研究報告/第75号

農業・園芸総合研究所研究報告

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第75号：2006年3月発行

第75号抄録
遺伝子組換え植物における導入遺伝子発現用プロモーターに関する研究
瀬尾直美
Studies of the Promoters for Transgene Expression in Transgenic Plants
Naomi SHIRASAWA-SEO

遺伝子組換え技術を利用した植物の改良において,導入遺伝子の発現を適切に制御するプロモーターの開発が必要不可欠である。遺伝子組換え植物では,予期しない導入遺伝子及びこれに相同性のある内在性遺伝子の発現抑制（ジーンサイレンシング）が起こることがあり,その機構を解明する必要がある。代表的な導入遺伝子発現用プロモーターとして広く用いられているカリフラワーモザイクウイルス由来35SRNAプロモーター（P35S）を改変し,高発現を可能にしたプロモーターが開発されている。本研究では,これをホタル由来ルシフェラーゼ遺伝子に連結し,導入したタバコで起きるジーンサイレンシングの様相を詳細に解析した。その結果,このジーンサイレンシングは,活発な細胞分裂により一旦解除されて,次世代には移行せず,生育中に新たに開始することが明らかとなった。また,P35S改変高発現プロモーターを用いて形質転換キクを作出したところ,全ての個体において導入遺伝子の発現が認められなかった。原因の解明を試みた結果,導入遺伝子のメチル化が関与していると考えられた。
予期しないジーンサイレンシングを回避するための一つの手段として,同一の植物形質転換用ベクター内に相同性のある遺伝子配列の反復使用を避けることが望ましい。しかしながら,現状では利用可能なプロモーターが限られているため,目的遺伝子と選抜マーカー用遺伝子に同じプロモーターを繰り返し使用することが多い。これを解決するために,P35Sと代替可能なプロモーターの開発を行った。遺伝子の解析を目的として既に単離・解析されている植物由来のプロモーターをデータベースで検索し,利用可能と考えられたシロイヌナズナ由来のトリプトファン合成酵素βサブユニット遺伝子のプロモーター（PTSB1）及びフィトクロームB遺伝子のプロモーター（PPHYB）を選出した。PTSB1及びPPHYBを各々GUS遺伝子及びカナマイシン耐性遺伝子に連結し,タバコに導入して実用性を評価したところ,これらはP35Sに匹敵するプロモーターとして有用であることが示された。PTSB1を抗菌性タンパク質であるエンバク由来チオニン遺伝子に連結し,カーネーションに導入したところ,得られた形質転換体においてカーネーション萎凋細菌病抵抗性が付与された。次に,新奇性・利用性の高いプロモーターを得ることを目的として,1本鎖DNAウイルスであるレンゲ萎縮ウイルスに由来する11種類のプロモーターについて,形質転換タバコにおける発現特性を明らかにした。これらのうち最も構成的に近い発現を示した細胞間移行タンパク質遺伝子のプロモーターPMC8の発現様式をさらに詳細に解析した結果,PMC8が双子葉植物のタバコだけでなく,単子葉植物のイネにおいても発現するP35Sと代替可能なプロモーターとして有用であることが示された。

［キーワード］
遺伝子組換え植物,プロモーター,導入遺伝子,ジーンサイレンシング,P35S

＜Summary＞

(1-1) Transgenic tobacco plants over-expressing luciferase (luc) gene driven by a modified P35S, frequently exhibit post-transcriptional gene silencing (PTGS) of luc. The silencing was observed over five generations and found not to be inherited but acquired by the next generation at a certain frequency. Luc imaging analysis of silenced plants revealed luc activity only in proliferating tissues such as shoot meristem and developing flower. The luc gene expression has been recovered from silencing before development of germ cells, excluding a possible recovery from the PTGS at meiosis. A systemic silencing signal transferred from older tissue likely induces gene silencing of younger tissues in which cell proliferation has been completed. Only seeds maintained Luc activity, probably because of isolation from the silencing signal by a possible partition from the parent placenta. Calli newly induced from the leaf pieces of silenced plants recovered from silencing and exhibited strong luc activity similar to nonsilenced leaves, further indicating that the silencing cannot be maintained in proliferating cells. Thus release from PTGS in proliferating cells is a possible mechanism for noninheritance of silencing.

