【作者】 李亚浩；

【导师】 刘大钧；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 由禾谷镰刀菌引起的小麦赤霉病是世界温暖潮湿和半潮湿地区广泛发生的一种严重性病害。该病发生后,不仅造成产量损失,而且病麦粒中含有多种危害人、畜健康的单端孢霉烯族毒素,其中脱氧血腐镰刀菌烯醇（deoxynivalenol,简称DON）危害最大,但是有关小麦抗DON遗传机制方面的研究报道较少。本研究以我国高抗赤霉病小麦品种望水白为试材,构建了DON诱导cDNA文库,并通过EST测序、半定量RT-PCR和染色体定位分析,旨在揭示DON诱导后小麦基因的表达特点,为小麦抗DON遗传育种研究提供新的信息。研究结果如下:选取抽穗后2d的望水白穗子,通过单花滴注接种DON(100mg.kg^-1),分别提取诱导24 h和48 h的RNA等量混合后,使用SMART文库构建试剂盒构建了DON诱导穗组织λ噬菌体cDNA文库。滴度测定表明,初始文库滴度为3×10⁶ pfu/mL,重组率达95%,能够代表mRNA的复杂度,扩增后的文库滴度为2×10⁹ pfu/mL;初始文库直接转入大肠杆菌BM25.8,经检测,其插入片段介于500～2000 bp,平均1053 bp。随机挑选96个克隆进行3′端测序,测序长度介于60-891 bp,平均393 bp。选取48条长度大于300 bp的EST在NCBI的GenBank中用BLAST软件进行比对后将其分成6类:（1）核糖体蛋白基因6条（13%）;（2）初级代谢与次生代谢相关基因8条（18%）;（3）DNA结合、RNA加工相关蛋白基因4条（9%）;（4）免疫或逆境相关蛋白基因5条（11%）;（5）未知功能基因9条（20%）;（6）未发现显著同源性的基因13条（29%）。根据这些序列,共设计17对新的小麦EST-PCR引物,并成功用于半定量RT-PCR扩增。以DON诱导0、6、12、24、48、72、96、120 h的望水白穗组织cDNA为模板,半定量分析发现,14个EST基因表现上调表达,1个基因表现下调表达,2个基因表达无差异。利用一套中国春缺体-四体进行染色体定位分析发现,clone1-1（具有SNF2结构域的蛋白基因）位于染色体7A,clone1-9（核糖体蛋白L9基因）位于2D,clone3-6（类金属硫蛋白基因）位于1A和1B。更多还原

【Abstract】 Fusarium head blight （FHB）, also called scab, caused mainly by Fusarium gramearwn, is a worldwide disease of wheat in the warm, semi-humid or humid regions, resulted in yield loss and deterioration of seed quality. In addition, the infected grains were usually contained with trichothecenes toxin, which is harmful to the health of human and animals. Of them, deoxynivalenol （DON） is one of the most harmful toxins. The genetic mechanism of DON resistance of wheat is unclear. In this paper, a new full cDNA library of scab-resistant wheat Wangshuibai induced by DON was constructed and some ESTs from the library were sequenced. Combined with the semi-quantitative RT-PCR and chromosome mapping, this research aimed at revealing the gene expressing patterns of wheat induced by DON and providing new information for breeding wheat with DON resistance. The results were as the following.Using 100 mg. kg^-1 DON, the spikes 2 d after heading of Wangshuibai were inoculated by single floret inoculation（SFI）. Total RNA was isolated from the spikes inoculated after 0, 6, 12, 24, 48, 72, 96 and 120 h, respectively, by TRIZOL method. Total RNA from the spikes inoculated after 24 and 48 h, were mixed equally and used to construct theλphage cDNA library as described by SMART cDNA library construction kit. The titer of the primary library was 3×10⁶ pfu/mL.The recombinant percent was 95%. The titer of the amplified library reached at 2×10⁹ pfu/mL. The primary library was directly introduced into the E. coli strain BM25.8 without amplification. The inserted fragments size of the positive clones varied from 0.5 kb to 2.0 kb with an average at 1053 bp.Ninty-six random cDNA clones were sequenced at 3’ end. The sequenced size ranged from 60 bp to 891 bp with an average of 393 bp. 48 ESTs over 300 bp were selected and analyzed by BLAST software in NCBI GenBank database. These sequences were divided into 6 groups. （1）6 ribosomal protein genes（13%）; （2）8 primary and secondary metabolism relative genes（18%）; （3） 4 DNA binding and RNA processing associated genes （9%）; （4） 5 immune associated and stress induced genes（11%）; （5） 9 unknown genes （20%）; （6）13 no BLAST hit ESTs （11%）.Seventeen pairs of EST-derived primers were developed based on these sequences. Analyzed by semi-quantitative RT-PCR using these markers, the expressing patterns of 17 EST genes at 0, 6,12, 24, 48, 72, 96 and 120 hs after inoculation with DON were revealed.14 genes were up-regulated, one gene was down-regulated and two genes were constitutive. Three EST genes were mapped on 4 chromosomes of wheat after amplified in a set of nulli-tetrasomic lines of Chinese Spring. Of them, Clonel-1（SNF2 domain-containing protein gene） was located on 7A. Clonel-9（a ribosomal protein L19 gene） was on 2D. Clone2-5, （a metallothionein-like protein gene） was on 1A and 1B.更多还原