黄芪UGPase cDNA克隆、分析及其在大肠杆菌中的表达

在已报道的UGPase的植物cDNA序列基础上,从膜荚黄芪( Astragalus membranaceus (Fisch.) Bunge)毛状根中分离了此酶的cDNA.此cDNA全长为1 831 bp,推测编码分子量为51.5 kD、等电点为6.01的由471个氨基酸残基组成的多肽.将此cDNA的开放阅读框载入质粒pET28(a)+并转入大肠杆菌( Escherichia coli ) BL21.SDS-PAGE表明此酶已经在 E.coli 中获得大量表达,表达量约为总细菌蛋白的40%.酶活分析表明,转化菌中UGPase 的活性比非转化菌高0.50～3.27倍,证明此cDNA可以在原核生物中获得表达.Northern blot表明UGPase在黄芪的根、茎、叶及毛状根中均有表达,在根及毛状根中表达量较高,证明了此酶主要分布于植物贮藏组织的报道.     

转化毛状根获得萝芙木生物碱的研究

药用植物萝芙木(Rauvolfia verticillata)的无菌苗被含有Ri质粒的发根农杆菌(Agro—bacterium rhizogenes)感染后,诱导出毛状根(Hairy root)。将毛状根分离,除菌后,在不合激素的Ms固体或液体培养基上培养,从296个株系中筛选出Rv 19、RV 26,RV 37和RV53Eq个生长速度快、分枝数量多等优良特性的无性系。对毛状根中次生代谢产物的提取和测定表明,四个无性系均含有原植物体所具有的吲哚生物碱。为应用转化毛状根技术获得植物次生代谢产物开辟了一条新的有效途径。     

FtMYB1转录因子调控苦荞毛状根黄酮醇合成的机理研究

R2R3-MYB转录因子在植物类黄酮合成中起重要的调控作用。本研究从川荞1号中克隆获得一个R2R3-MYB家族基因FtMYB1,酵母转录激活分析显示其具有转录激活活性,激素处理下的表达模式分析显示,FtMYB1基因能够被茉莉酸甲酯(MeJA)和脱落酸(ABA)诱导表达。在苦荞毛状根中过表达FtMYB1基因,结果表明转基因毛状根株系中总黄酮和芦丁含量显著低于野生型,且在黄酮醇合成途径下游分别编码黄酮醇合酶(FLS)和鼠李糖基转移酶(RT)的FLS和RT1基因表达量在转基因株系中显著降低,表明FtMYB1可能通过调控FLS和RT1的表达来抑制黄酮醇的生物合成。     

何首乌毛状根的诱导和培养(简报)

发根农杆菌（Agrobacteriumrhizogenes）侵染后,通过其Ri质粒的T－DNA片段在植物细胞基因组中整合,诱导形成毛状根（hairyroot）l’l。利用生长迅速、生产效能高而稳定的毛状根培养物生产药用植物的次生物质,已有不少报道「一习。但未见发根农杆菌对何首乌遗传转化的报道。何首乌（Polygonu。。ltij7orumThunb．）为常用中药,其块根为乌须发、悦颜色、益精养血和抗衰老的要药。本文报道发根农杆菌对何首乌遗传转化的实验结果,为今后用毛状根培养物生产何首乌的次生物质打下基础。l材料和方法细菌菌株及培养发根农杆菌（Agrobacteri…     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

