【作者】 郑雅楠；

【导师】 段玉玺；

【作者基本信息】 沈阳农业大学 ， 有害生物与环境安全， 2009， 博士

【摘要】 大豆胞囊线虫病是一种为害大豆生产的重要病害，其病原物大豆胞囊线虫（Heterodera glycines，Ichinohe，1952；Soybean Cyst Nematode SCN）是一类进化程度较高的植物寄生线虫，由于其生活史循环中具有“胞囊期”这个特殊的休眠阶段，使其对不良环境条件具有较高的抗性，特别能够适应冬季的低温。本文针对在大豆生产中SCN常遇的逆境因子，研究了不同胁迫条件对SCN的影响，以期从生理生化角度和分子角度揭示SCN抗逆境胁迫的机制，主要研究内容如下：1．通过测定大量和微量元素化合物在寄主体外对大豆胞囊线虫二龄幼虫（J2）存活、卵孵化的影响，明确了不同化合物对大豆胞囊线虫的抑制作用。其中KH₂PO₄、（NH₄）₂HPO₄和FeCl₃·6H₂O分别为大量元素化合物和微量元素化合物中对大豆胞囊线虫J2存活抑制作用较强的化合物。尤其是盆栽条件下，FeCl₃·6H₂O对大豆胞囊线虫繁殖有很强的抑制作用，（NH₄）₂HPO₄和FeCl₃·6H₂O对大豆根的生长有明显的促进作用。在对J2作用的LC50浓度下，绝大多数化合物对胞囊卵孵化具有很强的抑制作用。2．通过测定大豆田常用的8种除草剂对SCN在寄主体外存活和孵化的影响。得到了SCN敏感的除草剂为乙草胺和2,4-滴丁酯+异丙草胺，在田间喷施浓度下二者分别对SCNJ2的存活和卵孵化的抑制作用最强。进而测定了温室和田间乙草胺和2,4-滴丁酯+异丙草胺对SCN在大豆上繁殖的影响，证实了这两种除草剂对SCN的繁殖具有较强的抑制作用。且乙草胺和2,4-滴丁酯+异丙草胺对大豆生长均无不良影响。3．通过测定不同pH值条件下SCN J2的死亡率，明确了SCN J2对酸性和碱性环境均有很强的适应性。测定了温度、pH值、光照和土壤含水量对休眠SCN的影响，结果证明pH 9．0 KOH、pH 5．0H₂SO₄和pH 6．0 H₂MoO₄均能促进SCN卵的孵化。此外，SCN J2对低温和高温逆境均有一定程度的抗性，尤其是对低温有较强的适应能力，并且低温和高温锻炼能够显著地增强SCN J2对低温和高温胁迫的抗性。通过分析海藻糖与SCN耐低温、高温的关系，得出SCN体内海藻糖含量与其对低温的适应性显著相关，而与其耐热性无显著关系。在一定的范围内，温度的升高有助于SCN休眠的解除，且能够提高非休眠卵的孵化率；增加光照时间有助于SCN卵的孵化和打破休眠；在寄主体外，15％的土壤含水量最适合SCN卵孵化。4．首次对不同方法提取大豆胞囊线虫不同虫态基因组DNA进行了比较，结果表明，不同虫态的样品中以白色雌成虫制备的DNA质量较好，而以不同方法和不同虫态提取的DNA为模板进行扩增，其质量均不影响已知片段的扩增结果。采用基因组步移技术，从SCN全基因组中得到了SCN热激蛋白70（Hsp70）基因的DNA序列，长度为2841bp；再通过RT-PCR得到了长度为1950bp的SCN Hsp70基因（Hg-Hsp-70）编码区，其GeneBank登录号为FJ816100。根据已知序列设计引物，克隆到SCN热激蛋白90（Hsp90）的DNA序列，长3003bp；通过RT-PCR得到了长度为2163bp的SCN Hsp90基因（Hg-Hsp-90）全长编码区，二者的GeneBank登录号分别为FJ985783和FJ985784。5．利用pEASY-E1成功构建了SCN Hsp70和Hsp90的原核表达载体Hg70pEASY-E1和Hg90pEASY-E1并在大肠杆菌Transetta（DE3）中成功地进行了表达。以pET-30为载体，实现了SCN Hsp70的非融合表达；经Western blot检测，验证了原核表达获得的Hg-Hsp-70和Hg-Hsp-90蛋白确实属于Hsp蛋白家族（Hsps）。6．以actin基因作为内参照基因，采用半定量RT-PCR技术，对温度和化学胁迫前后，SCN体内Hsp70和Hsp90基因表达的差异进行了检测。结果表明，SCN Hsp70基因的过表达确为SCN耐胁迫的重要机制之一。SCN受到温度和化学胁迫后，体内的Hg-Hsp-70和Hg-Hsp-90基因的表达量均有不同程度的增加，但经高温胁迫后基因表达量的上升幅度高于低温胁迫后表达量的上升幅度；在绝大部分胁迫处理中，SCNHg-Hsp-90基因表达量的增幅都低于Hg-Hsp-7D基因。利用显微镜对虫体进行观察发现，SCN受到化学胁迫后，体壁发生不同程度的缢缩现象，有的胁迫处理后虫体头部口针明显突出体外。酶标仪对海藻糖和海藻糖酶测定的结果显示，化学胁迫使SCN体内海藻糖含量上升，且随着胁迫时间的增加，海藻糖含量呈先上升后下降的趋势；海藻糖酶活性随着胁迫时间的增加而升高。说明在化学胁迫下，SCN自身通过海藻糖的合成和分解对机体进行调控，以适应或抵抗逆境胁迫。7．通过测定不同休眠阶段SCN体内生理生化物质的含量，明确了休眠期SCN体内物质变化的动态规律。即SCN休眠期间的糖醇积累型属海藻糖积累型，同时总糖、糖原和可溶性蛋白也参与休眠SCN体内物质代谢，为SCN休眠期消耗提供了能量，甘油与SCN休眠及抗寒能力密切相关。证实了SCN休眠程度越深，体内氨基酸总含量越多，休眠程度浅或处于活动状态时，氨基酸含量较少。游离亮氨酸、组氨酸和异亮氨酸与休眠过程中的生命活动有一定的关系，且水解苏氨酸、丝氨酸、谷氨酸、甘氨酸和丙氨酸在SCN休眠过程中与氨基酸的代谢有关。SDS-PAGE电泳结果显示，在各个休眠阶段SCN全蛋白种类无明显变化。海藻糖酶、糖原磷酸化酶（Gpase）、山梨醇脱氢酶（NAD-SDH）、磷酸果糖激酶（PFK）、丙酮酸激酶（PK）和果糖1-6-二磷酸羧醛酶（FBP）均与SCN休眠密切相关，进入休眠SCN体内海藻糖酶活性迅速升高，Gpase活性也显著升高，有利于海藻糖所需的碳源的供给，而PFK、PK、FBP和NAD-SDH四种酶活性均显著降低，休眠解除后活性回升。更多还原

【Abstract】 Soybean cyst nematode （Heterodera glycines Ichinohe, 1952, SCN） is one of the mostdestructive pest of soybean. Because of the special stage-dormancy during its life-history,SCN can overcome stress environment, especially long cold winter. Experiencing longperiods of evolution, SCN is able to adapt to various environments and conditions. Westudied on effects of adverse environmental factors, which were common in soybeancropping, on SCN. In order to reveal the anti-stress mechanism of SCN, many physiologicaland biochemical