摘要
取在１／２ＭＳ＋ ＮＡＡ０－０２ ｍｇ／Ｌ 培养基中发育４ ～６ 周的欧洲黑杨转Ｂｔ 基因植株１５ｎ１９２ 试管苗幼叶，在ＭＳ＋ ＢＡ０－２ ｍｇ／Ｌ＋ ＮＡＡ０－０２ ｍｇ／Ｌ培养基中培养１６ｈ ，分别用基因枪法和叶盘法转化ＴＡ２９Ｂａｒｎａｓｅ嵌合基因，其Ｂａｒｎａｓｅ 基因的转化率依次为１６－１ ％ 和２３ ％ ，再生株数分别为１７５／１２ 和７１／１０ 。试验表明，选择发育适宜的叶片，利于目的基因转化后的植株再生。进一步用ＳＤＳ 法提取转基因植株总ＤＮＡ，经ＰＣＲ 检测表明：转化植株基因组中扩增出的ＤＮＡ 片段与Ｂａｒｎａｓｅ 基因的大小一致；Ｓｏｕｔｈｅｒｎ Ｂｌｏｔ 分析出转化后的欧洲黑杨基因组中携有Ｂａｒｎａｓｅ 基因片段，进行Ｄｏｔ Ｂｌｏｔ 检测，转化植株有一明显的杂交斑，并证实为阳性转化植株，并通过试管培养，结合林木常规育种方法，获得了转Ｂａｒｎａｓｅ 基因的抗虫欧洲黑杨转基因植株。
Chimaeric TA29 Barnase gene was introduced into anti insect transgenic Populus nigra of good quality and high yield by Agrobacterium tumefaciens(A.t)and biolistics transformation from tender leaves of 15 n 192 tube plantlets, which growing at the medium 1/2MS+NAA0 2?mg/L for 4～6 weeks, the leaves was cultured with MS+BA0 2?mg/L+NAA0 02?mg/L about 16 hours. The transformation frequency of Barnase gene by biolistics and A.t was 16 1 percent and 23 percent respectively, the number of repropagation plantlets were 175/12 and 71/10 respectively. The experiment showed that, the suitable developing leaves were selected, which was good for plantlets regeneration after Barnase gene transformation. Further more, we extracted the total DNA from transgenic plants by SDS method. The PCR analysis showed that, the DNA fragment from trangenic genome was amplified were completely identical with the Barnase gene; Southern Blot analysis showed that the transgenic plants genome carry about with Barnase gene fragment; Dot Blot analysis showed that the transgenic plants passes a cross spot clearly, and it was positive transformed plants further. The trans Barnasegenic plants of anti insect \%Populus nigra \%were obtained by the tissue culture and the methods of normal forest tree breeding.
出处
《林业科学》
CAS
CSCD
北大核心
2000年第1期28-32,共5页
Scientia Silvae Sinicae
基金
国家自然科学基金
关键词
抗虫转基因
欧洲黑杨
Barnase基因
雄性不育
The anti insect transgenic Populus nigra, Barnase gene, Male sterility, Bacillus thuringiensis insecticidal crystal protein(B.t)gene, Pollen pollution
