颗粒结合型淀粉合成酶过量表达显著提高黄芪毛状根中黄芪甲苷含量(英文)  

Enhanced Astragaloside IV Biosynthesis in Hairy Roots of Astragalus Membranceus by Over-Expression of Granule Bound Starch Synthase

作　　者：吴晓俊[1] 杜旻[2] 王子艳[1] 胡之璧[1] 

机构地区：[1]上海中医药大学中药研究所,上海201203 [2]中国科学院上海巴斯德研究所,上海200025

出　　处：《药物生物技术》2012年第6期485-490,共6页Pharmaceutical Biotechnology

基　　金：Supported by National Natural Science Foundation of China(30100237);Foundation of China State Administration of Traditional Chinese Medicine(02-03ZQ01,02-03ZQ02);Shanghai Board of Health Foundation(2002Q005);Program of Shanghai Pujiang Talent Plan(11PJ1408800);Program of Shanghai Talent Developmental Plan(2010009)

摘　　要：黄芪甲苷(Astragaloside IV,ASI)是药材黄芪中的活性成分之一。由于目前对于黄芪皂苷代谢途径知之甚少,使得通过直接转入皂苷母核合成相关基因从而提高黄芪毛状根中黄芪甲苷含量的方法不能施行。考虑到皂苷中的糖基侧链,增加活性葡萄糖代谢流入也许可以增加黄芪甲苷的最终转化效率。为了验证这个假说,该文将ADP-葡萄糖代谢相关基因颗粒结合型淀粉合成酶(Granule-bound starch synthase,GBSS)转入黄芪毛状根中,分子鉴定表明其基因已经整合进黄芪毛状根基因组,并且在mRNA水平增加了表达;GBSS酶活性分析表明,转基因株系平均酶活增加了约21.8倍;PAS染色结果表明,转基因株系中的多糖成分显著增加;与此同时,ASI含量亦提高了4倍。研究结果表明,通过转入活性糖代谢相关基因方法提高皂苷产量的方法切实可行。Astragaloside IV （ASI） is one of the bioactive saponins isolated from the root of Astragalus membranaceus. Due to the limited knowledge of ASI metabolic pathway,it is unfeasible currently to increase its production in hairy roots of A. membranaceus by genetic manipulation of enzymes directly involved in the synthesis of agloycone of the molecule. However considering the glycosyl group, alteration of glucose influx might enhance the final transformation of ASI. To testify the hypothesis, granule-bound starch syn- thase （GBSS） ,one of the enzymes involved in ADP-glueose metabolism,was over-expressed in the hairy roots under the control of the constitutive CaMV 35S promoter. Molecular identification, enzyme activity assay, and PAS staining were used, respectively, to con- firm the successful transformation and expression of GBSS. HPLC analysis was conducted to assess the effect of transformation on ASI biosythesis. Transgenic hairy roots differed from control roots in terms of phenotype. PCR results showed that rolC gene from Agrobac- terium rhizogenes integrated into genomes of the transgenic hairy roots. Further southern blotting and RT-PCR analysis confirmed the integration of GBSS into hairy roots at both DNA and mRNA levels. GBSS enzyme activity assay suggested the successful expression of the enzyme. Transgenic hairy root lines had a 21.8-fold increase in enzyme activities on the average. PAS staining showed that pol- ysaccharide components had been elevated significantly in the transgenic lines. Compared with control lines, transgenic hairy roots had accumulated 50% more biomass. Meanwhile, production of ASI was approximately 4-fold increased and reached 14. 44 mg/g （w/w）. These results indicated that over-expression of GBSS regulated ASI biosynthesis in hairy roots of A. membranaceus and possibly by influencing the metabolic influx of ADP-glucose and driving it towards the accumulation of ASI. The present study provides an alter- native approach to modulate saponin productivity in plants by genet

关 键 词：黄芪 黄芪甲苷 颗粒结合型淀粉合成酶 毛状根 皂苷 糖代谢 

分 类 号：Q78[生物学—分子生物学]