沙棘hrh-miRn458靶向转录因子WRI1调控油脂合成

doi:10.11983/CBB22004

植物学报 ›› 2022, Vol. 57 ›› Issue (5): 635-648.DOI: 10.11983/CBB22004

沙棘hrh-miRn458靶向转录因子WRI1调控油脂合成

于淼¹, 阮成江¹^,^*(), 丁健¹, 李景滨¹, 卢顺光², 温秀凤²

¹大连民族大学资源植物研究所, 大连 116600
²水利部沙棘开发管理中心, 北京 100038

收稿日期:2022-01-06 接受日期:2022-03-18 出版日期:2022-09-01 发布日期:2022-09-09
通讯作者: 阮成江
作者简介:*E-mail: ruan@dlnu.edu.cn
基金资助:
国家自然科学基金(32071799);国家自然科学基金(32111540255);西藏中央引导地方项目(XZ202201YD0025C);水利部沙棘开发管理中心项目(2022-zg-kj-018);大连民族大学-西藏农牧学院联合基金(DLMZ-NMXY2021001)

Hrh-miRn458 Regulates Oil Biosynthesis of Sea Buckthorn via Targeting Transcription Factor WRI1

Yu Miao¹, Ruan Chengjiang¹^,^*(), Ding Jian¹, Li Jingbin¹, Lu Shunguang², Wen Xiufeng²

¹Institute of Plant Resources, Dalian Minzu University, Dalian 116600, China
²Seabuckthorn Development Management Center, Ministry of Water Resources, Beijing 100038, China

Received:2022-01-06 Accepted:2022-03-18 Online:2022-09-01 Published:2022-09-09
Contact: Ruan Chengjiang
About author:*E-mail: ruan@dlnu.edu.cn

摘要/Abstract

摘要： 转录因子WRI1 (WRINKLED 1)在油脂合成过程中发挥重要调控作用, 其转录和翻译水平调控机理以及下游的靶基因现已基本明确, 但尚未见其转录后调控的报道。为探讨沙棘(Hippophae rhamnoides) hrh-miRn458与转录因子WRI1之间的靶向关系, 并阐明其在果肉和种子发育过程中的表达模式, 通过生物信息学方法预测hrh-miRn458成熟体序列及其与候选靶基因WRI1的结合位点, 采用双荧光素酶报告基因检测和RNA pull down技术相结合的方法验证hrh-miRn458与WRI1基因之间的靶向关系, 并应用qRT-PCR方法分析hrh-miRn458与WRI1在沙棘不同发育期果肉和种子油脂合成过程中的表达变化。结果表明, 生物信息学预测WRI1基因的CDS区第309-327位与hrh-miRn458成熟体序列的15个碱基互补; 荧光检测显示pmirGLO-WRI1-WT+hrh-miRn458 mimics显著抑制荧光素酶活性(P<0.001); RNA pull down实验证实hrh-miRn458与WRI1存在互作关系; 不同发育期果肉和种子中hrh-miRn458的表达量总体呈先下降后上升趋势, 其靶基因WRI1的表达量则呈先上升后降低趋势; 同一时期, 果肉中hrh-miRn458的表达量明显低于种子, 而果肉中的WRI1表达量则明显高于种子。综上, 沙棘hrh-miRn458靶向转录因子WRI1, 且二者在果肉和种子油脂合成过程中存在负调控关系。研究结果为深入理解沙棘种子油脂合成机制及培育高油品种提供了参考依据。

