梁山慈竹DfMYB3基因克隆及启动子分析

doi:10.7525/j.issn.1673-5102.2021.05.011

植物研究 ›› 2021, Vol. 41 ›› Issue (5): 729-737.doi: 10.7525/j.issn.1673-5102.2021.05.011

梁山慈竹DfMYB3基因克隆及启动子分析

杜恬恬¹, 李会萍¹, 王博雅¹, 欧倩¹, 黄艳¹, 曹颖¹, 胡尚连¹^,²()

^1.西南科技大学植物细胞工程实验室，绵阳 621010
^2.四川省生物质资源利用与改性工程技术研究中心，绵阳 621010

收稿日期:2020-12-29 出版日期:2021-09-20 发布日期:2021-07-05
通讯作者: 胡尚连 E-mail:hushanglian@126.com;hashanglian@126.com
作者简介:杜恬恬（1995—），女，硕士研究生，主要从事生物化学与分子生物学研究。
基金资助:
四川省应用基础研究项目(18YYJC0920);四川省“十三五”重点攻关项目(2016NYZ0038);四川省重点研发项目(2019YFN0005);环境友好能源材料国家重点实验室项目(19fksy0113)

Cloning and Promotor Analysis of DfMYB3 from Dendrocalamus farinosus

Tian-Tian DU¹, Hui-Ping LI¹, Bo-Ya WANG¹, Qian OU¹, Yan HUANG¹, Ying CAO¹, Shang-Lian HU¹^,²()

^1.Lab of Plant Cell Engineering Southwest University of Science and Technology，Mianyang 621010
^2.Engineering Research Center for Biomass Resource Utilizaiton and Modification of Sichuan Province，Mianyang 621010

Received:2020-12-29 Online:2021-09-20 Published:2021-07-05
Contact: Shang-Lian HU E-mail:hushanglian@126.com;hashanglian@126.com
About author:DU Tian-Tian（1995—），female，master，focused on biochemistry and molecular biology.
Supported by:
Applied Basic Research Project of Sichuan Province(18YYJC0920);13th Five-Year Key Project of Sichuan Province(2016NYZ0038);Key Research and Development Projects of Sichuan Province(2019YFN0005);Environmentally friendly Energy Materials of State Key Laboratory Project(19fksy0113)

摘要/Abstract

摘要：

基于梁山慈竹转录组数据库，以梁山慈竹叶片为材料，克隆得到一个MYB转录因子，命名为DfMYB3，其开放阅读框长度为1 287 bp，编码428个氨基酸，GenBank注册号为KY963358。保守结构域分析显示，DfMYB3有典型的SANT结构域，具有DNA-Binding结构域。系统进化树分析发现，DfMYB3与甘蔗、拟南芥、毛白杨等物种的R2R3-MYB转录因子聚集在一枝上。亚细胞定位结果显示，DfMYB3蛋白在细胞核以及细胞膜中均有表达，在细胞核上更为显著。通过染色体步移法克隆得到DfMYB3基因5′侧翼2 000 bp左右的序列。PlantCARE在线软件分析表明，该序列具有典型的启动子特征，并含有GA、ABA、MeJA等激素以及干旱等胁迫响应元件。100 μmol·L^-1 GA、100 μmol·L^-1 ABA处理梁山慈竹后，显著上调了DfMYB3基因的表达，表明DfMYB3基因能对GA及ABA处理产生应答。为探究DfMYB3启动子的功能，构建了DfMYB3启动子融合GUS基因的表达载体，并遗传转化烟草。在转基因烟草叶片及茎杆中都检测到了GUS信号，在叶脉处最为显著。

关键词: 梁山慈竹, MYB转录因子, 启动子, GA、ABA处理, GUS染色

Abstract:

Based on the transcriptome database of Dendrocalamus farinosus， a MYB transcription factor named DfMYB3 was cloned from the leave of D. farinosus. Its open reading frame length was 1 287 bp， encoding 428 amino acids， and GenBank accession Number was KY963358. The conserved domain analysis showed that DfMYB3 had the typical SANT domains and DNA-Binding domains. Phylogenetic tree analysis showed that DfMYB3 clustered with R2R3-MYB transcription factors in Saccharum， Arabidopsis and Populus. Subcellular localization showed that DfMYB3 protein was expressed in both the nucleus and cell membrane，and was more significant in the nucleus. The sequence of 5′ flanking of DfMYB3 gene about 2 000 bp was cloned by chromosome walking method. Analysis of PlantCARE online software showed that the sequence had typical promoter characteristics， and contained hormones responsive elements such as GA， ABA， MeJA as well as stress responsive elements such as drought. The expression level of DfMYB3 gene was significantly up-regulated after 100 μmol·L^-1 GA and 100 μmol·L^-1 ABA treatment， indicating that DfMYB3 gene was involved in response to GA and ABA treatments. In order to explore the function of DfMYB3 promoter， the expression vector of GUS gene fused with DfMYB3 promoter was constructed and genetically transformed into tobacco. The results showed that GUS signal was detected in both leaf and stem of transgenic tobacco， especially in the veins.

