利用激光微束穿刺法将外源基因导入小麦的研究

Introduction of Exogenous Genes Into Wheat Using Puncture Technique of Microbeam Laser

用激光微束穿刺法将携带有新霉素磷酸转移酶（ＮＰＴ－Ⅱ）基因的质粒ｐＪＩＴ１０１导入京花１号小麦幼胚细胞。方法是将小麦幼胚细胞进行高渗缓冲液预处理，用微米级的激光微束处理，然后在卡那霉素培养基上筛选出具抗性的愈伤组织及绿色小植株。第一年，从１５０个小麦幼胚中，在卡那霉素培养基上筛选出４株绿苗，取两株进行ＮＰＴ－Ⅱ酶活性分析，测到了ＮＰＴ－Ⅱ酶的活性。第二年重复实验，从２４５个小麦幼胚中，经筛选获得１株绿苗，进行了叶片ＤＮＡＰＣＲ扩增检测，转化的绿色小苗扩增出所导入的ＮＰＴ－Ⅱ基因编码的片段。结果表明，外源ＮＰＴ－Ⅱ基因已导入了小麦，并实现了整合表达。

The plasmid pJIT101 with NPT-Ⅱ was introduced into the embryo cells of wheat cultivar Jinghua No.1 using the technique of with puncture microbeam laser.The wheat embryo cells werepre-treated in hypertonic buffer,then irradiated with microbeam laser(micro grade),and cultured on the media with Km.In the first year,4 gre plantlets were screened out from 150 wheat embryos,and the enzyme analysis of 2 green plantlets revealed the activity of NPT-Ⅱ enzyme.The next year,1 green plantlet was screened out from 245 wheat embryos,and the DNA PCR amplified test of the plantlet leaves showed that the introduced NPT-Ⅱ gene was found in thetranformed plantlet and indicated that the exogenous NPT-Ⅱ gene had been introduced into wheat and the integrated expression had been accomplished.