枣树novel-miR16靶基因ZjTCP4鉴定及生物信息学分析

doi:10.3969/j.issn.1004-1524.20230488

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (3): 534-543.DOI: 10.3969/j.issn.1004-1524.20230488

枣树novel-miR16靶基因ZjTCP4鉴定及生物信息学分析

张露荷¹^,²(), 王多锋¹, 张德¹, 张广忠¹, 赵通¹, 吕斌燕¹, 张洋军¹, 李毅²^,^*()

1.甘肃省林业科学研究院,甘肃兰州 730020
2.甘肃农业大学林学院,甘肃兰州 730070

收稿日期:2023-04-12 出版日期:2024-03-25 发布日期:2024-04-09
作者简介:张露荷(1989—),女,甘肃天水人,博士,副研究员,主要从事经济林抗逆研究。E-mail:1030868271@qq.com
通讯作者: *李毅,E-mail: liyi@gsau.edu.cn
基金资助:
甘肃省林业科技计划项目(2016kj048);甘肃省林业和草原科技创新项目(kjcx202006);中央财政林业科技推广示范项目(〔2018〕ZYTG 4)

Identification and bioinformatics analysis of novel-miR16 target gene ZjTCP4 in Chinese jujube

ZHANG Luhe¹^,²(), WANG Duofeng¹, ZHANG De¹, ZHANG Guangzhong¹, ZHAO Tong¹, LYU Binyan¹, ZHANG Yangjun¹, LI Yi²^,^*()

1. Gansu Academy of Forestry Sciences, Lanzhou 730020, China
2. College of Forestry, Gansu Agricultural University, Lanzhou 730070, China

Received:2023-04-12 Online:2024-03-25 Published:2024-04-09

摘要/Abstract

摘要：

TCPs转录因子主要通过调节细胞增殖和植物激素途径来调控植物器官发生和形态构型,探究枣树ZjTCP4转录因子的序列特征及其表达特性,以期为TCP转录因子响应干旱胁迫的机理研究提供参考,也可以为后续对枣树分子育种奠定基础。novel-miR16的二级结构具有miRNA典型的发夹结构,为枣树miR319家族成员;靶基因ZjTCP4的降解位点多数都在novel-miR16结合位点的第10和11个核苷酸的位置;生物信息学分析表明,ZjTCP4基因的全长cDNA序列为1 386 bp,预测编码452个氨基酸的多肽,预测编码蛋白的等电点(pI)为6.51,分子量为49 091.55 u;应该是一个膜内或膜外蛋白,为非分泌性蛋白,为亲水性蛋白质,定位于细胞核;枣树ZjTCP4基因编码的氨基酸序列与拟南芥AtTCP4最为相似,与拟南芥AtTCP17,AtTCP18和AtTCP12的相似性也较高;枣树novel-miR16在响应干旱胁迫的过程中上调表达,而其靶基因ZjTCP4在干旱胁迫下下调表达。枣树novel-miR16通过靶向调控ZjTCP4转录因子响应干旱胁迫,干旱胁迫下枣树novel-miR16的上调表达导致靶基因ZjTCP4 (CCG006181)下调表达来提高枣树的耐旱性。

关键词: 枣树, 微RNA, TCP转录因子, 生物信息学, 亚细胞定位

Abstract:

TCPs transcription factor regulates plant organogenesis and morphological configuration mainly through regulating cell proliferation and plant hormone pathways, so as to explore the sequence characteristics and expression characteristics of ZjTCP4 transcription factor of Chinese jujube, in order to provide reference for the mechanism study of TCP transcription factor response to drought stress, and lay a foundation for the subsequent molecular breeding of jujube tree. The novel-miR16 secondary structure has the typical hairpin structure of miRNA and is a member of the jujube miR319 family. Most of the degradation sites of target gene ZjTCP4 were located at the 10th and 11th nucleotides of the novel-miR16 binding sites. Bioinformatics analysis showed that the full-length cDNA sequence of ZjTCP4 gene was 1 386 bp, predicted to encode 452 amino acid polypeptide, isoelectric point (pI) of 6.51, molecular weight of 49 091.55 u. It should be an intramembranous or extracellular protein, a non-secretory protein, a hydrophilic protein, located in the nucleus. The amino acid sequence encoded by ZjTCP4 gene of Chinese jujube is most similar to AtTCP4 of Arabidopsis thaliana, and has high similarity to AtTCP17, AtTCP18 and AtTCP12 of Arabidopsis thaliana. Novel-miR16 was up-regulated in response to drought stress, while its target gene ZjTCP4 was down-regulated under drought stress. Novel-miR16 can regulate ZjTCP4 transcription factor in response to drought stress. Under drought stress, the up-regulated expression of novel-miR16 leads to down-regulated expression of target gene ZjTCP4 (CCG006181) to improve drought tolerance of Chinese jujube.

