HBV母婴传播阻断及其易感性的相关性研究--《第三军医大学》2016年硕士论文
HBV母婴传播阻断及其易感性的相关性研究
【摘要】：乙型肝炎病毒(hepatitis B virus,HBV)感染呈现世界性流行,而我国HBV感染尤其普遍,由慢性乙型肝炎(chronic hepatitis B,CHB)发展而来的肝炎、肝衰竭、肝硬化、肝癌等严重威胁公众健康。每年全球约有65万人死于HBV感染所致的HBV相关疾病。HBV传播途径主要有血液传播(包括母婴传播)及性传播。其中母婴传播被公认为我国最重要的传播方式,其他国家也有30%~50%的HBV感染是通过母婴传播发生。当前新生儿出生后给予主被动免疫措施,约有6%~20%感染HBV的风险。近年来,随着核苷(酸)类药物(nucleos(t)ide analogues,NUCs)拉米夫定(lamivudine,LAM)、替比夫定(telbivudine,LdT)及替诺福韦(tenofovir disoproxil fumarate,TDF)广泛应用于CHB感染的妊娠妇女,阻断HBV垂直传播已取得满意成效。但关于何时用药、母乳喂养等问题仍存在争议。本研究在前期基础上进一步探讨LdT/TDF阻断HBV母婴传播的安全性和有效性。共收集2009年6月至2016年2月就诊于重庆西南医院的乙型肝炎病毒e抗原(hepatitis B e antigen,HBe Ag)阳性HBV DNA≥6 log10 IU/m L患者518例,其中LdT组311例(根据用药开始时间进一步分为:LdT 20~27组26例,LdT 28~32组260例,LdT33组25例),TDF组(28~32周开始用药)27例,180例孕期不愿意使用抗病毒药物者则为对照组,于孕20~38周开始抗病毒治疗。目前已证实钠离子-牛磺胆酸共转运多肽(Na+-taurochaolate cotransporting polypeptide,NTCP)是HBV感染的功能性受体,亦称为SLC10A1,由SLC10A1基因编码。既往多项研究表明SLC10A1基因单核苷酸多态性(single nucleotide polymorphism,SNP)位点变异与HBV易感性相关。但既往的HBV感染的宿主易感性研究忽视了环境和感染途径对HBV的影响,带有一定的局限性。因此基于母婴传播这一感染途径出发,研究HBV感染易感性将为我们认识HBV感染机制、药物开发提供新线索。本研拟在群体水平探讨HBV功能性受体SLC10A1基因SNP位点(rs2296651(p.Ser267Phe)、rs(p.Val166Phe)、rs(p.Ala64Thr)、rs(p.Ile223Thr)、rs(p.Lys314Glu)、rs(p.Ile279Thr)、rs(p.Gly158Ser)),2个内含子区域SNP位点(rs(c.356+1098CT)、rs(c.5683169AC)及位于位于5’端的rs7154439(c.-1956GA))变异对HBV母婴传播易感性的影响。共收集感染组247例(NUCs(-)免疫干预(+)55例、NUCs(-)免疫干预(-)192例),未感染组174例(NUCs(-)免疫干预(-)56例、NUCs(+)免疫干预(+)118例)HBe Ag阳性高HBV DNA水平母亲所生子代,采集入组人群的一般临床资料以及血液样本,提取基因组DNA,对c.800GA(p.Ser267Phe)等10个进行基因分型,并进行病例-对照关联研究分析。主要结果:1.LdT组经治疗后HBV DNA水平从基线水平7.34 log10 IU/m L降至分娩时的3.15log10 IU/m L,TDF组HBV DNA水平从基线7.40 log10 IU/m L降至分娩时的3.81 log10IU/m L,而对照组HBV DNA水平无明显下降。2.用药开始孕周越早者分娩前HBV DNA水平下降幅度越大,LdT 20~27,LdT28~32,LdT33组分娩前HBV DNA水平分别为1.61 log10 IU/m L、3.17 log10 IU/m L、4.14 log10 IU/m L。3.对照组脐带血低于检测下限率显著低于用药组(P0.05),LdT 20~27组11/11(100%),LdT 28~32组186/188(98.9%),LdT33组8/8(100%)。TDF组23/23(100%),对照组80/130(61.5%)。4.6月龄时,LdT/TDF用药组乙型肝炎病毒表面抗原(hepatitis B surface antigen,HBs Ag)阳性率为0,对照组为15.3%,差异显著(P0.05)。用药组脐血HBV DNA低于检测下限或者6月龄时HBs Ag阴性率均为100%(LdT20~27组:26/26;LdT28~32组:270/270;LdT33组:22/22;TDF组:23/23),明显高于对照组90/104(86.5%),差异有统计学差异(P0.05)。5.LdT 20~27、LdT 28~32、LdT33组分别有50%、44.5%、48%选择经阴道分娩,且分别有61.5%、39.4%、48%选择母乳喂养,TDF组44.4%经阴道分娩,11.1%选择母乳喂养,后代均未感染HBV,且无特殊不良反应。6.