experiments had been done, and the main results were summarized asfollow:1. The macroelement and microelement compounds were tested against the SCN, in vitroand in vivo on soybean. In vitro, KH₂PO₄ and （NH₄）₂HPO₄ were better treatments forreducing the number of surviving J2 and achieved the lowest value of LC50 of macroelementtreatments. Among microelement treatments, FeCl₃·6H₂O was the best treatment inhibiting J2survival with the lowest value of LC50. In glasshouse, FeCl₃·6H₂O suppressed SCNreproduction obviously and achieved the highest percentage of nematode inhibition. In thefield, all the tested compounds inhibited reproduction of the first generation of SCN, but hadno effect on the second generation. All treatments improved growth of root, and applicationof （NH₄）₂HPO₄ and FeCl₃·6H₂O gave better root growth （dry and fresh weighs） of soybean.2. The effects of eight kinds of herbicides were tested on survival of SCN J2 and eggshatching in the lab. Results showed that acetochlor and 2,4-D+propisochlor were effectiveherbicides on SCN. Mortality of J2 treated with acetochlor reached to the highest of all testedherbicides, and 2,4-D butyl ester+propisochlor was the best treatment to inhibit SCN eggshatching. Experiments in the glasshouse and in the fields showed that acetochlor and2,4-D+propisochlor significantly inhibited reproduction of SCN. These two herbicides had noharmful effect on growth of roots.3. The influence of different pH conditions on mortality of SCN J2 were tested, the resultsshowed that SCN could adapt to wide pH range. We also tested effects of temperature, pHvalue, light and soil water content on dormant SCN. The results showed that pH 9.0 KOH,pH 5.0 H₂SO₄ and pH 6.0 H₂MoO₄ improved eggs hatching of SCN. In addition, SCN J2 hadresistance to low and high temperature, especially to low temperature. Cold and thermalacclimation could improve the cold and thermal tolerant. By analyse the relationship between trehalose concentration in SCN and adverse temperature tolerance, a positive correlationbetween cold tolerance was observed. But we didn’t found the obvious correlation betweentrehalose content and thermal tolerance. Eggs hatching rate was improved with increasing oftemperature among 18℃-30℃, higher temperature and long illumination were help to breakdormancy of SCN. In vitro, 15% soil water content was proper to eggs hatching of SCN.4. DNA were extracted from different stages of SCN by means of different methods. Theelectrophoresis results showed that DNA extracted from female adult was better than thatfrom other tested state. As PCR amplification template, DNA extracted from different SCNstate by different tested methods, had no effect on PCR results. DNA sequence of SCN heatshock protein （Hsp70） gene, which length was 2841bp, was obtained from SCN total DNAgenome by genome walking technique. Based on the known 2841bp sequence, SCN Hsp70gene （Hg-Hsp-70） CDS sequence was obtained by RT-PCR, the length was 1950bp and theGenBank accession number was FJ816100. Based on the known SCN heat shock protein（Hsp90） gene