a week
＜水耕栽培条件＞
栽培条件：閉鎖温室 20℃、16時間明、湿度60％
栽培装置：フィールド水耕栽培装置（エスペックミック製）
栽培ベッド：ココピート（エスペックミック製）
養液：111日まで：マツザキ1号（マツザキアグリビジネス製）0.38g/l+マツザキ2号（マツザキアグリビジネス製）0.25/l、pH6.0、EC 0.73mS/cm
112日以降：マツザキ1号（マツザキアグリビジネス製）0.19g/l+マツザキ2号（マツザキアグリビジネス製）0.13/l、pH5.6、EC 0.32mS/cm
<Hydroponic cultivation conditions>
Cultivation conditions: closed greenhouse 20 ℃, 16 hours light, humidity 60%
Cultivation equipment: Field hydroponic cultivation equipment (manufactured by Especmic)
Cultivation bed: coco peat (made by Especmic)
Nutrient solution: Until 111 days: Matsuzaki No. 1 (manufactured by Matsuzaki Agribusiness) 0.38 g / l + Matsuzaki No. 2 (manufactured by Matsuzaki Agribusiness) 0.25 / l, pH6.0, EC 0.73 mS / cm
After 112th: Matsuzaki No. 1 (manufactured by Matsuzaki Agribusiness) 0.19g / l + Matsuzaki No. 2 (manufactured by Matsuzaki Agribusiness) 0.13 / l, pH5.6, EC 0.32mS / cm
以上の結果から、Type I遺伝子を有するセリバオウレンは、水耕栽培に特に適していることが判明した。 From the above results, it was found that seribaouren having a Type I gene is particularly suitable for hydroponics.
本発明によれば、水耕栽培に適したコプティス・ジャポニカを選抜することができ、そこで選抜されたコプティス・ジャポニカを水耕栽培することにより、生薬として有用なコプティス・ジャポニカを効率良く得ることができる。そのため、本発明は、漢方薬の黄連の製造に好適である。 According to the present invention, coptis japonica suitable for hydroponics can be selected, and by hydroponically cultivating the selected coptis japonica, it is possible to efficiently obtain coptis japonica useful as a crude drug. it can. Therefore, the present invention is suitable for producing Coptis chinensis, a Chinese herbal medicine.
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