母亲基线和分娩前CK水平用药组和对照组无统计学差异(P0.05)。LdT组和对照组6月~1岁的CK水平分别为146.08 IU/L、170.8 IU/L,高于两组1~3岁的CK水平127.67 IU/L、139.5 IU/L,且该年龄段的CK水平均高于成人水平(本研究母亲基线CK水平52.17~68.99 IU/L)。LdT组和对照组婴幼儿CK水平无统计学差异(P0.05)。7.本研究LdT 28~32组发现1例6月龄HBs Ag阴性、抗-HBs阴性但3岁时HBs Ag弱阳性(9.02 IU/ml),COBAS DNA 4.24E+4 IU/ml。经3个月恩替卡韦0.25mg/日口服治疗,其HBs Ag转阴且出现抗-HBs。8.SLC10A1基因编码区rs、rs、rs、rs、rs、rs在本研究中检测样本中未检测出变异,各组基因型频率分布无统计学意义。9.rs2296651、rs、rs、rs7154439均检测出突变位点,但感染组(NUCs(-)免疫干预(+))和未感染组(NUCs(-)免疫干预(+))比较各位点基因型分布频率无统计学差异(P0.05)。结论:1.高病毒载量HBe Ag阳性母亲孕中晚期应用LdT/TDF安全、有效。2.LdT/TDF用药时机以28~32周为宜,但超过33周仍应积极进行挽救应用。3.在NUCs联合主被动免疫措施条件下,经阴道分娩和母乳喂养并未增加传播风险。4.孕期短期应用LdT不会引起母婴CK明显升高。5.产后仍存在水平传播可能性,尤其见于未产生抗-HBs者。6.SLC10A1基因突变与HBe Ag阳性高病毒载量HBV母亲所生子代对HBV易感性无显著关联。
【学位授予单位】：第三军医大学【学位级别】：硕士【学位授予年份】：2016【分类号】：R714.251
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400-819-9993乙型病毒性肝炎合并丙型(丁型)病毒性肝炎其血清NPT水平--《第十届全军检验医学学术会议论文汇编》2005年
乙型病毒性肝炎合并丙型(丁型)病毒性肝炎其血清NPT水平
【摘要】：目的通过对乙型病毒性肝炎患者重叠感染其血清新蝶呤(NPT)的测定,观察混合病毒性感染的肝炎患者的血NPT的水平:方法用HAMBURG IBL公司的NPT ELISA试剂检测NPT:结果对照组、乙型肝炎组、丁型肝炎组和丙型肝炎组其血NPT分别为4.07±3.76、42.35±4.61、48.87±6.65、52.58±12.15,其中丙型肝炎组、丁型肝炎组与乙型肝炎组比较t、P分别为4.53、0.01,5.17、0.01;丁型肝炎组与丙型肝炎组比较t=1.32、p0.01:结论乙型病毒性肝炎合并感染的肝炎患者血清NPT水平比单独乙型病毒性肝炎的高,通过检测乙型病毒性肝炎合并感染的患者血清NPT值,对于了解合并感染的肝炎患者的病情有一定的临床价值。
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400-819-9993治疗成人慢性乙型肝炎新药——富马酸替诺福韦艾拉酚胺(Vemlidy&)Determination of Forsythin in Fengreganmao Granules by HPLC--《Anti-Infection Pharmacy》2016年05期
Determination of Forsythin in Fengreganmao Granules by HPLC
CHEN ZFood and Drug Safety Monitoring Center of C
Objective: To establish a method for the determination of forsythin in Fengreganmao granules by HPLC. Methods: Using column of ZORBAX Eclipse XDB-C18(4.6 mm×250mm, 5 μm). The mobile phase was methanol- water(35:65). The wavelength was at 230 nm. The flow rate was 1.0 m L/min. Calculate the content area using external standard method. Results: Forsythin concentration within 5.115~25.575 μg/m L showed a good linear relationship(r=0.999 6). Phillyrin average recovery was 98.3%(RSD=0.52%,n=6). Fengreganmao particles contain phillyrin of0.064 2 mg/g. Conclusions: The method is sensitive, accurate, reliable and repeatable.It is suitable for the determination of forsythin in Fengreganmao granules.
