花生β-1,3-葡聚糖酶基因启动子的克隆及功能分析
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Cloning and functional analysis of promoter of peanut β-1,3-glucanase gene
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    摘要:

    植物β-1,3-葡聚糖酶属于一种重要的植物病程相关蛋白(PR蛋白),容易受到激发子的诱导而积累。用1.5 mmol/L水杨酸 (Salicylic Acid,,SA)对花生品种花育20号幼苗进行诱导处理,提取RNA利用 Real-time PCR技术检测β-1,3-葡聚糖酶基因的表达量。结果表明经诱导后24h,β-1,3-葡聚糖酶基因的表达量达到最高,是未经SA诱导的1.8倍。根据花生β-1,3-葡聚糖酶基因 cDNA序列(GenBank JQ801335)设计三个嵌套的5'端特异引物,以花育20号基因组DNA为模板,利用TAIL-PCR方法扩增得到973bp的花生β-1,3-葡聚糖酶基因上游启动子片段, ,命名为Ah-Glu-Pro,,在NCBI网站注册序列号为GenBank KC290400。PLACE 和 PlantCARE 启动子在线预测分析表明,Ah-Glu-Pro序列中含有TATA-box和CAAT-box等核心元件,还含有病原菌及SA响应的顺式调控元件。根据Ah-Glu-Pro序列设计上下游引物,采用普通PCR法从花育20号基因组中扩增得到931bp的启动子序列,命名为Ah-Glu-P。将Ah-Glu-P取代pCAMBIA1301质粒中的CaMV35S启动子,构建植物表达载体 pCAMBIA1301-Ah-Glu-P。通过农杆菌介导法将 pCAMBIA1301-Ah-Glu-P 转化洋葱表皮细胞,经5.0 mmol/L SA诱导处理48h后进行GUS染色。结果表明:经SA诱导后的洋葱表皮细胞GUS染色显示为蓝色,未经诱导的洋葱表皮细胞GUS染色未显示蓝色,说明Ah-Glu-P是一个诱导型的启动子,可能含有对SA响应的顺式调控元件。

    Abstract:

    The plant β-1,3-glucanase belongs to one of the important pathogenesis-related proteins (PR-proteins), which could be accumulated when induced by elicitors. The seedlings of peanut cultivar Huayu20 were sprayed with 1.5 mmol/L Salicylic Acid (SA), then the total RNAs were extracted and the mRNA expression amount of β-1,3-glucanase gene were inspected by Real-time PCR. The expression amount of β-1,3-glucanase gene reached the highest after 24h induced by SA, which was 1.8 times higher than that of control not induced by SA. Three specific 5’ upstream primers were designed and synthesized according to peanut β-1,3-glucanase gene cDNA sequences (GenBank JQ801335), and the PCR amplification were conducted using the genomic DNA of Huayu20 as the template by TAIL-PCR method. A 973bp upstream promoter fragment was obtained and submitted in NCBI (GenBank KC290400), named by Ah-Glu-Pro. Promoter sequence analysis by PLACE and PlantCARE showed that the 973bp sequence contained some typical cis-elements, such as TATA box, CAAT box, pathogen and SA cis-acting regulatory element. A 931bp fragment was obtained and named Ah-Glu-P, using a pair of primers designed according to Ah-Glu-Pro. Ah-Glu-P then was inserted into pCAMBIA1301, replacing its CaMV35S promoter. The recombinant plasmid was named pCAMBIA1301-Ah-Glu-P and then transferred into onion epidermal cells by Agrobacterium EHA105-mediated transformation. GUS staining on the onion epidermal cells was conducted after 48h induced by 5.0 mmol/L SA. The transformed onion epidermal cells appeared blue when induced by SA, while the control cells not induced by SA didn’t appear blue, which indicated that the Ah-Glu-P could be an inducible promoter and might contain responsive element relative to SA.

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王合春,陈新利,隋炯明,等.花生β-1,3-葡聚糖酶基因启动子的克隆及功能分析[J].植物遗传资源学报,2013,14(5):864-870.

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  • 收稿日期:2013-01-15
  • 最后修改日期:2013-03-19
  • 录用日期:2013-08-09
  • 在线发布日期: 2013-08-21
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