木薯SDH蛋白的序列分析及其与MeH1.2关系的研究

Sequence Analysis of MeSDH Protein and Its Relationship with MeH1.2 in Cassava

【目的】山梨糖醇脱氢酶(SDH)在调控蔷薇科植物果糖和山梨醇转化中起重要作用,也参与植物对逆境的应答过程,木薯SDH功能的研究可以为培育优质木薯种质提供理论基础。【方法】以‘SC124’的cDNA为模板克隆木薯SDH基因,并利用实时荧光定量PCR分析木薯SDH基因的组织特异性及其对干旱、低温、PEG和ABA的响应模式。通过筛选木薯干旱和低温混合的酵母cDNA文库,并利用Y2H点对点及其双分子荧光互补(BiFC)实验确认与目标蛋白的关系。【结果】木薯SDH基因CDS全长1092 bp,编码364个氨基酸,与数据库中的序列无差异。MeSDH蛋白含有催化锌结合位点,NADP结合位点,结构锌结合位点,属于MDR超家族。烟草叶片表皮细胞瞬时表达显示MeSDH蛋白定位于细胞核。MeSDH基因的表达量在功能叶、幼嫩叶、须根和茎中依次降低。MeSDH基因受干旱、低温和PEG胁迫诱导在木薯叶片上调表达,在ABA处理后的木薯叶片和根系中都显著上调表达。文库筛选、Y2H点对点和BiFC实验证实MeH1.2与MeSDH互作。【结论】MeSDH基因可以响应多种胁迫上调表达,并可能在蛋白水平和MeH1.2共同作用。

【Objective】Sorbitol dehydrogenase(SDH)plays an important role in regulating the conversion between fructose and sorbitol in Roseviaceae plants,and also participates in plant resistance to stress.The study on the function of MeSDH gene may provide a theoretical basis for cultivating cassava germplasm with high yield and resistance to stress.【Method】MeSDH gene was cloned using‘SC124’cDNA as template,and the tissue specificity of MeSDH gene and its response pattern to drought,low temperature,PEG and ABA were analyzed by quantitative real-time PCR(RT-qPCR).MeH1.2 was used as bait to screen the mixed yeast cDNA library of cassava under drought and low temperature.Yeast two hybridization(Y2H)point-to-point and bimolecular fluorescence complementation(BiFC)experiments were further used to confirm the relationship between target proteins.【Result】The CDS length of MeSDH gene cloned from‘SC124’cDNA is 1092 bp,encoding 364 amino acids,and the sequence shows no difference with it in the database.MeSDH protein contains catalytic Zn binding site,NADP binding site and structural Zn binding site,belonging to the MDR superfamily.MeSDH protein is localized in the nucleus by transforming tobacco leaves.The expression of MeSDH gene is decreasing in the order of functional leaves,young leaves,fibrous roots and stems.The expression of MeSDH is up-regulated in the cassava leaves treated with drought,low temperature and PEG,and significantly up-regulated in the cassava leaves and roots treated with ABA.Yeast library screening,Y2H point-to-point and BiFC experiments confirm the interaction between MeH1.2 and MeSDH.【Conclusion】MeSDH gene is up-regulated in response to various stresses,and probably MeSDH and MeH1.2 work together.