【作者】 王凤让；

【导师】 宋凤鸣；

【作者基本信息】 浙江大学 ， 植物病理学， 2010， 硕士

【摘要】 锚蛋白(ANK)是生命体中一类庞大的类群,研究表明一些锚蛋白参与植物防卫反应的调控。本实验室前期研究发现水稻锚蛋白基因OsBIANKl在抗病反应中上调表达。为了研究OsBIANK1在抗病反应中的功能,构建了过量表达载体35S：：OsBIANK1,并利用根癌农杆菌介导的花序浸蘸法导入拟南芥中,获得35S：：OsBIANK1单拷贝株系,转代选取3个纯合株系(OsBIANK1-OE#17、#24和#29)。比较了野生型、OsBIANK1-OE植株对Pseudomonas syringae pv. tomato (Pst)DC3000和灰霉病菌(Botrytis cinerea)的抗病性变化。注射接种Pst DC3000、活体接种B. cinerea后,OsBIANK1-OE植株上病害显著轻于野生型。Pst DC3000侵染后,OsBIANK1-OE的Pst DC3000菌量显著低于野生型对照。OsBIANK1-OE#17、#24和#29植株中PR-1和PR-5基因表达上调；而B. cinerea侵染后,OsBIANK1-OE植株中PDF1.2和BIK1基因表达上调。此外,经水杨酸(salicylic acid, SA)和苯并噻二唑(benzothiadiazole, BTH)诱导2天后,再接种PstDC3000, OsBIANK1转基因拟南芥植株菌量低于野生型对照,且低于没有经SA和BTH诱导后的植株。在B. cinerea侵染后,OsBIANK1-OE植株叶片中仅有少量活性氧积累。这些结果表明,OsBIANK1在拟南芥中对不同类型病害的抗病反应中起正调控作用。亚硫酸盐氧化酶(sulfite oxidase, SO)是近几年在植物中得到证实的一个钼酶,在植物硫代谢中起着重要的作用,具有亚硫酸盐解毒功能。近期研究,都集中在SO基因的生化特性方面,抗病性方面报道至今未见报道。本研究中,我们分离鉴定了水稻中两个SO酶基因OsSO1和OsSO2。为了研究OsSO1和OsSO2在抗病反应中的功能,构建了过量表达载体35S::OsSO1,35S::OsSO2,并利用根癌农杆菌介导的花序浸蘸法导入拟南芥中,获得35S::OsSO1和35S::OsSO2单拷贝株系,转代选取纯合株系(OsSO1-OE#19、#23和OsSO2-OE#4)。比较了野生型、OsSO1-OE、OsSO2-OE植株对Pst DC3000和B. cinerea的抗病性变化。注射接种Pst DC300、活体接种B. cinerea后，OsSO1-OE和OsSO2-OE植株上病害显著轻于野生型。Pst DC300侵染后,OsSO1-OE和OsSO2-OE的Pst DC3000菌量显著低于野生型对照,OsSO1-OE和OsSO2-OE植株中PR-1和PR-5基因表达上调；而活体接种B. cinerea后,OsSO1-OE和OsSO2-OE植株中PDF1.2和BIK1基因表达上调。此外,经SA和BTH诱导2天后,再接种Pst DC3000, OsSO1和OsSO2转基因拟南芥植株菌量低于野生型对照且低于没有经SA和BTH诱导后的植株。在B. cinerea侵染后,OsSO1-OE和OsSO2-OE植株叶片中仅有少量活性氧积累。这些结果表明,OsSO1和OsSO2在拟南芥中对不同类型病害的抗病反应中起正调控作用,而且可能通过SA信号途径。更多还原

