烟草花粉管生长信号转导研究中一种简单的RNA提取方法_简报_英文_

烟草花粉管生长信号转导研究中一种简单的RNA提取方法_简报_英文_ 第 卷 第 期42 2 Vol,4 2, No,2 分 子 细 胞 生 物 学 报 年 月 20094 April 2009Journal of Molecular Cell Biology *烟草花粉管生长信号转导研究中一种简单的 简报提取方法RNA ,, 1** 1**2 1***方华红 马兆武 余光辉 芬 李 1 中南民族大学生命科学学院生物技术国家民委重点实验室武 汉 ,, , 430074, 2 河南师范大学生命科学学院 新 乡 ， 453007, 摘要 液氮研磨是一种从具有坚韧细胞壁的植物细胞中提取 的常规方法 然而 对 于 那 种 不 RNA 。 ， 易得到的少量的植物材料如花粉 这种方法的操作 显 得 尤 为 困 难 针 对 这 一 问 题 快递公司问题件快递公司问题件货款处理关于圆的周长面积重点题型关于解方程组的题及答案关于南海问题 本 论 文 描 述 了 ， 。 ， 一种不涉及液 氮研磨的一种简单的 提 取 方 法 改变萌发花粉管的渗透压使其破裂而释放 RNA , 抽提材料的用量并且提高了 的 抽 提 效 率 用 于 抽 提 的 花这一方法减少了 RNA。 RNA RNA ， RNA 粉的量可以少 至 一 个 花 药 后 续 的 分析 定性数据统计分析pdf销售业绩分析模板建筑结构震害分析销售进度分析表京东商城竞争战略分析 需要的花粉可以少至 毫 克 这一用量相当于从 。 ， RNA 10 50个烟草花药中得到的量 这一 提取方法的可靠性和可操作性为研究花粉管生长过程中对细胞 。RNA 外信号响应的基因时空 表 关于同志近三年现实表现材料材料类招标技术评分表图表与交易pdf视力表打印pdf用图表说话 pdf 达提供了有益的参考 。 关键词 基因表达 烟草 花粉管 提取, RNA 本 文 年 月 日 收 到 年 月 日 接 受 2008 9 1 。 2009 1 10 。 * 基 金 项 目 国家自然科学基金项目 资 助 中南民族大学自然科学基金 资 助 ,,3070042,、730,600343,, ,yzz07001,。 ** 同 为 第 一 作 者 。*** 通 讯 联 系 人 ， ma, yusheen@163.com E-il A SIMPLIFIED RNA EXTRACTION METHOD TO STUDY SIGNAL TRANSDUCTION IN TOBACCO POLLEN TUBE GROWTH 1* 2 1*1**FANG Hua HongMA Zhao LI FenYU Guang Wu Hui 1,Key Laboratory for Biotechnology tof he State E thnic Affairs Commssiion， College of Li fe Scienc，e South-Central Universitfory Natonalites， Wuhan 430074, ii2College of Li fe Scencei，s Henan Normal Un iversity， Xinxiang 45300 7, Key words, gene expression. Nicotiana Tabacum. pollen tubes. RNA extraction *Both authors ocntrbuted equay to this work. ill**Corresponding author， E-mail, yusheen@163.com Pollen， the male gamet，e plays apivota l role in needed for the pollen germination and elongatio，n plant sexual reproduction. Investigation focusedthe on as well as the componentsfor the interactionof pollen , 3 -6 , transcriptome of pollen has achieved great success. and pistils are containedin the maturepollen . To Complex signal transduction networks are produced elucidate the mechanisms underlying this elegant r-e from pollen landing on the stigma of female pistil s， productivecours e， in vitro germinating culture of coursing downthe ovule and releasing spermto fulfill pollen are often employed. Specificall y， the typical ,1，2,tip-growth of pollen makesit a good model systemto needed double fertilization In the courseof the . ,7,fertilization， all the enzymes and gene transcripts decipherthe complex signal transduction networks . In vitro tobacco pollen cultureis a popular modelthat erable spacein the cultivationof the plant materials. Moreove，r collection of pollen is tedious and time - maybe adapted dueto its rapid growthin a relatively consuming.A better alternative methodis to usesmall simple culture medium. During the investigation of quantity materials to study gene expression at the signal transduction network， sthe alteration of RNA RNA level. level as an important criterion shouldbe monitored as To extractRNA with small quantitiesof tobacco to when to study pollen tube growth in response to pollen， Nicotiana tabacumcultivar Petit HavanaS R- signals .Howeve，r obtaining special extracellular th ,9, 1 flowersat 12developmental stagewere otainebd. more pollen weight to extractRNA is a bottle -neck The plants were grownin the green houseat 8h/16h problemthat hindersthe analysisof gene expression dark /light circle under 2?5 condition. At this stage， level. Traditional RNA extraction basedon grinding the pollen had developed fully into its mature state with liquid nitrogenwill exaggeratethis questiongi v- ,Fig.1-A,. Becausethe tobacco floweris biggerthan en the small weightof pollen. It is a