【作者】 肖罗；

【导师】 戴良英； 谢丙炎；

【作者基本信息】 湖南农业大学 ， 植物病理学， 2008， 博士

【摘要】 FMRF酰胺类多肽（FMRFamide-like peptide,FLP）,是已知的最大和最多样的神经肽家族。FLP介导神经信号传递系统,与寄生线虫的运动和感觉功能密切相关。研究植物寄生线虫FLP,对于了解植物寄生线虫的致病机理,探讨防治植物寄生线虫新途径具有重要意义。β-1,4-葡聚糖酶的主要功能是降解纤维素,引起植物细胞形态和功能上的变化,从而提高线虫在寄主中的寄生能力。编码这些分泌物的基因被看作是寄生基因,克隆这些基因是了解线虫侵染机制的基础。因此,本研究分别从两大外来入侵的植物寄生性线虫松材线虫（Bursaphelenchusxylophilus）和相似穿孔线虫（Radopholus similis）中分离得到FMRF酰胺类多肽基因和葡聚糖酶基因,并进行了较详细的研究,主要研究如下:1.利用生物信息学筛选靶标基因的EST序列,采用RACE法从松材线虫体内获得了9个flp基因的cDNA全长和一个cDNA片段（Bx-flp-3b）,并进行深入分析。这9个flp基因分别是Bx-flp-1,Bx-flp-2,Bx-flp-3a,Bx-flp-6a,Bx-flp-6b,Bx-flp-6c,Bx-flp-6d,Bx-flp-12,Bx-flp-14,其登陆号分别为EU026161,EU930826,EF422867,FJ151415,FJ151416,FJ151417,FJ151418,EF422868,EF622046。从基因结构上分析发现Bx-flp-1由4个外显子和3个内含子组成:Bx-flp-3a由2个外显子和一个内含子组成;Bx-flp-6基因发生两种不同的剪辑方式,剪切方式分别发生在中间和C端,产生了4种不同的选择性剪切异构体。用Tail-PCR的方法分别得到3个基因的5’侧翼序列,大小分别为857bp、377bp和1630bp。2.利用原位杂交的方法将6个flp基因进行定位,发现Bx-flp-1和Bx-flp-3a主要在松材线虫的咽坏和头部表达,Bx-flp-2,Bx-flp-3b,Bx-flp-6a～d,Bx-flp-14主要在松材线虫的尾部表达;而Bx-flp-12的表达模式比较复杂。3.体外合成Bx-flp基因片段的dsRNA,利用浸泡法处理松材线虫,对其进行离体干扰效应验证。QPCR分析表明干扰后的靶标基因Bx-flp-1,Bx-flp-3a,Bx-flp-6转录水平明显降低,只达到对照的22%、41%、29%,达到差异显著（P＜0.05）。显微镜观测干扰后线虫活动能力明显下降,呈现僵直和假死状态。但是,40℃放置1hr后,僵直的线虫又能恢复活动能力。繁殖能力检测发现,干扰松材线虫Bx-flp-1,Bx-flp-3a,Bx-flp-6不能抑制其繁殖。4.用RT-PCR和RACE方法,获得了β-1,4-内切葡聚糖酶基因的cDNA全长,并将该基因命名为Rs-eng-1（GenBank登录号为EU414839）。此cDNA全长序列为1630bp,包括1个1404bp的完整ORF,编码含467个氨基酸的蛋白,其理论分子量与等电点分别为48.22KD,pI为6.04。序列分析表明含有糖基水解酶GHF5的保守结构域,N端具有22个氨基酸残基组成的信号肽,C端含有细菌式样的纤维素结合域（CBDⅡ）。原位杂交结果显示此基因由穿孔线虫的食道腺分泌。Southern分析为多拷贝基因。cDNA与基因组DNA重叠分析表明,此基因包含6个内含子,切割点符合5‘-GT...AG-3’的规律。进化分析表明,与细菌Bacillus subtilis和Erwina carotovora分泌的纤维素酶属于同一支,推测其来源于细菌的水平基因转移。5.分别构建了pET-28（a）-Rs-eng-1,pET-42（a）- Rs-eng-1,pGEX-6P-1-Rs-eng-1原核表达载体,转化大肠杆菌BL21,诱导表达,没有发现特异条带。引入EcoRⅠ和pstⅠ两酶切位点构建表达载体,获得表达菌株pMAL-c2X-Rs-eng-1（BL21）,诱导表达后出现包涵体。因为包涵体没有活性,故重新构建了酵母表达载体pPIC9K-Rs-eng-1,电击转化酵母细胞,利用甲醇诱导,发现Rs-eng-1在酵母中大量表达并分泌到胞外,SDS-PAGE检测表明表达蛋白的表观分子量约为60kD。并且表达的蛋白具有降解纤维素的活性。6.离体RNAi香蕉穿孔线虫Rs-eng-1基因。QPCR检测发现Rs-eng-1基因的mRNA丰度显著下降,其表达量只有对照的15%。纤维素酶活性测定结果显示,经dsRNA处理的线虫纤维素酶分解圈平均值为17cm,而对照为22cm,达到显著差异。繁殖率测定,发现RNAi能抑制香蕉穿孔线虫的繁殖,使其生育滞后。分离RNAi处理后接种的香蕉穿孔线虫,发现雄虫比例上升。7.通过植物介导沉默靶标基因对线虫进行RNAi活体效应研究。将Rs-eng-1基因片段分别正向和反向插入到载体中,构建了目的基因的dsRNA干扰植物表达载体pFGC5941-RSENG-RNAi。采用农杆菌介导法转化番茄,获得了dsRNA干扰表达载体的转基因番茄。对转基因番茄进行PCR扩增检测和Southern分析,证明外源基因已成功整合到植物基因组中,RT-PCR验证外源基因高效表达。将香蕉穿孔线虫接种到T₁代植株,45d后发现转基因植株的根系中香蕉穿孔线虫侵染的数目明显少于对照,并且干扰效果优于离体RNAi。将根结线虫2龄幼虫接种到T₁代植株,25d后检测根结数,发现表达Rs-eng-1基因dsRNA的番茄根结数明显减少,表明转基因番茄具有高效的抗线虫作用。更多还原

【Abstract】 FMFRamide-like peptides（FLPs） occur widely across the phylum Nematoda and FLPs involved neurotransmission plays a central role in plant nematode biology.FLPs appear integral to how these parasites move,feed,sense their environment and perhaps establish parasitism within the plant.Therefore,extensive study of FLPs genes and their functions will greatly improve our understanding of the nematode parasitism and might lead to novel and efficient ways in plant-parasitic nematodes control,β-1, 4-endoglucanase is a large class of cellulases