甘蓝型油菜抗虫转基因植株及其抗性分析

通过油菜子叶外植体－农杆菌共培养法将苏云金杆菌杀虫蛋白基因导入甘蓝型油菜,获得抗虫的转基因植株。带有１￣２ｍｍ子叶柄的油菜子叶经农杆菌感染后,共培养２￣３天,然后转移到附加１５ｍｇ／Ｌ卡那霉素的ＭＳ选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含２０￣５０ｍｇ／Ｌ卡那霉素的选择培养基上继代培养,再转移到含２５ｍｇ／Ｌ卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫蛋白基因为探针,进行１     

芥菜型油菜抗虫转基因植株及其后代株系的研究*

带有1～2mm子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加10～20mg/L卡那霉素的MS选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含30～50mg/L卡那霉素的选择培养基上继代培养,再转移到含20mg/L卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行Southern blot分子杂交,得到阳性结果。PCR分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系。     

亚麻下胚轴离体培养和转化的研究

报道了亚麻（Ｌｉｎｕｍ　ｕｓｉｔａｔｉｓｓｉｍｕｍ）的高频率植株再生和细胞转化。亚麻下胚轴切段培养在ＭＳＢ分化培养基上,诱导分化出不定芽。最佳的激素组合是ＢＡ２ｍｇ／Ｌ＋ＩＡＡ０．５ｍｇ／Ｌ,分化频率可达９７％。在附加ＢＡ１ｍｇ／Ｌ和ＮＡＡ０．０２ｍｇ／Ｌ的ＭＳＢ培养基上,亚麻下胚轴外植体的不定芽分化频率也可达９０％以上。用根癌农杆菌（Ａｇｒｏｈａｃｔｅｒｉｕｍ　ｔｕｍｅｆａｃｉｅｎｓ）感染亚麻下胚轴外植体,在含卡那霉素５０ｍｇ／Ｌ的选择培养基上筛选获得卡那霉素抗性苗,并检测到ＧＵＳ基因活性表达。表明它们是转化植株,转化频率约为２％。     

亚麻遗传转化体系的建立及几丁质酶基因导入的研究

报道了亚麻遗传转化体系的建立和几丁质酶基因对亚麻遗传转化的研究。亚麻下胚轴切段培养在不同激素浓度的ＭＳ培养基上,诱导分化出不定芽。最佳的激素组合是ＭＳ＋ＢＡ１ｍｇ／Ｌ＋ＩＡＡ０．５ｍｇ／Ｌ,分化频率可达９７％。亚麻的下胚轴经带有几丁质 根癌农杆菌感染后,在含有１００ｍｇ／Ｌ卡那霉素的选择分化培养基上,１４￣２１ｄ就能产生抗生小芽,小芽进一步伸长后可在１００ｍｇ／Ｌ卡那霉素的ＭＳ选择生根培养基（ＭＳ     

GUS基因在梨转化植株中的稳定表达

以获得的梨转GUS基因植株的试管苗为试材,转基因植株的叶片在含卡那霉素的再生培养基上可成功再生不定梢,这些不定梢在含卡那霉素的生根培养基上可诱导生根。再生不定梢经组织化学检测仍表现出GUS基因的表达。而非转基因植株的叶片在含卡那霉素的再生培养基上不能产生不定梢,显示外源基因在转基因植株中的表达在无性繁殖后代中是稳定遗传的。     

表达barstar基因及bar基因的转基因油菜的研究

从细菌Ｂａｃｉｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ｂａｒｎａｓｅ抑制剂ｂａｒｓｔａｒ的基因,构建了带有ＴＡ－２９基因５′调控区（－１３００－＋３）与ｂａｒｓｔａｒ基因编码区、ＣａＭＶ３５Ｓ启动子与除草剂抗性基因ｂａｒ两个表达框架的植物表达质粒ｐＢＢＳ。以“双低”油菜“５－４”的子叶柄为受体,通过农杆菌介导的遗传转化,获得了在含有１０ｍｇ／Ｌ卡那霉素和２０ｍｇ／ＬＰＰＴ的筛选培养基上再生的转基因植株。ＰＣＲ分析结果表明,ｂａｒｓｔａｒ基因已整合到油菜染色体上;Ｎｏｒｔｈｅｒｎｂｌｏｔ检测表明,ｂａｒｓｔａｒ基因及ｂａｒ基因在转基因植物中得到了正确的调控与表达。以转基因油菜“５－４”为父本授粉给表达ｂａｒｎａｓｅ基因的雄性不育植株,不育株能正常结实。     

