植物表皮蠟質(zhì)相關(guān)文獻(xiàn)閱讀

1、蠟質(zhì)相關(guān)文獻(xiàn)閱讀(1)(2)菊科千里光蠟質(zhì)合成相關(guān)蛋白The major cuticular components have been shown to be synthesized in the epidermis. Therefore, cloning of epidermis-specific genes could yield information to be used to isolate and characterize the enzymes involved in the cuticle biosynthesis. A subtractive cDNA library was

2、 prepared from Senecio odorus in which epidermis-specific cDNAs were enriched. Differential screening of the library using epidermal and non-epidermal probes revealed two cDNAs. One of them designated epi425 was identified, based on the sequence homology, as a member of a new class in the LTP gene f

3、amily and the other clone designated epi23 as a gene encoding an aldehyde decarbonylase. Northern blot analyses showed that epi425 and epi23 cDNAs hybridized with a transcript of about 600 and 2,100 nucleotides, respectively, from the epidermis but not from the non-epidermal tissues. Further charact

4、erization of these clones will provide more information on the mechanism of the cuticle biosynthesis.(3)Senecio odorus lipid transfer protein mRNA, 3' end GenBank: L33792.1lipid transfer protein Senecio odorus GenBank: AAA33934.1 千里光多年生草本。莖木質(zhì)細(xì)長，高約25米，曲折呈攀援狀，上部多分枝，有脫落性的毛。葉互生；橢圓狀三角形，或卵狀披針形，長710厘米，

5、寬3.54.5厘米，先端漸尖，基部戟形至截形，邊緣具不規(guī)則缺刻狀的齒牙，或呈微波狀，或近于全緣，有時(shí)基部稍有深裂，兩面均有細(xì)軟毛。頭狀花序頂生，排列成傘房花序狀，頭狀花序徑約1厘米；總苞圓筒形，苞片1012片，披針形或狹橢圓形，長56毫米，寬2毫米，先端尖，無毛或 少有細(xì)毛；周圍舌狀花黃色，雌性，約8朵，長約9毫米，寬約2毫米，先端3齒裂；中央管狀花，黃色，兩性，長約6.5毫米，先端5裂。瘦果圓筒形，長約3 毫米，有細(xì)毛；冠毛長約7毫米，白色?；ㄆ?0月到翌年3月。果期25月。生于路旁及曠野間?？煞駨倪@個(gè)蛋白入手?有基因序列嗎？在蠟質(zhì)合成路徑的什么位置？測蠟質(zhì)的方法：l 3.1. Collec

6、tion of epicuticular wax from raspberry plants, sample preparation and analysis by GC and GC±MS and conduct of biosassay with aphids有做過蠟質(zhì)成分與蚜蟲的關(guān)系嗎？ Details of plant growth, sample collection, analytical instrumentation, chromatographic conditions and ana-lytical methodology for chemical analysi

7、s of the wax by GC and GC±MS, are given in the preceding paper and by Shepherd et al. (1995a).(4)ll Surface wax from the top 3 cm of bolting stems, following removal of buds, flowers and siliques, was extracted in hexane for 30 s. Total intracellular and cuticular wax components were extracted

8、from epidermal peels by immersion of epidermal peels in hexane. To all extracts, 10 g of 17:1 FAME internal standard were added and the extracts were then dried under a stream of nitrogen gas. N,Obis (trimethylsilyl) trifluoroacetamide with 1% trimethylchlorosilane was added and derivatization of sa

9、mples proceeded at 80oC for 90 min, followed by analysis on an HP6890 GC system. The GC column was an HP-5 (30 m length, 0.32 mm capillary diameter) with helium as carrier gas. The wax program used an initial temperature of 140oC, increasing at 4oC min-1 to 320oC where it remained for 10 min. Wax co

10、mponents were identified by retention times, compared to known standards內(nèi)參是否容易獲得？, and quantified based on flame ionization detector peak areas, compared to the internal standard. Wax loads were expressed per unit surface area, which was calculated from stem diameters (measured microscopically on fr

11、ee hand sections) and 3 cm length. Results were confirmed by independent GC and GC-MS analyses using the methods as outlined in R. Jetter, S.Schäffer Plant Physiol. 126, 1725 (2001)(5).Analysis of CuticularWax Composition and LoadsCuticular waxes were extracted from the leaves (200 to 1000 mg)

12、andstems (200 mg) of 4-week-old plants in chloroform for 30 s at room temperature. n-Octacosane, docosanoic acid, and 1-tricosanol were added to the extracted chloroform solvent as internal standards. The solvent was subsequently evaporated under a gentle stream of nitrogen and redissolved in a mixt

13、ure of 100 mL of pyridine and 100 mL of bis-N,N-(trimethylsilyl)trifluoroacetamide. The wax mixtures were heated at 908Cfor 30 min to convert waxes into trimethylsilyl derivatives. Qualitative and quantitative composition analyses were conducted as described previously (Lee et al., 2009a, 2009b). Th

