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本文报道了用于获得人工雄性不育系的质粒pTABNBAR55和用于获得人工恢复系的质粒pTABSBAR23的构建。用PCR的方法从烟草(Nicotiana tabacum cv.Samsun)中扩增了花药绒毡层特异表达TA29基因上游—291～+12共303bp的片段,称作TA29启动子(简写作pTA29);从解淀粉芽孢杆菌(Bacillus amyloliquefaciens)中扩增了一种RNA酶及其特异抑制剂基因barnase和barstar。DNA序列分析表明,pTA29和barstar片段均与已发表的序列完全一致,barnase片段在3′端和已见报道的两种序列都有一个碱基的差异,但由此推断的氨基酸序列是一致的,不影响其在植物中的表达。把TA29启动子分别与barnase和barstar基因连接起来构成两个嵌合基因pTA29-barnase和pTA29-barstar,在这两个嵌合基因的3′端加上了260bp的Nos终止子(Nos.ter)以增强其在植物中的表达。然后将一个2.0kb的完整除草剂抗性标记“bar cassettee”插在含有这两个基因的质粒上,作为转化和制种过程的筛选标记。若将前者导入欲作杂交制种母本的植物中,可以获得雄性不育系;后者导入欲作父本的植物中,可获得恢复系。
Here we report the construction of plasmid pTABNBAR55 for obtaining male sterile lines and plasmid pTABSBAR23 for obtaining artificial restorer lines. A total of 303bp fragment of -291 ~ +12 upstream of TA29 gene was amplified from tobacco (Nicotiana tabacum cv.Samsun) by PCR and named as TA29 promoter (abbreviated as pTA29) Bacillus amyloliquefaciens amplified a RNase and its specific inhibitor gene barnase and barstar. DNA sequence analysis showed that both the pTA29 and barstar fragments were identical to the published sequences, the barnase fragment had one base difference between the 3 ’end and the two reported sequences, but the deduced amino acid sequence was consistent , Does not affect its expression in plants. The TA29 promoter was ligated with the barnase and barstar genes to form the two chimeric genes, pTA29-barnase and pTA29-barstar respectively, and a 260 bp Nos terminator was added to the 3 ’end of both chimeric genes. To enhance its expression in plants. A 2.0 kb full herbicide resistance marker, “bar cassettee,” was then inserted on the plasmid containing both genes as a selection marker for transformation and seed production. If the former is to be introduced into the plants which are to be used as the hybrid breeding seed, male sterile lines can be obtained, and the latter can be obtained from plants which are intended as parents.