银杏毛状根褐化机制的初步研究

该实验依据现有发根农杆菌侵染法,以银杏无菌苗叶片为外植体,对农杆菌诱导银杏毛状根的实验操作进行优化,并观察记录银杏毛状根继代生长情况,测定继代不同天数的毛状根组织内多酚含量、多酚合成相关酶基因和POD基因相对表达量,以探讨银杏毛状根褐化的生理机制。结果显示:(1)银杏农杆菌侵染最适预培养时间为3d、菌种为R1601、共培养基为White、侵染时间为30min,优化侵染操作后的银杏毛状根诱导率可达72.36%;银杏毛状根继代生长的最适IBA浓度为0.5mg/L。(2)银杏毛状根初始发根时颜色为乳白色,多在伤口处簇生,伸长生长时无向地性,不分叶正背面都能在伤口处诱导出根,且在除菌培养20d时毛状根长可达0.4～0.8cm;继代培养25d时毛状根为白色,继代35d时部分毛状根从根部至中部开始转黄,继代45d时大部分毛状根由白转黄,根部至中部开始有褐化出现。(3)继代35d时毛状根的PAL、C4 H、4CL和POD基因表达量分别为25d的68%、44%、51%、52%;继代45d时PAL、C4 H、4CL、POD基因表达量分别为35d的19%、75%、86%、105%。(4)继代35d、45d的银杏毛状根总酚类含量分别为17.14和13.78mg/g。研究发现,PAL基因表达量变化与其下游的两个关键酶基因(C4 H、4CL)表达量变化不一致,且PAL基因表达量的大幅降低并未对下游C4 H、4CL基因表达有明显影响,说明PAL基因表达量变化对其下游合成速率影响不大,可能不是银杏毛状根苯丙氨酸代谢途径的限速酶;而C4 H与4CL基因表达量变化趋势高度一致,说明C4H应是该途径中的限速酶之一;推测继代前期可能是由PPO作为关键酶参与毛状根褐化反应,导致后期毛状根生长受抑制,POD基因表达水平随之上升,POD参与褐化反应。     

发根农杆菌介导的菠菜毛状根遗传转化体系的建立

发根农杆菌(Agrobacterium rhizogenes)侵染植物后可诱导植物产生毛状根。菠菜(Spinacia oleracea)是常见的食用蔬菜, 目前尚未见菠菜毛状根的研究报道。经筛选得到适合诱导菠菜毛状根的发根农杆菌菌株LBA9402, LBA9402侵染菠菜外植体茎后, 毛状根的诱导率最高可达16%。菠菜毛状根呈白色, 具有丰富的根毛, 能在无外源激素的固体培养基上快速增殖生长。通过诱导菠菜毛状根产生愈伤组织并进行分化, 获得了菠菜毛状根的再生植株, 再生率为8%。此外, LBA9402可将含有Ri质粒的T-DNA和携带外源GFP基因的Ti质粒T-DNA共同导入外植体中。PCR检测和荧光显微观察结果显示, rolB及GFP基因在菠菜毛状根基因组中稳定表达, 共转化频率为50%。     

特异性过表达SQS1基因的甘草毛状根培养体系的建立

目的:建立根特异性过表达鲨烯合酶(SQS)基因的甘草毛状根培养体系。方法:构建根特异性过表达甘草SQS1基因的发根农杆菌ACCC10060工程菌;侵染甘草无菌苗胚轴,共培养48 h,以诱导毛状根的形成;多次除菌后,利用PCR法及测序法对诱导获得的甘草毛状根进行验证。结果:PCR结果显示扩增得到了长度约为730、580和1400 bp的基因片段,分别与烟草根特异性启动子TobRB7、发根农杆菌rolC基因和甘草SQS1基因长度一致;测序结果进一步确定了PCR扩增序列的正确性,从而证明了甘草根特异性过表达SQS1基因毛状根诱导成功。结论:获得了大量生长良好的特异性过表达SQS1基因的甘草毛状根,为研究功能基因SQS1与甘草酸次生代谢的相关性,及提高甘草毛状根中的甘草酸含量奠定了良好的实验基础。     

