摘要
将质粒pWR306中包含ED35s启动子、Omega元件及TNOS终止子的一段核苷酸序列定向克隆到质粒pCAMBIA1300,构建了植物中间表达载体pWR-II;将重组质粒pGEM-T-ALDH的醛脱氢酶(ALDH)基因片段定向克隆到pWR-II,构建了葡萄ALDH基因的植物过量表达载体pWR-II-ALDH。采用改进冻融法将其导入农杆菌EHA105,采用花序浸蘸法转化拟南芥,获得了潮霉素抗性的转化拟南芥植株。PCR检测证明外源基因已整合进拟南芥基因组,Real-timePCR结果表明ALDH基因在转化植株的表达量远高于野生种。转化植株和野生植株在形态上有一定的差异,说明葡萄ALDH基因在拟南芥中表达对其生长发育有一定影响。
A nucleotide sequence of plasmid pWR306 carrying promotor ED35s, Omega TMV translation enhancer and nopaline synthase terminator was specifically cloned into plasmid pCAMBIAI300 to construct the plant intermediate expression vector pWR-Ⅱ; the segment of aldehydehyde dehydrogenase gene from the recombinant plasmid pGEM-T-ALDH was specifically cloned into intermediate expression vector pWR-II to construct the plant overexpression vector pWR-Ⅱ-ALDH, which was introduced into Agrobacterium strain EHA105 by means of the improved freezing-thawing method and then was transformed into A rabidopsis thaliana by floral dip to obtain transformed plants. PCR analysis indicated that the ALDH gene was indeed integrated into the Arabidopsis thaliana genome,Real-time PCR analysis indicated that the expression of the ALDH gene in transformed plants was far more than that in wild plants. There were some phenotype differences between transformed plants and wild plants,which indicated that the A LDH gene＇s expression has an impact on the A rabidopsis thaliana＇ s growth.
出处
《果树学报》
CAS
CSCD
北大核心
2009年第1期37-42,共6页
Journal of Fruit Science
基金
国家自然科学基金(No.30571280)
关键词
中国野生葡萄
醛脱氢酶基因
过量表达载体
拟南芥
Chinese wild Vitis pseudoreticulata
Aldehydehyde dehydrogenase gene
Overexpression vector
A rabidopsis thaliana
