蓖麻<i>RcNAC100-like</i>基因克隆及表达特性分析

doi:10.7668/hbnxb.20194389

摘要/Abstract

摘要：

NAC(NAM,ATAF1/2,CUC2)基因是胁迫信号转导网络中重要的调节因子,克隆蓖麻NAC基因,研究其分子特征及表达特性,旨在为研究蓖麻NAC基因的潜在功能提供数据支持。采取RT-PCR技术克隆通篦5号的RcNAC100-like基因并分析其分子特征,包括生物信息学、亚细胞定位、表达模式和转录激活域分析。结果表明,RcNAC100-like基因的cDNA全长1 244 bp,包含1 086 bp的CDS,共推导361个氨基酸,该蛋白质的不规则卷曲和α-螺旋结构较多,是1个亲水的、非分泌性蛋白质;系统进化分析结果表明,RcNAC100-like蛋白质与木薯和橡胶树NAC蛋白质的亲缘关系最近,且motif组成及位置高度相似;RcNAC100-like蛋白的亚细胞定位结果与预测结果一致,定位在细胞核;RcNAC100-like启动子的顺式作用元件预测结果显示,该区域有多个环境响应类元件和生长发育类相关元件;基因表达模式分析结果表明,RcNAC100-like基因有组织表达特异性,在根中的相对表达量最高;另外,该基因能够对不利环境(干旱、盐、冷和ABA胁迫)迅速做出反应并积极地表达,说明RcNAC100-like基因可能是蓖麻对逆境响应的关键基因;转录激活试验结果表明,RcNAC100-like转录因子在酵母中具有转录激活活性。综上,RcNAC100-like基因可能在蓖麻的抗逆境过程中发挥重要作用。

关键词: 蓖麻, RcNAC100-like基因, 生物信息学, 克隆, 表达

Abstract:

The NAC(NAM,ATAF1/2,CUC2)gene is an important regulatory factor in the stress signal transduction network.Cloning the NAC gene in castor,studying its molecular characteristics and expression characteristics,aiming to provide data support for the potential function of the castor NAC gene.The RcNAC100-like gene of Tongbi No.5 was cloned by RT-PCR technology,and its molecular characteristics were analyzed,including bioinformatics,subcellular localization,expression patterns,and transcription activation domain analysis.The results showed that the full length of RcNAC100-like gene cDNA was 1 244 bp,including a 1 086 bp coding sequence(CDS),encoding 361 amino acids.The protein had more irregular coil and α-helix structures,and was a hydrophilic,non-secretory protein.Phylogenetic analysis showed that the RcNAC100-like protein was most closely related to NAC proteins in Manihot esculenta and Hevea brasiliensis,with highly similar motif composition and positioning.The subcellular localization of RcNAC100-like protein was consistent with the predicted results,located in the nucleus.The predicted cis-acting elements in the RcNAC100-like promoter region indicate the presence of multiple environmental response and growth-related elements.Expression pattern analysis showed that the RcNAC100-like gene had tissue-specific expression,with the highest relative expression level in the roots.Additionally,the gene could respond rapidly to adverse environments(drought,salt,cold,and ABA stress)and actively express,indicating that the RcNAC100-like gene might be a key gene in the castor's response to stress.Transcription activation assay results showed that the RcNAC100-like transcription factor has transcriptional activation activity in yeast.In summary,the RcNAC100-like gene may play an important role in the castor's resistance to adversity.

Key words: Castor, RcNAC100-like gene, Bioinformatics, Clone, Expression

李艳肖, 王丽娜, 朱贵爽, 刘鹏, 向殿军. 蓖麻RcNAC100-like基因克隆及表达特性分析[J]. 华北农学报, 2024, 39(3): 67-76. doi: 10.7668/hbnxb.20194389.

LI Yanxiao, WANG Lina, ZHU Guishuang, LIU Peng, XIANG Dianjun. Cloning and Expression Characteristics Analysis of RcNAC100-like Gene in Castor[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 67-76. doi: 10.7668/hbnxb.20194389.