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【Citations】
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Shen Xin,Zha Yani (Huaihua Institute for Drug Control,Huaihua,Hunan,China 418000);[J];中国药业;2008-09
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CHEN ZFood and Drug Safety Monitoring Center of C;[J];抗感染药学;2016-05
ZHAO DXU Shao-LI WZHU Shi-MAO Jun-Department of Pharmacy,85 Hospital of PLA;Department of Pharmacy,105 Hospital of PLA;;[J];东南国防医药;2015-06
PANG Hui-LI SFANG LMA Xing-LU Jia-LIU Zhi-Nanjing University of Chinese MAffiliated Hospital of Traditional Chinese Medicine of Jiangsu P;[J];中国实验方剂学杂志;2013-18
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PMCID: PMC2860387HHMIMSID: HHMIMS156333PMID: The lack of floral synthesis and emission of isoeugenol in Petunia axillaris subsp. parodii is due to a mutation in the isoeugenol synthase gene,1 ,2 ,3 ,3 ,2 and
1,*Irina Orlova2 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USAFind articles by Thomas J. Baiga3 Howard Hughes Medical Institute, Jack H. Skirball Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USAFind articles by Joseph P. Noel3 Howard Hughes Medical Institute, Jack H. Skirball Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USAFind articles by Natalia Dudareva2 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USAFind articles by Eran Pichersky1 Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Street, Ann Arbor, MI , USAFind articles by 2 Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN 47907, USA3 Howard Hughes Medical Institute, Jack H. Skirball Chemical Biology and Proteomics Laboratory, The Salk Institute for Biological Studies, 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA*
To whom correspondence should be addressed. Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University Avenue, Ann Arbor, MI . Tel. 1-734-936-3522, Fax: 1-734-647-0884,
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the published article.Floral scent has been extensively investigated in plants of the South American genus Petunia. Flowers of Petunia integrifolia emit mostly benzaldehyde, while flowers of Petunia axillaris subspecies axillaris emit a mixture of volatile benzenoid and phenylpropanoid compounds that include isoeugenol and eugenol. Flowers of the man-made species P. hybrida, a hybrid of P. integrifolia and P. axillaris, emit a similar spectrum of volatiles as P. axillaris subsp. axillaris. However, the flowers of P. axillaris subspecies parodii emit neither isoeugenol nor eugenol but contain high levels of dihydroconiferyl acetate in the petals, the main scent-synthesizing and scent-emitting organs. We recently showed that both isoeugenol and eugenol in P. hybrida are biosynthesized from coniferyl acetate in reactions catalyzed by isoeugenol synthase (PhIGS1) and eugenol synthase (PhEGS1), respectively, via a quinone methide-like intermediate. Here we show that P. axillaris subsp. parodii has a functional EGS gene that is expressed in flowers, but its IGS gene contains a frame-shift mutation that renders it inactive. Despite the presence of active EGS enzyme in P. axillaris subsp. parodii, in the absence of IGS activity the coniferyl acetate substrate is converted by a yet unknown enzyme to dihydroconiferyl acetate. By contrast, suppressing the expression of PhIGS1 in P. hybrida by RNAi also leads to a decrease in isoeugenol biosynthesis, but instead of the accumulation of dihydroconiferyl acetate, the flowers synthesize higher levels of eugenol.Keywords: pollination, plant volatile, scent, evolution, petuniaMany plant species produce and emit a mixture of scent compounds from their flowers to attract pollinators. Floral bouquets are usually a blend of aromatic compounds. Plants have evolved their floral scent to maximize the reproductive potential under specific ecological conditions and specific pollinators. It appears likely that individual chemical components of these scents allow pollinators, most often insects, to distinguish between even closely related plant species (). The analysis of variation in the floral scents among genetically closely related plant species may lead to a better understanding of the mechanisms underlying the generation of scent diversity. For example, the flowers of the California annual Clarkia breweri emit a mixture of volatiles that includes linalool while a closely related species, C. concinna, does not emit this compound. Notably, the genomes of both species contain a gene for linalool synthase but the gene is highly expressed in flowers of C. breweri but not in flowers of C. concinna. The emission of linalool from C. breweri flowers (and additional scent compounds, which contain eugenol and isoeugenol, missing from the C. concinna flowers) correlated with the attraction of a hawkmoth pollinator that does not regularly visit C. concinna flowers.The coevolution of scent and pollinators has also been studied in the South American genus Petunia. Petunia integrifolia flowers are pigmented, emit mostly benzaldehyde, and are pollinated by bees. Flowers of the related species P. axillaris are white, emit a mixture of several benzenoids and phenylpropenes, and are pollinated by various species of hawkmoths (, ). Moreover, some variation in scent composition among different accessions of P. axillaris was noted, and in particular it was observed that the flowers of the subspecies, P. a. parodii, did not emit eugenol and isoeugenol while P. a. axillaris flowers did ().Flowers of several lines of the man-made species P. hybrida, a hybrid of P. integrifolia and P. axillaris, have been examined for volatile emission. It has been found that they emit a similar spectrum of volatiles as Petunia axillaris subsp. axillaris (, ). In particular, most P. hybrida cultivars emit varying amounts of eugenol and isoeugenol. The synthesis of isoeugenol and eugenol was recently elucidated in P. hybrida cv. Mitchell. It was shown that isoeugenol was formed by the action of isoeugenol synthase 1 (PhIGS1) using coniferyl acetate as the substrate, which itself is biosynthesized from coniferyl alcohol and acetyl-CoA in a reaction catalyzed by coniferyl alcohol acetyltransferase (PhCFAT) (, ). The enzyme responsible for the synthesis of eugenol in P. hybrida flowers was also identified (). This protein, designated eugenol synthase 1 (PhEGS1), is homologous to PhIGS1, and it also uses coniferyl acetate as the substrate but biosynthesize eugenol (). also examined scent biosynthesis and emission in P. axillaris, confirming that flowers of several P. a. parodii accessions as well as some P. a. axillaris accessions emitted little or no isoeugenol and eugenol. They also demonstrated that the petals of these flowers, unlike the high isoeugenol-emitting P. a. axillaris accessions, contained high levels of dihydroconiferyl acetate (). Given the obvious similarity in chemical structure between dihydroconiferyl acetate and coniferyl acetate, the precursor of eugenol and isoeugenol,
