| 537 | 4 | 49 |
| 下载次数 | 被引频次 | 阅读次数 |
天然橡胶是事关国计民生和国家安全的重要战略物资，其主要成分为顺式-1,4-聚异戊二烯，主要产自巴西橡胶树(Hevea brasiliensis)。由于受到该树种热带生长特性限制，我国适合种植的区域有限，产能严重不足，开发新的天然橡胶资源已成为迫切需要解决的问题。杠柳(Periploca sepium)乳管分泌的乳汁含有顺式-1,4-聚异戊二烯，非常适用于我国作为天然橡胶生产的替代植物。通过对杠柳树枝(包括皮、枝、叶)进行转录组测序，成功克隆了两个对顺式-1,4-聚异戊二烯生物合成起关键作用的基因PsIPPI和PsCPT,即异戊烯基二磷酸异构酶(isopentenyl diphosphate isomerase, IPPI)和顺式-异戊二烯基转移酶(cis-prenyltransferase, CPT)基因；以杠柳茎段为外植体，通过农杆菌介导的转化，将这两个基因以单独或组合形式在杠柳植物体内过表达，结果发现其基因相对表达量最高可增加200倍以上，但单独过表达PsIPPI的转基因植株，其聚异戊二烯含量和分子量分布没有明显变化；单独过表达PsCPT的转基因植株，其聚异戊二烯含量增加了约5倍，且高分子部分的比例提升了约1.6倍；同时过表达PsIPPI和PsCPT的转基因植株，其聚异戊二烯含量增加了5～6倍，高分子部分的比例提升了约2倍。本研究对杠柳顺式-1,4-聚异戊二烯生物合成关键基因的功能做了初步分析，证实利用部分关键基因的过表达可以提高杠柳的天然橡胶产量和高分子量的比例。随着对杠柳有关顺式-1,4-聚异戊二烯生物合成机制、编码各种酶系的关键基因的不断挖掘，可望将杠柳培育成为生产天然橡胶的替代植物，为解决我国天然橡胶贫乏的工业瓶颈和丰富橡胶资源提供技术支撑。
Abstract：Natural rubber is a key strategic material that is vital to the people's livelihood, the national economy and security. Its primary component is one kind of isoprenoids, known as cis-1,4-polyisoprene. The source of natural rubber is almost exclusive produced from Hevea brasiliensis whose strict growing conditions limit its cultivation to specific tropical environments. The problem of insufficient capacity of natural rubber production become more and more urgent that lead to the search for alternative sources in China. We found that the milk secreted by the laticifers in Periploca sepium contains cis-1,4-polyisoprene, and considered that it may be very suitable for China as a substitute plant for natural rubber production. Two genes encoding PsIPPI and PsCPT, namely isopentenyl diphosphate isomerase and cis-isopentenyl transferase, which play key roles in the biosynthesis of cis-1,4-polyisoprene, were successfully cloned from the branches(including barks, stems and leaves) of P. sepium according to its transcriptome sequencing. Using stem segments as explants, the two genes were overexpressed alone or in combination by Agrobacterium-mediated transformation, and their expression level were increased sharply up to 200-fold. However, the polyisoprene content and the molecular weight distribution had no significant change in the transgenic plants overexpressing PsIPPI alone, while the polyisoprene content and the proportion of high molecular weight increased about 5-fold and 1.6-fold in those of transgenic plants overexpressing PsCPT alone, as well as increased about 5-fold to 6-fold and 2-fold in those of PsIPPI and PsCPT co-overexpressing transgenic plantlets. In current research, the functions of the key genes associated with the biosynthesis of cis-1,4-polyisoprene in P. sepium were preliminarily identified, and the possibility was proved that the yield and high molecular weight of natural rubber could be improved by overexpression of some key genes. With the progress in the research on cis-1,4-polyisoprene biosynthesis mechanism, more and more importent enzymes and their encoded genes will be discovered and characterized, it is expected that P. sepium will be developed as an alternative source for the production of natural rubber, and completely solve the bottleneck of natural rubber existing in China.
Archer B.L.,Audley B.G.,Cockbain E.G.,and McSweeney G.P.,1963,The biosynthesis of rubber.Incorporation of mevalonate and isopentenyl pyrophosphate into rubber by Hevea brasiliensis-latex fractions,Biochem.J.,89(3):565-574.