http://www.hbnxb.net/CN/Y2024/V39/I3/67

图/表 12

表1 引物序列及用途

Tab.1 Primer sequences and uses

引物名称 Primer names	描述 Description	引物序列(5'—3') Primer sequence(5'—3')
RcNAC100-like	基因克隆	F:CAAGAGAACAGTAGGGTTTTAGC
		R:CAGCGAGCCAAACCATTTTTC
q-RT-RcNAC100-like	RcNAC100-like表达水平	F:CTCTATCCACAACCTCCCCA
		R:CCGAAAGAACCTAACCTCAC
q-RT-Actin	Actin表达水平	F:TCCCTCAGTACGTTCCAGCA
		R:CACCTCCATACTCCTCCCT
P-RcNAC100-like	启动子克隆	F:TATATAATGCTTATTTTTAATATAG
		R:TTCTTTAAAGAGAAGAAAACGAGAG
35S∷RcNAC100-like-GFP	亚细胞定位	F:AGAACACGGGGGACGGATCCATGGAAAACATTTCTGGTCTTG(BamH Ⅰ)
		R:ACCATGGTGTCGACGTCGACGTAGTTCCAAAGACAATCAAGGTCC(Sal Ⅰ)
RcNAC100-like:1~423	转录激活活性检测	F:CATATGATGGAAAACATTTCTGGTCTTG(Nde Ⅰ)
		R:GGATCCTAATCTGTATTCATGCATGACCC(BamH Ⅰ)
RcNAC100-like:424~1 086	转录激活活性检测	F:CATATGGAGGGTAAATACTCTATCCAC(Nde Ⅰ)
		R:GGATCCTCAGTAGTTCCAAAGACAATC(BamH Ⅰ)

图1 RcNAC100-like基因的克隆 1.RcNAC100-like基因;M.DL2000 Marker。

Fig.1 Cloning of RcNAC100-like gene 1.RcNAC100-like gene;M.DL2000 Marker.

图2 RcNAC100-like基因推导的氨基酸序列

Fig.2 The deduced amino acid sequence of RcNAC100-like gene

图3 RcNAC100-like基因在染色体上的位置、共线性及结构域分析 A.RcNAC100-like基因的位置及共线性关系;B.RcNAC100-like蛋白质的结构域预测。

Fig.3 Location,collinearity and structural domain analysis of RcNAC100-like gene on chromosome A.Location and collinearity of the RcNAC100-like gene;B.Structural domain prediction of the RcNAC100-like protein.

图4 RcNAC100-like蛋白质的磷酸化位点预测

Fig.4 Prediction of phosphorylation sites for RcNAC100-like protein

图5 RcNAC100-like蛋白质的结构分析 A.跨膜结构域; B.信号肽结构;C.二级结构;D.三级结构(左)和拉式构象打分图(右)。

Fig.5 Structural analysis of RcNAC100-like protein A.Transmembrane domain;B.Signal peptide structure;C.Secondary structure;D.Tertiary structure(left)and pull-type conformation scoring diagram(right).

图6 RcNAC100-like蛋白质的进化及保守基序分析 A.NAC蛋白质的进化树分析;B.保守基序分析;C.结构域分析。

Fig.6 Evolution and conserved motif analysis of RcNAC100-like proteins A.Evolutionary tree analysis of NAC proteins;B.Conserved motif analysis;C.Structural domain analysis.

图7 RcNAC100-like启动子顺式作用元件分析 A.RcNAC100-like启动子克隆:1.RcNAC100-like启动子;M.DL2000 Marker。B.RcNAC100-like顺式作用元件预测。

Fig.7 Analysis of cis-acting elements in RcNAC100-like promoter A.RcNAC100-like promoter clone:1.RcNAC100-like promoter;M.DL2000 Marker.B.RcNAC100-like cis-acting element prediction.

图8 RcNAC100-like蛋白的亚细胞定位

Fig.8 Subcellular localization of RcNAC100-like protein

图9 RcNAC100-like基因的组织表达模式

Fig.9 Tissue expression profile of the RcNAC100-like gene

图10 RcNAC100-like基因的非生物胁迫表达模式不同小写字母表示处理间差异显著(P<0.05)。

Fig.10 The abiotic stress expression pattern of RcNAC100-like gene Different lowercase letters indicate significant differences among treatments(P<0.05).

图11 RcNAC100-like转录活性分析 A.pGBKT7、pGBKT7-FL、pGBKT7-C和pGBKT7-N分别是空载体、RcNAC100-like全长(1~1 086 bp)、N端(1~423 bp)和C端(424~1 086 bp)亚克隆示意图;B.从左至右依次是各载体在平板上的位置、SD/-Trp培养基和SD/-Ade/-His/-Trp+ X-α-gal培养基。

Fig.11 Analysis of RcNAC100-like transcriptional activity A.pGBKT7,pGBKT7-FL,pGBKT7-C,and pGBKT7-N represent the empty vector, RcNAC100-like full-length(1—1 086 bp),N-terminal(1—423 bp),and C-terminal(424—1 086 bp)subcloning schematic,respectively;B.From left to right are the positions of each vector on the plate,SD/-Trp medium,and SD/-Ade/-His/-Trp+ X-α-gal medium.

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Cloning and Expression Characteristics Analysis of RcNAC100-like Gene in Castor

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蓖麻RcNAC100-like基因克隆及表达特性分析

Cloning and Expression Characteristics Analysis of RcNAC100-like Gene in Castor

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