sequence, the 3003bp SCN Hsp90 gene （Hg-Hsp-90） sequence was obtained,which GenBank accession number was FJ985783. And the gene CDS was isolated from SCNRNA by RT-PCR. The total length of Hg-Hsp-90 CDS was 2166bp, and the GenBankaccession number was FJ985784.5. Prokaryotic expression vectors of SCN Hsp70 and Hsp90 were construsted bypEASY-E1 and successfully expressed in E.coli. Tansetta （DE3）, were namedHg70pEASY-E1 and Hg90pEASY-E1, respectively. And the Hsp70 was also expressed innon-fusion expression system built by vector pET-30. Both expressed Hg-Hsp-70 andHg-Hsp-90 were identified by western blot, the results showed that target proteins wasexpressed successfully.6. With actin gene as the inner control, the expression of Hg-Hsp-70 and Hg-Hsp-90 inSCN suffering from temperature stress and chemical stress were detected bysemi-quantitative RT-PCR. The results showed that over-expression of Hg-Hsp-70 was one ofimportant stress-tolerance mechanism. Expression quantity of both Hg-Hsp-70 andHg-Hsp-90 increased under stresses. The level of these gene-express increasing of SCNstressed by higher temperature was higher than that of SCN stressed by low temperature. Inmost stress treatments, the degree of increaseing of Hg-Hsp-90 expression was lower thanthat of Hg-Hsp-70. By means of microscopic observation, we found the body wall ofchemical-stressed SCN shrinked, and stylets of some stressed SCN breaked through the body obviously. Trehalose content and trehalase activity of stressed and unstressed SCN wereassayed by enzyme-labeled instrument. The results showed that both of trehalose content andtrehalase activity increased after stressed by chemiscals, and trehalose content changedregularily with increasing of treated time, suggesting that SCN adapted to the environmentalstress and resisted to stress with regulating synthesis and decomposition of trehalose.7. The dynamic regulation of metabolites was determined by assaying the physiological andbiochemical substances content of dormant SCN. The results showed that trehalose wasaccumulated during the dormancy period, and total sugar, glycogen and soluble proteinparticipated in metabolism of SCN, providing energy for dormant SCN. Glycerol content wasclosely related to cold tolerance of SCN. Data of amino acid showed that total amino acidscontent increased with the enhance of SCN dormant degree, while the amino acids content ofactive SCN was lower than that of dormant SCN. Contents of free leucine, histidine andisoleucine were related to life activities of dormant SCN, and hydrolysis threonine, serine,glutamate, glycine and alanine were related to metabolism of amino acids. SDS-PAGE resultsshowed that the kinds of total protein did not change between dormant SCN and active SCN.The activity of trehalase, Gpase、PFK、PK、FBP and NAD-SDH during SCN dormant periodwere assayed, the results showed that the activity of trehalase and Gpase activity significantlyincreased when SCN enter into dormant stage. But the activity of PFK、PK、FBP andNAD-SDH in dormant SCN were obviously lower than active SCN.更多还原