木薯MeMYB106基因克隆及其在叶片花青素合成中的功能

doi:10.13560/j.cnki.biotech.bull.1985.2025-0856

摘要/Abstract

摘要：

目的 MYB转录因子可参与植物类黄酮及花青素的生物合成，深入挖掘调控木薯叶片花青素合成的MYB转录因子并验证其功能，为深入解析花青素合成的分子机制及叶色改良提供理论依据。方法以华南9号（SC9）木薯叶片cDNA为模板克隆MeMYB106基因，在烟草中瞬时表达该蛋白明确其亚细胞定位，RT-qPCR技术分析MeMYB106在不同组织以及不同颜色叶片中的表达模式。利用病毒诱导的基因沉默技术（virus-induced gene silencing, VIGS）分析该基因在叶片花青素合成中的功能。采用酵母单杂交及双荧光素酶报告系统（dual luciferase reporter assay, Dual-LUC）检测MeMYB106对花青素还原酶（anthocyanidin reductase, ANR）基因MeANR的调控关系。结果从SC9叶片中克隆得到1个MeMYB106基因，其编码区为1 194 bp，编码氨基酸数量为398个。亚细胞定位显示MeMYB106定位于细胞核。组织特异性表达分析发现，MeMYB106在SC9叶片、腋芽、茎和块根中高表达，且在绿叶品种叶片中的表达显著高于紫叶品种。在SC9木薯中沉默MeMYB106发现沉默植株新生叶片颜色变紫，且不同沉默效率植株叶片紫色程度不同，沉默植株叶片中花青素含量显著高于空载体对照。进一步分析发现MeMYB106可结合MeANR启动子区域促进其表达，进而影响叶片花青素的生物合成。结论木薯MeMYB106转录因子可调控MeANR基因进而负向调控花青素的生物合成，沉默MeMYB106更有利于花青素积累。

关键词: 木薯, MeMYB106转录因子, 花青素, 基因克隆, 功能分析

Abstract:

Objective MYB transcription factors are primarily participate in the biosynthesis of flavonoids and anthocyanins. To explore MYB transcription factors in regulating anthocyanin biosynthesis in cassava leaves and verify its biological function would provide a theoretical basis for elucidating the molecular mechanisms of anthocyanin synthesis and leaves color improvement in cassava (Manihot esculenta Crantz). Method The coding sequence of MeMYB106 was cloned using cDNA from South China 9 (SC9) leaves as a template. Transient expression in tobacco was performed to determine its subcellular localization. qRT-PCR was applied to analyze the expression patterns of MeMYB106 in different tissues and differently colored leaves. Virus-induced gene silencing (VIGS) was employed to investigate the gene function in anthocyanin synthesis. Yeast one-hybrid assays and dual luciferase reporter assay were used to examine the regulatory relationship between MeMYB106 and anthocyanidin reductase (MeANR) gene. Result A MeMYB106 gene was cloned from SC9 leaves, with a coding region of 1 194 bp encoding 398 amino acids. The subcellular localization revealed that MeMYB106 was localized in the nucleus. Tissue-specific expression analysis showed that MeMYB106 was highly expressed in SC9 leaves, axillary buds, and stems, with significantly higher expression in green-leaves varieties compared with purple-leaves varieties. Silencing MeMYB106 in SC9 resulted in purple pigmentation in newly emerged leaves, with varying degrees of purple pigmentation depending on silencing efficiency. The anthocyanin content in the leaves of silenced plants was significantly higher than that in empty vector control. Further analysis demonstrated that MeMYB106 bound to the promoter region of MeANR to enhance its expression, thereby influencing anthocyanin biosynthesis in cassava leaves. Conclusion MeMYB106 transcription factor in cassava negatively regulate the biosynthesis of anthocyanins, and MeMYB106 positively regulate the expression of MeANR. Silencing MeMYB106 promotes anthocyanin accumulation in cassava leaves.

Key words: cassava, MeMYB106 transcription factor, anthocyanin, gene cloning, functional analysis

安飞飞, 罗秀芹, 蔡杰, 薛晶晶, 朱文丽. 木薯MeMYB106基因克隆及其在叶片花青素合成中的功能[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0856.

AN Fei-fei, LUO Xiu-qin, CAI Jie, XUE Jing-jing, ZHU Wen-li. Cloning of MeMYB106 in Cassava and Its Function in Anthocyanin Biosynthesis of Leaves[J]. Biotechnology Bulletin, doi: 10.13560/j.cnki.biotech.bull.1985.2025-0856.

