虎杖转录因子PcMYB2的表达特性和功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-1231

摘要/Abstract

摘要：

对虎杖R2R3-MYB转录因子PcMYB2进行转录活性鉴定和表达特性分析，并在转基因拟南芥和转基因毛状根中进行功能研究。利用酵母单杂交试验分析PcMYB2的转录活性；实时荧光定量PCR（RT-qPCR）技术检测虎杖中PcMYB2的表达模式；DMACA法检测PcMYB2转基因拟南芥和虎杖毛状根中的原花青素含量。酵母单杂试验表明，PcMYB2具有转录激活活性；RT-qPCR结果显示，PcMYB2在虎杖根、茎、叶中均有表达，在叶中表达量最高，并且ABA和H₂O₂外源处理可诱导叶片中PcMYB2的表达；与野生型拟南芥相比，转基因拟南芥种皮颜色加深，原花青素含量为野生型的1.8倍；与野生型毛状根相比，转基因毛状根DMACA染色更深，原花青素含量为野生型的2.3倍。PcMYB2具有转录激活活性，且促进植物原花青素的生物合成。

关键词: 虎杖, PcMYB2, 转录活性, 表达分析, 原花青素生物合成

Abstract:

This work aims to identify the transcriptional activity and expression profile of PcMYB2, encoding an R2R3-MYB transcription factor from Polygonum cuspidatum, and to evaluate the biological functions of PcMYB2 in transgenic Arabidopsis thaliana plant and transformed P. cuspidatum hairy roots. The yeast one hybrid was used to analyze transcriptional activity. And quantitative real time PCR（RT-qPCR）technique was applied to detect the tissue-specific expression profile of PcMYB2 in P. cuspidatum and its expression pattern in responses to stress treatments. The DMACA method was to determine the contents of proanthocyanidins in PcMYB2-transgenic A. thaliana and P. cuspidatum hairy roots. The yeast one hybrid experiment showed that PcMYB2 had transcriptional activity. The PcMYB2 gene was expressed in the roots, stems and leaves of P. cuspidatum, with the highest expression in the leaves, and the expression of PcMYB2 gene in the leaves was induced after application of ABA or H₂O₂. Transgenic A. thaliana presented an increase in the pigmentation of mature seed compared with the wild type, and the proanthocyanidins content was 1.8-fold higher than that of the wild type. Staining of the hairy roots with DMACA was darker compared with wild type, and proanthocyanidins content was 2.3-fold higher. Altogether, this study reveals that PcMYB2 has transcriptional activity and may promote the proanthocyanidins biosynthesis.

Key words: Polygonum cuspidatum Sieb. et Zucc., PcMYB2, transcriptional activity, expression analysis, proanthocyanidins biosynthesis

姜晴春, 杜洁, 王嘉诚, 余知和, 王允, 柳忠玉. 虎杖转录因子PcMYB2的表达特性和功能分析[J]. 生物技术通报, 2023, 39(5): 217-223.

JIANG Qing-chun, DU Jie, WANG Jia-cheng, YU Zhi-he, WANG Yun, LIU Zhong-yu. Expression and Function Analysis of Transcription Factor PcMYB2 from Polygonum cuspidatum[J]. Biotechnology Bulletin, 2023, 39(5): 217-223.

图/表 9

表1 引物序列信息

Table 1 Primer sequence information

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	退火温度 Annealing temperature/℃
35Sf3	CGGGATCCCATGGAGTCAAAGATTCA	55
35Sr3 PcMYB2-F PcMYB2-R	AAAACTGCAGAGTCCCCCGTGTTCTCTC CGCGGATCCGTATGGGGAGAAGAAA AAAACTGCAGGAAATTTCCGAGCCATCC	55
qPcActin-F1	GTCCCTGCCATGTATGTTGC	58
qPcActin-R1	ACCATCACCAGAATCCAGCA
qPcMYB2-F	GAGGAAGGGTGGAGAAATGA	58
qPcMYB2-R	TGCCCTTGTGCTGTATCTGA

