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为研究4-香豆酸辅酶A连接酶基因(4CL)在白桦木质素合成代谢过程中的组织特异性表达,利用染色体步移法克隆其启动子,用该启动子定向置换pBI121载体的35S启动子,构建重组载体P_(4CL)::GUS。利用瞬时转化法将重组载体转入白桦实生苗茎后进行GUS染色。结果显示:获得了4CL基因编码区起始密码子上游长1344 bp的启动子序列,该启动子除分布有TATA-box、CAAT-box等基本的转录起始元件外,还存在多个顺式作用元件序列位点,包括35S启动子作用元件ASF,参与脱落酸响应的顺式作用元件ABRE,参与茉莉酸甲酯响应的顺式调控元件CGTCA-motif,以及光反应元件G-Box、ACE、4CL-CMA2b等;启动子表达分析结果显示经过瞬时侵染的白桦茎段被染成蓝色。以上结果表明克隆获得的4CL基因启动子具有启动子表达活性,其可能参与了白桦木质部的发育。
In order to study the tissue-specific expression of 4-coumarate coenzyme A ligase gene (4CL) in the process of birch lignin biosynthesis, the promoter was cloned by chromosome walking and the 35S promoter of pBI121 vector was targetedly replaced by this promoter To construct the recombinant vector P_ (4CL) :: GUS. Transient transformation method was used to transfer the recombinant vector into the stem of white birch and then GUS stained. The results showed that the promoter sequence of 1344 bp upstream of the start codon of 4CL gene was obtained. In addition to basic transcription initiation elements such as TATA-box and CAAT-box, there are many cis Including ASF acting as a 35S promoter, cis-acting element involved in abscisic acid response, CGTCA-motif involved in the reaction of methyl jasmonate, and G-Box, ACE, 4CL-CMA2b, etc .; promoter expression analysis showed that the birch stem section transiently infected was dyed blue. The above results indicated that the 4CL gene promoter obtained by cloning possessed the promoter expression activity, which may be involved in the development of the xylem of birch.