AtDXS和AtDXR共表达对烟草萜类代谢产物的影响  

Effects of Co-expression of AtDXS and AtDXR on Terpene Metabolites in Tobacco(Nicotiana tabacum)

作　　者：张涵 冷璐[1] 祖庆学 聂忠扬 林松 成志军 李国明 陆引罡[1] ZHANG Han;LENG Lu;ZU Qing-Xue;NIE Zhong-Yang;LIN Song;CHENG Zhi-Jun;LI Guo-Ming;LU Yin-Gang(College of Agriculture,Guizhou University,Huaxi 550025,China;Kaiyang Branch of Guizhou Tobacco Company,Kaiyang 550300,China;Hunan China Tobacco Industry Co.,Ltd.,Changsha 410014,China)

机构地区：[1]贵州大学农学院,花溪550025 [2]贵州省烟草公司开阳分公司,开阳550300 [3]湖南中烟工业有限责任公司,长沙410014

出　　处：《农业生物技术学报》2022年第8期1480-1487,共8页Journal of Agricultural Biotechnology

基　　金：贵州省烟草公司贵阳市公司科技项目(筑烟科技{2019}2号);中国烟草总公司贵州省公司科技项目([中烟黔科(2018)07])。

摘　　要：1-脱氧-D-木酮糖-5-磷酸合酶(1-deoxy-D-xylulose-5-phosphate synthase,DXS)和1-脱氧-D-木酮糖-5-磷酸还原异构酶(1-deoxy-D-xylulose-5-phosphate reductoisomerase,DXR)分别是萜类化合物前体物质脱氧木酮糖-5-磷酸(1-deoxy-D-xylulose-5-phosphate pathway,DXP)合成途径的第1个和第2个限速酶,在烟草(Nicotiana tabacum)次生代谢过程中发挥着重要的调控作用。本研究以拟南芥(Arabidopsis thaliana)DXS和DXR基因为基础,选择目前植物基因表达最常用的启动子CaMV 35S(35S-promoter,originated from Cauliflower mosaic virus)和终止子NOS(nopaline synthase-terminator,derived from Agrobacterium tumefaciens),人工合成全基因序列DXS-NOS和35S-DXR,构建共表达载体p SH737-DXSDXR,通过农杆菌(Agrobacterium tumefacien)介导的遗传转化,成功获得DXS和DXR共表达的烟草转基因植株。通过GUS化学活性测定、PCR和Southern blot检测,证明该融合基因已成功导入烟草再生植株基因组并获得表达。RT-PCR检测结果显示,转基因植株的DXS基因表达较对照显著增加(P<0.05)。GCMS测定结果表明,转基因植株的新植二烯和腺毛分泌物均较对照有不同程度的增加(P<0.05)。本研究利用遗传改良技术为烟草育种提供了研究材料,为进一步利用代谢工程手段调控烟草萜类物质的生物合成提供参考依据。1-deoxy-d-xylulose-5-phosphate synthase (DXS) and 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) are the first and second rate limiting enzyme of the synthesis pathway of terpene precursor 1-deoxy-d-xylulose-5-phosphate (DXP),and play important regulatory roles in the secondary metabolism of tobacco (Nicotiana tabacum).In this study,based on the DXS and DXR in Arabidopsis thaliana the CaMV 35S promoter and NOS terminator,which are the most commonly used plant gene expression,were selected to synthesize the whole gene sequences DXS-NOS and 35S-DXR,and the co-expression vector pSH737-DXS-DXR was constructed.The tobacco transgenic plants co-expressing DXS and DXR were successfully obtained through Agrobacterium tumefacien-mediated genetic transformation.GUS chemica activity assay,PCR and Southern blot showed that the fusion genes had been successfully introduced into the genome of regenerated tobacco plants and expressed.RT-PCR showed that the DXS gene expression in transgenic plants was significantly higher than that of the control (P<0.05).The results of GC-MS showed that the neophytodiene and glandular hair secretion of transgenic plants increased in varying degrees compared with the control (P<0.05).This study provides research materials for tobacco breeding by using genetic improvement technology,and provides a reference basis for further regulating the biosynthesis of tobacco terpenoids by means of metabolic engineering.

关 键 词：烟草 1-脱氧-D-木酮糖-5-磷酸合酶基因(DXS) 1-脱氧-D-木酮糖-5-磷酸还原异构酶基因(DXR) 共表达 萜类代谢 

分 类 号：S752[农业科学—森林经理学]