基于过表达技术的烟草NtGGPPS1基因功能的研究
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摘要
将NtGGPPS1-T质粒和spCAMBIA1300载体分别双酶切(SalⅠ和KpnⅠ)后连接,转化,挑取单克隆,筛选得到植物超量表达载体spCAMBIA1300-NtGGPPS1,并通过农杆菌介导侵染栽培烟草K326,在获得阳性转基因烟草后,进行定量RT-PCR以定量分析烟草牻牛儿基牻牛儿基焦磷酸合成酶基因NtGGPPS1在转基因植株中的转录积累.同时,通过LC-MS和GC-MS检测转基因植株中质体色素和萜类化合物含量的变化.结果显示,与对照组相比,NtGGPPS1基因在编号为Nt1-OE1、2、3、4、7植株中表达上调,阳性转基因烟草中新黄质、紫黄质、叶黄素、叶绿素a、叶绿素b和β-胡萝卜素6种质体色素,以及烟叶中代表性二萜类物质α-西柏三烯二醇(α-CBD)和β-西柏三烯二醇(β-CBD)的含量明显增加.以上结果表明,NtGGPPS1基因参与了烟草中质体色素和二萜类化合物的生物合成.
NtGGPPS1-T and spCAMBIA1300 were similarly digested with SalⅠand KpnⅠ,ligated with T4 DNA ligase and then transformed into competent cells to construct overexpression vector of spCAMBIA1300-NtGGPPS1.The vector was transferred into K326 by agrobacterium-mediated transformation method.After transgenic tobacco plants had been obtained,quantitative RT-PCR was conducted to quantify the transcript accumulation of NtGGPPS1 in the plants.The contents ofα-andβ-cembrenediol and plastid pigments in NtGGPPS1 up-regulated plants were detected by LC-MS and GC-MS.The results clearly show that the expression level of NtGGPPS1 is increased in the leaves of transgenic lines numbered as Nt1-OE1,2,3,4 and 7.Compared with the control,the contents ofα-andβ-cembrenediol,neoxanthin,violaxanthin,lutein,chlorophyll a,chlorophyll b andβ-carotene increase significantly in transgenic plants.This indicates that NtGGPPS1 is involved in the biosynthesis ofα-andβ-cembrenediol and carotenoids in tobacco.
NtGGPPS1-T and spCAMBIA1300 were similarly digested with SalⅠand KpnⅠ,ligated with T4 DNA ligase and then transformed into competent cells to construct overexpression vector of spCAMBIA1300-NtGGPPS1.The vector was transferred into K326 by agrobacterium-mediated transformation method.After transgenic tobacco plants had been obtained,quantitative RT-PCR was conducted to quantify the transcript accumulation of NtGGPPS1 in the plants.The contents ofα-andβ-cembrenediol and plastid pigments in NtGGPPS1 up-regulated plants were detected by LC-MS and GC-MS.The results clearly show that the expression level of NtGGPPS1 is increased in the leaves of transgenic lines numbered as Nt1-OE1,2,3,4 and 7.Compared with the control,the contents ofα-andβ-cembrenediol,neoxanthin,violaxanthin,lutein,chlorophyll a,chlorophyll b andβ-carotene increase significantly in transgenic plants.This indicates that NtGGPPS1 is involved in the biosynthesis ofα-andβ-cembrenediol and carotenoids in tobacco.
引文
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[15]TIAN Y S,WANG B,PENG R H,et al.Enhancing carotenoid biosynthesis in rice endosperm by metabolic engineering[J].Plant Biotechnology Journal,2018,doi:10.1111/pbi.13059.
[2]李雪君,崔红,刘海礁,等.fps转基因烤烟类胡萝卜素及其降解产物的研究[J].中国烟草科学,2006,27(3):25-27.LI X J,CUI H,LIU H J,et al.Studies on cartenoids and aromatic components of the transgenic flue-cured tobacco expressing fps gene[J].Chinese Tobacco Science,2006,27(3):25-27.
[3]GUTBROD K,ROMER J,DRMANN P.Phytol metabolism in plants[J].Progress in Lipid Research,2019,pii:S0163-7827(18)30002-X.doi:10.1016/j.plipres.2019.01.002.
[4]HENRY L K,THOMAS S T,WIDHALM J R,et al.Contribution of isopentenyl phosphate to plant terpenoid metabolism[J].Nature Plants,2018,4(9):721-729.
[5]LAULE O,FURHOLZ A,CHANG H S,et al.Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana[J].Proceedings of the National Academy of Sciences of the United States of America,2003,100(11):6866-6871.
[6]BURKE C,CROTEAU R.Geranyl diphosphate synthase fromAbies grandis:cDNA isolation,functional expression,and characterization[J].Archives of Biochemistry&Biophysics,2002,405(1):130-136.
[7]BECK G,COMAN D,HERREN E,et al.Characterization of the GGPP synthase gene family in Arabidopsis thaliana[J].Plant Molecular Biology,2013,82(4):393-416.
[8]KOJIMA N,SITTHITHAWORN W,VIROONCHATAPAN E,et al.Geranylgeranyl diphosphate synthases fromScoparia dulcis and Croton sublyratus.cDNA cloning,functional expression,and conversion to a farnesyl diphosphate synthase[J].Chemical&Pharmaceutical Bulletin,2000,48(7):1101-1103.
[9]TAKAYA A,ZHANG Y W,ASAWATRERATANAKUL K,et al.Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis[J].Biochimica et Biophysica Acta(BBA)-Gene Structure and Expression,2003,1625(2):214-220.
[10]ENGPRASERT S,TAURA F.Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase fromColeus forskohlii Briq[J].BMC Plant Biology,2004,4(1):18.
[11]魏攀,孟利军,陈千思,等.烟草牻牛儿基牻牛儿基焦磷酸合成酶基因NtGGPPS1的克隆和功能分析[J].烟草科技,2016,49(4):8-15.WEI P,MENG L J,CHEN Q S,et al.Cloning and functional analysis of geranylgeranyl pyrophosphate synthase gene NtGGPPS1from Nicotiana tabacum[J].Tobacco Science&Technology,2016,49(4):8-15.
[12]白玲,刘凡,李锁平,等.农杆菌介导Barnase基因转化番茄[J].河南大学学报(自然科学版),2002,32(3):16-19.BAI L,LIU F,LI S P,et al.Agrobacterium mediated Barnase gene transfer of tomato(Lycopersicon esculentum)[J].JournaI of Henan University(Natural Science),2002,32(3):16-19.
[13]白杰英,庞晓斌,曾林,等.人表皮生长因子在烟草中的表达及其重组蛋白的初步鉴定[J].河南大学学报(医学版),2007,26(3):9-12.BAI J Y,PANG X B,ZENG L,et al.Expression of human epidermal growth flactor in tobacco plants and primary identification of the recombinant protein[J].JournaI of Henan University(Medical Science),2007,26(3):9-12.
[14]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J].Methods,2001,25(4):402-408.
[15]TIAN Y S,WANG B,PENG R H,et al.Enhancing carotenoid biosynthesis in rice endosperm by metabolic engineering[J].Plant Biotechnology Journal,2018,doi:10.1111/pbi.13059.