JP7313759B2 - How to increase tomato fruit - Google Patents

How to increase tomato fruit Download PDF

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JP7313759B2
JP7313759B2 JP2023500253A JP2023500253A JP7313759B2 JP 7313759 B2 JP7313759 B2 JP 7313759B2 JP 2023500253 A JP2023500253 A JP 2023500253A JP 2023500253 A JP2023500253 A JP 2023500253A JP 7313759 B2 JP7313759 B2 JP 7313759B2
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析豊 陳
伯軍 馬
淑容 沈
以霊 許
敏 李
浩天 陳
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Description

本発明は、トマト果実の増大を促進する遺伝子およびその使用に関し、作物分子遺伝分野に属する。 The present invention relates to genes promoting tomato fruit growth and uses thereof, and belongs to the field of crop molecular genetics.

トマト（Ｓｏｌａｎｕｍ ｌｙｃｏｐｅｒｓｉｃｕｍ）は、重要な園芸植物および経済作物として、世界中で広く栽培され、世界的な野菜および果物になっており、見た目の美しさ、味の良さ、栄養価の高さから、消費者に大変人気がある。トマトの果実の大きさと重量は、トマトの外観と収量に影響を与える重要な農業形質である。果実の直径と重量を大きくすることにより、トマトの収量が増加するだけでなく、視覚的な美学が向上し、果実の栄養含有量が増加し、市場価値と経済価値が高まる。そのため、新しい機能遺伝子を開発し、遺伝子編集技術を利用して大きいトマトの新品種を選別することは、比較的早く便利な方法である。 Tomatoes (Solanum lycopersicum) are widely cultivated around the world as an important horticultural plant and economic crop, and have become a global vegetable and fruit that are very popular with consumers for their aesthetic appeal, good taste and high nutritional value. Tomato fruit size and weight are important agronomic traits that affect tomato appearance and yield. Increasing fruit diameter and weight not only increases tomato yield, but also improves visual aesthetics, increases fruit nutrient content, and increases market and economic value. Therefore, developing new functional genes and using gene editing technology to select new varieties of large tomatoes is a relatively quick and convenient method.

本発明が解決しようとする技術的課題は、トマト果実の大きさを効果的に増大させ、果実の収量を向上させることである。 The technical problem to be solved by the present invention is to effectively increase the size of tomato fruits and improve the yield of fruits.

上述の技術的問題を解決するために、本発明は、トマトの遺伝子ＩＦＷ１（ｉｎｃｒｅａｓｅ ｆｒｕｉｔ ｓｉｚｅ ａｎｄ ｗｅｉｇｈｔ １）を提供し、それコードするヌクレオチド配列は、配列番号１に示される配列である。 In order to solve the above technical problems, the present invention provides a tomato gene IFW1 ( increase fruit size and weight 1), the encoding nucleotide sequence of which is the sequence shown in SEQ ID NO:1.

本発明は、上記遺伝子の使用をさらに提供する。トマト中でＩＦＷ１遺伝子をノックアウトすることでその果実の大きさを効果的に増大（顕著に増大）させ、収量を向上させることができる。 The present invention further provides uses of the above genes. Knocking out the IFW1 gene in tomato can effectively increase (significantly increase) the fruit size and improve yield.

本発明のトマト果実の増大におけるＩＦＷ１遺伝子の使用の改良：
ＩＦＷ１遺伝子の２つのノックアウト株は、ｉｆｗ１－１およびｉｆｗ１－２であり、ｉｆｗ１－１およびｉｆｗ１－２植物体中のＩＦＷ１遺伝子変異配列はいずれも配列番号２に示される配列である。 Improved use of the IFW1 gene in tomato fruit expansion according to the invention:
The two knockout strains of the IFW1 gene are ifw1-1 and ifw1-2, and the IFW1 gene mutation sequences in both ifw1-1 and ifw1-2 plants are the sequences shown in SEQ ID NO:2.

