玉米大斑病菌MAPK基因St IME2的基因组定位、蛋白质结构预测及表达分析

【目的】确定玉米大斑病菌(Setosphaeria turcica)MAPK基因St IME2在基因组中的位置;系统解析目的蛋白质St Ime2的结构特征;分析玉米大斑病菌St IME2在不同发育时期及不同胁迫条件下(温度、氧胁迫、高渗胁迫)的表达,为深入研究该基因的功能奠定基础。【方法】通过本地Blast搜索玉米大斑病菌基因组数据库,确定St IME2在基因组的精确位置;利用Prot Param在线分析St Ime2蛋白的理化性质,利用SOMPA在线软件预测St Ime2蛋白的二级结构。通过PHYRE2在线服务器对St Ime2蛋白的三维结构进行预测。利用半定量RT-PCR方法分析不同发育时期及不同胁迫条件下St IME2的表达。【结果】玉米大斑病菌St IME2为一类与酿酒酵母中Sc IME2具有较高同源性的基因,为在植物病原真菌中鲜有报道的MAPK基因。该基因的ID为98 105,位于scaffold_7正链的1 560 184—1 562 574位置,St Ime2蛋白具有MAPK类蛋白激酶的特征性保守结构域,其二级结构主要以α-螺旋和无规则卷曲为主,β-折叠较少且主要存在于N端,其三级结构具有1个较小的N端域和1个较大的C端域;该基因在病菌分生孢子时期表达量最高,附着胞发育时期表达量最低。将病菌置于不同温度条件下处理,发现培养温度为28℃时St IME2表达量达到最高。在高渗胁迫条件下,随Na Cl浓度的增加,St IME2的表达量逐渐增加,但在较高胁迫条件下(0.8 mol·L-1 Na Cl),该基因表达几乎被完全抑制。经H2O2胁迫处理后,St IME2的表达量随H2O2的浓度增加而增强,在10 mmol·L-1 H2O2的处理下表达量最高。【结论】玉米大斑病菌St IME2位于scaffold_7正链的1 560 184—1 562 574位置。St Ime2蛋白具有MAPK激酶的所有特征性保守结构域,为一类功能鲜有报道的MAPK蛋白激酶。该基因在玉米大斑病菌分生孢子时期表达量最高,推测可能在调控病菌分生孢子发育过程中起重要作用。该基因的表达水平可随培养温度的变化而变化,28℃表达量最高。该基因可能参与病菌的高渗胁迫及氧胁迫反应。

Analysis of the Genomic Location,Protein Structure Prediction and Expression of MAPK Gene StIME2 in Setosphaeria turcica

【Objective】The objectives of this study are to determine the location in genome, investigate the protein structure of StIME2 of Setosphaeria turcica, and to compare the expression level of StIME2 at different developmental stages and under different stress conditions, such as temperature, oxygen stress, hyperosmolar stress, hence laying a foundation for future studying functions of StIME2.【Method】 Blast search tool was used to determine the exact location of StIME2 in genome of S. turcica. Physical and chemical properties of StIme2 was analyzed by employing online software ProtParam, and secondary structure of StIme2 was predicted by applying Software SOMPA. Three-dimensional structure of StIme2 was doped out using PHYRE2 server and the structure model was evaluated using online software SAVES and the aim was to obtain Laplace image (Ramachandran Plot). StIME2 expression level was detected at different developmental stages and stress conditions based on semi-quantitative RT-PCR analysis. 【Result】The identity of StIME2 was 98 105, located between 1 560 184 and 1 562 574 in the positive-strand of scaffold_7 in the genome of S. turcica. StIme2 shared characteristic conserved domains of MAPK-like protein kinase, α-helices and random coils, which were its main elements of secondary structure of StIme2. β strands were less and mainly located in N-terminal in this structure. Three-dimensional structure of the protein was composed of a smaller N-terminal and a bigger C-terminal. The expression level of StIME2 was the highest in conidium developmental periods, and was the lowest during appressorium development stage. Under different stress conditions, the expression level of StIME2 appeared the highest when the incubation temperature was at 28℃. In hyperosmotic stress conditions, the expression level of StIME2 increased with the increase of NaCl concentration, but the expression level of the gene was almost completely inhibited at higher stress conditions (0.8 mol·L^-1 NaCl). Under oxygen stress conditions, in H₂O₂ stress treatment, the expression level of StIME2 was heightened with the increase of the concentration of H₂O₂. And the expression level of the gene was the highest under 10 mmol·L^-1 H₂O₂. 【Conclusion】 StIME2 was located between 1 560 184 and 1 562 574 in the positive-strand of scaffold_7 in the genome of S. turcica. The characteristics of StIme2 were analyzed, and the result showed that StIme2 shared all conserved domains of MAPK kinase of plant pathogenic fungi. However, unlike three types of MAPK genes, which were widely reported in plant pathogens, StIme2 was rarely reported. The expression level of StIME2 in conidium developmental period in S. turcica was the highest. Thus, the authors speculated that StIME2 could play an important role in regulating the developmental processes in spore of the pathogen. Moreover, the expression level of StIME2 changed with the temperature and the highest expression level was at 28℃. The gene may be also involved in osmotic stress and oxygen stress reaction of the pathogen.