图1 pGBKT7与pGBKT7-PcMYB2载体结构示意图

Fig. 1 Structure of pGBKT7 and pGBKT7-PcMYB2 vectors

图2 pCAMBIA 1380-35S-PcMYB2载体结构示意图 LB：转移DNA的左边界；t35S：花椰菜花叶病毒CaMV 35S终止子；hpt：潮霉素抗性基因 P35S-花椰菜花叶病毒CaMV 3SS启动子； tNOS：氧化氮合酶基因终止子；RB：转移DNA的右边界

Fig. 2 Structure of pCAMBIA 1380-35S-PcMYB2 vectors LB: Left border of transfer DNA. t35S: Cauliflower mosaic virus CaMV 35S terminator. hpt: Hygromycin resistance gene P35S - Cauliflower mosaic virus CaMV 3SS promoter. tNOS: Nitric oxide synthase gene terminator. RB: Right of transfer DNA boundary

图3 重组质粒pGBKT7-PcMYB2的验证 M：Marker；1：PCR扩增获得PcMYB2基因；2：质粒pGBKT7-PcMYB2双酶切

Fig. 3 Identification of pGBKT7-PcMYB2 M: Marker. 1: PCR amplifies PcMYB2 gene. 2: Digestion pGBKT7-PcMYB2

图4 PcMYB2的转录活性验证分析 A：pGBKT7-PcMYB2转化子；B：阴性对照pGBKT7转化子

Fig. 4 Transcriptional activity verification and analysis of PcMYB2 A: Transformant pGBKT7-PcMYB2. B: Negative control transformant pGBKT7

图5 PcMYB2的表达模式分析 A：不同组织PcMYB2基因表达量；B：外源信号分子处理下叶片PcMYB2的表达分析。**P< 0.01，下同

Fig. 5 Expression pattern analysis of PcMYB2 A : Expressions of PcMYB2 gene in different tissues. B : PcMYB2 expressions in leaves under exogenous signaling molecules. ** P < 0.01. The same below

图6 重组质粒pCAMBIA1380-35S-PcMYB2的验证 M：Marker；1：BamH I/ Pst I双酶切重组质粒；2： BamH I/ Nco I双酶切重组质粒

Fig. 6 Verification of recombinant plamid pCAMBIA1380-35S-PcMYB2 M: Marker. 1: Digestion recombinant plasmid by BamH I/Pst I. 2: Digestion recombinant plasmid by BamH I/ Nco I

图7 转基因株系的鉴定和表现型观察 A： RT-qPCR鉴定；B：拟南芥种皮颜色观察；C： DMACA染色前的毛状根；D：DMACA染色后的毛状根。CK1：野生型拟南芥；A1：转基因拟南芥；CK2：野生型毛状根；H1：转基因毛状根

Fig. 7 Identification and phenotypic observation of trans-genic lines A: RT-qPCR identification. B: Observation on the seed coat's color of A. thaliana. C: Hairy roots before DMACA staining. D: Hairy root after DMACA staining. CK1: Wild-type A.thaliana; A1: transgenic A.thaliana; CK2: wild-type hairy roots; H1: transgenic hairy roots

图8 原花青素含量的测定 CK1：野生型拟南芥；A1：转基因拟南芥；CK2：野生型毛状根；H1：转基因毛状根；*P < 0.05

Fig. 8 Determination of proanthocyanidins CK1: Wild-type A.thaliana; A1: transgenic A. thaliana; CK2: wild-type hairy roots; H1: transgenic hairy roots; *P < 0.05

