外文翻譯--亞硝酸鹽擴(kuò)增pcr引物系統(tǒng)的發(fā)展

1、<p>　　畢業(yè)設(shè)計(jì)(論文)外文資料翻譯</p><p>　　系 別： 電子信息系 </p><p>　　專 業(yè)： 自動(dòng)化 </p><p>　　班 級(jí) </p><p>　　姓 名： </p>

2、<p>　　學(xué) 號(hào)： </p><p>　　外文出處： 電子電器類外文文獻(xiàn) </p><p>　　附 件： 1. 原文； 2. 譯文 </p><p><b>　　年月</b></p><p>　　Development of PCR Primer Systems for

3、Amplification of Nitrite</p><p>　　Reductase Genes (nirK and nirS) To Detect Denitrifying</p><p>　　Bacteria in Environmental Samples</p><p>　　A system was developed for the detection

4、 of denitrifying bacteria by the amplification of specific nitrite reducetase gene fragments with PCR. Primer sequences were found for the amplification of fragments from both nitrite reductase genes (nirK and nirS) afte

5、r comparative sequence analysis. Whenever amplification was triedwiththese primers, the known nir type of denitrifying laboratory cultures could be confirmed. Likewise, the method allowed a determination of the nir type

6、of five laboratory s</p><p>　　Denitrification is a dissimilatory process of bacteria in which oxidized nitrogen compounds are used as alternative electron acceptors for energy production. The gaseous end pro

7、ducts NO, N2O, and N2 are released concomitantly. In the environment,denitrification is responsible for the release of fixed nitrogen into the atmosphere in form of N2 . It causes major nitrogen losses in agricultural so

8、ils to which fertilizers are applied. Accumulation of the greenhouse gases NO and N2O leads to the dest</p><p>　　In the present study, we report on the application of new primer systems for both types of nit

9、rite reductase genes. We used several different primer pairs to determine the nir type of denitrifying strains. Using samples from aquatic habitats, we amplified nir fragments and used the most reliable primer pairs for

10、nirK or nirS, respectively, to successfully detect, in these aquatic samples, different populations of denitrifying bacteria. </p><p>　　MATERIALS AND METHODS</p><p>　　Bacteria and growth conditi

11、ons. A variety of denitrifying and nondenitrifying bacterial strains were used to evaluate the specificity of designed PCR primers. All strains were grown aerobically at 27°C. For genomic DNA isolation, Pseudomonas,

12、 Alcaligenes, Ochrobactrum, Paracoccus, and Azospirillum strains and the denitrifying isolate IFAM 3698 were grown on nutrient broth (NB; Merck, Darmstadt, Germany). Rhizobium strains were grown on yeast extract medium .

13、 Hyphomicrobium zavarzinii IFAM ZV-622</p><p>　　Preparation of total DNA from an enrichment culture and four environmental samples. DNA was prepared from five samples. A 500-ml volume of medium 337-B1 with 0

14、.5% (wt/vol) KNO3 for the enrichment of denitrifying methylotrophic bacteria was inoculated with 100 ml of activated sludge from a sewage treatment plant near Plo¨n (Schleswig-Holstein, Germany). After 4 weeks at 28

15、°C under anaerobic conditions, 500 ml of the enrichment was again inoculated and kept under the same growth conditions. Six mo</p><p>　　PCR amplification of the nir genes. PCR amplifications from pure c

16、ultures and environmental samples were performed in a total volume of 50 ml containing 5 ml of 103 PCR buffer (500 mM KCl, 25 mM MgCl2, 200 mM Tris-HCl [pH 8.4], 0.1% Triton X-100), 200 mM each deoxyribonucleoside tripho

17、sphate, 1.0 U of Taq polymerase (5 U ml21; Appligene Oncor, Illkirch, France), 25 pmol (forgenomic DNA) or 35 pmol (for total DNA from environmental samples) of both primers (5 pmol ml21 each), and DNA (10 to 100 n</p

18、><p>　　Sequencing of amplified nir products. For DNA sequencing, amplified PCR products from pure cultures were purified with the QIAquick PCR purification kit (Qiagen, Hilden, Germany) as specified by the manu

19、facturer. DNA sequences were determined by direct sequencing of purified PCR products with the cycle-sequencing kit (GATC, Konstanz, Germany) and Thermosequenase 2.0 (Amersham, Braunschweig, Germany) as specified by the

20、manufacturers. Labeling was performed by terminating the polymerization with </p><p>　　Hybridization analysis of nir products from total DNA of environmental samples. Approximately 100 ng (pure cultures) or

21、250 ng (environmental samples) of PCR product was analyzed on an agarose gel (2%, wt/vol). After electrophoresis, the DNA was transferred onto a positively charged nylon membrane samples. Approximately 100 ng (pure cultu