根癌农杆菌对甘蓝型油菜的转化及转基因植株的再生

用根癌农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍ ｅｆａｃｉｅｎｓ）共培养法把外源基因导入甘蓝型油菜（Ｂｒａｓｓｉ－ｃａ ｎａｐｕｓＬ．）主要栽培品种“云北２ 号”,获得转基因植株。所用外植体为带有１—２ ｍ ｍ 子叶柄的完整子叶,根癌农杆菌为Ａ２０８ＳＥ（ｐＴｉＴ３７－ＳＥ, ｐＲＯＡ９３）。Ｔｉ质粒ｐＲＯＡ９３ 带有ＮＰＴⅡ及ＧＵＳ嵌合基因。共培养２ 天后转到附加２５ ｍ ｇ／Ｌ卡那霉素的分化培养基（ＭＳ＋ ４．５ ｍ ｇ／ＬＢＡＰ）上。ＡｇＮＯ３ 和羧苄青霉素促进芽的分化,头孢霉素则有抑制作用。最高转化频率为２７％ 。把分化出的茎芽切下,插入含有２５ ｍ ｇ／Ｌ卡那霉素的生根培养基中。羧苄青霉素不利于根的形成。把完整抗性植株移入盛土壤的盆中,生长状况良好。测定β－葡糖苷酸酶活性,８４％ 明显高于对照。以ＮＰＴⅡ基因作探针进行Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分析,证实外源基因已插入到植物细胞基因组中     

PEG法介导转化诸葛菜下胚轴原生质体获得转基因植株

采用诸葛菜无菌苗的下胚轴组织为材料,分离原生质体,在原生质体培养基中作液体浅层暗培养,植板率为５％,植株再生频率为１００％。作者进而开展了遗传转化研究。为研究ＰＥＧ介导转化诸葛菜原生质体的影响因素,通过瞬间表达,实验了ＰＥＧ法转化子叶原生质体的过程,在此基础上将分离纯化后的原生质体与带ＨＰＴ基因的质粒ＤＮＡ（ｐＢＩ２２２）混合,ＨＰＴ基因作选择标记,ＰＥＧ介导转化;重新收集转化后的原生质体,以５×１０４／ｍｌ的密度在原生质体培养基中作浅层培养;培养１０—１５天后用２５ｍｇ／Ｌ的潮霉素（ｈｙｇｒｏｍｙｃｉｎ）进行筛选,一月后出现少量细胞团,转入含潮霉素５０ｍｇ／Ｌ的扩增培养基扩增愈伤组织,进而转入含５０—１００ｍｇ／Ｌ潮霉素的分化培养基诱导分化成苗,分化率为１００％,转入生根培养基中生根成完整植株。抗性植株再生率为４×１０（－５）。在获得再生转基因植株后,以再生植株叶片为材料,进行Ｓｏｕｔｈｅｒｎｂｌｏｔ分子杂交,证实外源基因已稳定整合到植物基因组中并表达,再生转基因植株频率为１０（－５）。国内外首次转化诸葛菜属植物原生质体获得成功。     

植物抗毒素转化水稻和转基因植株的生物鉴定

用基因枪法转化了水稻（ＯｒｙｚａｓａｔｉｖａＬ．）６个材料的未成熟胚、成熟胚及胚性愈伤组织。质粒ｐＳＳＶｓｔ１和ｐＶＥ５＋是由葡萄中分离出的编码芪类合成酶的植物抗毒素基因与３５Ｓ或它自己的启动子组成。Ｇ４１８（１００～１５０ｍｇ／Ｌ）或潮霉素（５０ｍｇ／Ｌ）筛选后,经ＰＣＲ、Ｓｏｕｔｈｅｒｎｂｌｏｔ或Ｄｏｔｂｌｏｔ分析证明的转基因植株共５４株。对转基因植株及其后代进行了稻瘟病和白叶枯病的抗性鉴定。初步结果表明,芪类合成酶基因可以提高转基因植株及后代的抗性。     

基因枪轰击谷子幼穗获得转基因植株

以ＪＱ－７００型国产基因枪轰击豫谷２号谷子幼穗,在１５０ｍｇ／Ｌ卡那霉素选择培养基上筛选到９０８块抗性愈伤组织,其中,绿芽块愈伤５块,共分化出１６株绿苗,组织化学检测ＧＵＳ表达,获得３株阳性植株,Ｓｏｕｔｈｅｒｎ杂交证明１株为阳性。以轰击总外植体计算的转基因植株频率为０．０５％。     

Innateness and the Sciences

The concept of innateness is a part of folk wisdom but is also used by biologists and cognitive scientists. This concept has a legitimate role to play in science only if the colloquial usage relates to a coherent body of evidence. We examine many different candidates for the post of scientific successor of the folk concept of innateness. We argue that none of these candidates is entirely satisfactory. Some of the candidates are more interesting and useful than others, but the interesting candidates are not equivalent to each other and the empirical and evidential relations between them are far from clear. Researchers have treated the various scientific notions that capture some aspect of the folk concept of innateness as equivalent to each other or at least as tracking properties that are strongly correlated with each other. But whether these correlations exist is an empirical issue. This empirical issue has not been thoroughly investigated because in the attempt to create a bridge between the folk view and their theories, researchers have often assumed that the properties must somehow cluster. Rather than making further attempts to import the folk concept of innateness into the sciences, efforts should now be made to focus on the empirical questions raised by the debates and pave the way to a better way of studying the development of living organisms. Such empirical questions must be answered before it can be decided whether a good scientific successor – in the form of a concept that refers to a collection of biologically significant properties that tend to co-occur – can be identified or whether the concept of innateness deserves no place in science.