花生β-1,3-葡聚糖酶基因启动子的克隆及功能分析

Cloning and Functional Analysis of the Promoter Region ofβ-1,3-glucanase Gene in Peanut

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(SA)对花生品种花育20号幼苗进行诱导处理0.5、12、24、36、48和72 h后,提取RNA进行荧光定量检测β-1,3-葡聚糖酶基因(Ah-Glu)的表达量。结果表明,经诱导24 h后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。根据前期已克隆的花生β-1,3-葡聚糖酶基因序列(GenBank JQ801335),在其5'端设计3个嵌套的特异性引物扩增其上游启动子序列。以花育20号基因组DNA为模板,利用TAIL-PCR方法,扩增得到973 bp的花生β-1,3-葡聚糖酶基因启动子片段,在NCBI网站注册序列号为GenBank KC290400,并命名为Ah-Glu-Pro。PLACE和PlantCARE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,还含有病原菌及水杨酸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号扩增得到931 bp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 mmol/L SA诱导处理48 h后进行GUS染色。结果表明:经SA诱导后的洋葱表皮细胞GUS组织化学染色显示为蓝色,未经SA诱导的洋葱表皮细胞GUS组织化学染色未显示蓝色,说明Ah-Glu-P是一个可被诱导的启动子,可能含有对SA响应的顺式调控元件。

Plant 6-1,3-glucanase was one of the important pathogenesis-related proteins （PR-proteins） , which could be accumulated when induced by elicitors. The seedlings of peanut cultivar Huayu 20 were sprayed with 1.5 mmol/L SA for 0.5 h,12 h,24 h,36 h,48 h,and 72 h. And then their total RNAs were extracted and expres- sion level of 6-1,3-glucanase was inspected by real-time PCR. Results showed that the expression amount of β-1,3- glucanase gene reached the highest after 24 h induced by SA,which was 1.8 times higher than the control that not induced by SA. In order to clone the promoter sequence of 6-1,3-glucanase gene,three specific upstream primers were designed and synthesized according to peanut 6-1,3-glucanase gene cDNA sequences （GenBank JQ801335）. The PCR amplification were conducted using the genomic DNA of Huayu 20 as the template by TAIL-PCR method, and a 973 bp fragment was obtained and submitted in NCBI （GenBank KC290400） , named by Ah-Glu-Pro. Promot- er sequence analysis by PLACE and PlantCARE showed that the sequence contained some typical cis-elements,such as TATA box and CAAT box, and pathogen and SA cis-acting regulatory elements. A 931 bp sequence was obtainedand 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 transformed into onion epidermal ceils by Agrobacterium -mediated transformation. GUS staining was conducted after the onion epidermal cells were induced by 5 mmol/L SA for 48 h. These results showed the onion epidermal cells appeared blue when induced by SA,while the control not induced by SA did not appear blue. The transient ex- pression resuh showed that the Ah-Glu-P could be an inducible promoter and contain SA responsive element.