22、res) or 250 ng (environmental samples) of PCR product was analyzed on an agarose gel (2%, wt/vol). After electrophoresis, the DNA was transferred onto a positiv</p><p>　　DISCUSSION</p><p>　　The

23、genetic diversity of denitrifying bacteria in environmental samples can be investigated by different molecular methods. We describe herein the first steps required to detect denitrifying bacteria in aquatic habitats by t

24、he use of two distinct PCR systems for the nitrite reductase genes, nirK and nirS. Since denitrifiers are not defined by close phylogenetic relationship, an approach involving 16S rRNA molecules is not suitable for gener

25、al detection of this physiological group in the environm</p><p>　　several authors, since this is the key enzyme in the denitrification process. Antisera against the dissimilatory nitrite reductase (dNir) fro

26、m Pseudomonas stutzeri ATCC 14405 were highly specific and reacted with the immunizing strain and few other closely related bacteria . Less specific reactions could be obtained with combinations of antisera against hemet

27、ype dNirs from P. stutzeri JM300 and P. aeruginosa . When a variety of approximately 150 denitrifying strains of uncharacterized dNir type </p><p>　　in other heme-binding proteins . By using different combin

28、ations of primers and low-temperature stringency conditions in the PCR assays, amplification of nir fragments from both genes was possible for all denitrifying strains tested. The specificity of the PCR procedure was con

29、firmed, since specific products were not obtained when the primer combinations were used with nondenitrifying strains that could perform assimilatory respiration of NO3 2 or with strains possessing the gene coding for th

30、e </p><p>　　亞硝酸鹽擴(kuò)增PCR引物系統(tǒng)的發(fā)展</p><p>　　一個(gè)特定的亞硝酸鹽還原酶基因片段的PCR擴(kuò)增反硝化細(xì)菌檢測(cè)系統(tǒng)的開發(fā)。引物序列，發(fā)現(xiàn)從兩個(gè)亞硝酸鹽還原酶基因擴(kuò)增片段的序列比較分析后（nirK基因和NIRS）。已知的近紅外型硝化實(shí)驗(yàn)室培養(yǎng)可以得到證實(shí)。同樣，該方法允許NIR五個(gè)實(shí)驗(yàn)室培養(yǎng)菌株類型的決心。通過nirK基因擴(kuò)增產(chǎn)堿桿菌。nirS基因擴(kuò)增產(chǎn)堿 和反硝

31、化隔離。對(duì)于每個(gè)的兩個(gè)基因，成功擴(kuò)增引物組合至少有一個(gè)為所有測(cè)試菌株。未取得與種細(xì)菌或其他NIR型菌株特異性擴(kuò)增產(chǎn)物。隨后測(cè)序證實(shí)擴(kuò)增產(chǎn)物的基因。這些結(jié)果表明反硝化細(xì)菌在環(huán)境樣品的定性檢測(cè)的適用性酸堿度。這是每個(gè)nir基因在本研究開發(fā)的水生生物棲息地的總DNA的一個(gè)普遍擴(kuò)增引物組合。</p><p>　　脫氮是一氧化氮化合物作為替代能源生產(chǎn)的電子受體的細(xì)菌的異化過程。隨之而來的氣體終端產(chǎn)品一氧化氮，氧化亞氮，和氮

32、氣被釋放。在環(huán)境中，反硝化是負(fù)責(zé)釋放到大氣中的氮?dú)庑纬傻墓潭ǖ?。它?huì)導(dǎo)致在肥料應(yīng)用的主要農(nóng)業(yè)土壤的氮素?fù)p失。NO和N2O的溫室氣體的積累會(huì)導(dǎo)致臭氧層的破壞。此外，反硝化細(xì)菌引起的，其中硝化耦合硝化過程從廢水中含氮化合物的去除。環(huán)境污染物的生物修復(fù)技術(shù)，可實(shí)現(xiàn)反硝化條件下。反硝化細(xì)菌的系統(tǒng)發(fā)育多樣。他們屬于腸桿菌，厭氧菌和革蘭氏陽性菌，芽孢桿菌屬以外的其他所有主要的生理團(tuán)體除外。作為一個(gè)生理群的定義，這些兼性厭氧菌可以切換由氧氣，氮氧化物

