中国农业科学 ›› 2020, Vol. 53 ›› Issue (24): 5017-5026.doi: 10.3864/j.issn.0578-1752.2020.24.005
龙凤1(),王擎1(
),朱行1,王建霞1,申珅1,刘宁1,郝志敏1(
),董金皋1,2(
)
收稿日期:
2020-03-28
接受日期:
2020-05-10
出版日期:
2020-12-16
发布日期:
2020-12-28
通讯作者:
郝志敏,董金皋
作者简介:
龙凤,E-mail: 基金资助:
LONG Feng1(),WANG Qing1(
),ZHU Hang1,WANG JianXia1,SHEN Shen1,LIU Ning1,HAO ZhiMin1(
),DONG JinGao1,2(
)
Received:
2020-03-28
Accepted:
2020-05-10
Online:
2020-12-16
Published:
2020-12-28
Contact:
ZhiMin HAO,JinGao DONG
摘要:
【背景】隔膜蛋白Septin广泛存在于除植物以外所有真核生物中,是高度保守的GTP结合蛋白家族,被认为是继微管、微丝和中间纤维之后的第4种细胞骨架蛋白。病原真菌的Septin蛋白参与细胞极性的确定、形态塑造及与致病相关的形态转换。【目的】鉴定玉米大斑病菌(Setosphaeria turcica)Septin基因家族,并进一步分析其在不同发育时期的表达模式,为明确隔膜蛋白Septin与真菌侵染结构发育之间的关系打下基础。【方法】以玉米小斑病菌(Bipolaris maydis)中6个Septin蛋白的氨基酸序列为探针,在玉米大斑病菌数据库在线Blastp比对和关键词搜索,获得大斑病菌的候选Septin,对其基因结构、理化性质以及跨膜区结构等方面进行生物信息学分析。收集人造疏水介质诱导下侵染结构发育不同时期以及侵染感病寄主叶片不同时间的玉米大斑病菌材料,利用实时荧光定量PCR(real-time fluorescence quantitative PCR,RT-qPCR)技术系统分析Septin基因家族在玉米大斑病菌侵染结构形成不同阶段的转录水平。【结果】获得了玉米大斑病菌6个候选Septin,其中4个核心Septin,均含有G1、G3、G4基序,分别将其命名为StSep1、StSep2、StSep3、StSep4。在人造疏水介质诱导下,Septin表达水平均呈上升趋势。StSep1在芽管形成后期表达最活跃,其表达量达到分生孢子时期的25.69倍(P<0.01),StSep4的表达水平在附着胞发育后期到达高峰,随后表达逐渐下调。该基因家族在病菌侵染寄主叶片过程中的转录水平变化趋势与其在人造疏水介质诱导下的表现趋于一致。StSep1在接种后6 h转录水平极显著上调(P<0.01),随着时间延长,表达水平下降,StSep4在附着胞形成阶段表达活跃,表达量在接种后18 h达到高峰值,之后表达下调,但仍高于萌发初期。StSep2、StSep3在接种后18 h和24 h表达活跃,高于萌发初期。【结论】玉米大斑病菌基因组中含有4个核心Septin,StSep1和StSep4分别在芽管和附着胞形成时期活跃表达,结果可为进一步明确 Septin的功能及玉米大斑病菌的侵染调控机制提供依据。
龙凤,王擎,朱行,王建霞,申珅,刘宁,郝志敏,董金皋. 玉米大斑病菌Septin基因家族的鉴定与表达模式分析[J]. 中国农业科学, 2020, 53(24): 5017-5026.
LONG Feng,WANG Qing,ZHU Hang,WANG JianXia,SHEN Shen,LIU Ning,HAO ZhiMin,DONG JinGao. Identification and Expression Pattern Analysis of Septin Gene Family of Setosphaeria turcica[J]. Scientia Agricultura Sinica, 2020, 53(24): 5017-5026.
表1
RT-qPCR试验所用引物"
名称Primer | 序列Sequence (5′ to 3′) |
---|---|
β-Tubulin-F | GGGAACTCCTCACGGATGTTG |
β-Tubulin-R | TAACAAVTGGGCAAAGGGTCA |
StSep1F | ACTGCTGCCTGTTCTTCA |
StSep1R | TGGAACTCCTCCTTTATCC |
StSep2F | GATTCGGAGACCAGATTGA |
StSep2R | AGTAGGCGTGATGAAGTAGAG |
StSep3F | TGGCTCGGAGAAGGATGT |
StSep3R | GGGTTCGGATGAGGATGG |
StSep4F | GCTCATCCGCACCCACATG |
StSep4R | CATCTTCTGGAGCTTGGC |
表3
玉米大斑病菌Septin的信息"
基因 Gene | 基因登录号 Database accession number | 基因长度 Gene length (bp) | 蛋白质ID Protein ID | 大小 Size (aa) | 基因组定位 Genomic mapping |
---|---|---|---|---|---|
StSep1 | XP_008021140.1 | 1588 | 162710 | 341 | scaffold_1:3755824-3757411 (+) |
StSep2 | XP_008027140.1 | 1883 | 163440 | 375 | scaffold_3:81842-83724 (+) |
StSep3 | XP_008028687.1 | 1406 | 93639 | 381 | scaffold_4:2483454-2484859 (+) |
StSep4 | XP_008026163.1 | 1424 | 110191 | 432 | scaffold_21:190124-191547 (+) |
StSep5 | XP_008030793.1 | 1977 | 165795 | 475 | scaffold_8:185341-187317 (-) |
StSep6 | XP_008030826.1 | 2554 | 99177 | 793 | scaffold_8:379528-382081 (-) |
表4
Septin的基本理化性质"
特征蛋白 Characteristic protein | StSep1 | StSep2 | StSep3 | StSep4 |
---|---|---|---|---|
相对分子质量 Relative molecular mass (kD) | 39.1454 | 42.6070 | 44.0992 | 49.7026 |
PI | 7.20 | 5.07 | 8.54 | 6.56 |
(Asp+Glu) | 44 | 72 | 60 | 68 |
(Arg+Lys) | 44 | 53 | 63 | 66 |
分子式Molecular formula | C1733H2735N495O520S10 | C1851H2985N535O595S11 | C1938H3107N573O580S12 | C2179H3514N624O671S16 |
不稳定性系数 Coefficient of instability | 49.91 | 47.37 | 48.84 | 42.73 |
脂肪族氨基酸指数 Aliphatic amino acid index | 84.31 | 86.53 | 78.87 | 81.62 |
总平均亲水性 Total average hydrophilicity | -0.498 | -0.608 | -0.175 | -0.681 |
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