麻风树JcMYB27基因的克隆与功能分析

doi:10.3969/j.issn.1004-1524.20240956

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (8): 1658-1665.DOI: 10.3969/j.issn.1004-1524.20240956

麻风树JcMYB27基因的克隆与功能分析

王小慧(), 贾赛男, 冯佳宇, 尹馨悦, 刘子萱, 刘雯洁, 赵帅滢, 王姝婧, 唐跃辉()

周口师范学院生命科学与农学学院,河南周口 466001

收稿日期:2024-11-11 出版日期:2025-08-25 发布日期:2025-09-03
作者简介:王小慧(1994—),女,河南平顶山人,博士,讲师,研究方向为植物基因克隆与功能。E-mail:xhw2022tjin@163.com
通讯作者: *唐跃辉,E-mail:yhtang2005@163.com
基金资助:
河南省高等学校重点科研项目(24A180029);河南省大学生创新创业训练计划项目(202410478007)

Cloning and function analysis of JcMBY27 gene from Jatropha curcas

WANG Xiaohui(), JIA Sainan, FENG Jiayu, YIN Xinyue, LIU Zixuan, LIU Wenjie, ZHAO Shuaiying, WANG Shujing, TANG Yuehui()

College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, Henan, China

Received:2024-11-11 Online:2025-08-25 Published:2025-09-03
Contact: TANG Yuehui

摘要/Abstract

摘要： MYB转录因子在植物生长发育和次生代谢物合成中起重要的调控作用。为研究麻风树MYB转录因子JcMYB27的功能,本研究以麻风树为材料,通过反转录PCR(RT-PCR)技术克隆麻风树JcMYB27基因,使用实时荧光定量PCR(qRT-PCR)技术分析JcMYB27基因的表达模式,通过拟南芥原生质体瞬时转化技术分析JcMYB27的亚细胞定位,利用花序浸染法获得JcMYB27过表达拟南芥,并分析其在正常生长条件下的表型、根毛数量和5%蔗糖诱导下叶片的花青素含量。结果显示,JcMYB27基因的开放阅读框全长243 bp,编码80个氨基酸。表达模式分析显示,JcMYB27基因在麻风树根中表达量最高。亚细胞定位分析显示,JcMYB27定位于细胞核和细胞质。正常生长条件下,在拟南芥中过表达JcMYB27基因可以增加转基因拟南芥的根毛数量,降低叶片花青素含量。蔗糖诱导条件下,JcMYB27过表达拟南芥叶片的花青素含量显著低于野生型。此外,与野生型相比,JcMYB27基因过表达拟南芥中花青素合成相关基因DFR、LDOX和UF3GT的相对表达量显著降低。综上所述,JcMYB27基因在麻风树的根毛发育和花青素合成中发挥调控作用。

关键词: 麻风树, JcMYB27基因, MYB转录因子, 拟南芥, 根毛, 花青素

Abstract:

MYB transcription factors play an important regulatory role in plant growth, development and secondary metabolite synthesis. To investigate the function of the MYB transcription factor gene JcMYB27 in Jatropha curcas, this study used J. curcas as material, and cloned the JcMYB27 gene by reverse transcription polymerase chain reaction (RT-PCR), analyzed its expression patterns using quantitative real-time PCR (qRT-PCR), determined its subcellular localization through transient expression in Arabidopsis thaliana protoplasts, and generated JcMYB27-overexpression Arabidopsis thaliana plants by the floral dip method. Phenotypic analyses were conducted under normal growth conditions, including root hair number and leaf anthocyanin content under 5% sucrose induction. The results showed that the length of JcMYB27 open reading frame was 243 bp, encoding 80 amino acids. Expression pattern analysis showed that JcMYB27 was expressed most highly in J. curcas roots. Subcellular localization analysis demonstrated that JcMYB27 was localized in the nucleus and cytoplasm. Under normal growth conditions, overexpression of the JcMYB27 gene in A. thaliana increased root hair numbers while reducing the anthocyanin content in leaves. Under sucrose induction, JcMYB27-overexpression A. thaliana exhibited significantly lower leaf anthocyanin content compared with wild-type plants. Additionally, the expressions of anthocyanin synthesis-related genes DFR, LDOX and UF3GT in JcMYB27-overexpression A. thaliana were significantly lower than those in wild type. In conclusion, JcMYB27 gene plays a regulatory role in root hair development and anthocyanin biosynthesis in J. curcas.

Key words: Jatropha curcas, JcMYB27 gene, MYB transcription factor, Arabidopsis thaliana, root hair, anthocyanin

中图分类号:

S718.3

王小慧, 贾赛男, 冯佳宇, 尹馨悦, 刘子萱, 刘雯洁, 赵帅滢, 王姝婧, 唐跃辉. 麻风树JcMYB27基因的克隆与功能分析[J]. 浙江农业学报, 2025, 37(8): 1658-1665.

