【作者】 李海萍；

【导师】 储昭辉； 丁新华；

【作者基本信息】 山东农业大学 ， 植物病理学， 2011， 硕士

【摘要】 马铃薯晚疫病是一种导致马铃薯茎叶死亡和块茎腐烂的毁灭性病害。利用植物抗病基因培育抗病品种是当前生产上防控该病害的首选策略。本实验所利用的基因是从Solanum mochiquense中克隆的抗马铃薯晚疫病基因Rpi-mcq1.1、Rpi-mcq1.2和从Solanum venturii中克隆的Rpi-vnt1.1,它们都属于CC-NBS-LRR型的抗性基因,序列相似度高达82%,属于同一基因家族成员。通过改变结构域、启动子的方法,探索改良新基因的可行性是本研究的主要目标。同时,实验设计中还可以帮助寻找抗病基因中决定特异性识别病原菌的结构域,对进一步研究病原物与植物之间的互作起到一定作用。利用结构域互换的方法,使用融合PCR技术将Rpi-vnt1.1 CC-NB区和Rpi-mcq1.1 LRR区组合（简称V1M2）、Rpi-vnt1.1启动子区与Rpi-mcq1.1、Rpi-mcq1.2 CC-NB-LRR区组合（简称RpiV::mcq1.1和RpiV::mcq1.2）,构建了3个新的载体。将三个融合基因连入pCAMBIA1300载体中,转入农杆菌工程菌AglⅠ,采用茎段转化法将上述三个载体转化马铃薯栽培种Desiree,分别获得PCR检测为阳性的转基因植株13株、11株、25株,转化效率分别达到73%、61%、79%。同时将三个载体转入农杆菌工程菌LBA4404。采用叶盘转化法将上述三个载体转化本生烟Nicotiana benthamiana,分别获得PCR检测为阳性的转基因植株8株、7株、13株,转化效率分别达到37%、30%、41%。所获得转基因植株可用于离体接种马铃薯晚疫病菌,对转基因植株进行抗病性检测,以判断新抗病基因抗病谱变化以及与病原菌互作的结构域。采用本生烟瞬时表达技术,利用农杆菌GV3101将Rpi-vnt1.1、Rpi-mcq1.1、Rpi-mcq1.2、V1M2、RpiV::mcq1.1、RpiV::mcq1.2分别与无毒基因Avr2（E2、G3、G1）混合共侵染本生烟,结果显示Rpi-mcq1.1、V1M2、RpiV::mcq1.1、RpiV::mcq1.2与无毒基因Avr2的G3、G12菌株混合后产生反应一致,均有明显的HR反应,从而验证Rpi-mcq1.1识别病原物的位点在LRR结构域。尤其有趣的是,Rpi-mcq1.2在低水平表达下与Avr2不识别,但在换用Rpi-vnt1.1启动子后能和Avr2识别,与之前35S超表达结果相符,说明,Rpi-mcq1.2与Avr2的识别是处于弱蛋白质相互作用,为进一步分析Rpi-mcq1.1、Rpi-mcq1.2、Rpi-vnt1.1LRR结构域与病原无毒基因Avr2的识别奠定基础。同时,在实验结果中我们意外的发现瞬时表达Rpi-vnt1.1能激发本生烟产生HR反应。为寻找Rpi-vnt1.1自激活现象中发挥主效功能的结构域,我们构建了pCAMBIA1300+Rpi-vnt1.1启动子-CC-NB区、pBin19s+35s启动子+Rpi-vnt1.1CC-NBS区、pBin19s+35s启动子+Rpi-vnt1.1LRR区、pBin19s+35s启动子+Rpi-vnt1.1CC-NBS-LRR区、pBin19s+35s启动子+Rpi- mcq1.1CC-NBS区、pBin19s+35s启动子+Rpi- mcq1.1LRR区、pBin19s+35s启动子+Rpi- mcq1.1CC-NBS-LRR区等载体,通过本生烟瞬时表达技术,利用农杆菌GV3101侵染本生烟。实验结果显示只有Rpi-vnt1.1能自身激发本生烟产生HR反应。由此推测Rpi-vnt1.1自身激发本生烟产生HR现象可能与Rpi-vnt1.1自身启动子结构域有着必然的联系。为进一步解析相关自激活机制奠定基础。生长素（auxin）是一个至关重要的植物激素,调节植物生长发育过程中的多个方面。天然植物生长素的主要活性形式是吲哚-3-乙酸（indole-3-acetic acid, IAA）。由水稻白叶枯病菌引起的水稻白叶枯病是世界水稻最重要的病害之一,每年给世界粮食生产造成巨大的损失。近期研究表明IAA不仅调节水稻的生长发育,而且可作为白叶枯病菌侵染水稻的毒性因子。研究发现水稻白叶枯病菌也可产生IAA,水稻白叶枯病菌中可能存在一条新的植物病原菌IAA合成途径。本研究的目的是通过同源重组构建水稻白叶枯病菌预测的IAA合成基因突变体,鉴定水稻白叶枯病菌的IAA合成途径,并验证水稻白叶枯病菌产生的IAA在白叶枯病菌侵染水稻过程中所起的作用。以拟南芥IAA合成路径中相关蛋白的氨基酸序列通过BlastP搜索NCBI数据库,找到水稻白叶枯病菌PXO99基因组中存在的同源的腈水解酶家族基因PXO₀0867。利用同源重组突变法将PXO99菌株中的PXO₀0867基因突变,获得突变菌株PXO99△PXO₀0867。利用RT-PCR和基因序列测定法对突变菌株PXO99△PXO₀0867进行验证,确定该菌株中PXO₀0867基因发生了同源重组,导致该基因不能正常表达。通过高效液相色谱法检测用乙酸乙酯萃取得到的野生型PXO99及PXO99△PXO₀0867代谢产物中的IAA含量。结果显示PXO99△PXO₀0867中IAA含量低于PXO99。表明PXO₀0867基因在水稻白叶枯病菌PXO99中参与IAA的合成,但是从IAA改变量来看,该基因在IAA合成途径中所起的作用并不十分显著。利用转DR5-GUS的水稻品种日本晴为实验材料,接种PXO99及PXO99△PXO₀0867菌株,采用GUS组织染色法对各个时间段接种的水稻叶片进行染色。显微镜下观察染色结果显示,同时段的PXO99△PXO₀0867侵染的叶片染色较PXO99侵染的叶片颜色略浅,说明突变菌株PXO99△PXO₀0867诱导的水稻IAA合成的量比野生型对照PXO99要低。这一结果与突变菌株PXO99△PXO₀0867产生的IAA含量比对照略低的结果想吻合。通过全波长多功能酶标分析仪检测分析用PXO99△PXO₀0867和PXO99接种的水稻叶片的GUS活性。实验结果显示PXO99△PXO₀0867侵染的叶片激发值较PXO99侵染的叶片激发值低。研究结果与预期一致。同时将PXO99△PXO₀0867和PXO99接种不同水稻品种,进行病原致病性鉴定。实验结果显示,PXO99△PXO₀0867侵染的水稻病斑长度较短,初步说明IAA在病原菌侵染过程中起到一定的作用。综上所述,我们通过同源重组方法构建了PXO99中IAA合成相关基因的突变体,对其他相关基因通过此方法进行基因突变起到了指导作用,同时对我们了解水稻白叶枯病菌产生的IAA在白叶枯病菌侵染水稻过程中所起的作用,阐明水稻白叶枯病菌的致病机理都有重要的理论意义。更多还原