hypothesized that the lack of eugenol and isoeugenol synthesis and the synthesis of dihydroconiferyl acetate were linked, but no molecular mechanism has been identified to date.Here we present data that indicate that the IGS gene in the genome of P. a. parodii contains a mutation that renders the encoded IGS enzyme non-functional. This natural mutation in PapIGS leads to the production of a non-functional IGS enzymes, and despite the presence of EGS activity in the flowers, the coniferyl acetate substrate is mostly converted to dihydroconiferyl acetate by the action of an unidentified reductase. By contrast, experimentally reducing PhIGS1 expression in P. hybrida by using RNAi suppression leads to a reduction in isoeugenol synthesis and the concomitant increase in eugenol biosynthesis.Analysis of floral scents in Petunia axillaris parodiiWe obtained the same P. a. parodii line analyzed by
and confirmed that it does not emit isoeugenol or eugenol (). The flowers also do not emit dihydroconiferyl acetate (). On the other hand, extraction of the petal tissue with hexane revealed that the petals contained dihydroconiferyl acetate at a concentration of 183.5±4.6 ng/mgFW ().GC-MS analysis of volatile compounds from P. a. parodii flowers(a) Separation by GC of volatile compounds emitted from P. a. parodii petals and collected by headspace sampling system. Compounds were identified based on their mass spectra and retention time. 1, B 2, B 3, M 4, B 5, M 6, B 7, Benzyl salicylate (but, not coniferyl derivatives). Relative abundance is shown in arbitrary units, based on Total Ion Current (TIC) measurement. The GC program for this analysis was according to .(b) GC-MS analysis of hexane-soluble compounds extracted from P. a. parodii petals. The arrow shows the peak of dihydroconiferyl acetate, with its mass spectra shown in the inset. 6, B 7, Benzyl salicylate.(c) GC-MS of authentic dihydroconiferyl acetate (DCA) standard. Its structure is also shown.(d) GC-MS and structures of authentic isoeugenol (IEG) and eugenol (EG) standards.(e) GC-MS and structures of authentic coniferyl acetate (CA) standard.Sequence analysis of P. axillaris parodii isoeugenol synthaseTo determine the cause of the lack of isoeugenol biosynthesis in P. a. parodii, we first isolated cDNAs encoding IGS (PapIGS) from these plants by RT-PCR, using oligonucleotides specific for the 5′ and 3′ ends of the P. hybrida IGS1 sequence. cDNAs were obtained from multiple independent RT-PCR reactions, and their sequences all showed differences at eight nucleotide positions relative to the previously reported PhIGS1 cDNA (). Most significantly, sequence alignment demonstrated that two nucleotides, T and C at position 250 and 251 in PhIGS1, were substituted with a single A, creating a reading frame shift in the PapIGS gene which if expressed and translated would lead to a significantly shortened protein. Determination of the sequence of the PapIGS gene (accession FJ609836), which was obtained by PCR of genomic DNA, showed that this mutation occurs in the middle of the second exon.Sequence of P. a. parodii isoeugenol synthaseThe sequence of the PhIGS1 protein and its gene is shown in full. A dot represents a nucleotide in the PapIGS sequence that is identical to that found in PhIGS1. The position of the single nucleotide deletion in PapIGS, which leads to the frame shift, is indicated with a highlighted dash. The sequence of the abbreviated protein encoded by the PapIGS gene is shown in italics above the sequence of PhIGS1 only when it differs from it.The PapIGS cDNA was spliced into a bacterial expression vector and transferred to E. coli, and upon induction of gene expression followed by lysis of the bacterial cells and in vitro biochemical assays, no IGS activity was detected in the lysate. RT-PCR isolation of IGS cDNAs from several P. a. axillaris accessions yielded cDNAs that encoded proteins whose sequences were either identical to that of PhIGS1 or different at no more that 5 positions. We expressed all these cDNAs in the heterologous E. coli system and in all cases the P. a. axillaris proteins were enzymatically active ().Suppression of PhIGS1 in P. hybrida leads to emission of eugenolP. hybrida is a man-made hybrid obtained by crossing P. integrifolia and P. axillaris. Its flowers emit high levels of isoeugenol (12.0 nmol/gFW/h) and significantly lower levels of eugenol (0.8 nmol/gFW/h). Moreover, some P. hybrida cultivars are amenable to genetic engineering manipulations (). To test if suppression of the PhIGS1 gene might result in the accumulation of dihydroconiferyl acetate in P. hybrida flower tissues, we employed two RNAi constructs containing gene-specific fragments from either the 5′ or 3′ ends of the coding region under the control of the C. breweri linalool synthase promoter, which is highly active in flower tissues, but also displays low levels of activity in roots and stems (). Several independent transgenic P. hybrida cv. Mitchell lines were generated for each construct. Four independent lines were chosen for further analysis. Two lines expressing the PhIGS1 RNAi fragment from the 5′ end of the coding region (lines I-4 and I-14) and two independent transformed lines expressing the PhIGS1 RNAi fragment from the 3′ end of the coding region (lines II-4 and II-5) showed substantially reduced PhIGS1 transcript levels () but no significant differences in the transcript levels of PhEGS1 (). In line II-5, which showed the most severe decrease in IGS1 mRNA levels, to 32% of the wild-type levels, the measured IGS activity levels were 35% those of the wild-type levels (not shown), a corresponding reduction. Analysis of the floral volatiles emitted by these four lines revealed moderate to strong (45–90%) reduction in isoeugenol emission ( and ), with corresponding increases in eugenol emission ( and ) so that the combined emission output of isoeugenol and eugenol per flower remained very similar. Progeny of these four transgenic plants showed near identical decreases in isoeugenol emission and increases in eugenol emission with the corresponding parents, indicating that this change was heritable (). Hexane extraction of the petal tissue of these transgenic plants showed no accumulation of dihydroconiferyl acetate, and none was found in the headspace.The effect of decreasing the levels of PhIGS1 mRNA in Petunia hybrida petals on eugenol and isoeugenol emission(a and b) The relative PhIGS1 (a) and PhEGS1 (b) transcript levels in control and transgenic PhIGS1 RNAi petunia flowers. The relative mRNA transcript levels were analyzed in corollas of 48-hrs-old control and PhIGS1 RNAi petunia flowers by qRT-PCR and the expression levels of PhIGS1 and PhEGS1 in control plants were each set as 1. Each graph represents the average of three replications for each of two biological samples. Bars indicate SE.(c and d) Isoeugenol (c) and eugenol (d) emission in control and transgenic PhIGS1 RNAi petunia flowers. Floral volatiles were collected from 48-hrs-old petunia flowers using the closed-loop stripping method. For each plant, an average of five to eight independent measurements were obtained. Bars indicate SE.Metabolic profiling of volatile compounds emitted from P. hybrida flowers of control and PhIGS1 RNAi II-5 transgenic plants(a) Headspace analysis of control flowers.(b) Headspace analysis of PhIGS1 RNAi II-5 transgenic flowers.Collected headspace volatiles were analyzed by GC-MS. Compounds were identified based on their mass spectra and retention time. 1, internal standard (naphthalene); 2, 3, 4, 5, phenylethylbenzoate. Relative abundance is shown in arbitrary units, based on Total Ion Current (TIC) measurement. The GC program for this analysis was according to , resulting in longer retention times for the compounds than observed in .Isolation and characterization of eugenol synthase and coniferyl alcohol acetyltransferase genes from P. axillaris parodii and the proteins they encodeSince transgenic P. hybrida plants in which the PhIGS1 gene was suppressed did not produce dihydroconiferyl acetate but instead showed increased production of eugenol, it appears that reduction in IGS activity allowed the PhEGS1 enzyme molecules in the petal cells, whose concentration has not changed, better access to the coniferyl acetate substrate. To understand why an elevated production of eugenol was not observed in P. a. parodii flower, which also lack a functional IGS protein, we examined if P. a. parodii flowers possessed similar levels of CFAT and EGS transcripts and CFAT and EGS enzymatic activities to those found in P. hybrida. We therefore isolated cDNAs of PapCFAT and PapEGS by RT-PCR from P. a. parodii petals RNA and expressed these cDNAs in E. coli cells. The sequences of the proteins encoded by PapEGS and PapCFAT cDNAs differ in 2 and 9 positions, respectively, relative to PhEGS1 and PhCFAT1 proteins (). However, PapEGS displayed apparent Km and kcat values with coniferyl acetate of 155.6 &#x003M and 0.85 sec−1, respectively, that are very similar to those of PhEGS1 (). In addition, a coupled in vitro assay using PapEGS and PapCFAT led to the synthesis of eugenol ().GC-MS analysis of the reaction products in reactions catalyzed by P. a. parodii EGS and CFAT(a) The reaction mixture contained coniferyl acetate, NADPH, and purified PapEGS.(b) The reaction mixture contained coniferyl alcohol, NADPH, acetyl-CoA, and purified PapEGS and PapCFAT. The eugenol peak was identified by Mass Spectra (inset), and matched to the mass fragmentation pattern and retention time of authentic eugenol standard.Transcript levels of CFAT, IGS, and EGS were determined by qRT-PCR in both P. a. parodii and P. hybrida plants (). EGS expression levels were similar in both species. Levels of PhIGS1 transcripts were approximately 70% of those of PhEGS1, whereas levels of PapIGS transcripts were extremely low. The significant decrease in accumulation of the mutated PapIGS transcripts is similar to what has been observed in other systems where transcripts encoding non-functional proteins are quickly degraded (). In contrast, PapCFAT transcript levels were approximately 2-fold higher than levels of PhCFAT transcripts. CFAT, IGS and EGS activities in crude extracts from petals of both species were also measured. CFAT and EGS activities were found to be 4-fold and 1.5-fold higher in P. a. parodii than in P. hybrida (). As expected, petals of P. a. parodii had no IGS activity ().Detection of EGS, IGS, and CFAT transcripts in P. hybrida and P. a. parodii floral tissuesThe relative mRNA transcript levels were analyzed in corollas of 48-hrs-old flowers by qRT-PCR and the expression of PhEGS1 in P. hybrida plants was set as 1. Each bar represents the average of three replications.EGS, IGS, and CFAT activities in P. hybrida and P. a. parodii floral tissuesTotal activity levels were analyzed in corollas of 48-hrs-old flowers. Each bar represents the average of three replications. N.D. indicates no detectable activity.Pollination syndromes have been described in which a set of floral traits such as shape, pigmentation and scent (or lack thereof) render the flower most suitable to a specific type of pollinator, such as bees, hawkmoths, or birds (; ; ). There has been considerable discussion about how many genetic changes are required to affect a change in pollinator. Several studies on floral scent in C. breweri (; , ; ; ) have shown that the cause of high levels of synthesis and emission of linalool and several other scent compounds including eugenol and isoeugenol in C. breweri flowers, which render the flowers attractive to hawkmoth pollinators, is due to the high levels of expression of the genes that encode the scent-biosynthetic enzymes. At least in some cases, these genes also exist in the essentially non-scented, bee-pollinated close relative C. concinna, but they are not appreciably expressed in the flowers of the latter species.Studies on the evolution of floral scent in the Petunia genus have also been aimed at understanding the molecular mechanisms that control the biosynthesis of compounds important for attraction of pollinators, and how changes in these pathways occur over time.