prerequisite to that o f Arabidopsi，s the anthers can be easily col- seek a simple and convenient methodof RNA extrac- lected using forceps/tweezersThe. pollen was co v- tion. To address the difficulty in RNA extractio，n a ered within the dehiscent anthers ,Fi g.1 -B,. The methodthat entailsrapid RNA extraction from germ- i anthers were placed into a 1.5 mL centrifuge tube; nating tobacco pollen tubes in a small quantity of about 10 mg of pollen can be extractedfrom 50 to- nitrogen grinding is pollen in the absence of liquid bacco anthers ,Fig.1 -C,. describedin this paper. To minimize errorsin the experimen， t morepo l- Tobaccopollen collection lens were collected and evenly divided into equal The tiny size of the pollen makesit difficult to portions. This was done by shaking the centrifuge harvest to meet experimental quantities. Previous tubes filled with anthers up and down for several studies once used a gentle vacuum to cleaner collect ,8,Arabidopsispolle n. Howeve，r this occupied consi- dtimes， after whichthe mixture was poured onto- a pa Fig.1 Polen colecon， RNA extracon and RT-PCR deecon. lltititti A. The lognitudinal view of one fl ower of tobacco, B. The overview of one fl ower of tobacco, e llyow patches showed the poen of tobacco, C. Fowers of tobacco used ftor he coecton of poen, D. Poen obtaned, E. The discarded an ther lllllilllli septum iwthout pollen. Coins placed at the right are for s caling purposes. 期2 烟草花粉管生长信号转导研究中一种简单的 提 取 方 法RNA 175 per， thus sorting the pollen from the anther septum phases.R NA existedin the upper waterphas e， mak- ing up about 60% of the total lyses medium. Then , Fig.1 -D，E,. The remaining pollens were collected from the wall of the antherswith the use of a toottransferredh- the water phaseto a new 1.5 mL RNase free centrifuge tube.An equal volumeof 70% ethanol pick， and the anther septums were subsequentlydis- carded.Fig. 1D showsthat the weightof the collected , about 0.6 mL, was added to the water phaseand pollens from about 400 anthersis about178 mg; Fig. was shakenup and down gently. The mixture then was 1 -E shows the separated anther septum， which is transferred to the resin adhesion column ,in the collec- about 2/3 of the total weightof the anthers. The 0.5 tion tube, and spun at 10 000 rpm for 45 s. The and 1 Yuan coins indicate the relativeof size the col- flow -through was then discarded. This step was r e- lected pollenand anther septum.A total of 20 mg of peated twice as necessarydue to a greater amount of pollen is sufficient for RNA extraction. The collected medium obtained throughthe abovestep. The adhe- pollens were divided into different portions and aset- sion column was washedwith 500 μL protein remo-v side for different treatment methods investigation.and ing elusion , RE, ， and centrifugedat 12 000 rpm for Pollen gemination and RNA extraction 45 s. The flow-through was then discarded.A total of Traditional RNA extraction is needed to grind 700 μL washing medium was added and the tubes the materialsto about 100 mgin liquid nitrogenusing were spun for 1 min at 12 000 rpm. The flow-through mortar and pestle. To avoid this tedious procedur，e was also discarded. The above step waswith repeated the pollen was allowed to germinatefor about 3 h at 500 μL washing medium at the same condition and 25? in the dark. The tobaccopollen in vitro culture discardedthe flow-through. The adhesion column was , 10 , was ordinary and based on the previous mehtod . then put into the collection tube and centrifugedfor 2 The medium contained 2sucros0% ，e 1mmol/LC aCl， min at 12 000 rpm. To