水稻抗除草剂、抗稻瘟病基因转化及遗传特性研究
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摘要
本文对吉林省主栽水稻品种进行了组织培养特性的优化研究；应用基因枪法将抗BASTA除草剂基因(bar)和抗稻瘟病基因(植物抗毒素反二苯乙烯合成酶)转入吉林省主栽的高产、优质水稻品种中，获得了转基因水稻植株；对转抗稻瘟病基因水稻植株进行了抗稻瘟病性鉴定和遗传学分析，研究结果如下：
一、优化了吉林省主栽水稻品种(系)成熟种子胚的组织培养体系
1．不同品种(基因型)的愈伤组织诱导频率和分化频率差别很大，吉89-45、超产2号、丰优501和丰优307四个基因型的愈伤组织诱导频率分别为86.4％、88.7％、80.8％和81.6％；吉89-45、超产2号和丰优501分化率分别达到50.1％、45.8％和49.5％。
2．继代培养基中减少2，4二氯苯氧乙酸(2.4-D)用量到1.0mg/L，添加萘乙酸(NAA)1mg/L和激动素(KT)0.5mg/L可以改善水稻愈伤组织的生长状态。圆型，色泽浅黄，质地紧密，分散性好的胚性愈伤组织数量增多，提高了水稻愈伤组织的分化能力。
3．分化前干燥处理16小时，可以使水稻愈伤组织的分化率提高20.6％-37.2％。处理时间达到36小时后，愈伤组织的分化能力反而下降。
二、应用Gus基因(β-glucuronidase)的瞬间表达研究，优化了PDS-1000型基因枪转化吉林省主栽粳稻品种(Japonica)成熟胚来源愈伤组织的各种参数
1．在DNA－金弹与受体靶细胞距离为9厘米，轰击2次时，转化效
率最高，GUS基因的瞬间表效率达到88．2％；当轰击距离为6厘米时，
瞬间表达效率有所提高，但金粉对靶细胞产生破坏作用，转化效率降
低；轰击距离为12厘米时转化效率降低；
2．轰击距离相同，随着轰击次数的增多，GS S基因的瞬间表达效率
提高。轰击距离为9厘米时，轰击1、2、37！K时，GuS基因的瞬间表
达效率分别为 30．60％、88．25％和 90．04％。
三、获得了抗BASTA除草剂的转基因水稻植株
以水稻成熟胚来源的胚性愈伤组织为受体，应用基因枪法将质粒
0Dm03 含报告基因Gus和抗除草剂基因bar）转入北方早熟栽培粳
稻品种吉89－45中，获得了转基因水稻植株。经转基因植株叶片的除
草剂淙布试验表明，D；，－l，D。－2，D。－3三个转基囚植株水稻叶片颜色
没有变化，仍为绿色，表现对3ASTA除草剂的抗性，证明外源基囚在
转基因叶片中表达，使水稻叶片产生对BA盯A除草剂抗性。经pCR和
Southe，n杂交检测，证明抗除草剂基回己整合到水稻基因组中cD。－5
转基因植株中检测到429加的ba，基因目标带，但对除草剂敏感，外
源基因在D。－5植株中产生基因沉默现象。
四、获得了转抗稻瘟病基因的转基因水稻植株
应用基因枪法将抗稻瘟病基因转入吉林省主栽的高产、优质、稻瘟
病抗性弱的水稻品种超产2号和丰优201中。经PCR检测，转基因植
株中存在抗稻瘟病基因的目标带。
五、对转抗稻瘟病基因水稻进行了抗病性和遗传学分析
1．应用稻瘟病抱子对转抗稻瘟病基因水稻植株进行接种鉴定和
PCR检测技术，分析了抗稻瘟病基因在T；代和T。代转基因水稻中的遗
传特性和抗病性。研究表明，抗稻瘟性基因可以提高转基因水稻的稻
互且
瘟病抗性，外源基因可以通过有性世代遗传给下一代。抗稻瘟病基因
在T；代和L代未纯合的转基因水稻中呈3：1的盂得尔方式分离。获得
了抗稻瘟病性好，外源基因纯合的转基因水稻株系。
2．T；代转基因水稻植株农艺性状调查表明，基因枪法获得的转基
因后代产生大量变异。株高，分菜性，穗粒数，出穗日期和育性都产
生变异。
六、应用基因工程技术将抗水稻纹枯病基因（X821）连接到载体
（扯川阻A 1201）中
The tissue culture system for elite rice (Japonica) varieties (Fengyou 201 and Chaochan 2, high yield, good quality, but blast disease sensitive) cultivated in Jilin province was optimized. A herbicide and a rice blast disease resistant gene were delivered into those elite varieties. Herbicide and blast disease resistant transgenic rice plants were obtained. The blast resistance of transgenic rice plants and the inheritance of foreign gene in the progeny of those transgenic rice plants were analyzed. Results are as follows:
1.The tissue cultural system of mature embryos of elite cultivated rice varieties in Jilin province was optimized.
