链格孢菌苹果致病型的ATMT转化体系的建立

Development of the Agrobacterium tumefaciens Mediated Transformation System of Alternaria alternata Apple Pathotype

【目的】链格孢菌(Alternaria alternate)苹果致病型是苹果上的重要致病菌,可以侵染苹果叶片和果实。论文旨在建立农杆菌介导的链格孢菌苹果致病型高效稳定的分子转化体系,为在分子水平上研究链格孢苹果致病型病菌的致病机制提供技术支持。【方法】质粒pKO1-HPH是以pCAMBIA1300骨架为基础构建的穿梭质粒,含有潮霉素抗性基因,并在其多克隆位点上插入了绿色荧光蛋白基因,这个质粒在大肠杆菌和农杆菌细胞中都能够稳定地复制繁殖。将质粒pKO1-HPH采用冻融法转化到农杆菌菌株AGL1中,然后与链格孢苹果致病型菌株XP-1的分生孢子在诱导培养基上共培养进行基因转化,转化子在含有潮霉素的马铃薯-葡萄糖-琼脂培养基上进行筛选;在含有50μg·mL^(-1)潮霉素的马铃薯-葡萄糖-琼脂培养基上检测转化子细胞中抗性基因在转化子中的表达情况;用荧光显微镜检测绿色荧光蛋白基因在转化子细胞中的表达情况。在初步确认可以将绿色荧光蛋白和潮霉素B磷酸转移酶基因转入链格孢苹果致病型菌株中后,对菌落培养时间、转化前分生孢子的预处理方法对转化效率的影响进一步优化。转化子的稳定性采用分生孢子5次继代培养后,能否在含有潮霉素的马铃薯-葡萄糖-琼脂培养基上稳定生长来检测;采用PCR方法检测绿色荧光蛋白外源基因和潮霉素B磷酸转移酶基因是否存在于转化子基因组中;用Southern杂交检测外源基因在转化子基因组中插入的拷贝数;在苹果果实和叶片上接种检测转化子致病性的变化。【结果】将100μL孢子悬浮液(含105孢子)涂布于马铃薯-胡萝卜-琼脂平板上菌落培养36—48 h后,链格孢苹果致病型菌株XP-1可以产生足量的孢子用于农杆菌介导的分子转化。将分生孢子用无菌水洗下,在4℃处理6 h,再与含质粒pKO1-HPH的农杆菌进行诱导共培养,转化子在含有50μg·mL^(-1)潮霉素的马铃薯-葡萄糖-琼脂培养基上进行筛选,转化效率较好,107个分生孢子转化可获得约200个转化子。转化子经过5次继代培养,对潮霉素B的抗性没有改变。特异性引物PCR检测表明外源基因能够成功地整合到供试菌株的基因组中;Southern杂交表明外源基因多以单拷贝形式随机插入到链格孢苹果致病型菌株XP-1细胞的基因组中;转化子的菌丝和分生孢子在荧光显微镜下可以观察到明显的绿色荧光,说明荧光蛋白基因在菌丝及孢子中均能够成功表达。通过部分转化子的致病性试验,筛选获得数个在苹果果实和叶片上致病性都显著降低的菌株。【结论】建立了农杆菌介导的链格孢菌苹果致病型稳定高效的分子转化体系,为进一步开展此病菌与其寄主的分子互作研究打下基础。

【Objective】 Alternaria alternata apple pathotype is one of the important pathogens on apple which can infect leaves and fruits seriously. The objective of this study is to establish the Agrobacterium tumefaciens mediated transformation system of Al. alternata apple pathotype, provide a technical basis for study molecular mechanisms of pathogenesis. 【Method】 Vector pKO1-HPH is a shuttle plasmid which is constructed on the backbone of p CAMBIA1300 containing hygromycin phosphotransferase gene（hph） and GFP gene. Fungal transformation was carried out by co-incubation of Ag. tumefaciens strain AGL1 containing plasmid pKO1-HPH with conidia of Al. alternata apple pathotype. Transformants were screened at PDA plates containing hygromycin B. Hph gene expression was detected by incubation of transformants on PDA plates containing 50 μg·mL^（-1） hygromycin B, GFP was detected using Green fluorescence microscope. Influences of fungal cultural time and pretreatment method of conidia on transformation were evaluated. The stability of transformants was determined by growing on PDA plates containing hygromycin B after five-generation successive growth. PCR assay was used to confirm the existence of the hph gene and GFP gene in transformants. The copy number of inserted T-DNA in transformants was determined by Southern blot. Pathogenicity test of some transformants was performed on apple fruits and leaves. 【Result】 Spores of Al. alternata apple pathotype were formed enough when 100 μL of spore suspension（about 10~5 spores） was spread on PCA plate and incubated for 36-48 h. The maximum of transformation efficiency was up to 200 transformants/10~7 conidia when pretreatment of conidia at 4℃ for 6 h before transformation. The resistance to hygromycin B in transformants was stable after five successive generations. Results of specific PCR showed the exogenous genes（GFP and hygromycin B） were integrated into genome of Al.alternata apple pathotype strain and single copy insertion was detected in genome of majority of the transformants according to southern blot analysis. Green fluorescence was detected in the mycelia and spores under the fluorescent microscopy, indicating GFP was expressed in spores and hyphae of transformants. Results of pathogenicity test on fruits and leaves showed that the pathogenicity of several transformants was reduced.【Conclusion】Ag.tumefaciens mediated transformation system of Al.alternata apple pathotype is developed in this research and useful for further studies on molecular interaction of apple and Al.alternata apple pathotype.