关键词: 沙棘, 油脂合成, hrh-miRn458, 转录因子WRI1, qRT-PCR

Abstract: Transcription factor WRINKLED 1 (WRI1) plays important regulatory role in oil biosynthesis. Most studies focused on the regulation mechanism of its transcription, translation, and downstream target genes. Very few reports on its post transcriptional regulation. To explore the relationship between hrh-miRn458 and transcription factor WRI1 of sea buckthorn, and analyze their expression dynamics in oil biosynthesis of pulp and seeds, the bioinformatic method was used to predict the binding sites in the CDS region of the candidate target gene WRI1 by hrh-miRn458 mature sequence. Double luciferase reporter system and RNA pull down assay were used to verify whether hrh-miRn458 could target the CDS region of WRI1. The expression of hrh-miRn458 and WRI1 were analyzed using real-time fluorescent PCR technology in developing sea buckthorn pulp and seeds. The results showed that the positions between 309-327 of the CDS region of WRI1 gene were complementary to 15 bases of the mature hrh-miRn458 sequence. The vector of pmirGLO-WRI1-WT+hrh-miRn458 mimics significantly inhibited the luciferase activity (P<0.001), and RNA pull down assay further verified that hrh-miRn458 could pull down WRI1 in the system. In pulp and seeds of different developing stages, the expression level of hrh-miRn458 decreased at first and then increased, but the expression level of its target gene WRI1 first increased and then decreased. At the same developmental stage, the hrh-miRn458 expression level in pulp was less than that of seeds, but the expression level of its target gene WRI1 in pulp was more than that of seeds. Thus, sea buckthorn hrh-miRn458 may target the WRI1, and there is negatively regulative relationship between them. These data provide scientific references for further understanding the seed oil biosynthesis mechanism of sea buckthorn and breeding for high oil varieties.

Key words: sea buckthorn, oil biosynthesis, hrh-miRn458, transcription factor WRI1, qRT-PCR

于淼, 阮成江, 丁健, 李景滨, 卢顺光, 温秀凤. 沙棘hrh-miRn458靶向转录因子WRI1调控油脂合成. 植物学报, 2022, 57(5): 635-648.

Yu Miao, Ruan Chengjiang, Ding Jian, Li Jingbin, Lu Shunguang, Wen Xiufeng. Hrh-miRn458 Regulates Oil Biosynthesis of Sea Buckthorn via Targeting Transcription Factor WRI1. Chinese Bulletin of Botany, 2022, 57(5): 635-648.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22004

https://www.chinbullbotany.com/CN/Y2022/V57/I5/635

图/表 7

表1 qRT-PCR引物

Table 1 Primers for qRT-PCR

Gene name	Primer sequence (5′→3′)
UBQ5-F	GGCGAGTTTGACCTTCTTCTT
UBQ5-R	CCACCTTGTTCTTCGTCTCC
U6-F	GGAACGATACAGAGAAGATTAGC
U6-R	TGGAACGCTTCACGAATTTGCG
WRI1-F	AACGCTCATGGAAGAACAATAATC
WRI1-R	GCCTTGTGACTCCTTTGTAAATC
hrh-miRn458-F	GAGGAAGGAGAAGTCGAG

图1 不同发育期杂56果实和种子的表型(A)及含油率(B) 不同小写字母表示同一时期果肉和种子含油率在0.05水平差异显著。daa: 花后天数。Bar=3 mm

Figure 1 Phenotype of fruits and seeds of Za56 (A) and their oil contents (B) at different developmental stages Different lowercase letters indicate significant differences of oil content between pulp and seeds at the same developmental stage at the level of 0.05. daa: Days after anthesis. Bar=3 mm

图2 沙棘hrh-miRn458前体的二级结构和靶向WRI1基因的结合位点 (A) 前体的二级结构, 红色部分表示hrh-miRn458成熟体序列片段; (B) 靶向WRI1基因的结合位点, 竖线两端碱基表示靶向位点。

Figure 2 Precursor secondary structure of sea buckthorn hrh-miRn458 and binding sites in the targeted WRI1 gene (A) Precursor secondary structure, red indicates segments corresponding to the mature hrh-miRn458; (B) Binding sites in targeted WRI1 gene, the bases at both ends of the vertical line represent the target sites.

