致雄性不育基因—反义肌动蛋白基因和核糖核酸酶基因（Barnase）导入甘蓝的研究
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摘要
近几年来，随着分子生物学的发展，使利用基因工程技术创制雄性不育系成为可能。由于利用基因工程技术可以在较短的时间内创制雄性不育系以及相应恢复系，在一种作物上获得突破后，其应用可以迅速涵盖于其他作物。因而，近年来该技术在植物工程雄性不育的创制中被大量应用并已逐步走向商业化。结球甘蓝(Brassica oleracea. L)，属十字花科芸薹属甘蓝的一个变种。甘蓝的杂种优势明显，长期以来甘蓝主要利用自交不亲和系制种。但自交不亲和系的应用存在以下两方面的缺点：首先，长期连续自交会导致后代生活力的衰退，种子繁殖系数下降，且自交不亲和性还有可能减弱，从而使杂交率降低；其次，蕾期人工授粉，操作麻烦，效率低；且要花费大量的人力物力，增加了制种的成本。如果利用雄性不育系制种则可克服上述缺点。由于传统的植物雄性不育系选育得通过自然变异、人工诱变、远缘杂交等方法，并经过多代选育才有可能获得，费时费力。鉴于上述原因，本研究将致雄性不育的基因……反义肌动蛋白基因和Barnase基因分别与不同组织表达特异启动子构成嵌合基因后分别通过农杆菌介导转化甘蓝，以期获得雄性不育转基因甘蓝植株。主要的工作和结果如下：
1．甘蓝高频再生体系的建立
以甘蓝的下胚轴为外植体，以MS为基本培养基(添加0.7％的琼脂和3％的蔗糖)，加入不同浓度和组合的激素6-BA、NAA，筛选出下胚轴不定芽分化最佳培养基(MS+BA1.0mg/L+NAA0.2mg/L)；并比较了甘蓝下胚轴和子叶两种不同外植体的分化能力，下胚轴的分化频率高于子叶。同时进行了甘蓝不定芽的生根实验，在1/2 Ms培养基中添加不同浓度IAA，结果显示不论IAA浓度是多少，不定芽都能生根，只是生根的时间随着IAA的浓度的升高而缩短。
2．甘蓝外植体分化、不定芽生长以及不定芽生根对除草剂(PPT)的敏感性分析
盈努浩盆龄诊料述吝一一一一一~
在分化培养基中加入浓度为2.omg/L的PPT时，外植体愈伤组织形成明显受到抑制，形
成的愈伤组织也部分死亡:当加入的PPT浓度为2.smg/L时，外植体及部分形成的愈伤组织
全部逐渐白化死亡;当PPT浓度为3.Omg/L时，外植体未形成愈伤组织就白化死亡，因此确
定PPT 2.Slng/L的浓度作为外植体转化的筛选压.分化出的不定芽，转到P盯浓度为3.omg/L
的分化培养基中时，大部分会白化死亡，当PPT浓度为3.smg/L时，不定芽全部白化死亡;
在不定芽生根时，加入的P盯浓度为1.smg/L时，不定芽生根就受到抑制。因此，以PPT2.smg/L
为抗性不定芽分化的筛选压，以PPT3.smg/L作为抗性不定芽生长的筛选压，以PPTI.smg/L
作为抗性不定芽生根的筛选压。
3.甘蓝外植体高频转化体系的建立
在最佳培养基上试验了外植体的预培养时间、浸菌时间、共培养时间、选用何种抗生素
抑菌以及乙酸丁香酮(AS)等因素对转化率的影响，建立了甘蓝外植体的高频转化体系:以
6一7天的甘蓝下胚轴为外植体，分化培养基上预培养2天，在用Ms液体培养墓稀释的菌液中
感菌3一5分钟，共培养2天后，转入筛选培养基1(Ms+6一BA 1.omg/L+NAAo.Zmg/L+ PPT2.smg/L+
Ca比500呢/L)中筛选抗性愈伤组织和抗性芽，每两周换一次培养基。筛选出的抗性芽转到筛
选培养基2(MS+6一BA 1.omg/L十NAAO.Zmg/L+即T3.smg/L+Carb400mg/L)中筛选，并逐渐降
低Carb.的浓度。将在筛选培样基2中得到的抗性芽转入生根培养基(MS+I从0.lmg/L+
PPTI.sm以L+ CarbZoo雌/L)中进行生根筛选，逐步淘汰未转化的“假转化体”，得到具有P盯
抗性的甘蓝植株。
4.转基因植株的鉴定
提取转基因植株的总DNA，分别对其转化的目的基因(反义肌动蛋白基因，Barnase荃
因)，目的基因的启动子(TA29，N翎19)以及筛选基因(Bar基因)进行PCR扩增，转化植株均
扩增出相应目的大小片段，而非转化植株都呈阴性。结果证明致雄性不育的反义肌动蛋白墓
因和Barnase基因己被整合到甘蓝的基因组中。将经过PCR检测确认的甘蓝植株的叶片接种
入含PPT15Ing/1 MS培养基(Ms+B Al.0+NAAo.Zmg/l)中培养，25天后对照叶片黄化死亡，而转
化的甘蓝叶片未黄化，并且部分分化出愈伤组织:同时对甘蓝植株用含PPT4伽g/l溶液涂全
株的叶片，28天后对照死亡，而转化的甘蓝生长正常。上述结果显示转化植株具有抗除草剂
的特性。
In recent years, with the development of molecular biology, make it possible to get male-sterility line through genetic engineering. With genetic engineering, we can get male-sterility line and resume line. Moreover when it is succeed in one crop, then it can be supplied to other crops. So in recent years genetic engineering is being utilized broadly and stepwisely being tended towards into commerce. Brassica oleracea. L?it is belong to colewort of cruciferae, a