玉米大斑病菌SC35同源基因表达规律与互作分析

The Expression Pattern and Interaction Analysis of the Homologues of Splicing Factor SC35 in Setosphaeria turcica

【目的】获得玉米大斑病菌(Setosphaeria turcica)SC35类剪接因子的基因,并分析该基因家族之间的相互作用及在病菌不同生长发育时期与侵染过程中的表达规律,为明确剪接因子SC35家族与真菌生长发育的关系打下基础。【方法】以拟南芥(Arabidopsis thaliana)SC35编码的氨基酸序列为探针序列,在玉米大斑病菌全基因组数据库进行同源比对,获得玉米大斑病菌中潜在的SC35蛋白;利用生物信息学方法对其保守结构域、系统进化关系进行分析;分别收集接种在感病玉米叶片表面不同时间及玉米大斑病菌菌丝、分生孢子、芽管、附着胞及侵入丝等不同发育时期的材料,利用实时荧光定量PCR(qRT-PCR)技术分析在对玉米叶片侵染不同时间及不同发育时期可变剪接因子的转录水平;利用酵母双杂交技术明确玉米大斑病菌可变剪接因子之间的相互作用。【结果】在玉米大斑病菌中获得了8个潜在的SC35类蛋白基因,其编码的氨基酸具有典型的SR蛋白(Ser-Arg rich protein)结构域,而StSC1 C端具有2个RRM基序。8个基因位于染色体组的不同物理位置,不具有连锁关系。系统进化分析表明8个剪接因子分布于不同进化支,同源性较低。在病菌侵染寄主叶片过程中,StSC1、StSC2、StSC3、StSC4、StSC5、StSC6、StSC8在侵染18 h时表现为表达上调趋势,StSC7呈下调趋势,StSC4在6—18 h表现出较高表达活性;不同生长时期中,StSC1在病菌附着胞及侵染丝形成时期表达水平极显著上调(P<0.001),是分生孢子时期的24.44、8.25倍,其余7个可变剪接因子在病菌的生长过程中表达水平均呈下调趋势。酵母双杂交结果表明StSC4与StSC6、StSC3与StSC8、StSC3与StSC4、StSC8与StSC4有体外互作关系。【结论】在玉米大斑病菌侵染过程中及不同发育时期可变剪接因子的表达模式不同,StSC4在病菌整个侵染过程均活跃表达;StSC1、StSC4和StSC6对病菌附着胞和侵入丝的形成发挥比较重要的调控作用;StSC4与StSC6、StSC3与StSC8、StSC3与StSC4、StSC8与StSC4通过互作,调控剪接复合体的形成。

【Objective】The objective of this study is to obtain the homologous genes of the splicing factor SC35 in Setosphaeria turcica, and to analyze the interaction among them and the expression profiles during the different growth and development stages and infection process of the pathogen. It would lay the foundation for illustrating the relationship between the SC35 family members and fungal pathogenicity.【Method】Based on amino acid sequences of SC35 protein in Arabidopsis thaliana as probe sequences, online Blastp alignment was carried out in the S. turcica genome database to obtain candidate SC35 homologues. Then they were analyzed for conserved domain and phylogenetic relationship through bioinformatics procedures. The materials of S. turcica were collected at different infection stages on maize leaves and multiple developmental stages, such as hyphae, conidia, germ tubes, appressorium and penetration hyphae, to analyze the transcription levels of SC35 homologues through real-time quantitative PCR(qRT-PCR). And their interactions were verified in vitro by the yeast two-hybrid test.【Result】Eight SC35 genes of S. turcica were obtained, named as StSC1, StSC2, StSC3, StSC4, StSC5, StSC6, St SC7, and StSC8, respectively. All of them owned typical SR protein domains, besides StSC1 showed two RRM domains. They located at different physical locations of the genome and had no linkage. Phylogenetic analysis showed that the eight alternative splicing factors were distributed in different clades, with low homology. In the process of infection on maize leaves, gene StSC1, StSC2, StSC3, StSC4, StSC5, StSC6 and StSC8 were up-regulated, while StSC7 was down-regulated at 18 h after inoculation. St SC4 showed high expression activity at 6-18 h. In different development periods, the expression levels of StSC1 were extremely significant up-regulated(P<0.001) in the appressorium and the formation of penetration hyphae, which were 24.44 and 8.25 times higher than those in the conidial period, but the others were down-regulated during the development. The yeast two-hybrid proved that StSC4 interacted with StSC6, StSC3 with StSC8, StSC3 with StSC4, and StSC8 with StSC4. 【Conclusion】The expression patterns of SC35 are different in the infection process and multiple developmental stages of S. turcica. StSC4 is actively expressed throughout the infection process. StSC1, StSC4 and StSC6 play important regulatory roles during the formation of appressorium and penetration hyphae. StSC4 and StSC6, StSC3 and StSC8, StSC3 and StSC4, StSC8 and StSC4 interact to regulate the formation of splicing complexes.