【Abstract】 Ankyrin repeat-containing proteins are abundant protein classes functioning in protein-protein interactions. Ankyrin repeat proteins (ANK) have been reported to be involved in various biological processes of plant growth and development, transport and defense responses. Previously, we identified a gene, OsBIANK1, encoding for an ANK protein, was up-regulated in defense response in rice after infection by Magnaporthe grisea or treatment with benzothiadiazole (BTH). To study the function of OsBIANK1 in disease resistance response, I constructed an overexpression vector 35S::OsBIANK1, and introduced into wild-type Arabidopsis plants using floral dip transformation protocol.35S::OsBIANK1 transgenic lines with single copy of the transgene were obtained. Homozygous lines were identified from the 3rd generation through screening and three homozygous lines for 35S::OsBIANK1 (OsBIANK1-OE#17,#24and #29) were chosen for further work. Disease phenotypes of the wild-type, OsBIANK1-OE plants were compared after inoculation with Pseudomonas syringae pv. tomato (Pst) DC3000 and Botrytis cinerea. As compared with the wild-type plants, the OsBIANK1-OE plants showed a significant reduced susceptibility to the pathogens. Furthermore, growth of Pst DC3000 in leaves of the OsBIANK1-OE plants was significantly reduced. After inoculation with Pst DC3000, the expression of PR-1 and PR-5 genes was up-regulated in the OsBIANK1-OE plants. And after inoculation with B. cinerea, expression of PDF1.2 and BIK1 genes was up-regulated in the OsBIANK1-OE plants. Additionally, after two days of treatment with some well-known defense-related chemical inducers such as salicylic acid (SA) and benzothaidiazole (BTH), the OsBIANK1-OE plants also showed a significant reduced susceptibility to Pst DC3000, growth of Pst DC3000 in leaves of the OsBIANK1-OE plants was also significantly reduced and less than that without treatment with SA and BTH. Moreover, after Botrytis infection, accumulation of reactive oxygen species (H2O2 and superoxide anion) in leaves of the OsBIANK1-OE plants was lower than those of the wild type plants. These results indicate that OsBIANK1 is a positive regulator of defense responses in Arabidopsis thaliana against both necrotrophic and biotrophic pathogens.Sulfite oxidase (SO) is one of molybdenum(Mo) enzymes in plant. SO has been reported to be involved in sulphur metabolism in plants, and can detoxifies excessive sulfite. Information on functions of SO in plant defense response against pathogen infection is lacking. In this study, I identified two SO genes from rice. To study the function of OsSO1 and OsSO2 in disease resistance response, I constructed overexpression vectors 35S::OsSO1 and 35S::OsSO2 and introduced them into wild-type plants using floral dip transformation protocol.35S::OsSO1 and 35S::OsSO2 transgenic lines with single copy of the transgene were obtained. Homozygous lines were identified from the 3rd generation through screening and three homozygous lines for 35S::OsSO1 (OsSO1-OE#19,#23) and 35S::OsSO2(OsSO2-OE#4) were chosen for further work. Disease phenotypes of the wild-type, OsSO1-OE and OsSO2-OE plants were compared after inoculation with Pst DC3000 and B. cinerea. As compared with the wild-type plants, the OsSO1-OE and OsSO2-OE plants showed a significant reduced susceptibility to the patogens. Furthermore, growth of Pst DC3000 in leaves of the OsSO1-OE and OsSO2-OE plants was significantly reduced. After inoculation with Pst DC3000, the expression of PR-1 and PR-5 genes was up-regulated in the OsSO1-OE and OsSO2-OE plants. And after inoculation with B. cinerea, expression of PDF1.2 and BIK1 genes was up-regulated in the OsSO1-OE and OsSO2-OE plants. Additionally, after two days of treatment with SA and BTH, the OsSO1-OE and OsSO2-OE plants showed a significant reduced susceptibility to Pst DC3000, growth of Pst DC3000 in leaves of the OsSO1-OE and OsSO2-OE plants was also significantly reduced and less than that without treatment with SA and BTH. Moreover, accumulation of reactive oxygen species in leaves of the OsSO1-OE and OsSO2-OE plants after Botrytis infection was less than that in the wild type plants. These results indicate that OsSO1 and OsSO2 are involved in defense response against pathogen infection.更多还原