mutation of Brassica oleracea. L. The heterosis of cabbage is significantly notable, for the long time Brassica oleracea. L breeding get through self-incompatibility lines.but with self-incompatibility lines to breed, there are two sides disadventage: the first is inbreeding weaken for continual self-incompatibility,not only the coefficient of propagate will lower,but also the quality of self-incompatibility will be weaken.so that make the hybridize rate fall. The second is that bud stage should be pollination with manual work, do it is very annoyed
, and the efficient is low,the cost is very costliness,this will increase the cost in breed. If use male -stereile line to breed will overcome those disadvantage.Orthodox to get male -stereile line is through these ways of nature diversity manpower induce distant related hybridize and autocopulation hybridize in breeds.then may be get male -stereile line through breeding selection for several generations .
This article is to transfer male stereile gene-- Anti-actin gene and barnase gene which is linked with different special .anther or pollen promoter to Brassica oleracea. L through AgrobAnti-acterium tumefaciens-mediated. Anti-actin gene link with the promoter TA29 which is cloned from tobacco and the promoter NTM19 cloned from tobacco, then construct the chimaetic gene closed linkage with the gene BAR which expression can fastness herbicide PPT, Barnase gene is also construct gene expression vector through this way. Then transfer the two genes to cabbage through AgrobAnti-acterium-mediated transformation method . The main work and results are as below:
1 High efficiency regeneration system of Brassica oleracea. L
The high efficiency regeneration system is the base of plant genetic transformation. The explants was hypocotyls and MS was used as basic midium. By supplying different concentration of 6-BA, NAA, we got the optimum media which induced the adventitious bud differentiation of two Brassica oleracea. L explants types. The optimum media is MS+BA 1.0mg/L+NAA0.2mg/L.
we also compare two explants of cotyledons and hypocotyls in Brassica oleracea. L.Between the two explants types, the hypocotyls expressed the higher adventitious bud differentiation capacity.In the same time,we try on different concentration IAA on the 1/2 MS media,the result show that no way how much the concentration is ,the Explants can get root,but the time is longer with the concentration lower.
