三裂葉豚草銹菌論文：三裂葉豚草銹菌侵染機(jī)制DNA提取基因變異

1、三裂葉豚草銹菌論文：三裂葉豚草銹菌侵染機(jī)制 DNA 提取基 因變異【中文摘要】三裂葉豚草是重要的外來入侵生物，如何防治該雜 草已成為雜草學(xué)家和社會普遍關(guān)注的問題。利用專性寄生病菌是防治 三裂葉豚草的有效途徑。蒼耳柄銹菌三裂葉豚草?；?Pucci niaxan thii Schwei n. f.sp. ambrosiae-trifidae S.W.T. Batra)對三裂葉豚草表現(xiàn)出較好的生防潛能。為此，本論文對該銹菌的侵染機(jī)制 及在遼寧省的種群變異進(jìn)行了系統(tǒng)研究，為利用該菌防治三裂葉豚草 奠定理論基礎(chǔ)。本論文主要取得了如下三方面的研究結(jié)果：(1)利用人 工接種結(jié)合考馬斯亮藍(lán)染色技術(shù)研究蒼耳柄

2、銹菌三裂葉豚草?；?冬孢子萌發(fā)，擔(dān)孢子形成以及侵染過程。侵染結(jié)果表明，該銹菌的侵染 過程可以分為冬孢子的萌發(fā)、擔(dān)孢子的形成、侵染前、發(fā)育、侵染和 侵染后在寄主內(nèi)擴(kuò)展、冬孢子堆形成等階段。(2)比較研究了該銹菌 冬孢子 DNA 提取的 9種方法,其中 CTAB 鋼珠法、改良的微型電鉆法 以及 EZ-Kit 改良法獲得的基因組 DNA 經(jīng)檢測質(zhì)量較好。在此基礎(chǔ)上，利 I用 ITS-PCR 禾口 ISSR弓 I 物 UBC#835 各待用 DNA 進(jìn)行 PCF 擴(kuò)增檢測。 結(jié)果表明，上述 3 種方法提取的 DNA 適合于后續(xù)分子生物學(xué)要求。(3) 以 ISSR 和 SRAP 遺傳標(biāo)記方法對遼寧省

3、16 個銹菌群體進(jìn)行了遺傳多 樣性研究,11個 ISSR 引物和 13對 SRAP 弓物擴(kuò)增出多態(tài)位點(diǎn)百分?jǐn)?shù) 分別為 72.62%和 71.05%結(jié)果表明，在群體水平上 ISSR 法與 SRAP 法 獲得的基因多樣性指數(shù)為 0.0028 0.3933 和 0.2991 0.3869,Shannon s 信息指數(shù)為 0. 0047 0. 5703 和 0.4445 0.4750,Nei s 分析結(jié)果顯示 ISSR 和 SRAP 法的獲得的種群間的遺傳 分化系數(shù)(Gst)為 0.2537和 0.2330,分子方差分析(AMOVA顯示種群 間的遺傳變異占總變異的 5.70%和 4.60%,種群內(nèi)變異

4、占總變異的 94.30%和95.40%,表明銹菌在不同地理種群間存在較低水平的遺傳變 異，而在種群內(nèi)部存在廣泛的基因多樣性。通過UPGM 聚類圖顯示,SRAP 標(biāo)記分析結(jié)果與兩種標(biāo)記復(fù)合聚類分析的結(jié)果相近，說明SRAP 在銹菌的遺傳多樣性和親緣關(guān)系聚類分析中可能具有更高的可 信度。【英文摘要】 Ragweeds (Ambrosia L.) are alie n in vasive speciesfor China. Howto control the grasses has been closely noticed by weedscientists and public. It appear

5、s that it is an effective way to con trol thegrasses by absolute parasitic pathogens. Puccinia xanthii f. sp.ambrosia-trifidae S. W. T. Batra was a microcyclic rust fun gus forbiological con trol of gia nt ragweed, Ambrosia trifida. Thereafter thepaper is made to investigate its infection process an

6、d genetic variation inorder to provide a theoretical base for bioc on trol of the grasses by thefun gus. The paper con ducted the followi ng research aspects:(1) Thestudy illustrated the in fecti on process of P. xanthii f.sp.ambrosiae-trifidae by means of light microscopy associated withCoomassie B

7、rillia nt BlueR-250 sta ining method. The in fect ing process was divided intothe followi ng phases: teliospore germ in atio n, basidiospore formatio n,prepe netrati on developme nt, pen etrati on and postpenetrationdevelopment. The typicalinfectionstructuresin cluded germ tube, appressorium, pen et

8、ratio n peg, in traepidermalvesicle, primary hypha, in tracellular hyphae, M-haustorium (Chapterone).(2) The genomic DNA of P. xanthii f. sp. ambrosiae-trifidae wasextracted by nine methods from the teliospores of the rust fun gus. Theresults show that the methods of CTAB-steel balls, modified mini-

9、drill andmodified EZ-Kit were better than the other six methods due to their higherquality of DNA extraction with less impurity. The template DNAsamplesgen erated from these methods were detected by methods ofITS-PCRand ISSR-PCRwith the primer UBC#835.They all producedexpected band patter ns. The re

10、search con firms that the DNAsamplesgen erated from these three methods could be used for further molecularstudy of the rust fungus (Chapter two).(3) The gen etic diversity with inand among 16 populatio ns of P.xanthii f.sp. ambrosiae-trifidae from Liaoning province was inv estigatedusing ISSR and SRAP molecular markers. The perce ntage