1.1 The callus induction and differentiation efficiency of different genotypes is quite different. The callus induction efficiency of Ji 89-48, Chaochan 2, Fengyou 501, and Fengyou 307 could be as high as 86.4, 88.7, 80.8 and 81. 6 percent respectively. And the differentiation efficiency of Ji 89-45, Chaochan 2 and Fengyou 501 could be as high as 50.1, 45,8 and 49.5 percent respectively.
1.2 Reducing 2. 4-D concentration to 1 mg/L, adding NAA (1 mg/L) and KT (0. 5 mg/L) in the subculture medium can improve the growth of callus. And more yellowish, grain-shape cmbryogenic calli was obtained.
1. 3 Drying treatment of calli for 16h before differentiation could increase plant regeneration efficiency by 20.6-37.2 percent.
2. Parameters of Gene Gun (PDS-1000) transformation of the callus derived from mature embryo of elite rice varieties were studied by Gus gene transcient expression.
2. 1 When the distance between DNA-gold pellet and the target cells was 9 cm, bombard for 2 times, the transformation efficiency was the highest. The Gus gene transcient expression efficiency was as high as 88.2 percent.
2.2 When the distance was fixed, the Gus gene expression efficiency was increased with the increase of bombarded times. When the distance was 9 cm, and bombarded for 1, 2 and 3 times, Gus gene expression efficiency was 30.6, 88. 3 and 90.0 percent respectively.
3. A Herbicide resistant gene (bar) has been delivered into rice. And herbicide resistant transgenic rice plants were obtained.
Using the erabryogenic calli derived from mature embryo of elite rice varieties (high yield and early maturing) as the explant, several transgenic rice plants were obtained via Biolistic gene delivering method, bar gene had been inserted into rice genome demonstrated by PCR and Southern hybridization. Three transgenic rice plants (D 4. A grapevine stilbene synthase gene has been delivered into elite Japonica rice varieties wildly planted in Jilin province by Biolistic method. And rice blast resistant transgenic plants were obtained.
5. The blast resistance of transgenic rice plants was assayed by blast disease spore incubation. And the inheritance of grapevine stilbene synthase gene in Ti and T2 generation of transgenic rice was analyzed by PCR.
5. 1 The grapevine stilbene synthase gene could enhance the
blast resistance in transgenic rice plants, grapevine stilbene synthase gene could be transferred to the progeny, and showed 3:1 Memdelian segregation pattern.
5.2 The agronomic important trait of transgenic rice plants in T; generation showed great variation, such as plant hight, tillering ability, heading time and fertility. 6. The bacterial blight resistant, gene (Xa21) was reconstructed into vactor (pCAMBIA 1201) in order to transform rice.
一、优化了吉林省主栽水稻品种(系)成熟种子胚的组织培养体系
1．不同品种(基因型)的愈伤组织诱导频率和分化频率差别很大，吉89-45、超产2号、丰优501和丰优307四个基因型的愈伤组织诱导频率分别为86.4％、88.7％、80.8％和81.6％；吉89-45、超产2号和丰优501分化率分别达到50.1％、45.8％和49.5％。
2．继代培养基中减少2，4二氯苯氧乙酸(2.4-D)用量到1.0mg/L，添加萘乙酸(NAA)1mg/L和激动素(KT)0.5mg/L可以改善水稻愈伤组织的生长状态。圆型，色泽浅黄，质地紧密，分散性好的胚性愈伤组织数量增多，提高了水稻愈伤组织的分化能力。
3．分化前干燥处理16小时，可以使水稻愈伤组织的分化率提高20.6％-37.2％。处理时间达到36小时后，愈伤组织的分化能力反而下降。
二、应用Gus基因(β-glucuronidase)的瞬间表达研究，优化了PDS-1000型基因枪转化吉林省主栽粳稻品种(Japonica)成熟胚来源愈伤组织的各种参数
1．在DNA－金弹与受体靶细胞距离为9厘米，轰击2次时，转化效
率最高，GUS基因的瞬间表效率达到88．2％；当轰击距离为6厘米时，
瞬间表达效率有所提高，但金粉对靶细胞产生破坏作用，转化效率降
低；轰击距离为12厘米时转化效率降低；
2．轰击距离相同，随着轰击次数的增多，GS S基因的瞬间表达效率
提高。轰击距离为9厘米时，轰击1、2、37！K时，GuS基因的瞬间表
达效率分别为 30．60％、88．25％和 90．04％。
三、获得了抗BASTA除草剂的转基因水稻植株
以水稻成熟胚来源的胚性愈伤组织为受体，应用基因枪法将质粒
0Dm03 含报告基因Gus和抗除草剂基因bar）转入北方早熟栽培粳
稻品种吉89－45中，获得了转基因水稻植株。经转基因植株叶片的除
草剂淙布试验表明，D；，－l，D。－2，D。－3三个转基囚植株水稻叶片颜色
没有变化，仍为绿色，表现对3ASTA除草剂的抗性，证明外源基囚在
转基因叶片中表达，使水稻叶片产生对BA盯A除草剂抗性。经pCR和
Southe，n杂交检测，证明抗除草剂基回己整合到水稻基因组中cD。－5
转基因植株中检测到429加的ba，基因目标带，但对除草剂敏感，外
