摘要
【目的】膜内在水通道蛋白(NIPs)是细胞膜上运输水分子和一些小分子物质的膜蛋白,在植物生长以及抵御逆境胁迫等方面发挥着重要作用。温度和水分是影响竹子生长发育的重要环境因子,研究温度和干旱胁迫条件下毛竹NIP家族成员的表达模式,以揭示其在毛竹应答胁迫中的功能。【方法】利用生物信息学软件对毛竹基因组中NIP家族成员基因及其启动子序列进行系统分析,基于转录组数据分析基因在毛竹不同组织中的表达模式,并用实时荧光定量PCR(qPCR)技术检测基因在温度和干旱胁迫条件下的表达特征,构建2个NIP基因的酵母表达载体,分析它们的表达对酵母抗干旱和盐胁迫能力的影响。【结果】在毛竹基因组中共获得14个编码完整NIP蛋白的基因(PeNIP1-1—PeNIP1-6、PeNIP2-1—PeNIP2-4和PeNIP3-1—PeNIP3-4),含有3~5个内含子;在PeNIPs启动子区域中含有多种与胁迫、激素相关的调控元件;PeNIPs编码蛋白的氨基酸长度为235~297 aa,相对分子量为24.03~31.84 kDa;亚细胞定位预测显示所有PeNIPs均定位于细胞质膜上。共线性分析表明,有12个PeNIPs与水稻8个NIP基因存在27对片段重复,它们的非同义对同义取代比(Ka/Ks)均小于1,表明毛竹PeNIPs经复制后主要经历了较强的纯化选择。系统进化分析表明,来自毛竹和其他5个物种的NIP蛋白可分为3类(Ⅰ、Ⅱ、Ⅲ),毛竹在各类中的成员依次为6、4和4个。PeNIPs共包含10个保守基序,其中基序1、2和4为PeNIPs所共有。转录组表达谱热图分析表明,PeNIPs在不同组织中的表达存在一定的差异,如Ⅰ类的PeNIP1-3、PeNIP1-4和PeNIP1-5在根中表达,而在笋中几乎不表达;Ⅱ类的4个PeNIP2s以及Ⅲ类的PeNIP3-1和PeNIP3-2在根和笋中表达量较高。qPCR结果显示,随着胁迫时间的延长,4℃处理下8个基因上调表达,2个基因下调表达;42℃处理下,2个基因上调表达,4个基因下调表达;而干旱胁迫下3个基因上调表达。表达PeNIP1-1和PeNIP2-2的酵母在添加山梨醇或NaCl培养基上的生长优于对照。【结论】从毛竹中共鉴定出14个NIP家族基因成员(PeNIPs),各基因的分子特征、表达模式均存在着一定差异,说明它们在毛竹生长发育的不同阶段和响应环境胁迫中可能发挥着不同的作用;在酵母表达的PeNIP1-1和PeNIP2-2能够一定程度上提高酵母的抗干旱和盐胁迫能力,说明它们在毛竹应对逆境胁迫中可能发挥着重要作用。
【Objective】Nodulin 26-like intrinsic aquaporin proteins(NIPs)are transmembrane proteins essential for controlling the flow of water and other molecules,which play important roles in plant growth and stress tolerance.Temperature and water are important environmental factors for the growth and development of bamboo,the expression patterns of NIP family members of Moso bamboo(Phllostachys edulis)under temperature and drought stresses are of great significance to reveal their functions in response to stresses.【Method】A genome-wide analysis of NIP genes and their promoter sequences in Moso bamboo was conducted comprehensively using bioinformatics software.The tissue expression patterns of PeNIPs were analyzed using the transcriptome data,and their expression patterns under temperature and drought stresses were validated by quantitative real-time PCR(qPCR).Two yeast expression vectors of PeNIPs were constructed to analyze their effects on drought and salt stress tolerance of yeast.【Result】A total of 14 NIP genes were obtained from the genome of Moso bamboo(PeNIP1-1-PeNIP1-6,PeNIP2-1-PeNIP2-4 and PeNIP3-1-PeNIP3-4),of which the number of introns varied from 3 to 5.A variety of stress and hormone-related regulatory elements were found in the promoters of PeNIPs.The proteins encoded by PeNIPs had completely conserved domains of NIP family.The length of PeNIPs ranged from 235 aa to 297 aa with molecular weight of 24.03-31.84 kDa.The prediction of subcellular localization showed that all PeNIPs were localized on the plasma membrane.Collinearity analysis showed that a total of 27 pairs of fragments were found in 12 PeNIPs and 8 rice NIP genes.All the values of nonsynonymous and synonymous substitution ratios(Ka/Ks)were less than 1,indicating that these PeNIPs had undergone strong purification selection after replication in evolution.In the phylogenetic tree constructed with NIPs of P.edulis and other 5 species,all the NIPs were clustered into three groups(Ⅰ,Ⅱ,Ⅲ),which included 6,4,and 4 members of PeNIPs,respectively.A total of 10 conserved motifs were found in PeNIPs,of which motif 1,2 and 4 were common to all PeNIPs.Gene-specific expression analysis based on transcriptome data showed that there were some differences in the expression levels of different PeNIPs in 26 different tissues of Moso bamboo.For examples,PeNIP1-3,PeNIP1-4 and PeNIP1-5 of group I expressed in roots,but hardly expressed in bamboo shoots;four PeNIP2s of groupⅡas well as PeNIP3-1 and PeNIP3-2 of groupⅢhad higher expression levels in roots and shoots.The results of qPCR demonstrated that with the extension of stress duration,8 genes were up-regulated and 2 genes were down-regulated under 4℃stresses,and 2 genes were up-regulated and 4 genes were down-regulated under 42℃stresses.While under the drought stresses there were 3 genes up-regulated.The yeasts expressing PeNIP1-1 and PeNIP2-2 all grew better on the media supplemented with sorbitol or NaCl than the control.【Conclusion】Fourteen PeNIPs belonging to NIP family were identified in Moso bamboo.There were some differences in the molecular characteristics and expression patterns of PeNIPs,suggesting that they might play different roles in the growth and development of Moso bamboo and in response to environmental stress.The tolerance ability of yeasts expressing PeNIP1-1 and PeNIP2-2 were improved to a certain degree,indicating that they might have important roles in the stress response of Moso bamboo.
作者
朱成磊
杨克彬
徐秀荣
马霜
李晓佩
高志民
Zhu Chenglei;Yang Kebin;Xu Xiurong;Ma Shuang;Li Xiaopei;Gao Zhimin(Institute of Gene Science and Industrialization for Bamboo and Rattan Resources,International Center for Bamboo and Rattan Key Laboratory of National Forestry and Grassland Administration/Beijing for Bamboo&Rattan Science and Technology,Beijing 100102;Southwest University of Science and Technology,Mianyang 621010)
出处
《林业科学》
EI
CAS
CSCD
北大核心
2021年第1期64-76,共13页
Scientia Silvae Sinicae
基金
国际竹藤中心基本科研业务费专项资金项目(1632018005)
国家自然科学基金项目(31971736)。
关键词
毛竹
膜内在水通道蛋白
分子特征
基因表达
Phyllostachys edulis
nodulin 26-like intrinsic aquaporin protein
molecular characteristics
gene expression