本発明は、トマト中でＩＦＷ１遺伝子をノックアウトする方法をさらに提供する。この方法は、以下のステップを含む。
１）ＣＲＩＳＰＲ／Ｃａｓ９技術により、遺伝子編集される標的ｓｇＲＮＡ配列：５’－ＧＡＴＡＧＡＧＧＣＡＧＡＧＧＣＡＧＡＧＧ－３’を設計する。
２）ステップ１）で得られた配列を用いてプライマーを合成し、ＣＲＩＳＰＲ／Ｃａｓ９ベクターに構築する。
３）ステップ２）で得られたベクターを野生型トマト品種ＭｉｃｒｏＴｏｍに遺伝的変換し、対応する遺伝子組換え植物体を得る。前記遺伝子組換えトマト植物体からＩＦＷ１遺伝子がノックアウトされた植物体を同定する。 The present invention further provides a method of knocking out the IFW1 gene in tomato. This method includes the following steps.
1) Design a target sgRNA sequence to be gene-edited by CRISPR/Cas9 technology: 5′-GATAGAGGCAGAGGCAGAGG-3′.
2) Synthesize primers using the sequence obtained in step 1) and construct into CRISPR/Cas9 vector.
3) The vector obtained in step 2) is genetically transformed into a wild-type tomato cultivar MicroTom to obtain the corresponding transgenic plant. A plant in which the IFW1 gene is knocked out is identified from the transgenic tomato plant.

本発明の技術的解決策は、以下の通りである。
ＣＲＩＳＰＲ／Ｃａｓ９遺伝子編集技術により、ＩＦＷ１遺伝子のヌクレオチド配列（配列番号１）に基づいてＩＦＷ１遺伝子を特異的に標的とするｇＲＮＡ配列を合成し、対応するＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築し、野生型トマト品種ＭｉｃｒｏＴｏｍに遺伝的変換し、ゲノム中のＩＦＷ１遺伝子を特異的に編集し、遺伝子組換え植物体を取得し、遺伝子組換え植物体中のＩＦＷ１遺伝子に対してＰＣＲ増幅およびシーケンシングを行い、ＩＦＷ１遺伝子が得られた異なるノックアウト株ｉｆｗ１－１およびｉｆｗ１－２を同定する（図１）。ｉｆｗ１－１およびｉｆｗ１－２植物体におけるＩＦＷ１遺伝子の変異配列は、いずれも配列番号２である。果形を比較した結果、ＩＦＷ１遺伝子ノックアウト植物体の成熟果実の大きさ（図２、図３）および重量は、いずれも野生型対照品種（図４）よりも顕著に高く、トマト中でＩＦＷ１遺伝子をノックアウトすることにより果実中の生物量の合成の増加を効果的に促進でき、重要な育種価値を有する。 The technical solutions of the present invention are as follows.
By CRISPR/Cas9 gene editing technology, a gRNA sequence specifically targeting the IFW1 gene is synthesized based on the nucleotide sequence of the IFW1 gene (SEQ ID NO: 1), the corresponding CRISPR/Cas9 vector is constructed, genetically transformed into the wild-type tomato cultivar MicroTom, the IFW1 gene in the genome is specifically edited, the transgenic plant is obtained, and the IFW1 gene in the transgenic plant is subjected to PCR amplification and sequencing to obtain the IFW1 gene. We also identify different knockout strains ifw1-1 and ifw1-2 (FIG. 1). The mutated sequences of the IFW1 gene in ifw1-1 and ifw1-2 plants are both SEQ ID NO:2. As a result of comparing the fruit shape, the mature fruit size (Fig. 2, Fig. 3) and weight of the IFW1 gene knockout plant were both significantly higher than the wild-type control cultivar (Fig. 4). Knocking out the IFW1 gene in tomato can effectively promote the increase of biomass synthesis in fruit, and has important breeding value.

以下、図面を参照しながら本発明の具体的な実施形態をさらに詳しく説明する。
トマトＩＦＷ１遺伝子ノックアウト株のＣＲＩＳＰＲ／Ｃａｓ９標的部位およびシーケンシング結果である。 トマトＩＦＷ１遺伝子ノックアウト株と野生型対照ＭｉｃｒｏＴｏｍの果形との比較図である。 トマトＩＦＷ１遺伝子ノックアウト株と野生型対照ＭｉｃｒｏＴｏｍの果実の大きさの測定である。 トマトＩＦＷ１遺伝子ノックアウト株と野生型対照ＭｉｃｒｏＴｏｍの単一の果実の重量測定である。 図１～４において、ＷＴは野生型対照品種ＭｉｃｒｏＴｏｍを示し、ｉｆｗ１－１およびｉｆｗ１－２はＩＦＷ１遺伝子の２つの異なるノックアウト株を示す。図３および図４における数値は平均値±標準差であり、＊＊はトマトＩＦＷ１遺伝子ノックアウト株ｉｆｗ１－１（またはｉｆｗ１－２）と野生型（ＷＴ）対照ＭｉｃｒｏＴｏｍとの比較において、ｔ検定には顕著な有意差（Ｐ＜０．０１）が存在することを示す。 Specific embodiments of the present invention will be described in more detail below with reference to the drawings.
CRISPR/Cas9 target sites and sequencing results of tomato IFW1 gene knockout strain. Fig. 3 is a comparison of the fruit shape of the tomato IFW1 gene knockout strain and the wild-type control MicroTom. Measurement of fruit size of tomato IFW1 gene knockout strain and wild-type control MicroTom. Weight measurements of single fruit of tomato IFW1 gene knockout strain and wild-type control MicroTom. In Figures 1-4, WT indicates the wild-type control cultivar MicroTom and ifw1-1 and ifw1-2 indicate two different knockout strains of the IFW1 gene. The values in FIGS. 3 and 4 are the mean ± standard difference, and ** indicates that there is a significant difference (P<0.01) by t-test between the tomato IFW1 gene knockout strain ifw1-1 (or ifw1-2) and the wild-type (WT) control MicroTom.

