中国水仙LAR基因启动子的克隆及功能初步分析

Cloning and Functional Analysis of the Promoter of LAR Gene in Chinese Narcissus

为了解NtLAR基因的表达调控机制,该研究以中国水仙(Narcissus tazetta var.chinensis)‘金盏银台’DNA为模版,采用染色体步移法克隆了NtLAR基因起始密码子ATG上游启动子片段序列,测序结果显示,该克隆片段共995 bp(GenBank登录号:MH371155)。通过PlantCare数据库对获得的启动子序列顺式作用元件预测发现,NtLAR启动子序列中包含有大量顺式作用元件,如光反应元件ACE、G-box、GATA-motif、GT1-motif,激素响应元件CGTCA-motif、ABRE、TGACG-motif、TGA-element,胁迫响应元件和MYB结合位点MBS等。成功构建了植物表达载体pBI121-p NtLAR∷GUS和pGreenII 0800-p NtLAR-Luc。pBI121-p NtLAR∷GUS在烟草叶片的瞬时表达结果显示,克隆的启动子片段具有活性;pBI121-p NtLAR∷GUS在水仙不同组织器官的瞬时表达实验发现,NtLAR基因的表达具有组织特异型,其在鳞茎盘的表达量较高,在花瓣和副冠中的表达量较低;将pBI121-p NtLAR∷GUS分别和中国水仙R2R3-MYB转录因子NtMYB2、NtMYB5混合注射烟草叶片,GUS染色结果显示NtMYB2和NtMYB5并不能抑制NtLAR启动子的活性,定量PCR结果与GUS染色结果一致。采用pGreenII 0800-p NtLAR-Luc载体进行双荧光素酶实验进一步验证了GUS染色实验和定量PCR结果。

In order to understand the regulation mechanism of NtLAR gene,we cloned the 5′-terminal promoter sequence of NtLAR gene in this study using genome walking method from genomic DNA of Chinese narcissus(Narcissus tazetta var.chinensis cv.‘Jinzhanyintai’).The sequencing result showed that the cloned fragment was 995 bp in length(GenBank number MH371155).Cis-acting elements of the promoter were analyzed and predicted using plant care databases.Many cis-acting elements were found,such as light response element ACE,G-box,GATA-motif,GT1-motif,hormone response element CGTCA-motif,ABRE,TGACG-motif,TGA-element,stress corresponding element and MYB binding site MBS.Two expression vectors pBI121-p NtLAR∷GUS and pGreenII 0800-p NtLAR-Luc were constructed successfully.Transient expression ofpBI121-p NtLAR∷GUS in tobacco leaves showed that the cloned promoter fragment had activity.The transient expression results of pBI121-p NtLAR∷GUS in Chinese narcissus showed that NtLAR promoter had different activities in different tissues of Chinese narcissus.The expression level of p NtLAR∷GUS is higher in basal plates and very low in petal and corona.When tobacco leaves were agro-infiltrated with pBI121-p NtLAR∷GUS mixed with R2R3-MYB genes NtMYB2 and NtMYB5 respectively,GUS staining and qRT-PCR showed that the activity of NtLAR promoter could not be repressed by NtMYB2 or NtMYB5.Dural luciferase assay was also conducted in N.benthamiana leaves with pGreenII 0800-pNtLAR-Luc mixed with NtMYB2 or NtMYB5.The results of dual luciferase assay were consistent with GUS staining and qRT-PCR results.