33、作為終端電子受體在缺氧條件下保存時(shí)。亞硝酸鹽還原酶的異化反硝化過程中的關(guān)鍵酶。催化亞硝酸鹽還原為NO可以由兩個(gè)不同的亞硝酸鹽還原酶基因的產(chǎn)品：一個(gè)產(chǎn)品含有銅（nirK基因產(chǎn)品），和其他含有色素CD1（NIRS產(chǎn)品）。這兩個(gè)基因似乎發(fā)生在一個(gè)給定的應(yīng)變互斥，但在同一品種的不同品系已發(fā)現(xiàn)的這兩種類型的。雖然結(jié)構(gòu)不同，這兩種酶的類型，功能和生理等效。NIRS更廣泛地分布; nirK基因中只有30％的反硝化細(xì)菌的研究迄今發(fā)現(xiàn)的。然而，nirK基

34、因被發(fā)現(xiàn)在</p><p><b>　　材料與方法</b></p><p>　　細(xì)菌的生長條件。各種反硝化和菌株被用來評(píng)估設(shè)計(jì)的PCR引物的特異性。株生長需氧27°C。假單胞菌，產(chǎn)堿菌，副球菌，固氮螺菌菌株和反硝化隔離IFAM 3698基因組DNA的提取，增加營養(yǎng)肉湯（NB;默克公司，德國達(dá)姆施塔特）。培養(yǎng)基上生長的根瘤菌株（YEM [27]）。IFAM Z

35、V-622T 337-B1中型增長0.5％（體積/體積）甲醇。紅假單胞菌FSP。脫氮胰酪胨大豆肉湯（TSB; Difco公司實(shí)驗(yàn)室，底特律，密歇根州）種植，脫氮貧營養(yǎng)培養(yǎng)基上生長（PYGV [25]）輔以25‰的人工海水，蛋白胨酵母生長脫氮沒有維生素中提取的葡萄糖培養(yǎng)基，即PYGV。腸桿菌株生長盧里亞肉湯。基因組DNA提取。溶菌酶，蛋白酶K，十二烷基硫酸鈉（SDS）的治療之后氯仿抽提和隨后的乙醇沉淀基因組DNA，獲得純培養(yǎng)。分光光度計(jì)的

36、DNA制劑的純度和濃度測(cè)定。從富集培養(yǎng)和四個(gè)環(huán)境總DNA的制備樣品。</p><p>　　準(zhǔn)備從5個(gè)樣品的DNA。</p><p>　　中等量500毫升337-B1（重量/體積）與0.5％硝酸鉀硝化甲醇的富集細(xì)菌接種活性污泥從污水處理廠附近普羅¨N（石勒蘇益格 - 荷爾斯泰因州，德國）100毫升。4周后在28°C下厭氧條件下，500毫升的濃縮再次接種和相同的生長條件下保

37、存。半年后，細(xì)胞，離心收集（6,000 3克60分鐘，在4°C）和懸浮在400毫升雙蒸水。提取DNA，用Chelex 100。</p><p>　　1.5毫升從普羅¨N（石勒蘇益格 - 荷爾斯泰因州，德國）的污水處理廠的活性污泥量為顆粒（13600 3克）在4°C 10分鐘，沉淀空氣干燥和懸浮在0.85％NaCl溶液。提取DNA，其次是一個(gè)額外的十六烷基三甲基溴化銨（CTAB法; S

38、igma Aldrich公司，Steinheim，德國）沉淀步驟去除腐殖酸和碳水化合物。</p><p>　　地表水從克萊納普羅湖（30公升）“NER教廷（德國石勒蘇益格 - 荷爾斯泰因州，于1996年4月收集）纖維素過濾器（賽多利斯，轉(zhuǎn)到¨哥廷根，德國）通過過濾去除顆粒大于100毫米然后通過玻璃纖維過濾器（孔徑3毫米; Millipore公司，貝德福德，馬薩諸塞州），細(xì)胞收集上Durapore過濾器（

39、孔徑0.22毫米; Millipore公司）。震動(dòng)過濾湖水含有0.1 mM的EDTA（孔徑0.22毫米），100毫升（100轉(zhuǎn)5小時(shí)，在4°C），細(xì)菌細(xì)胞從過濾器中刪除。收獲細(xì)胞，然后通過離心（8000 3克45分鐘，在4°C）。風(fēng)干顆粒懸浮在10毫升（5％蔗糖，50 mM的EDTA，50毫米的Tris-HCl，pH值7.6]）設(shè)置緩沖。由的Smalla建議的修改由Gliesche等方法，在細(xì)胞裂解。暫停（20分鐘2

40、20℃）凍結(jié)和解凍（5分鐘在30°C）1冰鮮丙酮體積為30分鐘，并保持在冰上。顆粒（離心后，于4000 310分鐘克）干燥，懸浮在5毫升含5毫克的溶菌酶，在37°C孵育1小時(shí)的SET緩沖。DNA提取和純化由Gliesche等方法進(jìn)行。。</p><p>　　集中由切向流過濾（31）從10公升湖水湖Plussee，石勒蘇益格 - 荷爾斯泰因州，德國于1996年8月在9米的深度收集細(xì)胞。100毫升