14、e P values from each comparisonwere corrected for multiple tests using FDR control (Benjamini and Hochberg,1995).Analysis of Cutin Polyester MonomersRosette leaves of 4-week-old plants grown in soil were used to quantify cutin polyester monomers. Methyl heptadecanoate and v-pentadecalactone were add

15、ed as internal standards into the delipidated and dried leaves and then depolymerized by hydrogenolysis with LiAlH4 or by methanolysis with NaOCH3. Cutin polyesters were analyzed by gaschromatographymass spectrometry (GCMS-QP2010; Shimazu) with aHP-5 column (60 m, 0.32 mm inner diameter, film thickn

16、ess 0.1 mm;Agilent). The analysis system was maintained at 1108C. The temperature was increased to 3008C at a rate of 2.58Cmin21 and maintained at 3008C for 3 min.The MYB96 Transcription Factor Regulates Cuticular Wax Biosynthesis under Drought蠟質(zhì)成分分析：GC-MS氣象色譜-質(zhì)譜聯(lián)用技術(shù) 取第二至三片全展葉葉片，計(jì)算其表面積后，立即進(jìn)行蠟質(zhì)的提取。將葉

17、片置于室溫狀態(tài)下30mL氯仿中，30s后取出，再將處理后的葉片置于60氯仿中萃取30s，將兩次萃取的氯仿合并后，用氮吹儀吹干氯仿，稱取蠟質(zhì)質(zhì)量，計(jì)算單位面積蠟質(zhì)含量。在取樣品中加入5g正二十四烷作為內(nèi)參，然后將樣品轉(zhuǎn)入GC瓶，加入10l BSTFA和10l吡啶，70衍生1小時(shí)。液氮吹去BSTFA和吡啶。每個(gè)樣品加200l氯仿溶解。GC-MS分析。蠟質(zhì)組分用通用型VF-17MS毛細(xì)管柱，規(guī)格30m×0.25mm×0.25m,GC-MS儀為GCMS-QP2010。進(jìn)樣量1l,載氣為氦氣，柱流速為2ml/min恒流。進(jìn)樣溫度為280，50保持2min后，以40/min升溫至200

18、，保持2min；再以3/min升溫到320，該溫度保持25min。蠟質(zhì)組分可以通過離子峰出峰時(shí)間，從質(zhì)譜庫中檢索判定蠟質(zhì)成分。蠟質(zhì)含量依據(jù)峰面積與內(nèi)參比較進(jìn)行計(jì)算。單位內(nèi)蠟質(zhì)含量依據(jù)葉表蠟質(zhì)抽提面積進(jìn)行計(jì)算。(6)蠟質(zhì)合成路徑： Pathway was created on Thu Jun 2, 2011. Contributed by aracyc:Above-ground epidermal surfaces of vascular plants are covered by a lipophilic layer known as the cuticle. Plant

19、cuticles are composed of cutin (cutin biosynthesis) and cuticular wax. The major components of cuticular wax are very long chain fatty acids (chain length is greater than 18 carbon) and very long chain fatty acid derived aldehydes, alkanes, secondary alcohols, ketones, primary alcohols, and wax este

20、rs. The composition of cuticular wax varies among species and even within species in different tissues and at different developmental stages.【成分：長鏈脂肪酸，醛，烷烴，二級醇，酮，一級醇，蠟酯?！?#160; Parts of this pathway occur in:   cytosol細(xì)胞質(zhì)中     nucleus細(xì)胞核中   Legend:圖片看不清楚！Symbolsgene or metabolite

21、RNAprotein (complex)Symbol colors  cytosol  nucleusInteraction types+ regulation- regulationenzymaticcompositionother蠟質(zhì)合成相關(guān)基因：(7)(8)compounds (Aarts et al. 1995; Chen et al. 2003; Fiebig et al.2000; Hansen et al. 1997; Millar et al. 1999; Negruk et al.1996; Pruitt et al. 2000; St-Pierre et

22、 al. 1998; Toddet al. 1999; Xia et al. 1996, 1997; Xu et al. 1997; Zhanget al. 2005), whereas CER3, GL2, GL15, and WIN1/SHN1encode regulatory proteins (Aharoni et al. 2004; Broun et al.2004; Hannoufa et al. 1996; Moose and Sisco 1996; Tackeet al. 1995). Mutation in most of these genes showed altered

23、wax accumulation (Jenks et al. 2002). Co-suppression ofsome of the genes in Arabidopsis resulted in reduced wax onstems (Millar et al. 1999; Todd et al. 1999), and overexpressionof some of these genes in the Arabidopsismutants complemented the mutant phenotypes (Fiebig et al.2000; Hannoufa et al. 19

24、96). However, only a few reportsdiscuss the effect of over-expression of these genes in thewild-type background. Over-expression of the condensingenzyme gene CER6/CUT1 under the control of theCaMV35S promoter failed to promote wax deposition(Millar et al. 1999), whereas under the control of the epid

25、ermis-specific CER6 promoter, CER6/CUT1 overexpressionled to increased wax load in stems of Arabidopsis(Hooker et al. 2002). The only report on increased waxaccumulation in leaf tissues of Arabidopsis was on theover-expression of AP2/EREBP transcriptional activator(Aharoni et al. 2004; Broun et al.