showed that whereas in flowers of P. integrifolia, which typically emit only benzaldehyde and are pollinated by bees, the gene AN2 controls the production of floral anthocyanin pigments, P. axillaris accessions contain a mutated, inactive copy of this gene and as a consequence the flowers are white. On the other hand, flowers of P. axillaris subsp. axillaris emit many more benzenoid and phenylpropene volatiles than flowers of P. integrifolia, including eugenol and isoeugenol. It has also been shown that flowers of P. hybrida, the artificial hybrid between P. axillaris and P. integrifolia, emit all the volatiles typically emitted by flowers of P. a. axillaris and may be pigmented similarly to P. integrifolia. In P. hybrida the shikimate pathway is responsible for the synthesis of the precursors of the benzenoid scent compounds and is regulated by a myb transcription factor encoded by the gene ODORANT1 (). However, the activity of the ortholog of ODORANT1 in P. axillaris has not been investigated to date.Our data show that when IGS activity is reduced in P. hybrida, the coniferyl acetate substrate that would have been used by PhIGS1 is instead used by PhEGS1, leading to increased eugenol formation relative to control flowers. It is interesting that “wild-type” control (non-transgenic) flowers produce 15–30 fold more isoeugenol than eugenol, despite similar PhIGS1 and PhEGS1 transcript levels and kinetic parameters of the corresponding enzymes, with the kcat value of PhEGS1 being only 2-fold lower than that of PhIGS1 (). It is possible that the levels of the isoeugenol and eugenol synthesized reflect the levels of PhIGS1 and PhEGS1 enzymatic activities in non-transgenic flowers, indicating that synthesis of PhIGS1 and PhEGS1 is regulated post-transcriptionally. On the other hand, a close association between CFAT and IGS (i.e., channeling) may explain the preponderance of isoeugenol produced in these flowers.Since P. a. parodii lines from several natural habitats have been found to accumulate dihydroconiferyl acetate (), it is likely that the mutation in PapIGS is widespread in this subspecies and must therefore have occurred some time ago, before significant radiation of the species. While the identification of this natural mutation in the PapIGS gene provides a clear explanation for the lack of isoeugenol biosynthesis in this subspecies, we have not yet been able to determine why P. a. parodii plants do not make eugenol, nor identify the enzyme involved in the reduction of the coniferyl moiety to dihydroconiferyl. Our data indicate that CFAT as well as EGS are still active in flowers of P. a. parodii. Indeed, CFAT transcript levels as well as CFAT enzymatic activity in P. a. parodii petals are 2- and 4-fold higher than those in P. hybrida, respectively, while EGS activity (but not transcript levels) is also approximately 2-fold higher. Yet, in contrast to the outcome observed in P. hybrida flowers in response to artificial suppression of PhIGS1, the flowers of P. a. parodii do not make eugenol but instead they reduce coniferyl acetate (or coniferyl alcohol - dihydroconiferyl alcohol can be acetylated by PapCFAT, see ) to dihydroconiferyl acetate. An NADPH-dependent double-bond reductase that uses similar substrates has been identified in several plant species (), and it will be of interest to determine if a homologous enzyme is responsible for the synthesis of dihydroconiferyl acetate in P. a. parodii petals. However, while we were able to isolate several homologs of this gene from P. a. parodii by RT-PCR of petal cDNAs, none of the enzymes encoded by these genes were able to reduce coniferyl alcohol, coniferyl aldehyde, or coniferyl acetate (data not shown).GC-MS analysis of petal extracts indicate that P. a. parodii petals do not accumulate coniferyl alcohol or coniferyl acetate (), suggesting that the reduction of coniferyl acetate (or coniferyl alcohol) to the dihydro derivative is quick and that the enzyme responsible for it competes well with EGS for the substrate. Since the mutation in PapIGS occurred many generations ago, it is possible that selection for the upregulation of a double-bond reductase has occurred over time to favor the synthesis of dihydroconiferyl acetate over an increase in the levels of eugenol, perhaps due to interactions with specific pollinators. Given the apparent linkage between the floral