摘要
用竹节花黄斑驳病毒 (CommelinaYellowMottleVirus,CoYMV)启动子_gus嵌合基因和CaMV 35S_gus嵌合基因通过根癌土壤杆菌 (Agrobacteriumtumefaciens (SmithetTownsend)Conn)LBA44 0 4介导分别转入棉花 (Gossypiumhir sutumL .cv .Jingmian 7,J7G) ,诱导再生了棉花转基因植株。GUS活性的组织化学定位检测表明 ,在 5 97bpCoYMV启动子的驱动下 ,gus基因在棉花植株的叶、叶柄、苞叶、茎、根、下胚轴的维管组织及花的大部分组织的维管束中高表达。在含有较多维管组织细胞的叶脉和根中 ,CoYMV启动子_gus的表达活性与CaMV 35S_gus相当或更高 ,但在维管组织细胞含量相对较少的叶片、子叶和下胚轴中 ,CoYMV启动子_gus的表达活性仅为CaMV 35S_gus的 1/3～ 1/5。转CaMV 35S_gus基因棉花植株的叶脉与叶片GUS活性比例为 0 .5 ,而转CoYMV启动子_gus基因棉花约为 2 .0。随着转基因棉花的生长发育 ,gus基因在叶维管束中的表达活性有增强的趋势。以上结果表明CoYMV启动子在转基因棉花中具有维管束特异性。
Explants of cotton ( Gossypium hirsutum L. cv. Jingmian 7) were transformed with Agrobacterium tumefaciens (Smith et Townsend ) Conn LBA4404 harboring an expression cassette composed of CoYMV (Commelina Yellow Mottle Virus) promoter_ gus _nos terminator on the plant expression vector pBcopd2. Transgenic plants were regenerated and selected on a medium containing kanamycin. GUS (β_glucuronidase) activity assays and Southern blot analysis confirmed that the chimerical gus gene was integrated into and expressed in the regenerated cotton plants. Plant expression vector pBI121 was also transferred into the same cotton variety and the regenerated transgenic plants were used as a positive control in GUS activity analysis. Evidences from histochemical analysis of GUS activity demonstrated that under the control of a 597 bp CoYMV promoter the gus gene was highly expressed in the vascular tissues of leaves, petioles, stems, roots, hypocotyls, bracteal leaves and most of the flower parts while GUS activity could not be detected in stigma, anther sac and developing cotton fibers of the transgenic cotton plants. GUS specific activity in various organs and tissues from transgenic cotton lines was determined and the results indicated that the CoYMV promoter_ gus activities were at the same level or higher than that of CaMV 35S promoter_ gus in leaf veins and roots where the vascular tissues occupy a relatively larger part of the organs, but in other organs like leaves, cotyledons and hypocotyls where the vascular tissues occupy a smaller part of the organs the CoYMV promoter_ gus activity was only 1/3-1/5 of the CaMV 35S promoter_ gus activity. The GUS activity ratio between veins and leaves was averaged 0.5 for 35S_GUS plants and about 2.0 for CoYMV promoter_ gus transgenic plants. These results further demonstrated the vascular specific property of the promoter in transgenic cotton plants. An increasing trend of GUS activity in leaf vascular tissues of transgenic cotton plants developing from young to older was observed.
基金
国家"863"计划资助项目(Z17_01_01)
国际科学文化中心世界实验室部分资助
关键词
转基因棉花植株
CoYMV启动子
维管组织
GUS
transgenic cotton plant
Commelina Yellow Mottle Virus(CoYMV)promoter
vascular tissue
GUS