26、2004). Over-expression of WXP1 under the control of the CaMV35S promoter led toincreased cuticular wax loading on the leaf surfaces, reducedwater loss, and enhanced drought tolerance of transgenicalfalfa (Zhang et al. 2005). Transgenic expression of WXP1or of its paralog WXP2 in Arabidopsis also lea

27、ds to increased wax deposition and enhanced drought tolerance(Zhang et al. 2007).Wxp類似的基因能否克隆？在蠟質(zhì)合成中參與的上下游step是什么？CER1; 一些可能是蠟質(zhì)合成的調(diào)節(jié)基因, 如CER3。CER6 是目前唯一研究得較為清楚且功能明確的蠟質(zhì)基因, 它是延長C24 超長鏈脂肪酸必需的基因。CER6 在擬南芥整個(gè)生育期都有很高的表達(dá)量, 而且僅在表皮細(xì)胞中表達(dá), 唯一例外的是將要成熟的花粉中CER6 的mRNA 是在絨氈層中表達(dá)的。一些植株CER6 的過量表達(dá)導(dǎo)致擬南芥莖表皮蠟質(zhì)含量增加, 所以可以判斷C

28、ER6 的表達(dá)水平是擬南芥莖表皮蠟質(zhì)積累的控制因素之一。而在WIN1 過表的植株中, 這些蠟質(zhì)合成基因被誘導(dǎo), 其中CER1 的變化最明顯, KCS1 和CER2 也顯著增加。(9)參考文獻(xiàn)：1.Sturaro M, et al. (2005) Cloning and characterization of GLOSSY1, a maize gene involved in cuticle membrane and wax production. Plant Physiology 138(1):478.2.向建華 (2006) 水稻 WAX2 同源基因的克隆及遺傳轉(zhuǎn)化的研究. (湖南農(nóng)業(yè)大學(xué)碩

29、士學(xué)位論文).3.Pyee J (1996) Cloning of epidermis-specific cDNAs encoding a lipid transfer protein and an aldehyde decarbonylase from Senecio odorus. Journal of Plant Biology (Korea Republic).4.Shepherd T, Robertson G, Griffiths D, & Birch A (1999) Epicuticular wax ester and triacylglycerol compositio

30、n in relation to aphid infestation and resistance in red raspberry (Rubus idaeus L.). Phytochemistry 52(7):1255-1267.5.Pighin JA, et al. (2004) Plant cuticular lipid export requires an ABC transporter. Science 306(5696):702.6.王友華 (2010) 水稻 ERF 轉(zhuǎn)錄激活子 DRF2 調(diào)控葉表蠟質(zhì)合成. (中國農(nóng)業(yè)科學(xué)院).7.Samuels L, Kunst L, &am

31、p; Jetter R (2008) Sealing plant surfaces: cuticular wax formation by epidermal cells. Plant Biology 59(1):683.8.Islam MA, Du H, Ning J, Ye H, & Xiong L (2009) Characterization of Glossy1-homologous genes in rice involved in leaf wax accumulation and drought resistance. Plant Molecular Biology 7

32、0(4):443-456.9.李法蓮, 張淼, 朱彩霞, & 邵群 (2008) 植物表皮蠟質(zhì)的研究進(jìn)展及 WIN1 對植物表皮蠟質(zhì)的影響. 現(xiàn)代農(nóng)業(yè)科技 11.10.Zhang JY, Broeckling CD, Sumner LW, & Wang ZY (2007) Heterologous expression of two Medicago truncatula putative ERF transcription factor genes, WXP1 and WXP2, in Arabidopsis led to increased leaf wax accumul

33、ation and improved drought tolerance, but differential response in freezing tolerance. Plant Molecular Biology 64(3):265-278.11.Leide J, Hildebrandt U, Reussing K, Riederer M, & Vogg G (2007) The developmental pattern of tomato fruit wax accumulation and its impact on cuticular transpiration bar

34、rier properties: Effects of a deficiency in a beta-ketoacyl-coenzyme A synthase (LeCER6). Plant Physiology 144(3):1667-1679.12.Bergman D, Dillwith J, Zarrabi A, Caddel J, & Berberet R (1991) Epicuticular lipids of alfalfa relative to its susceptibility to spotted alfalfa aphids (Homoptera: Aphid

35、idae). Environmental entomology 20(3):781-785. Here we report the functional characterization of two putative ERF transcription factor genes WXP1and its paralog WXP2 from Medicago truncatula. Transgenic expression of WXP1 and WXP2 in Arabidopsis (ecotype Columbia) led to significantly increased cuticular wax deposition on leaves of 4-week-old and 6-week-old transgenic plants, assessed based on fresh weight or based on surface area. Both WXP1 and WXP2 transgenic p