甜菊醇糖苷生物合成关键基因的导入和鉴定分析

Transformation and Functional Identification of the Key Genes Associated with Steviol Glycosides Biosynthesis in Stevia rebaudiana

甜叶菊(Stevia rebaudiana Bertoni)生产的甜菊醇糖苷因具有高甜度、低热能、不参与人体内代谢兼具保健功能等特点,被誉为最有发展前途的新糖源。从甜叶菊叶片克隆了甜菊醇糖苷生物合成途径中的关键基因SrUGT85C2、SrUGT91D2m和SrUGT76G1,构建植物基因过量表达载体,以单独或组合的形式将这些基因导入到甜叶菊中,获得转基因植株。与野生型对照植株相比,单独导入SrUGT85C2的转基因植株中甜菊醇单糖苷含量提高,总糖苷、莱包迪苷A含量及占比没有明显变化;单独导入SrUGT91D2m的转基因植株中甜菊醇单糖苷含量显著降低,而甜菊醇双糖苷含量显著增加;单独导入SrUGT76G1的转基因植株中,总糖苷含量显著提高,莱包迪苷A含量达到10%以上,比对照提高了2倍,而甜菊糖苷含量减少了一半。3个基因组合同时导入的转基因甜叶菊植株与单独导入SrUGT76G1的转基因甜叶菊植株类似,其总糖苷、莱包迪苷A含量及其占比均显著提高。这些结果为以后通过分子生物学技术来调控甜菊醇糖苷生物合成关键基因的表达,培育莱包迪苷A含量高的高品质甜叶菊新品系提供了理论依据和技术方法。

Steviol glycosides produced by Stevia rebaudiana Bertoni are regarded as the most promising substitute for sucrose with a wide range of potential applications and high economic value due to their high sweetness,low caloric energy,non-participation in human metabolism and health care functions.Three key genes from Stevia leaves,SrUGT85C2,SrUGT91D2m and SrUGT76G1 in the biosynthetic pathway of steviol glycosides,were cloned,and overexpression vector of plant genes were constructed.In individual or in combined way,these genes were transferred into the S.rebaudiana,and the transgenic plants were obtained.Compared with the control of wild-type plant,the content of steviolmonoside increased in the transgenic plant transferred SrUGT85C2 alone,despite little changes in the total content of steviol glycosides,rebaudioside A,and the content ratio of rebaudioside A to the total steviol glycosides.In the transgenic plant transferred SrUGT91D2m alone,the content of steviolmonoside decreased,while the content of steviolbioside increased significantly.In the other hand,in the transgenic plant transferred SrUGT76G1 alone,the total amount of steviol glycosides significantly increased,especially the content of rebaudioside A reached to 10%,which was nearly two times than that in the control,whereas the content of stevioside reduced by half.The transgenic plant transferred the three genes in combination was similar to the transgenic plant transferred SrUGT76G1 alone,and the total content of steviol glycosides,rebaudioside A,and the content ratio of rebaudioside A to the total steviol glycosides increased significantly.These results provides the theoretical basis and technical methods for regulating the expressions of key genes in the biosynthesis of steviol glycosides and cultivating new high-quality S.rebaudiana strains with high content of rebaudioside A through molecular biology techniques.