J Integr Plant Biol. ›› 2001, Vol. 43 ›› Issue (1): 59-62.

• Research Articles • Previous Articles     Next Articles

Obtainment of Transgenic Tobacco Harboring phbA, phbB and phbC Genes by Twice Transformation

ZHANG Jing-Yu, YE Liang, LI Li, SONG Yan-Ru   

Abstract:

To avoid the long time required for conventional sexual crossing, transgenic tobacco (Nicotiana tabacum L.) plants harboring phbA gene (encoding 3-ketothiolase) were used as the target plant for the second transformation mediated by Agrobacterium tumefaciens (Smith et Townsend) Conn LBA4404 containing pZCB which was constructed by linking phbB (encoding acetoacetyl-CoA reductase), phbC (encoding PHB synthase) and ctp sequence to pBIB-HYG under the control of CaMV 35S promoter. The hygromycin resistant transformants were morphologically normal and stable integration of phbB and phbC was confirmed by PCR and PCR-Southern. Moreover, RT-PCR-DNA hybridization analysis showed that 6.67% of the transformed tobacco plants could express both phbB and phbC at transcriptional level.

二次转化获得整合phbA、PhB、phbC 基因的转基因烟草
张景昱 叶梁 李丽 宋艳茹*

(中国科学院植物研究所,北京100093)

摘要: 有导肽序列的phbB(编码乙酰乙酰CoA还原酶)和phbC(编码PHB合酶)连入pBIB_HYG得到组成型表达载体pZCB, 用冻融法转入根癌土壤杆菌(Agrobacterium tumefaciens (Smith et Townsend) Conn)并由其介导转化已整合且表达phbA(编码3-硫裂解酶)基因并具有卡那霉素抗性的转基因烟草(Nicotiana tabacum L.)。通过二次转化可避开传统杂交育种,在5个月内获得整合PHB合成所需3个基因的转基因烟草。所获转基因植株表型正常, 经PCR、PCR-outhern、RT-CR-NA杂交检测确定有50株烟草稳定整合phbB、phbC基因,其中6.67%的植株可在转录水平表达双基因。

关键词: 聚羟基丁酸酯;二次转化;转基因烟草

Key words: poly (3-hydroxybutyrate) (PHB), twice transformation, transgenic tobacco

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