elute RNA off of the column， 2 1.6mmol/L HBO， and 3mmol/LMES. The pH was a total of 50 μL of RNase -free water , providedby 33 adjustedto 5.8 with 1 mol/L Tris. The culture medium the kit, was extracted using a pipette the onto col- was sterilized before use. When the tubular structure umn membrane. ,pollen tube, emerged from pollen andits length was In the extraction of RNA procedur，e the lyses seen to be longer than that of the diameter of the solutioncan be directly addedinto the bottompellet pollen， the cells were collected via centrifuge for cells of centrifuge tubes. Gentle vortices can easily subsequent RNA extraction, Fig.2 A，B,. RNA ex- break the cells. One marked featureof the tubularcell traction can be conducted according to the workflow is that the cytoplasm is mainly located at the tip of ,1，2，7,using the Bioteke R NA pure rapid superpure total the pollen tube. When transferringthe cells from RNA extractionKit , Bioteke Corporatio， nBeijing,. the cultivation mediumto the lysessolutio n， they can The workflow for RNA extraction procedureis as fol- be easily brokendue to imbalancein osmoticpre s- lows, sure. With this method， we were able to easily ex- Spin the medium for 10 min at 12 000 rpm to tract RNA from the tubular structure cells without collect the germinatingpollen tubes. A total of 1 mL performingliquid nitrogengrinding. lyses buffer was added to the tubes which were Comparasion of different RNA extraction cappedand shakenup and down for 10 min to com- methods pletely lyse the cells. A total of 0.2 mL chloroform To evaluatethe effect of RNA extractio，n sam- , trichloromethan, ewas then addedto the centrifuge ples grindedwith liquid nitrogen extractionand those tube and was capped tightly, the mixture was shaken derived by direct extractionwere compared. It was for 15 s and allowed to sit for 3 min at roomtemper -a found that 28S and 18S rRNA bands were indicative of intact RNA， compared with the liquid nitrogen ture， and then centrifuged at 12 000 rpm for 10 min at 4 ? ， the samples were then divided into three grinding procedure , Fig.2 -C， lane 1,. The results clearly show that the 28SrRNA and 18SrRNA can be electrophoresis ,Fig. 2-C， lane 3,. Howeve，r the gel extracted from the germinating pollen ,tubesFig.2- C， bright RNA band was detected whenthe pollen sam- lane 2,. Moreove，r the band of RNA stained with ples were grinded , Fig.2 -C， lane 4,. We deduced Ethidium Bromide, EB, in the latter manipulationis that fresh pollen cannotbe easily brokenin the lystic brighter than that in the former ones. Thusthe result solution during the short extraction time due to the indicates that the yield of RNA in the formerdirect protection renderedby the stiff cell wall. To confirm extraction procedureis higherthan that of liquid ni- this， we mixed20 mg fresh pollen in the lystic solu- tion and stored it at -80? overnigh，t after which trogen grinding RNA extraction. This can be due to the inevitable lossof samples during the process of RNA was extracted. The result of gel electrophoresis liquid nitrogen grinding and transferof samples. on agarose demonstrates aRNA weak band ,Fig.2C， - lane 5,. Its RNA yield approximately equaled the The effect of RNA extraction from the fresh pollen withoutprior germination basedon the method yield of one anther polle n′ s RNA which was mentioned above furtherwas evaluated. The result allowed to germinatefor 3 h ,Fig.2C， lane 6,. This shows thatno RNA band was detectedvia the agarose result in-dicate s that the frostbite broughtby low temperature A B rbcS rRNA D Fig.2 Tobacco pollen tube