参考文献 27

[1]	Fan PH, Hostettmann K, Lou HX. Allelochemicals of the invasive neophyte Polygonum cuspidatum Sieb. & Zucc.(Polygonaceae)[J]. Chemoecology, 2010, 20(3): 223-227. doi: 10.1007/s00049-010-0052-4 URL
[2]	Li X, Liu JL, Chang QX, et al. Antioxidant and antidiabetic activity of proanthocyanidins from Fagopyrum dibotrys[J]. Molecules, 2021, 26(9): 2417. doi: 10.3390/molecules26092417 URL
[3]	Suganya M, Gnanamangai BM, Ravindran B, et al. Antitumor effect of proanthocyanidin induced apoptosis in human colorectal cancer(HT-29)cells and its molecular docking studies[J]. BMC Chem, 2019, 13(1): 21. doi: 10.1186/s13065-019-0525-7 pmid: 31384770
[4]	Wang JY, Zhang BX, Tian ST, et al. Ectopic expression of grape hyacinth R3 MYB repressor MaMYBx affects anthocyanin and proanthocyanidin biosynthesis and epidermal cell differentiation in Arabidopsis[J]. Hortic Environ Biotechnol, 2022, 63(3): 413-423. doi: 10.1007/s13580-021-00401-7
[5]	Zhao L, Song ZB, Wang BW, et al. R2R3-MYB transcription factor NtMYB330 regulates proanthocyanidin biosynthesis and seed germination in tobacco(Nicotiana tabacum L.)[J]. Front Plant Sci, 2022, 12: 819247. doi: 10.3389/fpls.2021.819247 URL
[6]	Zhang JK, Wang YC, Mao ZL, et al. Transcription factor McWRKY71 induced by ozone stress regulates anthocyanin and proanthocyanidin biosynthesis in Malus crabapple[J]. Ecotoxicol Environ Saf, 2022, 232: 113274. doi: 10.1016/j.ecoenv.2022.113274 URL
[7]	Gil-Muñoz F, Sánchez-Navarro JA, Besada C, et al. MBW complexes impinge on anthocyanidin reductase gene regulation for proanthocyanidin biosynthesis in persimmon fruit[J]. Sci Rep, 2020, 10(1): 3543. doi: 10.1038/s41598-020-60635-w pmid: 32103143
[8]	Passeri V, Martens S, Carvalho E, et al. The R2R3MYB VvMYBPA1 from grape reprograms the phenylpropanoid pathway in tobacco flowers[J]. Planta, 2017, 246(2): 185-199. doi: 10.1007/s00425-017-2667-y pmid: 28299441
[9]	李晓筱, 徐俊雄, 刘婵, 等. 虎杖MYB转录因子PcMYB2基因的克隆与原核表达[J]. 江苏农业科学, 2018, 46(23): 50-55.
	Li XX, Xu JX, Liu S, et al. Cloning and prokaryotic expression of MYB transcription factor PcMYB2 gene from Polygonum cuspidatum[J]. Jiangsu Agric Sci, 2018, 46(23): 50-55.
[10]	房志家, 陈婷, 郝贺龙, 等. 酿酒酵母转化方法的新探索[J]. 实验室研究与探索, 2012, 31(4): 5-8, 78.
	Fang ZJ, Chen T, Hao HL, et al. Exploration on transformation of saccharomy cescerevisiae[J]. Res Explor Lab, 2012, 31(4): 5-8, 78.
[11]	张新, 崔广艳, 陈翠娜, 等. 农杆菌介导DR1172基因转化拟南芥[J]. 基因组学与应用生物学, 2012, 31(6): 582-586.
	Zhang X, Cui GY, Chen CN, et al. Preliminary study on Agrobacterium-mediated transformation of DR1172 gene into Arabidopsis[J]. Genom Appl Biol, 2012, 31(6): 582-586.
[12]	段梦灵, 李鲁汉, 廖辉, 等. 发根农杆菌介导的虎杖转基因体系优化[J]. 现代农业科技, 2021(4): 46-50.
	Duan ML, Li LH, Liao H, et al. Optimization of Agrobacterium rhizogenes-mediated Polygonum cuspidatum transgenic system[J]. Mod Agric Sci Technol, 2021(4): 46-50.
[13]	Verdier J, Zhao J, Torres-Jerez I, et al. MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula[J]. Proc Natl Acad Sci USA, 2012, 109(5): 1766-1771. doi: 10.1073/pnas.1120916109 pmid: 22307644