ステップ１：トマトＩＦＷ１遺伝子がノックアウトされたＣＲＩＳＰＲ／Ｃａｓ９ベクターの構築
オンラインプロフェッショナルソフトウェア（ｈｔｔｐ：／／ｃｒｉｓｐｒ．ｍｉｔ．ｅｄｕ／）により、ＩＦＷ１遺伝子のコード配列（配列番号：１）においてＣＲＩＳＰＲ／Ｃａｓ９で編集される標的ｓｇＲＮＡ配列：５’－ＧＡＴＡＧＡＧＧＣＡＧＡＧＧＣＡＧＡＧＧ－３’を設計し、バイオテクノロジー企業で対応するプライマー配列：５’－ＴＧＡＴＴＧＡＴＡＧＡＧＧＣＡＧＡＧＧＣＡＧＡＧＧ－３’および５’－ＡＡＡＣＣＣＴＣＴＧＣＣＴＣＴＧＣＣＴＣＴＡＴＣＡ－３’を合成した。ＣＲＩＳＰＲ／Ｃａｓ９キット（Ｂｉｏｇｌｅ，Ｃｈｉｎａ）により対応するＣＲＩＳＰＲ／Ｃａｓ９ベクターを構築した。構築は製品マニュアルに従って操作した。 Step 1: Construction of CRISPR/Cas9 vector in which tomato IFW1 gene is knocked out Design the target sgRNA sequence to be edited with CRISPR/Cas9 in the coding sequence of IFW1 gene (SEQ ID NO: 1): 5'-GATAGAGGCAGAGGCAGAGG-3' by online professional software (http://crispr.mit.edu/), and corresponding primer sequence in biotechnology company: 5'-TGATTGATAGAG GCAGAGGCAGAGG-3' and 5'-AAACCCTCTGCCTCTGCCTCTATCA-3' were synthesized. The corresponding CRISPR/Cas9 vector was constructed by CRISPR/Cas9 kit (Biogle, China). The construction was operated according to the product manual.

ステップ２：ＣＲＩＳＰＲ／Ｃａｓ９ベクターで構築されたトマト遺伝的変換
Ｋｉｍｕｒａらの方法（Ｋｉｍｕｒａ Ｓ ｅｔ ａｌ，ＣＨＳ Ｐｒｏｔｏｃ，２００８）により、ステップ１で構築されたＣＲＩＳＰＲ／Ｃａｓ９ベクターをトマト品種ＭｉｃｒｏＴｏｍに遺伝的変換し、遺伝子組換えトマト植物体を得た。 Step 2: Tomato genetic conversion constructed with CRISPR/Cas9 vector By the method of Kimura et al. (Kimura S et al, CHS Protoc, 2008), the CRISPR/Cas9 vector constructed in step 1 was genetically converted to tomato cultivar MicroTom to obtain a genetically modified tomato plant.

