【作者】 张海燕；

【导师】 王刚；

【作者基本信息】 河南大学 ， 微生物学， 2011， 硕士

【摘要】 植物内生细菌是指能定殖于健康植物组织内部,并与寄主植物建立和谐联合关系的一类微生物。植物内生细菌具有促进植物生长,提高植物抗逆性等能力。目前,植物内生细菌的研究现已成为国内外研究的一大热点。利用小麦内生细菌防治植物土传病害逐渐以安全、有效等优点得到各国植病学家的广泛关注。生防菌生防机理的研究有助于开发稳定、高效、易保存的生防制剂,为大田应用奠定理论基础。分离生防相关基因,从分子水平上研究基因的功能,是阐述生防菌的生防机理的重要途径。本研究中所用的小麦全蚀病内生生防菌Serratia proteamaculans 336X是由本实验室从小麦根部分离得到的革兰氏阴性细菌,对小麦全蚀病的防效可达85%。内生生防菌336X具有产几丁质酶、蛋白酶等病原菌细胞壁降解酶的能力,也可以通过产生N-乙酰-高丝氨酸内酯(AHL)群体感应信号分子形成生物薄膜、铁载体、诱导系统抗性等途径发挥生防作用,并且能够在小麦根部高效定殖,具有运动性和趋化性等生防相关特征。为了研究内生生防菌336X的生防相关基因的功能,从而深入了解其发挥生防作用的分子机制,本研究从两个方向出发,一方面通过利用果聚糖蔗糖酶编码基因sacB表达时336X菌株对蔗糖的敏感性作为反向选择标记,构建小麦内生生防菌336X的环状载体遗传敲除体系,对与内生菌336X发挥生防作用相关的鞭毛运动性基因motA进行基因敲除,最后得到了4个motA基因的敲除突变株。同时,利用该方法对与336X菌株中趋化性相关基因CheW进行基因敲除,最后得到了3个CheW基因的敲除突变株,为该体系的可行性提供了有力的证据。另一方面,利用Red重组系统构建了小麦内生生防菌336X的线性载体遗传敲除体系。首先对内生生防菌336X进行了遗传改造,得到了工程菌336X-1,使得线性载体在336X-1中能够发生同源重组,并对工程菌336X-1进行了温度敏感性测定。进一步构建了内生菌336X产几丁质酶相关基因chiA的线性打靶基因片段C1-Km-C1,通过电击转化的方法将以上线性打靶基因片段导入到工程菌336X-1中,得到了2个几丁质酶A基因的敲除突变株。同时,构建了内生菌336X假定的植酸酶基因的线性打靶基因片段phy-Km-phy,利用该方法进行基因敲除,最后得到了4个phy因的敲除突变株,为该体系的可行性提供了有力的证据。最后,对两种敲除体系进行了比较,分析了它们各自的优缺点。以上研究为研究内生生防菌336X的生防相关基因的功能,进一步认识其生防作用的分子机制奠定了理论基础。更多还原

【Abstract】 Endophytic bacteria refers to a kind of microbial that can colonise in the healthy plant tissue and establish the harmonious relations with host plant. Endophytic bacteria can promote the growth of plants and improve plants resistance, etc. At present, Endophytic bacteria research has become a hot spot of research at home and abroad. Using endophytic bacteria on wheat to control plant soil-borne diseases, which is safe, effective etc, has become the various countries’ phytopathologists research hot spot.Studying on biocontrol mechanism of biocontrol strain found the theoretical basis of producting a stable, efficient and easy to save biocontrol agent and applying to fields. Separating biocontrol genes which is related to biocontrol and researching the functions of genes at the molecular level is an important way to find out biocontrol mechanism of biocontrol strain.Serratia proteamaculans 336X used in this research is Gram-negative bacteria separated from wheat root in our laboratory.It is a biological control agent against take-all disease of wheat. Biocontrol efficacy of 336X against wheat take-all disease can amount to 85%. Wheat endophytic bacteria 336X can produce chitinase and protease. Both of them are cell wall degrading enzymes.It also can form biofilm, iron carrier, Induction system resistance, etc. Serratia proteamaculans 336X shows well colonization in the wheat rhizosphere. It has the the ability of movement and chemotaxis which are associated with biocontrol ability.In order to study the functions of Genes which are related with biocontrol ability of endophytic bacteria 336X, further understand the biocontrol mechanism of biocontrol strain 336X.This study starts from two directions. On the one hand use the sacB gene that encodes levansucrase mark the carrier.When sacB gene express in 336X, it will has the sensitivity of sucrose. Construct ring carrier genetic knockout system of wheat endophytic bacteria 336X.With the methods knock out flagellar movement of related gene motA, get four motA knockout mutations. Similarly knock out chemotaxis genes CheW and get three CheW knockout mutations.All above provide a powerful evidence for the feasibility of the system.On the other hand, use Red Recombination System to construct linear carrier genetic knockout system of wheat endophytic bacteria 336X. At first get engineering bacterium 336X-1 through genetic transformation for 336X, which makes linear carrier in 336X-1 can occur. Further determine the homologous recombination 336X-1 on the temperature sensitivity. Construct the linear targeting C1-Km-C1 that is related with chiA gene.Through the electroporation method introduce the linear targeting fragment above into engineering bacterium 336X-l,and get two Chitinase A knockout mutations. Also, construct the linear targeting phy-Km-phy that is related with assume phytase gene of 336X. Through the knockout method above get four assume phytase gene knockout mutations.Above provide a powerful evidence for the feasibility of the system.Finally, compare the two knockout systems and analyze the advantages and disadvantages of each.The study above lay the theoretical foundation of researching the function of genes at the molecular level and stating biocontrol mechanism of biocontrol strain.更多还原