2 The best PPT concentration used to select the transformant plants
Explants were restrained from differentiating when 2.0mg/L PPY was added to the medium. When PPT concentration was 2.5mg/L, all of the explants and callus became white and dead. The adventitious bud put in medium contain 3.0mg/L PPT will be dead mostly, in PPT 3.5mg/l medium cause the adventitious bud all dead,but in root medium.PPT1 .5mg/L can cause the adventitious bud not generat root-So the best selective concentration of PPT is 2.5mg/L,The bud selective concentration of PPT is 3.5mg/L,the bud generat root selective concentration of PPT is 1.5mg/r .
3 Transformation system of cabbage
The procedures described were derived from numerous regeneration and transformation designed to test fAnti-actors that might affect shoot regeneration. Tested parameters include length of preculture length of inoculation with agrobAnti-acterium , length of co-cultivation select which antibiology and AS. Only those parameters producing the best result are described as below:
The cabbage explants which get from 6-7d were precultured on regeneration medium. After
1．甘蓝高频再生体系的建立
以甘蓝的下胚轴为外植体，以MS为基本培养基(添加0.7％的琼脂和3％的蔗糖)，加入不同浓度和组合的激素6-BA、NAA，筛选出下胚轴不定芽分化最佳培养基(MS+BA1.0mg/L+NAA0.2mg/L)；并比较了甘蓝下胚轴和子叶两种不同外植体的分化能力，下胚轴的分化频率高于子叶。同时进行了甘蓝不定芽的生根实验，在1/2 Ms培养基中添加不同浓度IAA，结果显示不论IAA浓度是多少，不定芽都能生根，只是生根的时间随着IAA的浓度的升高而缩短。
2．甘蓝外植体分化、不定芽生长以及不定芽生根对除草剂(PPT)的敏感性分析
盈努浩盆龄诊料述吝一一一一一~
在分化培养基中加入浓度为2.omg/L的PPT时，外植体愈伤组织形成明显受到抑制，形
成的愈伤组织也部分死亡:当加入的PPT浓度为2.smg/L时，外植体及部分形成的愈伤组织
全部逐渐白化死亡;当PPT浓度为3.Omg/L时，外植体未形成愈伤组织就白化死亡，因此确
定PPT 2.Slng/L的浓度作为外植体转化的筛选压.分化出的不定芽，转到P盯浓度为3.omg/L
的分化培养基中时，大部分会白化死亡，当PPT浓度为3.smg/L时，不定芽全部白化死亡;
在不定芽生根时，加入的P盯浓度为1.smg/L时，不定芽生根就受到抑制。因此，以PPT2.smg/L
为抗性不定芽分化的筛选压，以PPT3.smg/L作为抗性不定芽生长的筛选压，以PPTI.smg/L
作为抗性不定芽生根的筛选压。
3.甘蓝外植体高频转化体系的建立
在最佳培养基上试验了外植体的预培养时间、浸菌时间、共培养时间、选用何种抗生素
抑菌以及乙酸丁香酮(AS)等因素对转化率的影响，建立了甘蓝外植体的高频转化体系:以
6一7天的甘蓝下胚轴为外植体，分化培养基上预培养2天，在用Ms液体培养墓稀释的菌液中
感菌3一5分钟，共培养2天后，转入筛选培养基1(Ms+6一BA 1.omg/L+NAAo.Zmg/L+ PPT2.smg/L+
Ca比500呢/L)中筛选抗性愈伤组织和抗性芽，每两周换一次培养基。筛选出的抗性芽转到筛
选培养基2(MS+6一BA 1.omg/L十NAAO.Zmg/L+即T3.smg/L+Carb400mg/L)中筛选，并逐渐降
低Carb.的浓度。将在筛选培样基2中得到的抗性芽转入生根培养基(MS+I从0.lmg/L+
PPTI.sm以L+ CarbZoo雌/L)中进行生根筛选，逐步淘汰未转化的“假转化体”，得到具有P盯
抗性的甘蓝植株。
4.转基因植株的鉴定
提取转基因植株的总DNA，分别对其转化的目的基因(反义肌动蛋白基因，Barnase荃
因)，目的基因的启动子(TA29，N翎19)以及筛选基因(Bar基因)进行PCR扩增，转化植株均
扩增出相应目的大小片段，而非转化植株都呈阴性。结果证明致雄性不育的反义肌动蛋白墓
因和Barnase基因己被整合到甘蓝的基因组中。将经过PCR检测确认的甘蓝植株的叶片接种
入含PPT15Ing/1 MS培养基(Ms+B Al.0+NAAo.Zmg/l)中培养，25天后对照叶片黄化死亡，而转
化的甘蓝叶片未黄化，并且部分分化出愈伤组织:同时对甘蓝植株用含PPT4伽g/l溶液涂全
株的叶片，28天后对照死亡，而转化的甘蓝生长正常。上述结果显示转化植株具有抗除草剂
的特性。
In recent years, with the development of molecular biology, make it possible to get male-sterility line through genetic engineering. With genetic engineering, we can get male-sterility line and resume line. Moreover when it is succeed in one crop, then it can be supplied to other crops. So in recent years genetic engineering is being utilized broadly and stepwisely being tended towards into commerce. Brassica oleracea. L?it is belong to colewort of cruciferae, a mutation of Brassica oleracea. L. The heterosis of cabbage is significantly notable, for the long time Brassica oleracea. L breeding get through self-incompatibility lines.but with self-incompatibility lines to breed, there are two sides disadventage: the first is inbreeding weaken for continual self-incompatibility,not only the coefficient of propagate will lower,but also the quality of self-incompatibility will be weaken.so that make the hybridize rate fall. The second is that bud stage should be pollination with manual work, do it is very annoyed
