河北农业大学学报
河北農業大學學報
하북농업대학학보
JOURNAL OF AGRICULTURAL UNIVERSITY OF HEBEI
2001年
2期
69-70
,共2页
杨希才%刘国胜%王文慧%田波%居玉玲%罗智敏
楊希纔%劉國勝%王文慧%田波%居玉玲%囉智敏
양희재%류국성%왕문혜%전파%거옥령%라지민
无毒基因%晚疫病%转基因马铃薯%抗性
無毒基因%晚疫病%轉基因馬鈴藷%抗性
무독기인%만역병%전기인마령서%항성
晚疫病菌(Phytophthora infestans)引起的马铃薯晚疫病是危害全球马铃薯生产的严重病害.通过基因工程方法把外源基因导入植物体内以增强抗病性被证明是一条行之有效的途径.应用植物基因工程技术将具有能激活植物自身防御系统的无毒基因与适合于植物背景、非专一性的病原物诱导启动子组合成嵌合基因构建到植物表达载体中.通过农杆菌或基因枪的介导转化植物,可筛选出高效广谱的抗真菌和细菌病害的转基因植株.本研究从病原细菌Pseudomonas syringae PV. tomato中获得的无毒基因avrD (0*93 kb) 和从病原真菌Phytophthora parasitica中获得的无毒基因Elicitin(0.294 kb)分别与非专一性病原物诱导启动子Pill和BG组成含2个嵌合基因(Pill-avrD, BG-Elicitin)的植物表达载体pYH144和pYHEt.通过农杆菌LBA4404介导转化马铃薯,其中用pYH144载体转化2个品种(克新1号,2号),用pYHEt载体转化3个品种(Desiree,克新2号,4号),通过组织培养分别获得潮霉素(Hygromycin B)标记的转基因马铃薯试管苗.将转基因试管苗扩繁,应用马铃薯脱毒微型种薯生产技术获得转无毒基因微型薯,在温室(15～25℃和湿度高)条件下,观察转无毒基因马铃薯植株中对晚疫病菌自然感染的抗性.1998年和1999年(每年的3-5月)的温室试验初步表明:用avrD和elicitin基因分别转化的转无毒基因马铃薯植株都具有较明显的对晚疫病菌侵染的抗性,大部分转基因植株不表现或表现轻微的感病症状,对照植株(未转化)则表现明显的感病症状.转基因植株生长正常,且在感染后期(恢复期)生长良好.对照植株在恢复期生长弱和缓慢.在获得较多数量的转无毒基因马铃薯微型种薯的时候,将进行人工接种晚疫病菌和田间种植试验,从中筛选出抗真菌病和细菌病的转基因马铃薯株系.
晚疫病菌(Phytophthora infestans)引起的馬鈴藷晚疫病是危害全毬馬鈴藷生產的嚴重病害.通過基因工程方法把外源基因導入植物體內以增彊抗病性被證明是一條行之有效的途徑.應用植物基因工程技術將具有能激活植物自身防禦繫統的無毒基因與適閤于植物揹景、非專一性的病原物誘導啟動子組閤成嵌閤基因構建到植物錶達載體中.通過農桿菌或基因鎗的介導轉化植物,可篩選齣高效廣譜的抗真菌和細菌病害的轉基因植株.本研究從病原細菌Pseudomonas syringae PV. tomato中穫得的無毒基因avrD (0*93 kb) 和從病原真菌Phytophthora parasitica中穫得的無毒基因Elicitin(0.294 kb)分彆與非專一性病原物誘導啟動子Pill和BG組成含2箇嵌閤基因(Pill-avrD, BG-Elicitin)的植物錶達載體pYH144和pYHEt.通過農桿菌LBA4404介導轉化馬鈴藷,其中用pYH144載體轉化2箇品種(剋新1號,2號),用pYHEt載體轉化3箇品種(Desiree,剋新2號,4號),通過組織培養分彆穫得潮黴素(Hygromycin B)標記的轉基因馬鈴藷試管苗.將轉基因試管苗擴繁,應用馬鈴藷脫毒微型種藷生產技術穫得轉無毒基因微型藷,在溫室(15～25℃和濕度高)條件下,觀察轉無毒基因馬鈴藷植株中對晚疫病菌自然感染的抗性.1998年和1999年(每年的3-5月)的溫室試驗初步錶明:用avrD和elicitin基因分彆轉化的轉無毒基因馬鈴藷植株都具有較明顯的對晚疫病菌侵染的抗性,大部分轉基因植株不錶現或錶現輕微的感病癥狀,對照植株(未轉化)則錶現明顯的感病癥狀.轉基因植株生長正常,且在感染後期(恢複期)生長良好.對照植株在恢複期生長弱和緩慢.在穫得較多數量的轉無毒基因馬鈴藷微型種藷的時候,將進行人工接種晚疫病菌和田間種植試驗,從中篩選齣抗真菌病和細菌病的轉基因馬鈴藷株繫.
