虎杖PcMYB1启动子的克隆及其活性分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1319

摘要/Abstract

摘要：

获得药用植物虎杖（Polygonum cuspidatum）PcMYB1启动子,并分析其活性,为进一步研究虎杖转录因子PcMYB1的功能奠定基础。通过hiTAIL-PCR方法,从虎杖叶片基因组DNA中克隆PcMYB1启动子并进行生物信息学分析。分别构建由全长启动子或5'端缺失启动子驱动GUS的重组表达载体P1∷GUS、P2∷GUS、P3∷GUS和P4∷GUS。将重组表达载体转入发根农杆菌中进行虎杖毛状根转化试验,以转化的阳性毛状根为材料,通过GUS组织化学染色分析启动子的活性。成功获得2 884 bp的PcMYB1启动子序列（GenBank登录号MT811057）。该启动子具有真核生物启动子必需的核心元件TATA-box和CAAT-box,还含有茉莉酸甲酯和低温响应元件以及光响应、厌氧应答等相关的顺式调控元件。重组表达载体经PCR鉴定,证实已构建成功。转化的虎杖毛状根染色之后显蓝色,但同对照相比颜色较浅,说明全长启动子或5'端缺失启动子都具有驱动GUS的活性,但启动活性都比CaMV35S启动子的启动活性弱。PcMYB1的启动子被成功克隆,其具有驱动下游GUS表达的活性。

关键词: 虎杖, PcMYB1, 启动子, 序列分析, GUS活性

Abstract:

This work aims to obtain PcMYB1 promoter from Polygonum cuspidatum and analyze its activity,and thus to lay a basis for further investigating the functions of promoter PcMYB1 promoter. hiTAIL-PCR was used to clone the PcMYB1 promoter from the leave genome DNA of P. cuspidatum,and bioinformatics analysis of it was conducted. The full-length promoter P1（+1 to -2 884）and a series of progressive 5' truncated promoters P2（+1 to -2 259）,P3（+1 to -1 807）and P4（+1 to -1 362）were fused with GUS gene,and the recombinant expression vector P1∷GUS,P2∷GUS,P3∷GUS,P4∷GUS were constructed,respectively. Each recombinant expression vector was transferred into Agrobacterium rhizogenes to transform the hairy root of P. cuspidatum,and the transformed positive hairy root was used as the material. GUS histochemical staining was performed to analyze the activity of the promoter. The 2 884 bp promoter sequence（GenBank accession number：MT811057）of the promoter was obtained. The promoter contained several core fragments（TATA-box and CAAT-box）essential for eukaryotic promoters,and also cis-elements that were responsive to MeJA and low-temperature and cis-acting regulatory elements that were associated with light and anaerobic responses. The successful constructions of the recombinant vectors were confirmed via PCR. The transgenic hairy roots of P. cuspidatum can be stained to have blue,but the color was light compared to the control,that demonstrated that both the full-length promoter and the other 5' truncated promoters drove the GUS gene expression,but the driving ability was lower than that of CaMV35S promoter. In sum,PcMYB1 promoter is successfully cloned and it shows the activity of driving the expression of downstream GUS.

Key words: Polygonum cuspidatum Sieb. et Zucc., PcMYB1, promoter, sequence analysis, GUS activity

林艳丽, 覃建兵, 伍翔, 王岩岩, 潘佑找, 柳忠玉. 虎杖PcMYB1启动子的克隆及其活性分析[J]. 生物技术通报, 2021, 37(5): 48-55.

LIN Yan-li, QIN Jian-bing, WU Xiang, WANG Yan-yan, PAN You-zhao, LIU Zhong-yu. Cloning and Activity Analysis of PcMYB1 Promoter from Polygonum cuspidatum[J]. Biotechnology Bulletin, 2021, 37(5): 48-55.