图3 沙棘hrh-miRn458前体成熟体序列比较(A)及HrWRI1与其它物种同源序列的系统进化关系(B)

Figure 3 Sequence alignment of mature sequence of sea buckthorn hrh-miRn458 precursor (A) and phylogenetic relationship of HrWRI1 with its homologous genes in plants (B)

图4 pmirGLO-WRI1-WT/MUT+hrh-miRn458序列鉴定及荧光素酶活性分析 (A) pmirGLO-WRI1-WT/MUT+hrh-miRn458 PCR产物序列, 黄色部分为突变序列; (B) pmirGLO-WRI1-WT/MUT质粒酶切鉴定(a: XhoI酶切质粒; b: 野生型DNA质粒; c: NheI酶切质粒; d: 突变型DNA质粒); (C) 荧光素酶活性分析(1: pmirGLO- WRI1-WT+mimics NC质粒; 2: pmirGLO-WRI1-WT+hrh-miRn458 mimics质粒; 3: pmirGLO-WRI1-MUT+mimics NC质粒; 4: pmirGLO-WRI1-MUT+hrh-miRn458 mimics质粒)。*** 差异极显著(P<0.001); ns: 无显著差异(P>0.05)。

Figure 4 Sequences identification of pmirGLO-WRI1-WT/MUT+hrh-miRn458 and activity analysis of dual luciferase (A) Sequences of PCR products of pmirGLO-WRI1-WT/MUT+hrh-miRn458 from plasmid, yellow letters indicated mutation sequence; (B) Identification of pmirGLO-WRI1-WT/MUT plasmid digestion (a: Plasmid digested by XhoI enzyme; b: Plasmid DNA of WT; c: Plasmid digested by NheI enzyme; d: Plasmid DNA of MUT); (C) Activity analysis of dual luciferase (the numbers of 1, 2, 3 and 4 represents plasmid pmirGLO-WRI1-WT+mimics NC, pmirGLO-WRI1-WT+hrh-miRn458 mimics, pmirGLO-WRI1-MUT+mimics NC and pmirGLO-WRI1-MUT+hrh-miRn458 mimics, respectively.). *** Significant difference at the level of P<0.001; ns means no significant difference (P>0.05).

图5 hrh-miRn458及WRI1表达水平的qRT-PCR检测 (A) 探针组和NC组富集hrh-miRn458的表达水平; (B) 探针组和NC组富集WRI1的表达水平。 **和***分别表示NC组和探针组富集hrh-miRn458与WRI1间表达在0.01和0.001水平上差异显著。

Figure 5 qRT-PCR detection of hrh-miRn458 and WRI1 expression (A) Expressions of hrh-miRn458 enriched by probe and NC groups; (B) Expression of WRI1 enriched by probe and NC groups. * and *** indicated significant differences of expression of hrh-miRn458 and WRI1 between NC and probe groups at the level of 0.01 and 0.001, respectively.

图6 沙棘不同发育期种子和果肉中hrh-miRn458和WRI1的表达变化 (A) 沙棘种子中hrh-miRn458和WRI1的表达; (B) 沙棘果肉中hrh-miRn458和WRI1的表达。不同小写字母表示同一时期种子和果肉中hrh-miRn458与WRI1表达量间在0.05水平上差异显著。daa: 花后天数

Figure 6 Expression of hrh-miRn458 and WRI1 in sea buckthorn seeds and pulp at different developmental stages (A) Expression of hrh-miRn458 and WRI1 in sea buckthorn seed; (B) Expression of hrh-miRn458 and WRI1 in sea buckthorn pulp. Different lowercase letters indicate significant differences of oil content between hrh-miRn458 and WRI1 of seeds and pulp at the same developmental stage at the level of 0.05. daa: Days after anthesis

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沙棘hrh-miRn458靶向转录因子WRI1调控油脂合成

Hrh-miRn458 Regulates Oil Biosynthesis of Sea Buckthorn via Targeting Transcription Factor WRI1

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