源基因在D。－5植株中产生基因沉默现象。
四、获得了转抗稻瘟病基因的转基因水稻植株
应用基因枪法将抗稻瘟病基因转入吉林省主栽的高产、优质、稻瘟
病抗性弱的水稻品种超产2号和丰优201中。经PCR检测，转基因植
株中存在抗稻瘟病基因的目标带。
五、对转抗稻瘟病基因水稻进行了抗病性和遗传学分析
1．应用稻瘟病抱子对转抗稻瘟病基因水稻植株进行接种鉴定和
PCR检测技术，分析了抗稻瘟病基因在T；代和T。代转基因水稻中的遗
传特性和抗病性。研究表明，抗稻瘟性基因可以提高转基因水稻的稻
互且
瘟病抗性，外源基因可以通过有性世代遗传给下一代。抗稻瘟病基因
在T；代和L代未纯合的转基因水稻中呈3：1的盂得尔方式分离。获得
了抗稻瘟病性好，外源基因纯合的转基因水稻株系。
2．T；代转基因水稻植株农艺性状调查表明，基因枪法获得的转基
因后代产生大量变异。株高，分菜性，穗粒数，出穗日期和育性都产
生变异。
六、应用基因工程技术将抗水稻纹枯病基因（X821）连接到载体
（扯川阻A 1201）中
The tissue culture system for elite rice (Japonica) varieties (Fengyou 201 and Chaochan 2, high yield, good quality, but blast disease sensitive) cultivated in Jilin province was optimized. A herbicide and a rice blast disease resistant gene were delivered into those elite varieties. Herbicide and blast disease resistant transgenic rice plants were obtained. The blast resistance of transgenic rice plants and the inheritance of foreign gene in the progeny of those transgenic rice plants were analyzed. Results are as follows:
1.The tissue cultural system of mature embryos of elite cultivated rice varieties in Jilin province was optimized.
1.1 The callus induction and differentiation efficiency of different genotypes is quite different. The callus induction efficiency of Ji 89-48, Chaochan 2, Fengyou 501, and Fengyou 307 could be as high as 86.4, 88.7, 80.8 and 81. 6 percent respectively. And the differentiation efficiency of Ji 89-45, Chaochan 2 and Fengyou 501 could be as high as 50.1, 45,8 and 49.5 percent respectively.
1.2 Reducing 2. 4-D concentration to 1 mg/L, adding NAA (1 mg/L) and KT (0. 5 mg/L) in the subculture medium can improve the growth of callus. And more yellowish, grain-shape cmbryogenic calli was obtained.
1. 3 Drying treatment of calli for 16h before differentiation could increase plant regeneration efficiency by 20.6-37.2 percent.
2. Parameters of Gene Gun (PDS-1000) transformation of the callus derived from mature embryo of elite rice varieties were studied by Gus gene transcient expression.
2. 1 When the distance between DNA-gold pellet and the target cells was 9 cm, bombard for 2 times, the transformation efficiency was the highest. The Gus gene transcient expression efficiency was as high as 88.2 percent.
2.2 When the distance was fixed, the Gus gene expression efficiency was increased with the increase of bombarded times. When the distance was 9 cm, and bombarded for 1, 2 and 3 times, Gus gene expression efficiency was 30.6, 88. 3 and 90.0 percent respectively.
3. A Herbicide resistant gene (bar) has been delivered into rice. And herbicide resistant transgenic rice plants were obtained.
Using the erabryogenic calli derived from mature embryo of elite rice varieties (high yield and early maturing) as the explant, several transgenic rice plants were obtained via Biolistic gene delivering method, bar gene had been inserted into rice genome demonstrated by PCR and Southern hybridization. Three transgenic rice plants (D
5. The blast resistance of transgenic rice plants was assayed by blast disease spore incubation. And the inheritance of grapevine stilbene synthase gene in Ti and T2 generation of transgenic rice was analyzed by PCR.
5. 1 The grapevine stilbene synthase gene could enhance the
blast resistance in transgenic rice plants, grapevine stilbene synthase gene could be transferred to the progeny, and showed 3:1 Memdelian segregation pattern.
5.2 The agronomic important trait of transgenic rice plants in T; generation showed great variation, such as plant hight, tillering ability, heading time and fertility. 6. The bacterial blight resistant, gene (Xa21) was reconstructed into vactor (pCAMBIA 1201) in order to transform rice.
引文
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