만역병균(Phytophthora infestans)인기적마령서만역병시위해전구마령서생산적엄중병해.통과기인공정방법파외원기인도입식물체내이증강항병성피증명시일조행지유효적도경.응용식물기인공정기술장구유능격활식물자신방어계통적무독기인여괄합우식물배경、비전일성적병원물유도계동자조합성감합기인구건도식물표체재체중.통과농간균혹기인창적개도전화식물,가사선출고효엄보적항진균화세균병해적전기인식주.본연구종병원세균Pseudomonas syringae PV. tomato중획득적무독기인avrD (0*93 kb) 화종병원진균Phytophthora parasitica중획득적무독기인Elicitin(0.294 kb)분별여비전일성병원물유도계동자Pill화BG조성함2개감합기인(Pill-avrD, BG-Elicitin)적식물표체재체pYH144화pYHEt.통과농간균LBA4404개도전화마령서,기중용pYH144재체전화2개품충(극신1호,2호),용pYHEt재체전화3개품충(Desiree,극신2호,4호),통과조직배양분별획득조매소(Hygromycin B)표기적전기인마령서시관묘.장전기인시관묘확번,응용마령서탈독미형충서생산기술획득전무독기인미형서,재온실(15～25℃화습도고)조건하,관찰전무독기인마령서식주중대만역병균자연감염적항성.1998년화1999년(매년적3-5월)적온실시험초보표명:용avrD화elicitin기인분별전화적전무독기인마령서식주도구유교명현적대만역병균침염적항성,대부분전기인식주불표현혹표현경미적감병증상,대조식주(미전화)칙표현명현적감병증상.전기인식주생장정상,차재감염후기(회복기)생장량호.대조식주재회복기생장약화완만.재획득교다수량적전무독기인마령서미형충서적시후,장진행인공접충만역병균화전간충식시험,종중사선출항진균병화세균병적전기인마령서주계.
Potato late blight caused by Phytophthora infestans is a serious and destructive disease worldwide. To transform exogenous genes to plant and enhance its resistance to diseases by genetic engineering methods has been proved to be an effective way. Using plant genetic engineering techniques, a chimeric gene is constructed by combining an avirulent gene which can activate plant autologous defensive system with a non-specific pathogen induced promoter which is suitable to plant background, and inserted into a plant expressive vector. Then it can be transformed to plant by Agrobacterium tumefaciens or microparticle bombardment. Transgenic plant with effective and extensive resistance can be screened. In this study, an avirulent gene avrD (0.93 kb) isolated from a pathogenic bacteria Pseudomonas syringae PV. tomato and another avirulent gene Elicitin (0.294 kb) isolated from a pathomycete Phytophthora parasitica were ligated respectively with the non-specific pathogen induced promoters Pill and BG to construct plant expressive vectors pYH144 and pYHEt containing the two chimeric genes (Pill-avrD, BG-Elicitin). They were then transformed to potatoes by Agrobacterium tumefaciens LBA 4404. The vector pYH144 was transferred into two potato varieties (Kexin No.1 and No.2), and the vector pYHEt was transferred into three potato varieties (Desiree, Kexin No.2 and No.4). Transgenic potato tube seedlings labelled with Hygromycin B resistance were obtained by tissue culture. Then the transformed seedlings were proliferated. Transgenic potato minitubers were produced by a method of producing virus free potato minitubers. The resistance of the transgenic potato plant naturally infected P. infestans was recorded in greenhouse conditions (15～20℃, with high humidity). The greenhouse experiments in 1998 and 1999 (from March to May every year) primarily showed that the potato seedlings transformed with avrD gene or Elicitin gene have significant resistance to potato late blight, and most of them have no or slight symptoms of the disease, while the control (non-transgenic) potato seedlings have manifest symptom of the disease. The transgenic potato seedlings grow normal, and grow better in the late phase (the recovery phase), while the control potato seedlings grow weaker and slower in the recovery phase. When we have got enough number of the trans-avirulent gene minitubers, we will inoculate P. infestans to the transformed potato plant in the field, and select the transgenic potato plant with high resistance to fungal and bacterial diseases.