Archer B.L.,and Audley B.G.,1987,New aspects of rubber biosynthesis,Bot.J.Linn.Soc.,94(1-2):181-196.
Asawatreratanakul K.,Zhang Y.W.,Wititsuwannakul D.,Wititsuwannakul R.,Takahashi S.,Rattanapittayaporn A.,and Koyama T.,2003,Molecular cloning,expression and characterization of cDNA encoding cis-prenyltransferases from Hevea brasiliensis.A key factor participating in natural rubber biosynthesis,Eur.J.Biochem.,270(23):4671-4680.
Chen M.W.,2016,The current situation and coping strategies of China′s natural rubber industry,Issues in Agricultural Economy,37(10):91-94.(陈明文，2016,我国天然橡胶产业发展形势与因应策略，农业经济问题，37(10):91-94.)
Chen R.,Gyokusen M.,Nakazawa Y.,Su Y.Q.,and Gyokusen K.,2010,Establishment of an Agrobacte-rium-mediated transformation system for Periploca sepium Bunge,Plant Biotechnol.,27(2):173-181.
Chen R.,Harada Y.,Bamba T.,Nakazawa Y.,and Gyo-kusen K.,2012,Overexpression of an isopentenyl diphosphate isomerase gene to enhance trans-polyisoprene production in Eucommia ulmoides Oliver,BMC Biotechnol.,12:78.
Chen R.,Zhang H.,Zhang R.,Hu J.,Zhou T.,An S.Y.,and Lin Z.,2018,A plasmid system for construction of plant multiple gene expression vectors,Molecular Plant Breeding,16(4):1138-1146.(陈任，张虹，张芮，虎娟，周涛，安韶雅，林哲，2018,用于植物多基因表达载体构建的质粒系统，分子植物育种，16(4):1138-1146.)
Dennis M.S.,and Light D.R.,1989,Rubber elongation factor from Hevea brasiliensis.Identification,characterization,and role in rubber biosynthesis,J.Biol.Chem.,264(31):18608-18617.
Hepper C.M.,and Audley B.G.,1969,The biosynthesis of rubber from hydroxyl-methyl-glutaryl coenzyme A in Hevea brasiliensis latex,Biochem.J.,114:379-386.
He C.H.,Mo Y.Y.,and Liu R.J.,2020,Forecasting of natural rubber production capacity in China (2019-2025),Issues of Forestry Economics,40(3):320-327.(何长辉，莫业勇，刘锐金，2020,中国天然橡胶生产能力预测分析(2019-2025年),林业经济问题，40(3):320-327.)
Kajiwara S.,Fraser P.D.,Kondo K.,and Misawa N.,1997,Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli,Biochem.J.,324:421-426.
Ko J.H.,Chow K.S.,and Han K.H.,2003,Transcriptome analysis reveals novel features of the molecular events occurring in the laticifers of Hevea brasiliensis (Para rubber tree),Plant Mol.Biol.,53(4):479-492.
Lichtenthaler H.K.,1999,The 1-deoxy-d-xylulose-5-phosphate pathway of isoprenoid biosynthesis in plants,Annu.Rev.Plant Physiol.Plant Mol.Biol.,50:47-65.
Liu R.J.,Mo Y.Y.,Yang L.,Wu W.,and He C.H.,2022,Re-recognition and advice on the strategic role of natural rubber industry in China,China Tropical Agriculture,104(1):13-18.(刘锐金，莫业勇，杨琳，伍薇，何长辉，2022,我国天然橡胶产业战略地位的再认识与发展建议，中国热带农业，104(1):13-18.)
Liu X.,2017,Molecular cloning and functional analysis of key genes PsIPPI and PsCPT associated with cis-1,4-polyisoprene biosynthysis in Periploca sepium,Thesis for M.S.,Ningxia University,Supervisor:Chen R.,pp13-18.(刘祥，2017,杠柳顺式-1,4-聚异戊二烯生物合成关键基因PsIPPI和PsCPT的克隆及功能分析，硕士学位论文，宁夏大学，导师：陈任，pp13-18.)