bouquet and pollinators in the Petunia genus, it will be of interest to determine if P. a. parodii flowers are visited by different pollinators than P. a. axillaris.Plant materials and growth conditionP. axillaris parodii, P. hybrida cv. Mitchell (Ball Seed, West Chicago), and the transgenic petunia plants were grown in the greenhouse or growth chamber with a 16 h light period (supplemented to 100 &#x003mol.m−2.s−1) and a temperature of 21 °C, and an 8 h dark period at 16 °C.Preparation of dihydroconiferyl acetateTo synthesize 3-(4-hydroxy-3-methoxyphenyl)propyl acetate (Dihydroconiferyl acetate), dihydroconiferyl alcohol (182.2 mg, 1.0 mmol), Candida antarctica lipase B (25 mg), vinyl acetate (401 &#x003l, 5.0 mmol), and dry diethyl ether (50 ml, 0.2M) were added into a 125 ml Erlenmeyer flask at room temperature. The reaction was stirred at room temperature for 2 hrs. The solution was filtered through glass wool and concentrated in vacuo. The reaction yielded a colorless oil (222 mg, quantitative yield). 1H NMR (500 MHz, CDCl3) δ 6.83 (d, J = 8.6 Hz, 1H), 6.68 (m, 2H), 5.52 (br s, 1H), 4.08 (t, J = 6.6, 2H), 3.87 (s, 3H), 2.61 (t, J = 7.3 Hz, 2H), 2.06 (s, 3H), 1.92 (dt, J = 6.6, 7.3 Hz, 2H); 13C NMR (125 MHz, CDCl3) δ 171.2, 146.4, 143.8, 133.1, 120.9, 114.3, 110.9, 63.8, 55.8, 31.8, 30.4, 21.0. LCMS [M+Na]+ calculated for C12H16NaO4: 247.09, found: 247.17.Enzyme assaysThe reaction to detect CFAT activity contained 50 mM MES/KOH, pH 6.5, 1 mM acetyl-CoA, 0.5 mM coniferyl alcohol, and 75 &#x003l crude protein (=10.0 mgFW, for crude enzyme assays) or 5.9 &#x003g PapCFAT (reaction A). The reaction for EGS activity (total vol. 150 &#x003l) contained 50 mM MES/KOH, pH 6.5, 1 mM NADPH, 0.5 mM coniferyl acetate, and 3.5 &#x003g PapEGS (for the product identification) or 75 &#x003l crude protein (=10.0 mgFW, for crude enzyme assays). For the coupled reaction with PapCFAT and PapEGS, 3.5 &#x003g PapEGS and 1 mM NADPH were added into reaction A. The crude enzyme solution was prepared from 48-hrs-old flowers by grinding under the protein extraction buffer (50 mM BisTris/HCl, pH7.0, 5 mM Na2S2O5, 10 mM β-mercaptoethanol, 1% polyvinylpyrrolidone, 10% glycerol, and 100 mM phenylmethanesulphonylfluoride), followed by centrifugation at 12,000 × g for 10 min. Each enzyme reaction was carried out at room temperature for 30 min, and the reaction solution was extracted with 1 ml of hexane and concentrated with the mild stream of N2 gas. A concentrated fraction (2 &#x003l) was injected to GC-MS for analysis as described by .qRT-PCRTo determine transcript levels, total RNA was isolated from petal tissues 2 hrs before the dark period. For the relative expression analysis of petunia genes, 48 hrs-old petunia corolla tissues of control and PhIGS1 RNAi transgenic plants were used. In all cases, total RNA was extracted from collected tissues as previously described (). The RNA was subjected to DNase treatment using the DNA-free kit (Ambion), and first-strand cDNA was synthesized by Superscript II reverse transcriptase (Invitrogen) with poly-T primers in parallel with a negative control reaction in which no Superscript II reverse transcriptase was added. The qPCR reactions utilizing SYBR-Green I dye (Molecular Probes), Taq polymerase (New England Biolad), fluorescein (Bio-Rad), the specific primers (), and a dilution series of each cDNA standards, were performed as previously described ().Table 1Primer used in this studyConstructDirectionPrimer sequence (5′→3′)qRT-PCRFor CFATCFAT/F1ForwardGATGTCCTCACCAGTCGTGTCTACTCFAT/R1ReverseGAATCGTCAATACCTCCCTCTGCTFor EGSEGS/F1ForwardGGATGGAAACGCTAAGGCTGTAEGS/R1ReverseCTCCCAAATAGCCACAAGCTCAFor IGSIGS/F1ForwardGAGCAACTGAAGCAGCAGGAATACIGS/R1ReverseGTAGGCTGCGACATCTTCCTCATAFor UBQUBQ/F1ForwardCAGACCAGCAGAGGCTGATTTTUBQ/R1ReverseGACGAAGCACCAAGTGAAGAGTAGcDNA amplification (For C-terminal His-tag expression)For CFATCFAT/F2ForwardATGGGAAACACAGACTTTCATGTGACFAT/R2ReverseATAAGTAGCAGTAAGGTCCAAATFor EGSEGS/F2ForwardATGGCTGAGAAAAGCAAAATTCEGS/R2ReverseGGCAAAGTGACTAAGGTACTCCFor IGSIGS/F2ForwardATGACTACTGGGAAGGGAAAAATAIGS/R2ReverseAGTGGATGGTTGGGCATAAGTTGCCAATTTTRNAi constructsFor LIS promoterLIS/F1ForwardGAGCTCGCGGCCGCAAGCTTATCTAATAATGTATCAAAALIS/R1ReverseCTCGAGCCCGGGATGGTTGTCTTGTFor 5′IGSIGS/F2ForwardATGACTACTGGGAAGGGAAAAATA5′IGS/R1ReverseATCATGTTCACTAAGCTCTCCFor 3′IGS3′IGS/F1ForwardTCTGAACAAGAAATCATCAAAC3′IGS/R1ReverseTTAAGTGGATGGTTGGGCATAAGGeneration