41、細(xì)胞懸液加入MilliQ水200毫升; Chelex 100（28）提取DNA，并與CTAB進(jìn)一步純化。</p><p>　　沉積物DNA（克萊納普羅湖“NER見;收集在1996年4月），是由與一個(gè)額外的蛋白酶K處理方法（50毫升20毫克ML21解決方案），面包車Elsas和Smalla 分離后與SDS的潛伏期。nir基因的PCR擴(kuò)增。從單純的文化和環(huán)境樣品進(jìn)行PCR擴(kuò)增量在50毫升含5 PCR緩沖

42、液103毫升（500毫米氯化鉀，氯化鎂，25毫米200毫米的Tris-HCl [pH值8.4，0.1％曲拉通X-100）的總200毫米，1.0 Taq聚合酶鈾（5üML21 Appligene Oncor，伊爾基希，法國），25 pmol（forgenomic DNA），或35 pmol的兩個(gè)引物（從環(huán)境樣品總DNA）（5 pmol ML21每個(gè)每個(gè)deoxyribonucleoside磷酸）和DNA（10至100納克）。&l

43、t;/p><p>　　變性步驟5分鐘后，在95°C時(shí)，“著陸”聚合酶鏈反應(yīng)（熱循環(huán)2400;在Perkin-Elmer，Branchburg，新澤西州）。這包括變性步驟30秒在95°C間，40秒一步的引物退火，延伸40秒，在72°C。經(jīng)過30個(gè)循環(huán)，最后7分鐘的潛伏期為72°C間進(jìn)行。在第10個(gè)循環(huán)，退火溫度降低0.5°ç每一個(gè)周期開始，在45°C

44、，直到它到達(dá)觸地得分在40°C。額外的20個(gè)周期進(jìn)行了退火溫度為43°C。電泳分析擴(kuò)增產(chǎn)物在2％（重量/體積）與溴化乙錠（0.5毫克liter21）15分鐘，染色的瓊脂糖凝膠（勃林格殷格翰公司，德國）。測(cè)序擴(kuò)增近紅外產(chǎn)品。用于DNA測(cè)序，從純培養(yǎng)擴(kuò)增PCR產(chǎn)物純化QIAquick PCR純化試劑盒（Qiagen，希爾登，德國），由生產(chǎn)廠家指定的。直接測(cè)序的PCR產(chǎn)物純化循環(huán)測(cè)序試劑盒（的GATC，康斯坦茨，德國）和T

45、hermosequenase 2.0（Amersham公司，Braunschweig，德國），由廠家指定的DNA序列測(cè)定。標(biāo)簽進(jìn)行終止與生物素標(biāo)記的dideoxynucleoside三磷酸聚合。經(jīng)過4分鐘的變性步驟94°C時(shí)，30個(gè)循環(huán)變性30秒94°</p><p><b>　　討論</b></p><p>　　可以由不同的分子生物學(xué)方法研究環(huán)境

46、樣品中的反硝化細(xì)菌的遺傳多樣性。這里，我們描述了反硝化細(xì)菌檢測(cè)水生生物棲息地，由兩個(gè)截然不同的PCR系統(tǒng)使用的亞硝酸鹽還原酶基因，nirK基因和NIRS的第一步。由于反硝化不密切的親緣關(guān)系的定義，涉及16S rRNA分子的做法是不適合這種生理組一般在環(huán)境檢測(cè)。rRNA基因定位探頭的使用已成功地應(yīng)用于迄今只有探索脫氮活性污泥法社會(huì)的菌株和特定群體。一個(gè)更通用的方法來檢測(cè)環(huán)境樣品中的所有反硝化細(xì)菌可能是生理基因或酶作為分子標(biāo)記的使用。為此，

47、亞硝酸鹽還原酶，其基因已被用于幾位作者，因?yàn)檫@是在反硝化過程中的關(guān)鍵酶。對(duì)異化亞硝酸鹽還原酶（dNir）斯氏假單胞菌ATCC 14405抗血清高度特異性和反應(yīng)與免疫接種的應(yīng)變和其他一些密切相關(guān)的細(xì)菌。那么具體的反應(yīng)，可以得到組合血清對(duì)斯氏假單胞菌JM300和綠膿桿菌（hemetype dNirs。當(dāng)多種約150的未知dNir的類型反硝化菌株對(duì)銅產(chǎn)堿cycloclastes dNir這個(gè)組合和抗血清篩選，90％的菌株可以擁有的血紅素型或銅