百子莲ApCathB启动子克隆及其对超低温保存的响应分析

Cloning of ApCathB Promoter from African lily(Agapanthus praecox ssp. orientalis) and Its Response to Cryopreservation

组织蛋白酶B基因(ApCathB)是影响百子莲胚性愈伤组织超低温保存冻存率的关键基因,为探究ApCathB如何响应超低温保存复合逆境,利用染色体步移法扩增获得ApCathB的启动子序列,使用PlantCARE在线软件分析该启动子中含光、激素和非生物胁迫相关的顺式作用元件。将ProApCathB::GUS表达载体转化拟南芥,获得转基因植株,GUS组织化学染色表明ApCathB启动子在拟南芥幼苗时期和小花等部位活性较高,在成熟叶中较低。转基因拟南芥萌发60 h幼苗在超低温保存处理过程中,GUS基因和蛋白的表达量在脱水处理后显著提高,暗示ApCathB启动子受到脱水处理的诱导进而响应超低温保存复合逆境,这为进一步研究ApCathB在百子莲超低温保存中的功能及其相关调控机制提供了数据参考。

ApCathB is a key gene in regulating the relative survival rate of cryopreservation of African lily(Agapanthus praecox ssp. orientalis) embryogenic callus. For the purpose of exploring how ApCathB respond to the compound stress in cryopreservation, the promoter sequence of ApCathB was cloned using genome walking method. ApCathB promoter contained many cis-elements, such as photo response, hormone response and stress response cis-elements analyzed by PlantCARE online software. The plant expression vector ProApCathB::GUS was transformed to Arabidopsis, using transgenic Arabidopsis to detect the promoter activity. The glucuronidase(GUS)activity showed that the activity of ApCathB promoter was higher in seedling and young flowers, and lower in mature leaves. In the cryopreservation process of ProApCathB::GUS overexpressed Arabidopsis seedlings germinated for 60 h, both of the gene and protein level of GUS were elevated after dehydration treatments. This result demonstrated that the ApCathB promoter was activated by dehydration treatment during responding to the compound stress in cryopreservation. This result laid the foundation for further study of the function of ApCathB and its regulatory mechanisms during the cryopreservation of African lily.