of RNAi silencing constructsPhIGS1 RNAi constructs driven by the C. breweri LIS (linalool synthase) promoter constructed essentially as described in . The promoter was amplified using the forward and reverse primers indicated in , and the 1038-bp fragment was transferred to pART27 (), modified for GATEWAY cloning (Invitrogen). Two PhIGS1 RNAi constructs were made, one with a fragment from the 5′ end of the gene and another with a fragment from the 3′ end of the gene. These RNAi constructs were made by first amplifying fragments of the PhIGS1 cDNA corresponding to bases 1&#x and bases 725&#x by PCR with the specific primers listed in
and separately splicing them into the GATEWAY entry vector pENTR/D-TOPO (Invitrogen). Each fragment was then separately spliced into the destination vector (pART27 with the LIS promoter) in forward and reverse positions flanking the intron. Transgenic petunia plants were obtained via Agrobacterium tumefaciens strain EHA105 carrying a pLISG-PhIGS1 RNAi construct using the standard leaf disk transformation method (). Rooted plants were screened for the presence of the LIS-promoter by PCR with the specific primers indicated in . PCR-positive T0 plants were transferred to the greenhouse and analyzed for floral headspace volatiles. The level of silencing in each transgenic plant was measured by qRT-PCR as described above. The T0 transformants were also self-pollinated manually and the T1 seeds were analyzed for segregation by germination on MS medium supplemented with kanamycin (150 mg/L).Analysis of volatiles from petunia flowersFloral volatiles were collected from control and PhIGS1 RNAi flowers, using a closed-loop stripping method under growth chamber conditions (21°C, 50% relative humidity, 150 &#x003mol.m−2.s−1 light intensity, and 12 h photoperiod) as described previously (). We collected floral headspace from detached P. axillaris parodii flowers (48-hrs old) using a push-pull headspace collection system (). Five petunia flowers were placed in a 9.0 l glass vessel and the emitted volatiles were collected for 24 hrs by using a trap containing 125 mg Porapak Q (0.65 × 1.5 cm, 80/100 mesh, Alltech) using a vacuum pump at a flow rate of 1.0 l/min. After headspace collection, the volatiles were eluted from the Porapak Q filter with 3 ml of hexane, concentrated by a mild nitrogen stream, and analyzed by GC-MS (). Dihydroconiferyl acetate was extracted from petals using hexane, as previously described (). GC-MS analysis was preformed as described in
and .Supp Fig 1Supplementary Figure 1: Sequence and enzymatic properties of IGS from several P. axillaris accession lines.(163K, pdf)Supp Fig 2Supplementary Figure 2: Emission levels of isoeugenol and eugenol in progeny of the first generation PhIGS1 RNAi transgenic plants.(26K, pdf)Supp Fig 3Supplementary Figure 3: Comparison of protein sequences from P. a. parodii and P. hybrida EGS and CFAT genes.(74K, pdf)Supp Fig 4Supplementary Figure 4: Comparison of CFAT activities from P. a. parodii using coniferyl alcohol (CA) and dihydroconiferyl alcohol (DCA).(21K, pdf)Supp Legends(39K, doc)We thank Dr. Cris Kuhlemeier (University of Bern, Bern, Switzerland) and Dr. Tom Gerats (Radboud University, Nijmegen, Netherlands) for providing seeds of P. axillaris. This work was supported by National Science Foundation grant 0718152 to E.P., by National Science Foundation grant 0718064 to J.N.P., by National Research Initiative of the U.S. Department of Agriculture Cooperative State Research, Education, and Extension Service grant -16207, the National Science Foundation/USDA-NRI Interagency Metabolic Engineering Program grant 0331333 to N.D., and by a grant from the Fred Gloeckner Foundation, Inc to N.D. T.K. was supported in part by JSPS Postdoctoral Fellowships for Research Abroad (Japan Society for the Promotion of Science, Japan).Ando T, Nomura M, Tsukahara J, Watanabe H, Kokubun H, Tsukamoto T, Hashimoto G, Marchesi E, Kitching IJ. Reproductive isolation in a native population of Petunia sensu Jussieu (Solanaceae) Ann Bot. 2001;88:403–413.Dexter R, Qualley A, Kish CM, Ma CJ, Koeduka T, Nagegowda DA, Dudareva N, Pichersky E, Clark D. Characterization of a petunia acetyltransferase involved in the biosynthesis of the floral volatile isoeugenol. Plant J. 2007;49:265–275.
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