WANG Xueyan,YANG Fan,ZHU Pingyu,et al.Cloning and functional analysis of the promoter of CsCML25 gene related to determinate growth in cucumber[J].Journal of Nanjing Agricultural University,2020,43(4):621-628.[doi:10.7685/jnau.201911041]





Cloning and functional analysis of the promoter of CsCML25 gene related to determinate growth in cucumber
王雪艳 杨帆 朱拼玉 任跃波 王团团 李季 陈劲枫
南京农业大学作物遗传与种质创新国家重点实验室/园艺学院, 江苏 南京 210095
WANG Xueyan YANG Fan ZHU Pingyu REN Yuebo WANG Tuantuan LI Ji CHEN Jinfeng
State Key Laboratory of Crop Genetics and Germplasm Enhancement/College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
cucumberdeterminate growthCsCML25promoter function
[目的] 本文旨在通过对黄瓜有限生长调控基因CsCML25启动子功能的研究,揭示该基因在黄瓜有限生长过程中的转录调控机制。[方法] 以黄瓜自交系材料CCMC的叶片DNA为模板,克隆CsCML25上游的全长启动子序列,并对其顺式调控元件进行分析;构建CsCML25基因过表达载体以及CsCML25启动子驱动GFP报告基因的表达载体,通过农杆菌介导遗传转化法将其转化拟南芥后,观察过表达植株表型并利用荧光显微镜观察GFP的表达规律;采用RT-qPCR分析该CsCML25启动子驱动的GFP报告基因在IAA和6-BA处理后以及在调控拟南芥有限生长基因TFL1影响下的表达变化。[结果] CsCML25启动子全长2 979 bp,包括4个MYB转录因子结合位点、1个WUS结合位点以及多个细胞分裂素、生长素和光响应元件。过量表达CsCML25基因能够造成拟南芥顶端花融合现象,形成与拟南芥突变体tfl1-13相似的有限生长表型。CsCML25启动子驱动GFP在茎尖分生组织、叶原基、花原基以及花瓣等组织部位表达。RT-qPCR结果表明:IAA和6-BA处理能够显著影响CsCML25启动子的转录激活功能;拟南芥有限生长突变体tfl1-13杂交试验显示,TFL1的失活会降低CsCML25启动子驱动GFP表达的能力。[结论] CsCML25基因是调控有限生长的关键基因,在茎尖分生组织和幼嫩器官中特异性表达,其启动子转录活性受细胞分裂素和生长素的抑制;突变体杂交试验也证明有限生长调控基因TFL1可能通过调控CsCML25启动子的转录活性,进而影响植株的形态建成。
[Objectives] The aim of this study was to reveal the transcriptional regulation mechanism by means of analyzing the function of CsCML25 gene that involved in cucumber determinate growth.[Methods] The DNA from leaves of CCMC cucumber self-inbred line were used as templates. The full-length promoter sequence upstream of CsCML25 was cloned(PCsCML25). The cis-regulation elements of CsCML25 promoter were analyzed by bioinformatics method. CsCML25 overexpression vector(pLP100-35S-CsCML25) and the recombinant expression vector that GFP reporter gene driving by CsCML25 promoter were constructed(PCsCML25-GFP). The recombinant vectors were introduced into Arabidopsis thaliana by Agrobacterium-mediated method. The phenotype of overexpressed plants and the temporal and spatial expression of GFP were observed with fluorescence microscope. RT-qPCR was employed to analyze the changes of GFP gene expression level after PCsCML25-GFP transgenic plants were treated with IAA,6-BA and crossed with tfl1-13 mutant.[Results] CsCML25 promoter overall length was 2 979 bp,including four MYB binding sites,one WUS binding site and many numerous auxin,cytokinin and lights responsive cis-acting elements. The overexpression of CsCML25 in A.thaliana could lead to flowers fuse in the tip of inflorescence that resulted in shoot determinate growth. The phenotype was similar to the A.thaliana determinate growth mutant tfl1-13. GFP reporter gene driven by PCsCML25 was intensely expressed in vegetative shoot apical meristem,leaf primordia,young floral buds and petals. RT-qPCR results showed that the transcriptional activities of CsCML25 promoter were significantly influenced by IAA and 6-BA treatment. Meanwhile,hybrid with determinate growth mutant tfl1-13 result showed that GFP expression of PCsCML25 was lower because TFL1 was inactivated.[Conclusions] CsCML25 gene was a determinate growth regulatory gene that mainly expressed in shoot apical meristems and young organs,and the transcriptional activity of CsCML25 promoter was inhibited by IAA and 6-BA. Mutant hybridization tests also demonstrated that determinate growth regulatory genes TFL1 might participate in regulation of expression of CsCML25 gene and affected the formation of plant architecture.


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