甜菜保卫细胞应答NaCl胁迫气孔变化及富集优化

Stomatal changes and enrichment optimization of sugar beet guard cells in response to NaCl stress

气孔由两个特殊分化的保卫细胞构成,在植物抗逆境胁迫过程中发挥着重要作用。以具有优良抗逆能力的甜菜M14品系为研究对象,通过对活体植株叶片气孔孔径观察方法的比较,确定先采用撕取法获取表皮后再进行固定的方法更适合于甜菜保卫细胞的观察。采用该方法对盐胁迫前后活体植株叶片气孔孔径进行观察,发现200 mM NaCl胁迫处理下甜菜M14品系气孔响应最佳时间点为20 min,400 mM NaCl胁迫处理下气孔响应最佳时间点为30 min。此外,进一步对甜菜M14品系保卫细胞的富集进行了方法优化,通过比较透明胶带撕取法和搅拌机搅碎法,经过对叶肉细胞特异性表达基因ATPase-1的qRT-PCR鉴定发现其在叶片中的表达量是富集后保卫细胞中的70倍,最终确定采用搅拌机搅碎法获取表皮后再经4.2%纤维素酶R10、0.15%离析酶R10及0.2%果胶酶Y23的协同处理可实现甜菜保卫细胞快速富集,以满足后续分子生物学及蛋白质组学实验的需求。

The stomata are composed of two specially differentiated guard cells and play an important role in plant resistance to stress.In this study,sugar beet M14 line with excellent resistance to stress was used as research materials.By comparing the stomatal aperture observation methods of living plants,it was determined that the method of obtaining the epidermis by the tearing method and then fixing was more suitable for the observation of sugar beet guard cells.Subsequently,this method was used to observe the stomatal pore size of living plants before and after NaCl stress.It was found that the optimal point for stomata response of sugar beet M14 line was 20 min and 30 min under 200 mM NaCl and stress 400 mM NaCl respectively.In addition,the method of enrichment of guard cells of sugar beet M14 line was optimized by comparing the transparent tape tearing method to the mixer pulverization method with treated by 4.2%cellulase R10,0.15%segregation enzyme R10 and 0.2%pectinase Y23.The relative expression level of the mesophyll cell-specific gene ATPase-1 by qRT-PCR in leaves was 70 times higher than guard cells after enrichment.This rapid enrichment method of sugar beet guard cells would meet the needs of molecular biology and proteomics experiments subsequently.