J Integr Plant Biol. ›› 2002, Vol. 44 ›› Issue (4): 441-445.

• Research Articles • Previous Articles     Next Articles

Cloning Whole Cellulose-Synthesizing Operon (ayacs Operon) fromAcetobacter xylinumand Transforming It into Cultivated Cotton Plants

LU Ying Chun, WEI Gang, ZHU Yu Xian*   

Abstract:

The gram negative bacterium Acetobacter xylinum synthesizes an extracellular ribbon of cellulose microfibrils that possess unique structural and mechanical properties when compared to higher plant cellulose. All four genes in the cellulose synthesizing operon (ayacs operon) of A. xylinum Ay201 were amplified by polymerase chain reaction (PCR) using oligonucleotide primers designed according to published acsoperon sequence of A. xylinum ATCC 53582. Alignmentof the two operons showed that they were highly homologous (98% similarity, 97% identity). AcsA and acsB gene were cloned in pCAMBIA 1301 vector while acsC and acsD were cloned in pCOB302 3 under the control of CaM 35S promoter. The constructs were introduced into cotton by the pollen tube pathway method and seeds obtained from putative transgenic plants were germinated on media containing hygromycin and phosphinothricin (PPT). Five seedlings out of 934 seeds were proved to contain all four foreign genes by PCR amplification. This is the first time that a whole operon encoding four different bacterial enzymes with various biological functions is transformed into cultivated cotton plants.

木醋杆菌纤维素合成操纵子的克隆及棉花转化

卢迎春  魏刚  朱玉贤*

(北京大学生命科学学院,北京,100871)

革兰氏阴性菌木醋杆菌(Acetobacter xylinum (Brown) Yamada)合成一种由纤维素微纤丝组成的胞外带状物.与高等植物纤维素相比,它具有独特的结构和机械性能.根据从木醋杆菌ATCC 53582克隆的acs纤维素合成操纵子序列设计引物, PCR的方法从木醋杆菌Ay201中克隆了ayacs纤维素合成操纵子的全部4个基因.序列比较发现,两者高度同源.将连上CaMV 35S启动子的acsAacsB克隆到植物表达载体pCAMBIA 1301,acsCacsD克隆到pCOB302-3.然后通过花粉管通道法转化棉花(Gossypium hirsutum)胚珠,收获的种子在含有卡那霉素和除草剂的双抗培养基上进行筛选.PCR检测发现934粒种子中有5棵植株含有全部4个基因.这是首次将编码4个功能蛋白的细菌操纵子成功地转入棉花.

关键词 木醋杆菌 纤维素合成操纵子 棉花转化

通讯作者 Email: zhuyx@water.pku.edu.cn

 

Key words: Acetobacter xylinum, cellulose synthesizing operon, cotton transformation

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