摘要
通过PCR扩增获得了包含多聚半乳糖醛酸酶(PG)全部阅读框架的1.5kb cDNA,经限制酶酶谱和部分序列分析鉴定无误后,将其以反方向插入含两个增强子的35S启动子和Nos3＇端之间,构建成表达PG反义RNA的双元载体,经农杆菌途径转化番茄品种“丽春”,获得了60株抗卡那霉素再生植株,经PCR检测,证明有2/3的再生植株有外源PG基因导入,成熟果实的PG粗提液的SDS-PAGE分析表明:若干株系中PG蛋白量较对照有不同程度的下降。PG活性亦同步下降,其中一个株系3~#,PG酶活下降了93％。这些结果表明外源PG基因的反方向导入有效地抑制了内源PG基因的表达。
We have amplified a 1. 5kb PG cDNA including its complete open reading frame, using two primers specific to PG gene and PCR technique. Its fidelity and correctness were confirmed by restriction mapping and partial DNA sequencing. Then the PG gene was inserted into a binary vector, pBin437, in an inverted orientation between the 35S promoter with duplicated enhancers and the Nos3' transcriptional termination sequence , and the resulting plasmid pBPG was introduced into LBA 4404. The A, tumefa-ciens harboring pBPG was used to transform tomato plants. Sixty kanamycin-fresistant transformants were selected. PCR detection showed two-third of the transformed plants tested (30 plansts) contain the exogenous PG gene. The PG activity assay and SDS-PAGE analysis of proteins from ripening fruit cell wall showed that the PG activity in transgenic tomato plants were inhibited in different extent. One of them, plant 3# , gave only 7% PG activity compared with the control (100%). These results suggest that the expression of the anti-PG gene has effectively inhibited the expression of endogenous PG gene. The effect of low-level PG activity on tomato fruit ripening and storage is being investigated.
出处
《生物工程学报》
CAS
CSCD
北大核心
1994年第2期96-102,共7页
Chinese Journal of Biotechnology
基金
国家"八五"攻关
国家自然科学基金
关键词
多聚半乳糖
醛酸酶
转基因番茄
Polygalacturonase (PG), antisense RNA, transgenic tomato