ステップ３：遺伝子組換えトマト中のＩＦＷ１遺伝子のシーケンシング分析
遺伝子組換えトマト植物体の葉を０．１ｇとり、液体窒素で粉砕した後、６００μｌ抽出液（１５．７６ｇＴｒｉｓ－ｃｌ，２９．２２ｇＮａＣｌ，１５．０ｇＳＤＳ粉末に超純水を１Ｌまで加え、ｐＨ＝８．０に調整）を加え、６５℃で６０ｍｉｎインキュベートした。２００μｌの５Ｍ ＫＡＣを加え、均一に混合した後、氷浴で１０ｍｉｎ冷却した後、５００μｌクロロホルムを加え、均一に混合し、１００００ｒｐｍで５分間遠心分離し、上清を取り、５００μｌイソプロパノールを加え、均一に混合し、１２０００ｒｐｍで３ｍｉｎ遠心分離し、上清を捨て、７５％エタノールで沈殿を洗浄し、１２０００ｒｐｍで３分間遠心分離し、上清を捨て、逆さまにしてＤＮＡを１５分間乾燥させた後、３０μｌ純水を加えてＤＮＡを溶解した。 Step 3: Sequencing analysis of IFW1 gene in transgenic tomato 0.1 g of transgenic tomato plant leaves were pulverized with liquid nitrogen, then 600 μl of extract (15.76 g Tris-cl, 29.22 g NaCl, 15.0 g SDS powder was added to 1 L of ultrapure water to adjust the pH to 8.0) and incubated at 65° C. for 60 min. Add 200 μl of 5M KAC, mix evenly, cool in an ice bath for 10 minutes, add 500 μl of chloroform, mix evenly, centrifuge at 10000 rpm for 5 minutes, remove the supernatant, add 500 μl of isopropanol, mix evenly, centrifuge at 12000 rpm for 3 minutes, discard the supernatant, wash the precipitate with 75% ethanol, and wash at 12000 rpm. After centrifuging at pm for 3 minutes, discarding the supernatant, turning the tube upside down to dry the DNA for 15 minutes, 30 μl of pure water was added to dissolve the DNA.

ＩＦＷ１遺伝子ＰＣＲ増幅用の上流プライマー５’－ＡＡＣＧＴＴＣＡＡＣＧＧＡＣＡＡＴＣ－３’および下流プライマー５’－ＣＡＡＴＡＡＡＧＴＡＣＡＣＣＡＣＡＴ－３’を合成し、遺伝子組換えトマト植物体およびその対照品種ＭｉｃｒｏＴｏｍのゲノムＤＮＡをテンプレートとし、２×Ｔａｑ ＰＣＲ Ｍａｓｔｅｒ Ｍｉｘ（ＴＩＡＮＧＥＮ社）を用いてＩＦＷ１遺伝子に対してＰＣＲ増幅を行った。ＰＣＲ増幅系は、２×Ｔａｑ ＰＣＲ ＭａｓｔｅｒＭｉｘ１０μｌ、上流プライマーおよび下流プライマー（１０μＭ）それぞれ１μｌ、テンプレートＤＮＡ １μｌ（＜１μｇ）、無菌水７μｌ（合計２０μｌ）であった。ＰＣＲ増幅プログラムは、予備変性：９４℃、５分間；変性：９４℃、３０秒；アニーリング：５５℃、３０秒；伸長：７２℃、３５秒；３５×サイクル；伸長：７２℃、１０分間であった。 An upstream primer 5'-AACGTTCAACGGACAATC-3' and a downstream primer 5'-CAATAAAGTACACCACAT-3' for IFW1 gene PCR amplification were synthesized, and the genomic DNA of the transgenic tomato plant and its control variety MicroTom were used as templates, and PCR was performed on the IFW1 gene using 2 × Taq PCR Master Mix (TIANGEN). The PCR amplification system was 10 μl 2×Taq PCR MasterMix, 1 μl each of upstream and downstream primers (10 μM), 1 μl template DNA (<1 μg), 7 μl sterile water (20 μl total). The PCR amplification program was pre-denaturation: 94°C, 5 min; denaturation: 94°C, 30 sec; annealing: 55°C, 30 sec; extension: 72°C, 35 sec;

ＰＣＲ生成物に対してシーケンシング分析を行った後、ＩＦＷ１遺伝子が成功にノックアウトされた２つの株ｉｆｗ１－１およびｉｆｗ１－２を同定した。この２つの株の植物体におけるＩＦＷ１遺伝子コード領域にはいずれも２つの塩基が欠失することで（図１）、ＩＦＷ１遺伝子にフレームシフト変異が発生し、この遺伝子機能の喪失が引き起こされた。ｉｆｗ１－１およびｉｆｗ１－２株植物体におけるＩＦＷ１遺伝子のヌクレオチド配列は、配列番号２に示される配列である。 After performing sequencing analysis on the PCR products, two strains ifw1-1 and ifw1-2 were identified in which the IFW1 gene was successfully knocked out. Deletion of two bases in the coding region of the IFW1 gene in both plants of these two strains (Fig. 1) caused a frameshift mutation in the IFW1 gene, causing loss of this gene function. The nucleotide sequence of the IFW1 gene in ifw1-1 and ifw1-2 strain plants is the sequence shown in SEQ ID NO:2.