【Abstract】 Potato late blight is a devastating disease which leads to the rot of potato leaf,stem and tuber. Currently, cultivation resistant variety derived from plant resistant gene is the first tactic for control the disaese. In this study, we used three late blight resistance genes, which belong to the same gene family of CC-NBS-LRR type, shared with about 82% identities with amino acid sequence. Both Rpi-mcq1.1 and Rpi-mcq1.2 were cloned from Solanum mochiquense, and Rpi-vnt1.1 was cloned from Solanum venturii . By changed with Rpi-vnt1.1 gene promoter, domain swapping of the Rpi-mcq1.1 and Rpi-mcq1.2, we goal to seek after resistance improvement and to explore the domain that controlled the recognization between R gene and pathogen effector.Firstly, three new construts have been made with overlapping PCR technique and domain swap tactic. The V1M2 was combinated with CC-NB motif of Rpi-vnt1.1and LRR motif of Rpi-mcq1.1. RpiV::mcq1.1 was made by replaced the native promoter with Rpi-vnt1.1 promoter . RpiV::mcq1.2 was made by replaced with Rpi-vnt1.1 promoter as RpiV::mcq1.1.Three new genes were ligated into pCAMBIA1300, and mobilised into Agrobacterium strain AglⅠ, then introduced into suceptible potato variety Desiree and Nicotiana benthamiana. For each construct V1M2, RpiV::mcq1.1 and RpiV::mcq1.2, 13, 11 and 25 positive individuals were conducted with potato Desiree regarded as 73%, 61% and 79% of transformation efficiency respectively. The amout of 8, 7 and 13 positive individuals were conducted in N. benthamiana with 37%, 30% and 41% of transformation efficiency respectively. All the materials will be tested for inoculation Phytophthora infestans, to test whether their resistant spectrum have changed or not, and to detect whether they have been affected to recognize the Avr2.By using Agrobacterium mediated transient expression system on N. benthamiana, Rpi-mcq1.1, V1M2, RpiV::mcq1.1 and RpiV::mcq1.2 were indicated to recognize the Avr2 （G3, G12） after co-infiltration with GV3101 carried each resistance gene and Avr2. The results support to make aconclusion that it is LRR motif of Rpi-mcq1.1 correspond for the recognization but not the CC-NBS motif. More interestingly, we found that Rpi-mcq1.2 was turns to recognize to Avr2 after replaced with Rpi-vnt1.1 promoter which couldn’t trigger HR on N. benthamiana after co-infiltration with it original gene and Avr2. Together with the results that Rpi-mcq1.2 can recognize to Avr2 after dedrived with 35S promoter, we suggest that the interaction between Rpi-mcq1.2 and Avr2 is a weakly interaction, maybe the dosage-dependent reaction. This is basis for analysising interaction betwern Rpi-mcq1.1, Rpi-mcq1.2, Rpi-vnt1.1 LRR domain and avirulence gene Avr2.Meanwhile, we surprise to found that Rpi-vnt1.1 can auto-activate the HR on response in N. benthamiana. In order to explore the function motif response for this function, we constucted a series of vector as below which included pCAMBIA1300+Rpi-vnt1.1 promoter -CC-NB, pBin19s+35 promoter+ Rpi-vnt1.1CC-NBS, pBin19s+35s promoter + Rpi-vnt1.1 LRR, pBin19s+35s promoter+ Rpi-vnt1.1 CC-NBS-LRR, pBin19s+35s promoter+Rpi-mcq1.1 CC-NBS, pBin19s+35s promoter+ Rpi-mcq1.1LRR and pBin19s+35s promoter+ Rpi-mcq1.1 