, and the efficient is low,the cost is very costliness,this will increase the cost in breed. If use male -stereile line to breed will overcome those disadvantage.Orthodox to get male -stereile line is through these ways of nature diversity manpower induce distant related hybridize and autocopulation hybridize in breeds.then may be get male -stereile line through breeding selection for several generations .
This article is to transfer male stereile gene-- Anti-actin gene and barnase gene which is linked with different special .anther or pollen promoter to Brassica oleracea. L through AgrobAnti-acterium tumefaciens-mediated. Anti-actin gene link with the promoter TA29 which is cloned from tobacco and the promoter NTM19 cloned from tobacco, then construct the chimaetic gene closed linkage with the gene BAR which expression can fastness herbicide PPT, Barnase gene is also construct gene expression vector through this way. Then transfer the two genes to cabbage through AgrobAnti-acterium-mediated transformation method . The main work and results are as below:
1 High efficiency regeneration system of Brassica oleracea. L
The high efficiency regeneration system is the base of plant genetic transformation. The explants was hypocotyls and MS was used as basic midium. By supplying different concentration of 6-BA, NAA, we got the optimum media which induced the adventitious bud differentiation of two Brassica oleracea. L explants types. The optimum media is MS+BA 1.0mg/L+NAA0.2mg/L.
we also compare two explants of cotyledons and hypocotyls in Brassica oleracea. L.Between the two explants types, the hypocotyls expressed the higher adventitious bud differentiation capacity.In the same time,we try on different concentration IAA on the 1/2 MS media,the result show that no way how much the concentration is ,the Explants can get root,but the time is longer with the concentration lower.
2 The best PPT concentration used to select the transformant plants
Explants were restrained from differentiating when 2.0mg/L PPY was added to the medium. When PPT concentration was 2.5mg/L, all of the explants and callus became white and dead. The adventitious bud put in medium contain 3.0mg/L PPT will be dead mostly, in PPT 3.5mg/l medium cause the adventitious bud all dead,but in root medium.PPT1 .5mg/L can cause the adventitious bud not generat root-So the best selective concentration of PPT is 2.5mg/L,The bud selective concentration of PPT is 3.5mg/L,the bud generat root selective concentration of PPT is 1.5mg/r .
3 Transformation system of cabbage
The procedures described were derived from numerous regeneration and transformation designed to test fAnti-actors that might affect shoot regeneration. Tested parameters include length of preculture length of inoculation with agrobAnti-acterium , length of co-cultivation select which antibiology and AS. Only those parameters producing the best result are described as below:
The cabbage explants which get from 6-7d were precultured on regeneration medium. After
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