Key words: Dendrocalamus farinosus, MYB transcription factor, promotor, GA and ABA treatments, GUS staining

中图分类号:

S795.9

杜恬恬, 李会萍, 王博雅, 欧倩, 黄艳, 曹颖, 胡尚连. 梁山慈竹DfMYB3基因克隆及启动子分析[J]. 植物研究, 2021, 41(5): 729-737.

Tian-Tian DU, Hui-Ping LI, Bo-Ya WANG, Qian OU, Yan HUANG, Ying CAO, Shang-Lian HU. Cloning and Promotor Analysis of DfMYB3 from Dendrocalamus farinosus[J]. Bulletin of Botanical Research, 2021, 41(5): 729-737.

图/表 8

表1

图1

图2

图3

表2

DfMYB3启动子元件分析

元件 Site name	物种 Organism	位置 Position	链 Strand	评分 Matrix score	序列 Sequence	功能 Function
ABRE	拟南芥Arabidopsis thaliana	705	+	5	ACGTG	cis-acting element involved in the abscisic acid responsiveness
ABRE	大麦Hordeum vulgare	976	_	9	CGTACGTGCA	cis-acting element involved in the abscisic acid responsiveness
AuxRR-core	烟草Nicotiana tabacum	537	-	7	GGTCCAT	cis-acting regulatory element involved in auxin responsiveness
Box 4	欧芹Petroselinum crispum	96	+	6	ATTAAT	part of a conserved DNA module involved in light responsiveness
CGTCA-motif	大麦Hordeum vulgare	191	+	5	CGTCA	cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif	大麦Hordeum vulgare	813	-	5	CGTCA	cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif	大麦Hordeum vulgare	604	-	5	CGTCA	cis-acting regulatory element involved in the MeJA-responsiveness
CGTCA-motif	大麦Hordeum vulgare	854	-	5	CGTCA	cis-acting regulatory element involved in the MeJA-responsiveness
G-Box	豌豆Pisum sativum	704	-	6	CACGTT	cis-acting regulatory element involved in light responsiveness
GARE-motif	甘草Brassica oleracea	1017	-	7	TCTGTTG	gibberellin-responsive element
GATA-motif	拟南芥Arabidopsis thaliana	1574	-	7	GATAGGA	part of a light responsive element
GT1-motif	拟南芥Arabidopsis thaliana	673	-	6	GGTTAA	light responsive elemen
MBS	拟南芥Arabidopsis thaliana	841	+	6	CAACTG	MYB binding site involved in drought-inducibility
MBS	拟南芥Arabidopsis thaliana	1045	+	6	CAACTG	MYB binding site involved in drought-inducibility
P-box	水稻Oryza sativa	775	-	7	CCTTTTG	gibberellin-responsive element
TCA-element	烟草Nicotiana tabacum	1108	-	9	CCATCTTTTT	cis-acting element involved in salicylic acid responsiveness
TGACG-motif	大麦Hordeum vulgare	191	-	5	TGACG	cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	Hordeum vulgare	813	+	5	TGACG	cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	Hordeum vulgare	604	+	5	TGACG	cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	Hordeum vulgare	854	+	5	TGACG	cis-acting regulatory element involved in the MeJA-responsiveness

表2

图4

图5

图6

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引物名称 Primer name	引物序列（5′→3′） Primer sequences（5′→3′）
DfMYB3-F	ATGGGGAGGCATTCTTGTTG
DfMYB3-R	CTAGATATGCTCAAAAGATA
RT-DfMYB3-F	AAGCAACGGTATGGTATTCGAGCCA
RT-DfMYB3-R	GCACATATCCCTTCCATGTTGAATT
Tublin-F	GCCGTGAATCTCATCCCCTT
Tublin-R	TTGTTCTTGGCATCCCACAT
DfMYB3-F（KpnⅠ）	GGGGTACCCCATGGGGAGGCATTCTTGTTG
DfMYB3-R（HindⅢ）	CCCAAGCTTGGGGATATGCTCAAAAGATA
P3	TTGCTTGTAGCAACAAGAATGC
P2	TGTTCCCAAGGACAGCATGGAG
P1	TTTCAGGGCGATTCTTATCATA
pDfMYB3-F（HindⅢ）	CCAAGCTTGGTATCGAGTTTCGTGTTCCCAAG
pDfMYB3-R（XbaI）	GCTCTAGAGCTTCGGTGGAATGCACAACCTGA

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梁山慈竹DfMYB3基因克隆及启动子分析

Cloning and Promotor Analysis of DfMYB3 from Dendrocalamus farinosus

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