图/表 8

表1 引物序列

Table 1 Primer sequences

引物名称Primer name	引物序列Prime sequence（5'-3'）	酶切位点Enzymatic cleavage site
LAD1-1	ACGATGGACTCCAGAGCGGCCGC（G/C/A）N（G/C/A）NNNGGAA
LAD1-2	ACGATGGACTCCAGAGCGGCCGC（G/C/T）N（G/C/T）NNNGGTT
LAD1-3	ACGATGGACTCCAGAGCGGCCGC（G/C/A）（G/C/A）N（G/C/A）NNNCCAA
LAD1-4	ACGATGGACTCCAGAGCGGCCGC（G/C/T）（G/A/T）N（G/C/T）NNNCGGT
AC1	ACGATGGACTCCAGAG
SP0-1	AAATTTCCACGCTTGAGATCAGGGC
SP1-1	ACGATGGACTCCAGTCCGGCCCCCACATCGAAGAAGTCCAGCAGCT
SP2-1	GCTTTCTCACAACATGGAGACCTACCC
P0-F	GATGGACTCCAGAGCGGCC
P0-R	TTCTCGAAACTTACCAATC
P1-F	AAAACTGCAGGATGGACTCCAGAGCGGCC	PstⅠ
P2-F	AAAACTGCAGGAGTCTCATACCGTCCAA	PstⅠ
P3-F	AAAACTGCAGACGATTAGACCCGTTTGG	PstⅠ
P4-F	AAAACTGCAGTACCTCCATCCTCCTTGT	PstⅠ
pro-R	CGCGGATCCTTCTCGAAACTTACCAATC	BamHⅠ
Hyg-F	GAAGTGCTTGACATTGGGGAGT
Hyg-R	AGATGTTGGCGACCTCGTATT
GUS-F	GTCGCGCAAGACTGTAACCA
GUS-R	CGGCGAAATTCCATACCTG

图1 PcMYB1启动子系列缺失片段表达载体构建示意图

Fig.1 Schematic representation of expression vector construction of serial deletion fragments of PcMYB1 promoter

图2 PcMYB1的hiTAIL-PCR扩增产物电泳分析 M：Marker;A：预扩增产物;B：第一轮hiTAIL-PCR产物;C：第二轮hiTAIL-PCR产物

Fig.2 Analysis of PcMYB1 by electrophoresis of hiTAIL-PCR amplification products M ：Marker. A: Preamplification products. B: First round hiTAIL-PCR product.C: Round 2 hiTAIL-PCR products

图3 PcMYB1启动子的分析黑框标注为顺式作用元件;“+1”为翻译起始位点

Fig.3 Analysis of PcMYB1 promoter Black boxes are annotated as cis-acting elements. “+1” is the translation initiation site

表2 PcMYB1启动子顺式作用元件预测分析

Table 2 PcMYB1 promoter cis-acting element prediction analysis

名称 Name	识别序列 Identification of sequence	起始端位点 Start site	功能 Function
AE-box	AGAAACAA	-2100	部分光响应元件
AT1-motif	AATTATTTCTTATT	-640	部分光响应元件
I-box	GTATAAGGCC	-1410	部分光响应元件
TCT-motif	TCTTAC	-281;-1296	部分光响应元件
G-Box	CACGTC	-425-;-683	光响应元件
GT1-motif	GGTTAA	-1600;-1 984	光响应元件
ARE	AAACCA	-500;-522;-1 924;-2 479	厌氧诱导顺式作用元件
CGTCA-motif	CGTCA	-1 021;-1 147;-1 808	茉莉酸甲酯响应元件
TGACG-motif	TGACG	-1 072;-2 713	茉莉酸甲酯响应元件
LTR	CCGAAA	-503;-531;-2 812	低温诱导顺式作用元件
CAAT-box	CCAAT/CAAAT	-23;-710;-1 452;-1 783;-2 098;2 745	启动子和增强子常见顺式调控元件
TATA-box	TATA	-214;-599;-1 302;1 465;-1 735;-1 882;-1 948;-1 993;-2 202;-2 271;-2 484;-2 781	转录起始元件

