報告書番号

U94041

タイトル（和文）

分子育種法による高鉄含量レタスの作出（１）フェリチン遺伝子のレタスへの導入

タイトル（英文）

BREEDING OF HIGH IRON LETTUCE(LACTUCA SATIVAL.)(1)PRODUCTION OF (1)PRODUCTION OF TRANSGENIC LETTURCE PLANTS CONTAINING SOYBEAN FERRITIN GENE

概要 （図表や脚注は「報告書全文」に掲載しております）

フェリチン（鉄貯蔵蛋白質）遺伝子を改変し，葉中で恒常的にフェリチンを合成させ，その中に鉄を貯えることによりレタスの鉄含量を高めることを検討した。（１）サザンブロット分析によりレタスフェリチン遺伝子は検出されたが，ダイズ等のフェリチン遺伝子を活性化するような鉄刺激に対する発現応答は認められなかった。（２）ＣａＭＶの３５Ｓプロモーターにより転写制御を受けるように改変したダイズフェリチンｃＤＮＡを持つベクターｐＢＧ－１を構築した。ｐＢＧ－１をアグロバクテリウムを介してレタスへ導入し，カナマイシン（５０μｇ／ｍｌ）を含むＭＳ培地で選抜したところ，耐性を示す固体を得た。それらの個体からＤＮＡを抽出し，ＰＣＲ分析した結果，形質転換レタスであることが認められた。

概要 （英文）

VEGETABLE FACTORIES MAKE IT POSSIBLE TO GROWHIGH-QUALITY VEGETABLES THROUGH OUR THE YEAR.LETTUCE(LACTUCA SATIVA L.)IS A MAIN PRODUCT OF VEGETABLE FACTORIES.IF IRON-ENRICHED LETTUCE CAULD BE CREATED BY GENE MANIPULATION IT WAULD PROVIDE MINERALS AND HAVE ADDITIONAL VALUE AS A NEW SALAD VEGETABLE. WE TRIED GENE TRANSFER FROM SOYBEAN TO LETTUCE OF THE FERRITIN PROTEIN WHICH CONTAINS MORE THAN 4000 IRON ATOMS.LETTUCE HAS ITS OWN FERRITIN GENE PROVED BY SOUTHERN BLOT ANALYSIS OF ITS DNA USING THE SOYBEAN FERRITIN GENE FRAGMENT AS A PROBE.HOEVER,CHANGING THE IRON CONCENTRATION IN THE HYDROPONIC CULTURE MEDIUM DID NOT INDUCE EXPRESSION OF THE FERRITIN GENE. THEREFORE,TO INCREASE THE IRON CENTENT WE TRANSFERRED A MODIFIED FERRITIN GENE TO LETTUCE.FERRITIN CDNA WAS CLONED BY RT-PCR(REVERSE TRANSCRIPTASE-POLYMERIZE CHAIN REACTION)FROM MRNA ISOLSTED FROM SOYBEAN(GLYCINE MAX MERR)LEAVES AS A TEMPLATE.THE CDNA HAD 96% HOMOLOGY WITH A KNOWN SEQUENCE AND INCLUDED AN OPEN READING FRAME.A NEW VECTOR PLASMID PBG-1,DERIVED FROM PBI121 WAS CONSTRUCTED(12.8KP).IT CONTAINED SOYBEAN FERRITIN CDNA UNDER CONTROL OF CAULIFLOWER MOSAIC VIRUS 35S PROMOTER AND ALSO CONTAINED NEOMYCIN PHOSPHOTRANSFERASE II(NPT II)GENE.THE VECTOR WAS TRANSFERRED TO SEGMENTS OF LETTUCE LEAF VIA AGROBACTERIUM TUMEFACIENS STRAIN LBA4404.THE EXPLANTS WERE SELECTED BY KANAMYCIN(50MICRO G/ML).AFTER 40 DAYS,2% OF KANAMYCIN-RESISTANT LETTUCES HAD SURVIVED.THE PRESENCE OF TRANSFERRED GENES WAS TESTED BY PCR WITH THEIR GENOMIC DNAS.TWO BANDS DERIVED FROM INTRODUCED FOREIGN NPT II AND THE FERRITIN GENE WERE OBSERVED IN ALL RESISTANT LETTUCES.

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