百子莲类枯草杆菌蛋白酶基因ApSBT的克隆及表达特性分析

Expression characteristics analyses and cloning of ApSBT gene encoding a subtilisin-like protease from Agapanthus praecox

类枯草杆菌蛋白酶(subtilisin-like proteases, SBT)是一类与植物发育、防御反应相关的多功能丝氨酸蛋白酶。本文以百子莲(Agapanthus praecox ssp.orientalis)胚性愈伤组织为供试材料,利用RACE技术克隆出全长的ApSBT基因为2 316 bp,编码771个氨基酸。预测ApSBT蛋白为82.69 kDa,是从属于Peptidases S8家族的亲水蛋白。同源序列对比分析可知,百子莲ApSBT蛋白与棕榈科和禾本科等单子叶植物的SBT遗传进化关系较近。百子莲丛生芽经过4种单一的非生物(渗透、盐、氧化及内质网)胁迫处理12和24 h后,实时荧光定量PCR检测发现ApSBT基因的相对表达量对4种胁迫均有响应,其中胁迫12 h时的表达量最高,分别为对照的12.00、4.25、3.20和2.50倍。通过单一胁迫实验发现,在10 mmol·L^(-1) H2O2诱导下的转基因拟南芥幼苗死亡率高达78.57%,说明ApSBT主要响应了氧化胁迫。

The subtilisin-like protease is a multifunctional serine protease related to plant development and defense response. The ApSBT gene was cloned from Agapanthus praecox ssp. orientalis using RACE technology. The full-length sequence of ApSBT gene was 2 316 bp, encoded 771 amino acids. The relative molecular mass of ApSBT protein was 82.69 kDa, belonging to the Peptidases S8 family. Through phylogenetic analysis, the evolution of ApSBT protein obeys the classification of monocotyledons and dicotyledons, and is more closely related to the monocotyledons, such as the family of Palmae and Gramineae. In order to understand the molecular function of ApSBT gene, A. praecox embryogenic callus was treated with four single abiotic(osmotic, salt, oxidation and endoplasmic reticulum) stresses for 12 and 24 h. The relative expression of ApSBT gene was detected by real-time quantitative PCR(qRT-PCR). The results showed that ApSBT gene was responsive to all treatments, especially to osmotic stress. The expression level reached the highest at 12 h under all stress, which were 12.00, 4.25, 3.20 and 2.50 times as much as the control under the osmotic, salt, oxidative and endoplasmic reticulum stress respectively. Through two single oxidative stress experiments to verify the growth status of transgenic Arabidopsis at one week seedling, the results showed that the mortality rate of transgenic Arabidopsis was higher than the wild type. Particularly under 10 mmol·L-1 H2 O2 treatment, the death rate increased to 78.57%, which was significantly higher than the wild type seedlings. That indicated the ApSBT mainly responded to tolerate oxidative stress.