Liu X.,Liu S.Y.,Hu J.,Zhang H.,Zhang Z.P.,and Chen R.,2017,Eucommia-rubber analysis from Eucommia ulmoides Oliver using three solvents assisted sonication,Chinese Journal of Tropical Crops,38(4):723-727.(刘祥，刘淑瑛，虎娟，张虹，张自萍，陈任，2017,超声波辅助3种溶剂分析测定杜仲胶含量，热带作物学报，38(4):723-727.)
Oh S.K.,Kang H.,Shin D.H.,Yang J.,Chow K.S.,Yeang H.Y.,Wagner B.,Breiteneder H.,and Han K.H.,1999,Isolation,characterization,and functional analysis of a novel cDNA clone encoding a small rubber particle protein from Hevea brasiliensis,J.Biol.Chem.,274(24):17132-17138.
Okada K.,Kasahara H.,Yamaguchi S.,Kawaide H.,Kamiya Y.,Nojiri H.,and Yamane H.,2008,Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant deve-lopment in Arabidopsis,Plant Cell Physiol.,49(4):604-616.
Ramos-Valdivia A.C.,van der Heijden R.,and Verpoorte R.,1997,Isopentenyl diphosphate isomerase:a core enzyme in isoprenoid biosynthesis.A review of its biochemistry and function,Nat.Prod.Rep.,14(6):591-603.
Rodriguez-Concepcion M.,and Boronat A.,2002,Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastid.A metabolic milestone achieved through genomics,Plant Physiol.,130(3):1079-1089.
Skilleter D.N.,and Kekwick R.G.,1971,The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis,Biochem.J.,124(2):407-417.
Tanaka Y.,1989,Structure and biosynthesis mechanism of natural polyisoprene,Prog.Polym.Sci.,14(3):339-371.
Tangpakdee J.,and Tanaka Y.,1998,Why rubber trees produce polyisoprenes-a possible role of natural rubber in the Hevea tree.J.Rubber Res.,1(2):77-83.
Wang Y.C.,Qiu C.X.,Zhang F.,Guo B.H.,Miao Z.Q.,Sun X.F.,and Tang K.X.,2009,Molecular cloning,expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense,Biosci.Rep.,29(2):111-119.
Wu H.L.,Duan C.F.,Nie Z.Y.,Li Y.,Yu B.,and Zeng R.Z.,2009,Research progress in rubber particle of rubber tree(Hevea brasiliensis Muell.Arg.),Chinese Journal of Tropical Crops,30(7):1044-1049.(吴华玲，段翠芳，聂智毅，黎瑜，于波，曾日中，2009,巴西橡胶树橡胶粒子研究进展，热带作物学报，30(7):1044-1049.)
Xiang Q.L.,Xia K.C.,Dai L.J.,Kang G.J.,Li Y.,Nie Z.Y.,Duan C.F.,and Zeng R.Z.,2012,Proteome analysis of the large and the small rubber particles of Hevea brasiliensis using 2D-DIGE,Plant Physiol.Biochem.,60:207-213.
Yang Q.H.,Liu K.J.,Liu L.,Wang F.,Li H.B.,and Xie Q.L.,2021,Research progress on molecular regulation mechanism of NR biosynthesis,China Rubber Industry,68(10):784-788.(杨起航，刘坤杰，刘乐，王斐，李鸿彬，谢全亮，2021,天然橡胶生物合成分子调控机制的研究进展，橡胶工业，68(10):784-788.)
Zhao P.J.,An F.,Lin W.F.,Xie G.S.,and Mo Y.Y.,2012,Proposal for developing alternative nature rubber producing crops and techniques,Chinese Agricultural Science Bulletin,28(34):124-130.(赵平娟，安锋，林位夫，谢贵水，莫业勇，2012,大力开展巴西橡胶树替代产胶植物及技术研发的建议，中国农学通报，28(34):124-130.)
基本信息:
DOI：10.13417/j.gab.041.001079
中图分类号:S793.9
引用信息:
[1]路国栋,刘祥,刘慧,等.杠柳顺式-1,4-聚异戊二烯生物合成关键基因PsIPPI和PsCPT的克隆及功能分析[J].基因组学与应用生物学,2022,41(05):1079-1090.DOI:10.13417/j.gab.041.001079.
基金信息:
宁夏回族自治区重点研发计划重大(重点)项目(2019BFG02011); 国家自然科学基金(31960065)共同资助
2021-06-01
2021
2021-12-30
2022-09-03
2022
2
2022-07-10
2022-07-10
2022-07-10