secreted by parasitic nematodes that can degrade cellulose to alter the structure and function of the plant cells.The encoding genes of these secretions,so called parasitism genes,have been thought to greatly enhance the parasitism of the nematodes in the plant.Our study isolated and analyzed the full-length cDNA of flp and ENG genes from two alien invasive plant-parasitic nematodes Bursaphelenchus xylophilus and Radopholus similis,respectively.The study provided possible targets for anti- plant-parasitic nematodes drugs.The main results were as follows:1.Based on the EST sequences of the target genes from the gene databases.9 full-length cDNA and 1 cDNA fragment of flp genes（Bx-flp-3b） from B.xylophilus were RACE amplified and analyzed.The 9 obtained flp genes are designated Bx-flp-1, Bx-flp-2,Bx-flp-3a,Bx-flp-6a,Bx-flp-6b,Bx-flp-6c,Bx-flp-6d,Bx-flp-12,Bx-flp-14, respectively,with Genebank access number EU026161,EU930826,EF422867, FJ151415,FJ151416,FJ151417,FJ151418,EF622046,respectively.According to the gene structure analysis,Bx-flp-1 was composed of four 4 exons and 3 introns;Bx-flp-3a was composed of 2 exons and 1 intron;Bx-flp-6 had two different editing mechanisms, one in middle and the other in the C terminus,resulting 4 different selectively edited isomeric products.The 5 flanking sequences of three genes were also obtained by Tail-PCR,with length of 857 bp,377 bp,and 1630 bp,respectively.2.Six flp genes from B.xylophilus were localized by in-stiu hybridization and the result revealed that Bx-flp-1 and Bx-flp-3a were expressed in the pharyngeal ring and the head;Bx-flp-2,Bx-flp-3b,Bx-flp-6a～d and Bx-flp-14 were expressed mainly in the tail;the expression patter of Bx-flp-12 was very complicated. 3.RNA interference（RNAi） experiments were performed to study the functions of the flp genes,dsRNA fragments were synthesized in vitro and the B.xylophilus nematodes were treated by soaking.Q-PCR was used to compare the differences between the treated and control groups.The results showed that the transcription level of the target genes with treatment was significantly reduced（p＜0.05%） to 22%,41% and 29%,respectively.The microscopic observations showed that the mobility of the interfered nematodes was remarkably decreased,appearing to be in rigor and apparent death.However,after incubation at 40℃for 1 h,the mobility could be recovered.The reproductive capability assay demonstrated that the interference of Bx-flp-1,Bx-flp-3a and Bx-flp-6 could not inhibit the reproduction rate.4.The full length cDNA sequence ofβ-1,4-endoglucanase gene was obtained by RT-PCR and RACE from R.similes,named Rs-eng-1（GenBank:EU414839）.It consisted of 1630 bp,with a 1404 bp ORF encoding a 467 amino acid protein.The putative protein,with theoretical molecular weight 48.22 kDa and pI 6.04,was classified as a member of cellulase（glycosyl hydrolase family 5）,and had a 22 amino acids long singal sequence at N terminus and a high similarity