Cloning and Functional Study of UePkaC Gene in Ustilago esculenta
WANG Pei-You*, ZHANG Ya-Fen*, GE Qian-Wen, XIA Wen-Qiang, CUI Hai-Feng, YU Xiao-Ping, YE Zi-Hong**
Zhejiang Provincial Key Laboratory of Biometrology and Inspection Quarantine/College of Life Sciences, China Jiliang University, Hangzhou 310018, China
摘要菰黑粉菌(Ustilago esculenta)是一种二态性真菌,具有侵染菰(Zizania latifolia)植株的能力,并可使植株茎部膨大形成茭白。二型态转换对菰黑粉菌的侵染能力及致病性至关重要。促丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号级联途径和环化腺苷酸介导的蛋白激酶A (cyclic adenosine monophosphate-protein kinase A, cAMP-PKA)信号转导途径是真菌二型态转换的核心。本研究发现,外源添加cAMP后单倍体出现多位点芽殖现象,且融合菌丝生长受到影响。进一步克隆得到了编码菰黑粉菌PKA催化亚基的基因UePkaC (PkaC like protein in Ustilago esculenta)基因(GenBank No.KR870334.1)。序列分析结果显示,该基因全长1 346 bp,有1个内含子,两个外显子构成大小为1 233 bp的开放阅读框,编码410个氨基酸。该氨基酸序列与其他物种中PKA催化亚基的氨基酸序列同源,如大麦坚黑粉菌(U. hordei)、玉米瘤黑粉菌(U. maydis)。表达模式分析发现：UePkaC在菌丝融合生长过程中上调表达;对UePkaC缺失突变体表型分析发现其变长且无融合生长能力和致病性。进一步研究发现,UePkaC突变后无法形成接合管,且信息素合成基因表达受到显著抑制,表明UePkaC基因通过调控a基因的表达从而影响接合管的形成与细胞融合。以上结果表明,UePkaC基因在菰黑粉菌单倍体生长及二型态转换中起着关键作用。该研究为探讨菰黑粉菌的致病机理提供了基础资料。
Abstract：Ustilago esculenta is a dimorpic fungi with the ability to infect Zizania latifolia and causes stem enlargement. The fungal dimorphism is vital for the infection ability and pathogenicity of U. esculenta.The mitogen-activated protein kinase (MAPK) cascades and cyclic adenosine monophosphate-protein kinase A(cAMP-PKA) pathway are the core of signaling pathways in regulation of the fungal dimorphism. In this study, the phenomenon of multi-budding growth of cells after the addition of cAMP was found, and the hyphal growth was affected during the mating process. Furthermore, a gene encoding PKA catalytic submit (GenBank No. KR870334.1) was cloned. Sequence analysis results showed that the gene had a total length of 1 346 bp, with 1 intron and 2 exons forming 1 233 bp open reading frame, encoding 410 amino acids. This amino acid sequence was homologous to the amino acid sequence of PKA catalytic subunit in other species, such as U. hordei and U. maydis. The expression pattern analysis showed that UePkaC was up-regulated in the process of mating. The phenotype analysis of the UePkaC deletion mutants showed that they were longer and had no mating ability and pathogenicity. Furthermore, the ability of conjunction tube was lost in UePkaC mutant during mating and the expression of pheromone synthesis gene was significantly inhibited, indicating that UePkaC affects the conjugation tube formation and mating process by regulating the expression of a genes.The above results showed that UePkaC gene played a key role in the haploid growth and dimorphism of U. esculenta, which provided basic data for studying the pathogenic mechanism of U. esculenta.
王培又, 张雅芬, 葛倩雯, 夏文强, 崔海峰, 俞晓平, 叶子弘. 菰黑粉菌UePkaC基因的克隆及功能研究[J]. 农业生物技术学报, 2020, 28(8): 1477-1489.
WANG Pei-You, ZHANG Ya-Fen, GE Qian-Wen, XIA Wen-Qiang, CUI Hai-Feng, YU Xiao-Ping, YE Zi-Hong. Cloning and Functional Study of UePkaC Gene in Ustilago esculenta. 农业生物技术学报, 2020, 28(8): 1477-1489.
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