图4 重组载体的PCR鉴定 M：Marker;P1：P1∷GUS扩增产物;P2：P2∷GUS扩增产物;P3：P3∷GUS扩增产物;P4：P4∷GUS 扩增产物

Fig.4 PCR identification of recombinant vectors M ：Marker. P1: P1∷GUS amplification product. P2: P2∷GUS amplification product. P3 ：P3∷GUS amplification product. P4: P4∷GUS amplification product

图5 转基因毛状根的Hyg（A）和GUS（B）检测 M：Marker;WT：野生型毛状根;35S：35S∷GUS 转基因毛状根;P1：P1∷GUS转基因毛状根;P2：P2∷GUS转基因毛状根;P3：P3∷GUS转基因毛状根;P4：P4∷GUS转基因毛状根

Fig.5 Hyg (A) and GUS (B) detection of transgenic hairy roots M ：Marker. WT: Wild-type hairy roots. 35S: 35S∷GUS transgenic hairy roots. P1 ：P1∷GUS transgenic hairy roots. P2: P2∷GUS transgenic hairy roots. P3: P3∷GUS transposition. P4: P4∷GUS transgenic hairy roots

图6 转基因毛状根的GUS组织化学染色 WT：野生型毛状根;35S：35S∷GUS转基因毛状根;P1：P1∷GUS转基因毛状根;P2：P2∷GUS 转基因毛状根;P3：P3∷GUS转基因毛状根;P4：P4∷GUS转基因毛状根

Fig.6 GUS histochemical staining of transgenic hairy roots M ：Marker. WT: Wild-type hairy roots. 35S: 35S∷GUS transgenic hairy roots. P1 ：P1∷GUS transgenic hairy roots. P2: P2∷GUS transgenic hairy roots. P3: P3∷GUS transposition. P4: P4∷GUS transgenic hairy roots