反义4CL与UGPase双价基因在烟草中的转化及表达分析

构建了携带紫穗槐尿苷二磷酸葡萄糖焦磷酸化酶(UDP-glucose pyrophosphorylase, UGPase)基因和4-香豆酸: 辅酶A连接酶(4-coumarate: coenzyme A ligase, 4CL)反义基因的双价基因植物表达载体, 用热激法转化至根癌农杆菌LBA4404中, 并用制备的农杆菌工程菌进行了烟草转化。PCR及Southern 杂交结果证实, 双价基因已成功整合到烟草基因组中。综纤维素和硫酸木质素含量测定结果显示, 转基因烟草纤维素含量增加, 木质素含量减少,表明双价基因能够有效表达。     

芪合酶基因Fm-STS在何首乌毛状根中的过量表达及dsRNA干扰

目的:建立一套探究植物基因功能的方法体系,验证由芪合酶基因保守序列通过RACE扩增技术在何首乌中得到的基因Fm -STS的功能.方法:由含CaMV 35S启动子驱动的gfp基因的植物转基因表达基础质粒pBIN-35S-GFP构建过表达质粒pBIN-35S-STS-GFP(阳性)和双链RNA干扰重组质粒pBIN-35S-正向-反向-GFP(阴性),并同空白表达质粒pBIN-35S-GFP(空白)均导入野生型发根农杆菌ATCC15834中,转化何首乌外植体,诱导生成毛状根并培养,对毛状根进行高效液相色谱分析以及实时荧光定量检测.结果:在过表达组、空白组和干扰组中毛状根中发根农杆菌Ri质粒中的rolB基因和外源基因gfp均有表达,高效液相色谱法分析芪合物二苯乙烯苷含量依次为4.67mg/g、2.18mg/g和0.65 mg/g,在mRNA水平上测试荧光定量检测基因Fm-STS表达量:RNAi组是空白组的1/433.53,过表达组是空白组的2.41倍.结论:结果表明过量表达与双链RNA干扰相结合在植物基因功能研究中有良好的应用,何首乌中芪合酶Fm-STS是二苯乙烯苷主要的合成酶.     

Gene for a protein capable of enhancing lateral root formation.

Analysis of genes preferentially expressed in hairy roots caused by infection with Agrobacterium rhizogenes has provided insights into the regulation of lateral root formation. A hairy root preferential cDNA, HR7, has been cloned from hairy roots of Hyoscyamus niger. HR7 encodes a novel protein partially homologous to a metallocarboxypeptidase inhibitor and is expressed exclusively in the primordium and base of lateral roots in hairy roots. Overexpression of HR7 in transgenic roots of H. niger dramatically enhances the frequency of lateral root formation. The results of this study indicate that expression of HR7 plays a critical role in initiating lateral root formation.     

Changes in gene expression during hairy root formation by<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> infection in ginseng

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.     

Candidate genes involved in tanshinone biosynthesis in hairy roots of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> revealed by cDNA microarray

Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.     

Cloning of cDNA for UDP-glucose pyrophosphorylase and the expression of mRNA in rice endosperm

Rice endosperm UDP-glucose pyrophosphorylase (UGPase) cDNA clones were isolated by screening a lambda ZAP II library prepared from poly (A(+)) RNA of japonica rice (cv Sasanishiki) endosperm with a probe of potato UGPase cDNA. One cDNA clone, possessing about 1,700 nucleotides, contained the complete open reading frame of rice UGPase. At the nucleotide-sequence level, the UGPase cDNA of rice endosperm had high homology with the UGPase cDNA of barley endosperm (84%) and potato tuber (71%). The calculated molecular weight (50 kDa) agrees with the value determined by SDS-PAGE (51 kDa). At the amino-acid sequence level, rice UGPase has high homology with the UGPase of barley (92%) and potato (85%). The enzyme contained conserved sequence elements which are thought to be involved in substrate binding and catalytic activity. A Southern-blot analysis indicated that the gene existed as a single copy. Expression of the enzyme in rice endosperm examined by Northern-blot analysis was high at 10-15 days after heading.     

Anti-solasodine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants

We constructed a recombinant antibody fragment—single chain fragment-variable (scFv) antibody—derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.Communicated by F. Sato     

Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.