图/表 9

表1 载体构建、实时荧光定量PCR及测序引物

Table 1 Primers designed for vector construction, qRT-PCR and sequencing

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	用途 Application
MeMYB106-F	ATGGGTCGATCGCCATGCTG	基因克隆 Gene clone
MeMYB106-R	TTAGAAGATGGGCGAATCTG	基因克隆 Gene clone
qPCR-MeMYB106-F	AATACTGTCGGTGGTCCTA	荧光定量PCR RT-qPCR
qPCR-MeMYB106-R	CTGAGAATGTTAGAGAAGATGTTG
qPCR-MeActin-F	TGATGAGTCTGGTCCATCCA
qPCR-MeActin-R	CCTCCTACGACCCAATCTCA
SubN-MeMYB106-F	agtggtctctgtccagtcctATGGGTCGATCGCCATGCTG	亚细胞定位 Subcellular localization
SubN-MeMYB106-R	ggtctcagcagaccacaagtTTAGAAGATGGGCGAATCTG
pGreen-SubN-F	CATGGTCCTGCTGGAGTTCGTG
pGreen-SubN-R	ACCGGCAACAGGATTCAATC
VIGS-MeMYB106-F	agtggtctctgtccagtcctGCCGCAGCGGTCTCTTGATG	基因沉默 Gene silencing
VIGS-MeMYB106-R	ggtctcagcagaccacaagtATATGATGATCTGAATTTCC
pCsCMV-F	TGGGCGCTAATTAGTTTACTGCA
pCsCMV-R	GGTCAAGACGGCTCAACTCTTCA
MeANR-promoter-F	agtggtctctgtccagtcctAAAAAGAATGAGAAAATT	启动子扩增 Promoter amplification
MeANR-promoter-R	ggtctcagcagaccacaagtTTGCAATTTTCCTTAAGG	启动子扩增 Promoter amplification
pNC-GADT7-F	TAATACGACTCACTATAGGG	酵母单杂交 Yeast one-hybrid
pNC-GADT7-F	AGATGGTGCACGATGCACAG
pNC-AbAi-F	CCTTCTGTTCGGAGATTACC
pNC-AbAi-R	TGCTACAAAGGACCTAATGT

图1 木薯MeMYB106基因PCR扩增结果M：DL5 000 marker，1-3：MeMYB106基因扩增产物

Fig. 1 Results of PCR amplification product of MeMYB106 gene in cassava (Manihot esculenta Crantz)M: DL5 000 marker. 1-3:Amplified product of MeMYB106 gene

图2 MeMYB106的亚细胞定位

Fig. 2 Subcellular localization of MeMYB106

图3 MeMYB106基因的表达模式分析A：MeMYB106在不同颜色木薯叶片中的相对表达量；B：MeMYB106在SC9不同组织中的相对表达量。*P<0.05，下同

Fig. 3 Analysis of expression profiles of MeMYB106A: Relative expressions of MeMYB106 in the leaves with different color. B: Relative expressions of MeMYB106 in different tissues of SC9. *P<0.05. The same below

图4 MeMYB106沉默植株表型及花青素含量鉴定A：MeMYB106沉默植株表型；B：对照植株和沉默植株中MeMYB106的表达水平；C：对照植株和沉默植株叶片总花青素含量

Fig. 4 Phenotype characterization and anthocyanin content analysis in MeMYB106-silenced plantsA: Phenotype of MeMYB106-silenced plants. B: Relative expressions of MeMYB106 in control plants and silenced plants. C: Total anthocyanin contents of leaves in control plants and silenced plants

图5 木薯MeANR基因启动子扩增结果M：DL5 000 marker，1-3：MeANR启动子扩增产物

Fig. 5 Amplified results of MeANR gene promoter in cassavaM: DL5 000 marker. 1-3:Promoter of MeANR gene amplification product

图6 诱饵菌株最低AbA抑制浓度筛选

Fig. 6 Screening of minimal concentration of AbA of bait yeast strain

图7 酵母单杂交证明转录因子MeMYB106和MeANR启动子互作

Fig. 7 Y1H demonstrating the interaction of transcription factor MeMYB106 with MeANR promoter

图8 Dual-LUC验证转录因子MeMYB106对MeANR基因的调控关系

Fig. 8 Dual-LUC validating the regulatory relationship between transcription factor MeMYB106 and MeANR

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