ステップ４：トマト果実の大きさの測定
同定されたＩＦＷ１遺伝子ノックアウト株ｉｆｗ１－１、ｉｆｗ１－２および野生型対照品種ＭｉｃｒｏＴｏｍを温室（２５℃、照明１６時間、暗所８時間）で植え付けた。トマト果実が熟した後、各品種からランダムに３株を選択し、各株から３つの熟した果実を取って果柄を除去した。ノギスを用いて各トマト果実の最大直径（ｃｍ）を測定し、各品種果実の直径の平均値を計算し、測定結果についてｔ検定によりｉｆｗ１－１（またはｉｆｗ１－２）ノックアウト株と野生型対照との間の有意性を分析した。得られた結果として、ＩＦＷ１遺伝子ノックアウト株ｉｆｗ１－１およびｉｆｗ１－２の果実の直径はいずれも野生型対照よりも顕著に大きく（図２、３）、それぞれ対照品種よりも２３．２％および２５．９％増大した。 Step 4: Tomato Fruit Size Measurement The identified IFW1 gene knockout strains ifw1-1, ifw1-2 and the wild-type control variety MicroTom were planted in a greenhouse (25° C., 16 hours light, 8 hours dark). After the tomato fruit ripened, 3 plants were randomly selected from each variety, and 3 ripe fruits from each plant were taken and the peduncle removed. The maximum diameter (cm) of each tomato fruit was measured using a vernier caliper, the average value of the fruit diameter of each variety was calculated, and the significance of the measurement results between the ifw1-1 (or ifw1-2) knockout strain and the wild-type control was analyzed by t-test. The results obtained showed that the fruit diameters of both IFW1 gene knockout strains ifw1-1 and ifw1-2 were significantly larger than the wild-type control (FIGS. 2, 3) and increased by 23.2% and 25.9% respectively over the control cultivar.

ステップ５：トマト果実の重量測定
ステップ４で培養したトマト果実を取り、電子天秤で各トマト果実の重量（ｇ）を秤量し、各品種の果実重量の平均値を計算した。測定結果について、ｔ検定によりｉｆｗ１－１（またはｉｆｗ１－２）ノックアウト株と野生型対照との間の有意差を分析した。得られた結果として、ＩＦＷ１遺伝子ノックアウト株ｉｆｗ１－１およびｉｆｗ１－２果実の重量はいずれも野生型対照よりも顕著に大きく（図４）、それぞれ対照品種よりも５１．７％および５７．４％増大した。 Step 5: Weight measurement of tomato fruit The tomato fruit cultured in step 4 was taken, and the weight (g) of each tomato fruit was weighed with an electronic balance to calculate the average fruit weight of each variety. Measurement results were analyzed for significant differences between ifw1-1 (or ifw1-2) knockout strains and wild-type controls by t-test. The results obtained showed that fruit weights of IFW1 gene knockout strains ifw1-1 and ifw1-2 were both significantly larger than wild-type controls (FIG. 4), increasing 51.7% and 57.4% over control cultivars, respectively.

以上の説明は、本発明のいくつかの具体的な実施例に過ぎない。本発明は、以上の実施例に限定されず、たくさんの変形例を含む。当業者が本発明の開示内容から直接導出または想到できる全ての変形は、いずれも本発明の保護範囲に含まれる。 The above descriptions are only some specific examples of the present invention. The present invention is not limited to the above examples, but includes many variations. All variations that can be directly derived or conceived by a person skilled in the art from the disclosure of the present invention shall fall within the protection scope of the present invention.

Claims (2)

トマト果実を増大させる方法であって、
トマト中でＩＦＷ１遺伝子をノックアウトすることによりトマト果実の大きさを増大させ、収量を向上させる工程を含み、
前記ＩＦＷ１遺伝子でコードされるヌクレオチド配列は配列番号１に示される配列であることを特徴とする、方法。 A method for increasing tomato fruit comprising:
increasing tomato fruit size and yield by knocking out the IFW1 gene in tomato;
A method , wherein the nucleotide sequence encoded by said IFW1 gene is the sequence shown in SEQ ID NO:1. 前記ノックアウトにより得られるトマトＩＦＷ１遺伝子の変異配列が、配列番号２に示される配列であることを特徴とする、請求項１に記載の方法。
 2. The method according to claim 1, wherein the mutant sequence of the tomato IFW1 gene obtained by knockout is the sequence shown in SEQ ID NO:2.
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