CC-NBS-LRR. After N. benthamiana transient expression assay, still only Rpi-vnt1.1 with its native promoter has the property. The results indicated that the promoter and LRR motif was invovled in this self auto-activation. Those resluts will provide to further studies.Auxin is an essential plant hormone. Auxin regulates plant growth and development. The main activity of the natural form of auxin is indole-3-acetic acid, IAA. Rice bacterial blight disease caused by Xanthomonas oryza pv. oryza,is the most devastating diseases of rice worldwide. This diseases cause tremendous yield loss each year. Recent studies have shown that IAA not only regulate the growth and development of rice, also as virulence factors when bacterial blight and bacterial infection.Preliminary study have shown Xanthomonas oryza pv. oryza can produce IAA. There may be have a new synthesis pathway of IAA in plant pathogenic bacteria. The purpose of this research is the built of mutant from the method of homologous recombination. Identification the pathway of IAA biosynthesis in Xoo may help us text the function of IAA in the process of pathogen infect rice. Our study may have deep meaning in pathogenesis of Xoo.According to IAA biosynthesis pathway related the amino acid sequence databases of Arabidopsis thaliana in the NCBI BlastP, found the genome Xoo PXO99 homologous gene PXO₀0867 （Nitric Oxide Hydrolase Gene Family）. We construction PXO99 mutation via homologous recombination PXO ₀0867. Using PR-PCR and gene sequencing method to verificate PXO99 mutant strain, confirmed the accuracy of strain genotypy.We extracted IAA with ethyl acetate . By high performance liquid chromatography ,we texted the IAA content of metabolites in the wild-type PXO99 and PXO99△PXO₀0867 . The results showed the IAA content of PXO99△PXO₀0867 is slightly less than PXO99. It proved PXO₀0867 gene have effect in PXO99, but not play an important role in IAA biosynthesis pathway .we take transgene Dr5-GUS rice leaves as the experimental material, inoculated PXO99 and PXO99△PXO₀0867. By GUS staining method and microscope,we examine the stain of rice leaves. We found PXO99△PXO₀0867 infected leaves infected have lighter color than PXO99. This result consistent with PXO99△PXO₀0867 has lower-level produced of IAA.Multifunctional enzyme analysis the transgene Dr5-GUS rice leaves that inoculated PXO99 and PXO99△PXO₀0867. The results showed PXO99△PXO₀0867 has lower stimulate value than PXO99. Consistent with the expected results.While PXO99△PXO₀0867 and PXO99 inoculated rice leaves of different varieties to rice lesion analysis. The experimental results, the rice has slight disease course when infected PXO99△PXO₀0867 the initial description of IAA in the process of pathogen infection plays a role.In summary, we have constructed PXO99 mutantion by homologous recombination method. This method plays a guiding role for other genes. Meanwhile the result of this study play a role in finding the action of IAA produced by Xoo when Xoo infect rice, and it is helpful for us finding the pathogenesis of Xoo.更多还原