参考文献 18

[1]	Sharma S, Holme I B, Dionisio G, et al. Cyanidin based anthocyanin biosynjournal in orange carrot is restored by expression of AmRosea1 and AmDelila, MYB and bHLH transcription factors[J]. Plant Molecular Biology, 2020,103(4/5):443-456. doi: 10.1007/s11103-020-01002-1 URL
[2]	李竹韵, 焦子迅, 陈阳, 等. 黄芪AmMYB44基因的克隆与表达模式分析[J]. 基因组学与应用生物学, 2019,38(8):3605-3613.
	Li ZY, Jiao Zx, Chen Y, et al. Cloning and expression analysis of the AmMYB44 gene from Astragalus membranaceus[J]. Genomics and Applied Biology, 2019,38(8):3605-3613.
[3]	许锋, 孙楠楠, 张威威, 等. 银杏GbMYBF2启动子克隆及序列分析[J]. 贵州农业科学, 2014,42(4):16-20.
	Xu F, Sun NN, Zhang WW, et al. Cloning and sequencing of the GbMYBF2 promoter from Ginkgo biloba[J]. Guizhou Agricultural Sciences, 2014,42(4):16-20.
[4]	朱小亚, 崔璀, 周宏骏, 等. 丹参转录因子SmMYB7的克隆及表达模式分析[J]. 基因组学与应用生物学, 2015,34(2):365-372.
	Zhu XY, Cui C, Zhou HJ, et al. Cloning and expression analysis of the transcription factor SmMYB7 from Salvia miltiorrhiza Bunge[J]. Genom Appl Biol, 2015,34(2):365-372.
[5]	柳忠玉, 雷健, 李晓筱, 等. 虎杖转录因子PcMYB1基因的克隆及原核表达[J]. 河南农业科学, 2018,47(1):96-102.
	Liu ZY, Lei J, Li XX, et al. Cloning and prokaryotic expression of transcription factor PcMYB1 gene from Polygonum cuspidatum[J]. Journal of Henan Agricultural Sciences, 2018,47(1):96-102.
[6]	徐俊雄, 覃建兵, 王岩岩, 等. 虎杖MYB转录因子PcMYB1的表达特性和功能研究[J]. 中草药, 2020,51(3):726-732.
	Xu JX, Qin JB, Wang YY, et al. Expression and function analysis of transcription factor PcMYB1 from Polygonum cuspidatum[J]. Chinese Traditional and Herbal Drugs, 2020,51(3):726-732.
[7]	Liu YG, Chen YL. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. Bio Techniques, 2007,43(5):649-654.
[8]	Jefferson RA. Assaying chimeric genes in plants:The GUS gene fusion system[J]. Plant Molecular Biology Reporter, 1987,5(4):387-405. doi: 10.1007/BF02667740 URL
[9]	Wang H, Liu W, Qiu F, et al. Molecular cloning and characterization of the promoter of aldehyde dehydrogenase gene from Artemisia annua[J]. Biotechnol Appl Biochem, 2017,64(6):902-910. doi: 10.1002/bab.2017.64.issue-6 URL
[10]	宋菊, 林丽梅, 尤鹏升, 等. 刺五加FPS基因DNA序列和启动子的克隆与分析[J]. 分子植物育种, 2017,15(7):2584-2589.
	Song J, Lin LM, You PS, et al. Cloning and analysis of FPS gene DNA and promoter in Eleutherococcus senticosus[J]. Molecular Plant Breeding, 2017,15(7):2584-2589.
[11]	王虹, 李萌, 马东明, 等. 阳春砂萜类合酶AvTPS1启动子的克隆及其活性分析[J]. 中草药, 2019,50(9):2181-2187.
	Wang H, Li M, Ma DM, et al. Cloning and activity analysis of AvTPS1 promoter of terpene synthase from Amomum villousm[J]. Chinese Traditional and Herbal Drugs, 2019,50(9):2181-2187.
[12]	张晓东, 李彩霞, 赵青蓉, 等. 滇龙胆环烯醚萜氧化酶基因及启动子的克隆与生物信息学分析[J]. 中草药, 2018,49(18):4386-4392.
	Zhang XD, Li CX, Zhao QR, et al. Cloning and bioinformatics analysis of iridoid oxidase gene in Gentiana rigescens[J]. Chinese Traditional and Herbal Drugs, 2018,49(18):4386-4392.
[13]	Shen XJ, Wang YY, Zhang YX, et al. Overexpression of the wild soybean R2R3-MYB transcription factor GsMYB15 enhances resistance to salt stress and Helicoverpa Armigera in transgenic Arabidopsis[J]. Int J Mol Sci, 2018(12):3958-3977.
[14]	李甜甜, 李丽丽, 陈利红, 等. 水稻茉莉酸甲酯和白叶枯病菌诱导型启动子的克隆及功能鉴定[J]. 分子植物育种, 2018,16(3):689-695.
	Li TT, Li LL, Chen LH, et al. Cloning and functional identification of MeJA and bacterial blight-induced promoter in rice[J]. Molecular Plant Breeding, 2018,16(3):689-695.
[15]	Zhang K, Logacheva MD, Meng Y, et al. Jasmonate-responsive MYB factors spatially repress rutin biosynjournal in Fagopyrum tataricum[J]. J Exp Bot, 2018,69(8):1955-1966. doi: 10.1093/jxb/ery032 URL
[16]	Lai B, Li XJ, Hu B, et al. LcMYB1 is a key determinant of differential anthocyanin accumulation among geno types, tissues, developmental phases and ABA and light stimuli in Litchi chinensis[J]. PLoS One, 2014,9(1):e86293. doi: 10.1371/journal.pone.0086293 URL
[17]	Czemmel S, Stracke R, Weisshaar B, et al. The grapevine R2R3-MYB transcription factor VvMYBF1 regulates flavonol synjournal in developing grape berries[J]. Plant Physiology, 2009,151(3):1513-1530. doi: 10.1104/pp.109.142059 pmid: 19741049
[18]	Wang H, Xin H, Guo J, et al. Genome-wide screening of hexokinase gene family and functional elucidation of HXK2 response to cold stress in Jatropha curcas[J]. Mol Biol Rep, 2019,46(2):1649-1660. doi: 10.1007/s11033-019-04613-0 URL