转化酶抑制子(InvInh)在甘蔗蔗糖代谢中的功能研究

Sugarcane is one of the most important sugar and energy crops in the world. Sucrose invertase (INV) is the key enzyme that controls sucrose decomposition in higher plants, and plays a key role in the transport, storage and distribution of sucrose. The activity of INV enzyme is regulated by the level of transcription and translation. However, Invertase inhibitor (InvInh) is an important factor in its post-translational regulation, which inhibits the activity of INV enzyme by combining with INV. However, the molecular mechanism of sucrose metabolism was unknown.. Based on our previous gene expression and functional research on the sugarcane INV family members (SAI、CIN and NI) at the early stage, This proposal is going to further screen and clone sugarcane InvInh gene. Our laboratory has cloned 7 invertase inhibitor genes (SoInvInh) in stems of sugarcane through RT-PCR and RACE-PCR. By constructing the prokaryotic and yeast expression vector of SoInvInh, the antibody of SoInvInh will be prepared and the expression of InvInh gene in sugarcane will be detected.Sense and RNAi interference plant expression vector of SoInvInh will be constructed and transformed into Arabidopsis thaliana and sugarcane. The results will illustrate InvInh's interaction mechanism with INV and its role in the process of sucrose accumulation, and provide a reference for actively controlling the sucrose accumulation in sugarcane.

甘蔗是世界上最重要的糖料作物。蔗糖转化酶（INV）是高等植物体内控制蔗糖分解的关键酶，在蔗糖的转运、贮藏和分配中起关键作用。INV酶活性受转录和翻译水平的双重调控，而转化酶抑制子（InvInh）是其翻译后一个重要调控因子，通过与INV结合成复合物来抑制INV酶活性，但InvInh在甘蔗蔗糖积累过程中的调控作用及其机制尚不清楚。. 本研究在前期对甘蔗INV家族成员基因表达和功能研究基础上，进一步筛选出7个甘蔗InvInh基因，并通过构建原核和酵母表达载体，制备抗体，检测InvInh在甘蔗体内的表达情况。同时，构建InvInh基因正义和RNAi干扰植物表达载体，将正义表达载体导入拟南芥，并正、反导回甘蔗中。通过转基因植株性状表现，结合InvInh基因表达特性验证甘蔗SoInvInh基因的功能，阐明甘蔗糖分积累过程中InvInh与INV的互作机制，为能动调控甘蔗糖分积累提供依据。

甘蔗是主要的糖料作物。转化酶抑制子（InvInh）通过与酸性转化酶结合成复合物而抑制转化酶活性，而蔗糖浓度、pH值和SnPK磷酸化是影响二者结合的重要调控因子。本研究从甘蔗转录组数据库中筛选SoInvInh家族成员，分析基因表达和家族蛋白生物学特性；优化原核表达体系，纯化SoInvInh蛋白，进行体外抑制试验；构建植物表达载体，通过转化甘蔗和拟南芥进行基因功能研究。结果表明从甘蔗中克隆到5个SoInvInh家族基因，分别是SoInvInh1、SoInvInh2、SoInvInh3、SoInvInh4和SoInvInh5，在根、茎、叶、芽和花序中均能检测到家族成员基因的表达，但基因表达模式不同。SoInvInh家族成员蛋白N端都含有4个不对称的α螺旋结构和4个保守的Cys位点。重组SoInvInh1蛋白能抑制花序和叶中可溶性酸性转化酶（SAI）活性，而SoInvInh2蛋白能抑制茎、叶和花序中细胞壁结合转化酶（CIN）活性。高、低糖甘蔗品种茎中蔗糖含量与SAI酶活性显著负相关，而伸长期和成熟期茎中蔗糖含量与CIN活性都呈显著正相关，相关性分析表明SoInvInh1和SoInvInh3基因表达量与SAI酶活性显著负相关，SoInvInh2和SoInvInh5与CIN酶活性显著负相关，这种相关性具有组织和发育阶段特异性。转SoInvInh2基因甘蔗和拟南芥试验表明，过表达SoInvInh2基因，能抑制CIN酶活性，不利于蔗糖跨膜运输，导致蔗糖分含量降低，从而影响植物的生长发育和形态特征。转基因甘蔗伸长期茎中CIN酶活性与SoCIN2和SoCIN4基因表达量显著正相关，与SoCIN5基因表达量显著负相关，而SoInvInh2能抑制那一种SoCIN家族成员的酶活性，还需要进行酵母双杂交试验鉴定。本研究初步阐明了甘蔗SoInvInh与酸性转化酶的互作机制，为调控植物体内糖分积累机制提供依据。