[14]	Wei JB, Yang JF, Jiang WB, et al. Stacking triple genes increased proanthocyanidins level in Arabidopsis thaliana[J]. PLoS One, 2020, 15(6): e0234799. doi: 10.1371/journal.pone.0234799 URL
[15]	Liu JY, Osbourn A, Ma PD. MYB transcription factors as regulators of phenylpropanoid metabolism in plants[J]. Mol Plant, 2015, 8(5): 689-708. doi: 10.1016/j.molp.2015.03.012 pmid: 25840349
[16]	Shan TL, Rong W, Xu HJ, et al. The wheat R2R3-MYB transcription factor TaRIM1 participates in resistance response against the pathogen Rhizoctonia cerealis infection through regulating defense genes[J]. Sci Rep, 2016, 6: 28777. doi: 10.1038/srep28777
[17]	Bai QX, Duan BB, Ma JC, et al. Coexpression of PalbHLH1 and PalMYB90 genes from Populus alba enhances pathogen resistance in poplar by increasing the flavonoid content[J]. Front Plant Sci, 2020, 10: 1772. doi: 10.3389/fpls.2019.01772 URL
[18]	Wang S, Wu HL, Cao XX, et al. Tartary buckwheat FtMYB30 transcription factor improves the salt/drought tolerance of transgenic Arabidopsis in an ABA-dependent manner[J]. Physiol Plant, 2022, 174(5): e13781.
[19]	Wei QH, Liu YY, Lan KE, et al. Identification and analysis of MYB gene family for discovering potential regulators responding to abiotic stresses in Curcuma wenyujin[J]. Front Genet, 2022, 13: 894928. doi: 10.3389/fgene.2022.894928 URL
[20]	Wei QH, Luo QC, Wang RB, et al. A wheat R2R3-type MYB transcription factor TaODORANT1 positively regulates drought and salt stress responses in transgenic tobacco plants[J]. Front Plant Sci, 2017, 8: 1374. doi: 10.3389/fpls.2017.01374 pmid: 28848578
[21]	王霜, 雒晓鹏, 姚英俊, 等. 苦荞R2R3-MYB转录因子调控原花青素生物合成的研究[J]. 西北植物学报, 2019, 39(11): 1911-1918.
	Wang S, Luo XP, Yao YJ, et al. Characterization of an R2R3-MYB transcription factor involved in the synthesis of proanthocyanidins from Tartary buckwheat[J]. Acta Bot Boreali Occidentalia Sin, 2019, 39(11): 1911-1918.
[22]	Liu CG, Jun JH, Dixon RA. MYB5 and MYB14 play pivotal roles in seed coat polymer biosynthesis in Medicago truncatula[J]. Plant Physiol, 2014, 165(4): 1424-1439. doi: 10.1104/pp.114.241877 URL
[23]	Jin Z, Jiang WB, Luo YJ, et al. Analyses on flavonoids and transcriptome reveals key MYB gene for proanthocyanidins regulation in Onobrychis viciifolia[J]. Front Plant Sci, 2022, 13: 941918. doi: 10.3389/fpls.2022.941918 URL
[24]	An JP, Xu RR, Liu X, et al. Jasmonate induces biosynthesis of anthocyanin and proanthocyanidin in apple by mediating the JAZ1-TRB1-MYB9 complex[J]. Plant J, 2021, 106(5): 1414-1430. doi: 10.1111/tpj.v106.5 URL
[25]	张慧文, 张玉, 马超美. 原花青素的研究进展[J]. 食品科学, 2015, 36(5): 296-304. doi: 10.7506/spkx1002-6630-201505052
	Zhang HW, Zhang Y, Ma CM. Progress in procyanidins research[J]. Food Sci, 2015, 36(5): 296-304.
[26]	Luo XP, Zhao HX, Yao PF, et al. An R2R3-MYB transcription factor FtMYB15 involved in the synthesis of anthocyanin and proanthocyanidins from Tartary buckwheat[J]. J Plant Growth Regul, 2018, 37(1): 76-84. doi: 10.1007/s00344-017-9709-3 URL
[27]	Akagi T, Ikegami A, Yonemori K. DkMyb2 wound-induced transcription factor of persimmon(Diospyros kaki Thunb.), contributes to proanthocyanidin regulation[J]. Planta, 2010, 232(5): 1045-1059. doi: 10.1007/s00425-010-1241-7 URL