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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 β-1, 3-glucanase belongs to an important plant disease-related protein (PR protein), easily induced by the exciton and accumulation. After treated with 1.5 mmol / L salicylic acid (SA) for 0.5, 12, 24, 36, 48 and 72 h, the seedlings of peanut Huayu 20 were induced with RNA for fluorescence quantitative detection of β-1,3-glucan Expression level of carbohydrase gene (Ah-Glu). The results showed that the expression of β-1, 3-glucanase gene reached the highest level after induction for 24 h, which was 1.8 times of that induced by SA. According to the pre-cloned peanut β-1,3-glucanase gene sequence (GenBank JQ801335), 3 nested specific primers were designed at the 5 ’end to amplify the upstream promoter sequence. Using the genomic DNA of Huayu 20 as a template, a 973 bp peanut β-1,3-glucanase gene promoter fragment was amplified by TAIL-PCR and registered at the NCBI website as GenBank KC290400 and named Ah-Glu-Pro. Online prediction analysis by PLACE and PlantCARE showed that the Ah-Glu-Pro sequence contains the core elements of TATA-box and CAAT-box, as well as the cis-regulatory elements of pathogen and salicylic acid. The upstream and downstream primers were designed according to Ah-Glu-Pro sequence. The 931 bp promoter sequence was amplified from Huayu 20 by the ordinary PCR and named as Ah-Glu-P. Ah-Glu-P was substituted for the CaMV35S promoter in plasmid pCAMBIA1301 to construct plant expression vector pCAMBIA1301-Ah-Glu-P. The pCAMBIA1301-Ah-Glu-P was transformed into onion epidermal cells by Agrobacterium-mediated transformation. After being treated with 5 mmol / L SA for 48 h, GUS staining was performed. The results showed that the GUS histochemical staining of onion epidermal cells induced by SA showed blue, GUS histochemical staining of onion epidermal cells without SA induced no blue, indicating that Ah-Glu-P can be induced to start It may contain cis-regulatory elements that respond to SA.