(1-2) Transgenic chrysanthemum [Dendranthema grandiflorum (Ramat.) Kitamura] (2n=54) plants containing the β-glucronide (GUS) gene or oat thionin (Asthi1) gene driven by a modified P35S (PE7ΩI::GUS, PE7ΩI::Asthi1, PEl2Ω::GUS, PEl2Ω::Asthi1), were generated using transformation mediated by Agrobacterium. However, the expression of the introduced gene in these transgenic plants was not detected. When PE7ΩI::GUS chrysanthemum plants were treated by 5’-azacytidine, some of them showed GUS expression chimerically. This result indicated that this transgene inactivation was concerned with methylation. Transgenic wild chrysanthemum [D. boreale (Makino) Ling (2n=18)] plants containing PEl2Ω::GUS were also produced and assayed by histochemical GUS analysis. The GUS staining in these plants was detected in trichomes and vascular bundles. These results indicated that a modified P35S was not suitable for the expression of the introduced genes in transgenic chrysanthemum.

(2-1) To search for strong promoters that confer constitutive expression of transgenes, the promoters of an Arabidopsis tryptophan synthase protein β subunit gene (PTSB1) and a phytochrome B gene (PPHYB) as alternatives to the 35S RNA promoter (P35S) of Cauliflower mosaic virus were evaluated. Characteristics of the Soybean chrolotic mottle virus promoter (PNCR) were also studied for comparison. In transgenic calli, GUS gene fused with PTSB1, PPHYB and PNCR showed 50% or more of the activity of P35S. To drive the NPTII marker gene, the four promoters were similarly useful. In generated transgenic tobacco plants, both PTSB1 and PPHYB were active in all tissues tested, and superior to P35S in the leaves. The four promoters differed slightly in their tissue-specific expression, but were expressed constitutively, indicating that PTSB1 and PPHYB as well as PNCR are useful as strong and constitutive promoters as alternatives to P35S for genetic manipulation of plants.

(2-2) The oat thionin gene (Asthi1) driven by PTSB1 was introduced into carnation (Dianthus caryphyllus L.) via Agrobacterium-mediated gene transfer. Leaf bases of three cultivars of carnation (‘Scania’, ‘Percian Pink-Sim’ and ‘U. Conn. White Sim’) were infected with Agrobacterium harboring the binary vector with PTSB1::Asthi1 and hygromycin genes, and cultured on the medium containing hygromycin. Independent 99 lines resistant to hygromycin were regenerated and the introduction of the transgene was confirmed in 11 lines by polymerase chain reaction. Transgenic carnation plants exhibited the resistance against infection with Burkholderia caryophylli which caused bacterial wilt, and the levels of resistance correlated with those of the thionin gene transcript.

(3-1) Characteristic natures of promoter regions associated with each of the eleven single strand DNA components of Milk vetch dwarf virus (MDV) have been examined in tobacco plants. Predicted promoter regions of MDV component 1 to 11 (C1 - C11) were isolated and fused with GUS reporter gene to assess promoter activity. In transgenic calli, the promoter of MDV C4, C5, C6, C7 and C8 generated a stronger level of GUS expression than P35S. In leaves of generated transgenic tobacco plants, the promoter of C5 and C8 conferred comparable or more of the GUS activity of P35S. Histochemical GUS analysis showed that the promoters of C4, C5, C6, C7 and C9 were active in phloem and meristem. The promoter of C8 is active in most cell types. Almost no activities were generated by the promoters of C1, C2, C3, C10 and C11, which encode putative replication-associated proteins, in transgenic tobacco calli and plants.

(3-2) The activity of a predicted promoter PMC8 from Milk vetch dwarf virus was evaluated by comparing it with the Cauliflower mosaic virus 35S RNA promoter (P35S) and PNCR, a promoter from Soybean chlorotic mottle virus. When the GUS fusion gene was introduced into tobacco, PMC8 showed a similar expression profile to P35S but with a more intense expression in proliferating tissues. The usefulness of PMC8 was confirmed by driving NPTII for selection of kanamycin-resistant tobacco plants with improved transformation efficiency. PMC8 was also effective in transgenic rice plants. Thus, PMC8 is useful as an alternative to P35S in both dicotyledonous and monocotyledonous plants, especially for gene expression in proliferating tissues.