Key words: Chinese jujube, microRNA, TCP transcription factor, bioinformatics, subcellular localization

中图分类号:

S665.1

张露荷, 王多锋, 张德, 张广忠, 赵通, 吕斌燕, 张洋军, 李毅. 枣树novel-miR16靶基因ZjTCP4鉴定及生物信息学分析[J]. 浙江农业学报, 2024, 36(3): 534-543.

ZHANG Luhe, WANG Duofeng, ZHANG De, ZHANG Guangzhong, ZHAO Tong, LYU Binyan, ZHANG Yangjun, LI Yi. Identification and bioinformatics analysis of novel-miR16 target gene ZjTCP4 in Chinese jujube[J]. Acta Agriculturae Zhejiangensis, 2024, 36(3): 534-543.

图/表 8

图1 枣树novel-miR16二级发夹结构红色部分表示novel-miR16成熟序列。

Fig.1 The secondary hairpin structures of novel-miR16 in Chinese jujube Mature novel-miR16 sequences are in red.

表1 枣树novel-miR16的靶基因预测

Table 1 Predicted target genes of novel-miR16 in Chinese jujube

novel-miR16序列 Sequence of novel-miR16	靶基因 Target Acc	靶基因注释 Target gene annotation
UUGGACUGAAGGGAGCUCCC	CCG006181	transcription factor TCP4-like

图2 枣树novel-miR16靶基因的验证

Fig.2 Verification of novel-miR16 target mRNA in Chinese jujube

图3 PCR产物电泳检测 M,2 000 bp ladder Marker;1,PCR 产物。

Fig.3 Result of electrophoresis of PCR products M, 2 000 bp ladder Marker; 1, PCR products.

图4 ZjTCP4基因编码的蛋白质生物信息分析图 A,ZjTCP4基因编码的蛋白质三级结构预测;B,ZjTCP4基因编码的蛋白质跨膜结构域分析图;C,ZjTCP4基因编码的蛋白质信号肽预测;D,ZjTCP4基因编码蛋白质翻译后加工修饰预测;E,ZjTCP4基因编码的蛋白质疏水性分析。

Fig.4 Bioinformatics analysis of protein encoded by ZjTCP4 gene A, Tertiary structure prediction of protein encoded by ZjTCP4 gene;B, Transmembrane domain analysis of protein encoded by ZjTCP4 gene; C, Signal peptide prediction of protein encoded by ZjTCP4 gene;D, Post-translational modification prediction of protein encoded by ZjTCP4 gene; E, Hydrophobicity analysis of protein encoded by ZjTCP4 gene.

图5 ZjTCP4基因编码的蛋白质系统进化树

Fig.5 Phylogenetic tree of protein encoded by ZjTCP4

图6 ZjTCP4基因的亚细胞定位图从左到右分别为 CK/基因的 GFP 绿色荧光蛋白、CHI、明场和3个通道的叠加照片。

Fig.6 Subcellular localization of the ZjTCP4 protein From left to right, superimposed photos of GFP green fluorescent protein, CHI, bright field and three channels of CK/gene, respectively.

图7 枣树novel-miR16及基因靶ZjTCP4在干旱胁迫下的表达分析同一基因不同时间点间没有相同小写字母表示差异显著(P<0.05)。

Fig.7 The expression level of novel-miR16 and ZjTCP4 under drought stress in Chinese jujube No identical lowercase letters between different time points of the same gene indicate significant differences (P<0.05).

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枣树novel-miR16靶基因ZjTCP4鉴定及生物信息学分析

Identification and bioinformatics analysis of novel-miR16 target gene ZjTCP4 in Chinese jujube

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