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马尾松PmWRKY164基因的克隆及耐低磷功能分析

Cloning and Low Phosphorus Tolerance Function Analysis of PmWRKY164 from Pinus massoniana
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摘要 WRKY转录因子在植物应答逆境胁迫中扮演了重要角色。本研究克隆了马尾松(Pinus massoniana)的PmWRKY164基因(GenBank No. MH579749),该基因的cDNA全长1 977 bp,开放阅读框(ORF) 1 164 bp,编码387个氨基酸,具有WRKYGQK七肽保守结构域和C2H2(C-X4-5-C-X23-HXH)锌指结构域,属于WRKY家族第Ⅱb类成员。系统进化树分析表明,PmWRKY164与红豆杉(Taxus wallichiana var. chinensis, AEW91476.1)、欧洲云杉(Picea abies, ACA04888.1)的亲缘关系较近;分别以2种不同磷(phosphorus, P)浓度下胁迫处理的马尾松cDNA作为模板,对PmWRKY164的时空表达进行分析,结果表明,PmWRKY164在马尾松根茎叶中均有表达,叶中表达量最高,呈上调表达,与超氧化物歧化酶(superoxide dismutase, SOD)、过氧化物酶(peroxidase, POD)、酸性磷酸酶(acid phosphatase, Apase)3种酶的活性表达具有较高的相似性;构建pBWA(V)HS-PmWRKY164植物过表达载体,经遗传转化获得20株PmWRKY164过表达转基因植株。选取T1代3个转基因株系(Line-2, Line-3, Line-8)和野生型(wild type, WT)烟草株系,用4种不同浓度的Hogland营养液进行磷胁迫,结果表明,在P2低磷(营养液磷含量0.1 mmol/L)胁迫下,除了丙二醛(malondialdehyde, MDA)含量低于野生型之外,磷含量、Apase活性、POD活性及SOD活性在转基因烟草中均显著高于野生型(P<0.05);而正常磷P3 (1 mmol/L)及高磷P4 (10 mmol/L)条件下,转基因与野生型植株并无明显差异。本研究为进一步探讨PmWRKY164的功能,以及为马尾松耐低磷胁迫的分子机制提供新的信息与思路。 WRKY transcription factors play important roles in various stress response. However, the phosphorus (P) deficiency in the tropical and subtropical forest soils has severely posed challenges for the productivity. Therefore, screening the related low-P stress genes, as well as to reveal the molecular regulation mechanisms of low phosphorus tolerance were necessary, which will provide a theoretical basis for the molecular breeding and create novel germplasms with high quality and stress resistance. WRKY transcription factors (TFs), a plant-specific TF family, play important and unique roles in biological regulations involving in stress defenses, development, and metabolite synthesis, however, little has been known about their roles in response to phosphorus starvation in masson pine (Pinus massoniana). To further understand their roles in low phosphorus (P) stress, currently, the full-length sequence of PmWRKY164 was cloned by rapid amplification of cDNA ends (RACE) methodology. Homologous analysis, multiple alignments, as well as related bioinformatics analysis were performed. Quantitative real-time PCR (qRT-PCR) was used to detect the temporal and spatial expression patterns of PmWRKY164. Finally, the pBWA(V)HS-PmWRKY164 expression vector was introduced into tobacco through Agrobacterium-mediated procedure. The result showed that PmWRKY164 (GenBank No. MH579749) was obtained, whose full-length cDNA was 1 977 bp and the corresponding lengths of open reading frames (ORF) was 1 164 bp, which encoded 387 amino acids, including a single WRKYGQK conserved domain and C2H2 (C-X4-5-C-X23-HXH) zinc-finger motif and belonged to GroupⅡb of WRKY family. Homology analysis showed that PmWRKY164 had higher similarity with other plants in the conservative region of the WRKY family. Phylogenetic analysis revealed that PmWRKY164 was mostly close to the WRKY transcription factor of Taxus wallichiana var. chinensis and Picea abies. Spatiotemporal expression analysis showed that PmWRKY164 constitutively expressed in root, stem and le
作者 王庆竹 尚先文 汤纬玮 李慧平 文晓鹏 范付华 WANG Qing-Zhu;SHANG Xian-Wen;TANG Wei-Wei;LI Hui-Ping;WEN Xiao-Peng;FAN Fu-Hua(Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang 550025, China;College of Forestry, Guizhou University, Guiyang 550025, China;Institute for Forestry resources & Environment of Guizhou, Guizhou University, Guizhou University, Guiyang 550025, China)
出处 《农业生物技术学报》 CAS CSCD 北大核心 2019年第6期1016-1024,共9页 Journal of Agricultural Biotechnology
基金 国家重点研发计划(2017YFD0600303-4) 国家自然科学基金(No. 31660185) 贵州省科学技术基金(黔科合基础[2016]1051).
关键词 马尾松 低磷胁迫 WRKY转录因子 功能分析 Pinus massoniana Low phosphorus stress WRKY transcription factor Functional analysis
作者简介 通讯作者:范付华,fhfan1@gzu.edu.cn.
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