RNA extraction and RT-PCR detection A. Pollen grains without germination, B. Tobacco pollen tubes of pollen germination for 3h， Bar=60μm, C. Efficiency assay of RNA extraction, M indicates 1kb DNA ladder, Lane 1. RNA extracted fromPo llen tubes germination for 3h with liquid nitrogen g rinding, Lane 2. RNA extracted from Po llen tubes ge rmination for 3h without liquid nitrogen gr inding, Lane 3. RNA extracted from llpoen grains without germination without liquid nitrogen g rinding; Lane 4. RNA extracted from ll poen grains without germination but with liquid nitrogen g rinding; Lane 5. RNA extracted from one an′thes rpollen grains without germination and without liquid nitrogen g rinding, but with prior storage at -8 0? overnight in the lystic solution, Lane 6. RNA extraction from one anthe′s r pollen tubes which wasallo wed to germinate for 3h. D. RT-PCR results of MAPK gene in the pollen tubes with 28 PCR cycles. M indicates DNA marker, the molecular weights from top to bottom ar000e 2 kb， 1 000 kb， 750kb and 500 kb， respectvey. Lane 1. contro, poen grans; Lane 2. poen germnaton for 6h. rbcS was set as the illllillii internal control， the same qua ntity of rRNA was used as the template for the reverse tr anscription. 期2 烟草花粉管生长信号转导研究中一种简单的 提 取 方 法RNA 177 causedthe breakingof only a small portion of pollen this direct RNA extraction method without liquid ni- trogengrindin g， the yield of RNA will be enoughfor broken and cytoplasmicRNA release. subsequent molecular manipulation w evenith a small Key signal componen RT-PCR assay t quantityof pollen. Usingthis method， about50 μg of To assaythe effectivenessof this methodin ex- tracting RNA， the expressionof the geneof Mitogentotal RNA from 20 mg of pollen canbe obtained.For - pollen that is not easily accessibl，e the quantity of activated protein kinase , MAP K, was determined. pollen that can be used for RNA extraction can be MAPK cascade pathway is the important decoderand minimizedto 10 mg or 5 mg accordingto this method. regulatorof extracellular signalsto the transmissionto Though pollen from one anther corresponds to about nucleus.It links various extra -and intracellularsti m- ,11，12,0.45 mg and is enoughfor RNA extractio，n it is not uli to a wide rangeof cellular responses. Previous recommended to use such small quantity because of studies indicate that MAPK ,NCBI gene accession the difficulty in subsequent molecular manipulations. number X83880, also plays an important role in ,13,This methodcan be adaptedin extractingR NA from pollen germinationand pollen tube growth . In our pollens coming from other plantspecies. experiment， a two-step RT-PCR was conducted Acknowledgements dur- ing pollen tube growth to validate the This work was supportbyed the Natural Science Founda- expression of MAPK gene. The designof the primers tion of South Central University for Nationalities , yzz07001, -,14,was based on Primer 3.0 online programme. The and sponsoredby the National Natural Science Foundation of result shows that China , 30700 472,. We thank Drs. Lianghuan Qu and J ing 500 bp fragmentof the geneof MAPK was detectedat Zhao for greenhouse plant care aandintena mnce. We are deeply 6 h of cultivation , Fig.2D,. The result showed that indebted to the research center of ethniec di cmine biotechnolo- gy for t he use of the charge -coupled device , CCD, coupled this gene expression level was obviously enhanceddur- microscope. ing the cultivation process. The significant physiolog- i cal role of this geneis currently under investigation. 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