to a bacterial type cellulose-binding domainⅡat C terminus.Southern blot analysis showed that there were mμLtiple copies of Rs-eng-1 within R.simile genome.In situ hybridization showed RS-ENG-1 was secreted from R.simile’s esophageal gland cells.Genomic analysis suggested Rs-eng-1 contained six introns demarcated by 5’-GT...AG-3’ in the splicing sites and phylogenic analysis suggested that Rs-eng-1 had a strong homology to Bacillus.subtilis and Erwina carotovora,which might be indicative of an ancient horizontal gene transfer.5.Prokaryotic expression system was tried first to express Rs-eng-1.Rs-eng-1 was sub-cloned into vector pET28a,pET42a and pGEX-6p-1,respectively,using EcoRⅠand NotⅠrestriction sites.After transformed into E.coli BL21 component cells and induced by IPTG,no specific bands were observed on SDS-PAGE gels.A different expression vector pMAL-c2x/Rs-eng-1 was thus constructed using EcoRⅠand PstⅠrestriction sites and transformed into E.coli BL21 component cells.After induced by IPTG,it started to produce inclusion bodies based on SDS-PAGE analysis.The failure of the above trials suggested that Rs-eng-1 coμLd not be expressed in prokaryotic expression systems.Considering the eukaryotic origin of Rs-eng-1,a eukaryotic expression vector pPIC9K/Rs-eng-1 was constructed and transformed into yeast cells by electroporation.After induced by methanol,Rs-eng-1 had a large amount of extracellular expression and showed an expected apparent molecular size of 60 kD on SDS-PAGE gel.The expressed Rs-eng-1 also showed cellulase activity.6.The function of Rs-eng-1 was also analyzed by RNAi experiments.Q-PCR resμLts demonstrated that the mRNA abundance of Rs-eng-1 was significantly decreased,only 15%compared to the control.CellμLase activity assays showed that the average size of the decomposition circles of the treated nematodes was 17 cm, significantly different from that of 22 cm of the control nematodes.The reproductive capability assay suggested RNAi of Rs-eng-1 could defer the reproduction of R.similis and increase the proportion of the male nematodes.7.Plant expressing vector of Rs-eng-1 fragment was successfully constructed and applied to tomatoes transformation.We obtained many hygromycin-resistant transgenic plants expressing dsRNA by Agrobacterium tumefaciens EHA105 mediated transformation with plasmid pFGC5941-RSENG-RNAi.PCR analysis showed that transgenic plants could amplify the target gene fragment,and southern blot indicated that the Rs-eng-1 fragment was integrated into the genomes of the tomato plants. RT-PCR showed that the target gene was effectively expressed in tomato plants. Compared with the control group,the number of root knots in transgenic tomatoes was significantly less 25 dpi when J2 were inoculated to T₁ plants,suggesting that the transgenic plants exhibited strong resistance to J2.The method described here provided a new way for the control and prevention of plant-parasitic nematodes.更多还原