玉米大斑病菌分生孢子形成的影响因素及GATA转录因子家族的表达分析

摘要

[目的]玉米大斑病(northern leaf blight of corn)是由玉米大斑病菌(Setosphaeria turcica)引起的一种威胁玉米生产的重要叶部病害.本研究旨在确定影响玉米大斑病菌分生孢子产量的主要因素;明确GATA家族成员基因在分生孢子形成时期的表达特征,为深入解析调控病菌发育及致病的分子机制打下基础.[方法]以玉米大斑病菌野生型菌株01-23为试材,探索1 3种不同培养基(乳糖酪蛋白琼脂培养基、马铃薯葡萄糖琼脂培养基、玉米茎秆葡萄糖琼脂培养基、玉米茎秆琼脂培养基、玉米叶片葡萄糖琼脂培养基、玉米叶片琼脂培养基、玉米籽粒葡萄糖琼脂培养基、玉米籽粒琼脂培养基、玉米粉葡萄糖琼脂培养基、玉米粉琼脂培养基、查氏培养基、基本培养基、水琼脂培养基)、5种碳源(乳糖、葡萄糖、蔗糖、果糖、麦芽糖)、以乳糖酪蛋白琼脂培养基为基础培养基,以5 g·L-1的量分别添加不同的氮源(牛肉膏、蛋白胨、NH4Cl、KNO3、(NH4)2SO4、NH4NO3)、pH(4、5、6、7、8、9)、温度(15、20、25、30、35℃)及光照强度(1 500、3 000、6 000、9 000 1x)等条件对分生孢子产量的影响;进一步利用实时荧光定量PCR (qRT-PCR)技术,分析病菌菌丝形成时期和分生孢子形成时期GATA家族5个成员基因的表达量.[结果]利用单因素分析法确定了影响玉米大斑病菌分生孢子产量的主要因素,发现产孢量最大的培养条件为玉米叶片葡萄糖琼脂培养基,碳源为乳糖,培养温度为25℃,pH 8,光照条件为光暗交替各12 h,光照强度为6 000 1x.另外,还发现在培养基中添加KNO3可显著提高分生孢子产量.对GATA家族5个基因的相对表达水平分析表明,与菌丝时期相比,在分生孢子形成时期GATA2的表达量明显上调,其他基因(GATA1、GATA3、GATA4、GATA5)表达量均显著下调.[结论]利用单因素分析法确定了影响玉米大斑病菌分生孢子产量的最佳培养条件;根据GATA家族5个基因相对表达分析结果,推测GATA2可能参与了玉米大斑病菌的分生孢子形成.%[Objective] Northem leaf blight of corn is an important leaf disease and its agent is Setosphaeria turcica.The objectives of this study are to determine the main influencing factors in conidium yield and the expression characteristics of GATA transcription factor gene family at conidium formation stage in S.turcica.The research will lay a foundation for further illuminating the molecular mechanism of regulating growth,development and pathogenicity in S.turcica.[Method] S.turcica wild-type strain 01-23 was used as test material in the study.The main factors which affecting conidium yield was analyzed as follows.The media were lactose casein agar,potato dextrose agar,corn stalk dextrose agar,corn stalk agar,corn leaf dextrose agar,corn leafagar,corn niblet dextrose agar,corn niblet agar,corn starch dextrose agar,corn starch agar,czapek,minimal medium,and water agar.The carbons were lactose,glucose,sucrose,fructose,and maltose.Nitrogen sources were beef jelly,peptone,NH4C1,KNO3,(NH4)2SO4,NH4NO3,which were added in lactose casein agar medium respectively and the content was 5 g in per liter medium.The pHs were 4,5,6,7,8,and 9.The temperatures were 15,20,25,30,and 35℃.The light intensities were 1 500,3 000,6 000,and 9 000 lx,respectively.Quantitative real-time PCR (qRT-PCR) technology was employed to analyze the expression characteristics of GATA family members during conidium formation period.[Result] The optimum conditions for conidia yield in S0 turcica based on single factor method were as follows.The most appropriate medium was maize leaf dextrose agar medium,pH was 8,25℃,6 000 lx light intensity (12 hours light and 12 hours dark).In addition,it was found that the addition of KNO3 in lactose casein agar medium significantly increased conidial yield.GATA2 significantly enhanced its expression level while other GATA family members,such as GATA1,GATA3,GATA4,GATA5,cut down their expression level at conidium formation stage compared with mycelium development stage.[Conclusion] The optimum culture condition of S.turcica conidium yield was determined based on single factor method in the study.According to the relative expression analysis of 5 genes in GATA family,it was speculated that GATA2 may be involved in the formation of conidia in S.turcica.