WANG Xiaohui, JIA Sainan, FENG Jiayu, YIN Xinyue, LIU Zixuan, LIU Wenjie, ZHAO Shuaiying, WANG Shujing, TANG Yuehui. Cloning and function analysis of JcMBY27 gene from Jatropha curcas[J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1658-1665.

图/表 7

表1 本研究所用引物

Table 1 The primers used in this study

基因名称Gene name	正向引物序列Forward primer sequences(5'-3')	反向引物序列Reverse primerr sequences(5'-3')
qRT-PCR引物qRT-PCR primers
JcMYB27	GGTTGGAGAGAGGTGGTCGTTA	TCATCTCTCAGTTGATGATGAGCT
DFR	TGGTCGGTCCATTCATCACAACG	CTTGGCGGCTGCTTGTTCGTATA
LDOX	TCTACGAGGGCAAATGGGTCACT	TCCGGCAACGGCTTAAGAACAATC
UF3GT	ATCGAATGAATCGTCAAGCATGAG	TGAGGGATAGAGATGGTGTGGAAAG
CHS	AAGTTGGTCTCACCTTCCATCTCCT	TGTCGCCCTCATCTTCTCTTCCTT
JcActin	TAATGGTCCCTCTGGATGTG	AGAAAAGAAAAGAAAAAAGCAGC
AtActin2	GCACCCTGTTCTTCTTACCG	AACCCTCGTAGATTGGCACA
基因克隆引物Gene coding primers
JcMYB27	GCCATACTTCCCATACCTCTAA	CTGGGTGACTAAACTTCATCTCT

图1 JcMYB27基因的克隆及其同源蛋白的氨基酸序列和系统发育树 A,JcMYB27基因PCR产物电泳图;B,JcMYB27及其同源蛋白MYB结构域氨基酸序列分析;C,系统发育树分析;D,JcMYB27蛋白和拟南芥中同源蛋白全长氨基酸序列比对。

Fig.1 Cloning of JcMYB27 and amino acid sequence and phylogenetic tree of its homologous proteins A. Electrophoresis analysis of JcMYB27 PCR products; B. Amino acid sequence analysis of JcMYB27 and its homologous protein MYB domain; C, Phylogenetic tree analysis; D, Full-length amino acid sequence alignment of JcMYB27 protein and homologous proteins in Arabidopsis thaliana.

图2 JcMYB27基因在麻风树幼苗中的器官表达特异性

Fig.2 Organ-specific expression of the JcMYB27 gene in seedlings of Jatropha curcas

图3 JcMYB27蛋白在拟南芥原生质体中的亚细胞定位

Fig.3 Subcellular localization of JcMYB27 protein in Arabidopsis thaliana protoplasts

图4 拟南芥植株的表型和根毛 A,JcMYB27基因的表达情况;B,28 d拟南芥的表型;C,拟南芥的根毛表型;D,拟南芥的开花时间;E,拟南芥的单株产量。WT,野生型拟南芥;OE1、OE2、OE3为JcMYB27过表达拟南芥。

Fig.4 Phenotype and root hairs of Arabidopsis thaliana plants A, Expression of JcMYB27 gene; B, Phenotype of 28-day-old Arabidopsis thaliana; C, Root hair phenotype of Arabidopsis thaliana; D, Flowering time of Arabidopsis thaliana; E, Yield per plant of Arabidopsis thaliana. WT, Wild-type Arabidopsis thaliana; OE1, OE2 and OE3, JcMYB27 overexpressing Arabidopsis thaliana.

图5 拟南芥植株表型和叶片的花青素含量 A,拟南芥在1/2 MS培养基和含5%蔗糖的1/2 MS培养基的生长情况;B,正常生长条件下(1/2 MS培养基)拟南芥叶片的花青素含量;C,5%蔗糖诱导下(含5%的1/2 MS培养基)拟南芥叶片的花青素含量。**表示与野生型相比差异显著(p<0.01)。

Fig.5 Phenotype of Arabidopsis thaliana plants and the anthocyanin content in leaves A, Growth status ofArabidopsis thaliana on 1/2 MS medium and 1/2 MS medium containing 5% sucrose; B, Anthocyanin content in leaves of Arabidopsis thaliana under normal growth conditions (1/2 MS medium); C, Anthocyanin content in leaves of Arabidopsis thaliana under 5% sucrose induction (1/2 MS medium containing 5% sucrose). **indicates significant difference compared with the wild type (p< 0.01).

图6 拟南芥叶片中花青素合成相关基因的相对表达量 **表示与野生型相比差异显著(p<0.01)。

Fig.6 Relative expression levels of anthocyanin biosynthesis-related genes in Arabidopsis thaliana leaves ** indicates significant difference compared with the wild type (p<0.01).

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麻风树JcMYB27基因的克隆与功能分析

Cloning and function analysis of JcMBY27 gene from Jatropha curcas

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