拟南芥中过表达野菊miR396a基因增强其耐盐性

doi:10.16420/j.issn.0513-353x.2021-0007

摘要/Abstract

摘要：

从野菊（Chrysanthemum indicum）中克隆了cin-miR396a前体序列及其启动子,对前体序列进行比对与进化分析,对启动子顺式作用元件进行分析,并研究了cin-miR396a前体序列对拟南芥的遗传转化及对其耐盐性的影响。结果表明,cin-MIR396a长度为145 bp,含有完整的茎环结构,与拟南芥ath-MIR396a进化关系最近。启动子区具有光信号、茉莉酸甲酯、赤霉素、水杨酸、脱落酸等多种响应元件,推测cin-MIR396a参与多种逆境胁迫下的应答及调控。在拟南芥中过表达cin-miR396a前体可以提高盐胁迫下种子发芽率以及成苗期的耐盐性,cin-miR396a在拟南芥响应盐胁迫中发挥正调控作用。

关键词: 野菊, miR396, 拟南芥, 转基因, 盐胁迫

Abstract:

cin-miR396a precursor sequence and its promoter were cloned from Chrysanthemum indicum;the precursor sequence alignment and evolutionary analysis were performed;the promoter was analyzed for cis-acting elements. The genetic transformation of the cin-miR396a precursor sequence to Arabidopsis thaliana was further carried out,and the salt tolerance of transgenic Arabidopsis thaliana was analyzed. The results showed that cin-MIR396a was 145 bp in length,contained an intact stem-loop structure,and was evolutionarily closest to A. thaliana ath-MIR396a. The promoter region has multiple response elements such as light signal,methyl jasmonate,gibberellin,salicylic acid,and abscisic acid,and it is hypothesized that cin-MIR396a is involved in response and regulation under a variety of adversity stresses. Overexpression of the cin-miR396a precursor in A. thaliana can improve seed germination under salt stress and salt tolerance at the adult seedling stage,and cin-miR396a plays a positive regulatory role in A. thaliana in response to salt stress.

Key words: Chrysanthemum indicum, miR396, Arabidopsis thaliana, transgenic, salt stress

中图分类号:

S682.11

刘筱玮, 夏斌, 李子葳, 杨宇佳, 陈斌, 周蕴薇, 何淼. 拟南芥中过表达野菊miR396a基因增强其耐盐性[J]. 园艺学报, 2022, 49(4): 816-826.

LIU Xiaowei, XIA Bin, LI Ziwei, YANG Yujia, CHEN Bin, ZHOU Yunwei, HE Miao. Overexpression of Chrysanthemum indicum miR396a Gene in Arabidopsis Enhances Its Salt Tolerance[J]. Acta Horticulturae Sinica, 2022, 49(4): 816-826.

图/表 10

参考文献 29

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用途 Usage	引物名称 Primer name	引物序列（5′-3′） Sequence
前体基因克隆 Precursor gene cloning	MIR396a-F	TCTAGACGCTATTGTTTTACACACAAGTGTG
	MIR396a-R	CCCGGGCTGTGTTGATTCTTTCTCTATCTT
启动子克隆 Promoter cloning	P-miR396a-F	GCTCTCCATCTTGTTTCTCGTC
转化植株PCR验证 PCR assay of the transgenic plants	P-miR396a-R	GCAGTTCAAGAAAGCTGTGGAAG
	35S-F	TCCCACTATCCTTCGCAAGAC
	miR396a-R	CTGTGTTGATTCTTTCTCTATCTT
实时荧光定量PCR	athU6-F	CGATAAAATTGGAACGATACAGA
Fluorescent quantitative real-time	athU6-R	ATTTGGACCATTTCTCGATTTGT
PCR（qRT-PCR）	cin-miR396aF	TTCCACAGCTTTCTTGAACTG

用途 Usage	引物名称 Primer name	引物序列（5′-3′） Sequence
前体基因克隆 Precursor gene cloning	MIR396a-F	TCTAGACGCTATTGTTTTACACACAAGTGTG
	MIR396a-R	CCCGGGCTGTGTTGATTCTTTCTCTATCTT
启动子克隆 Promoter cloning	P-miR396a-F	GCTCTCCATCTTGTTTCTCGTC
转化植株PCR验证 PCR assay of the transgenic plants	P-miR396a-R	GCAGTTCAAGAAAGCTGTGGAAG
	35S-F	TCCCACTATCCTTCGCAAGAC
	miR396a-R	CTGTGTTGATTCTTTCTCTATCTT
实时荧光定量PCR	athU6-F	CGATAAAATTGGAACGATACAGA
Fluorescent quantitative real-time	athU6-R	ATTTGGACCATTTCTCGATTTGT
PCR（qRT-PCR）	cin-miR396aF	TTCCACAGCTTTCTTGAACTG

顺式作用元件 cis-acting elements	功能 Function	野菊 Chrysanthemum indicum	拟南芥 Arabidopsis thaliana	水稻 Oryza sativa
A-box	顺式作用调节元件cis-Acting regulatory element	+	-	-
ABRE	脱落酸应答元件cis-Acting element involved in ABA responsiveness	+	+	+
ACE	光应答元件cis-Acting element involved in light responsiveness	-	-	+
AE-box	部分光应答元件Part of a light responsive element	-	-	+
ARE	厌氧诱导应答元件cis-Acting element essential for the anaerobic induction	+	+	-
Box 4	部分光应答元件Part of a light responsive element	+	+	-
BoxⅡ	部分光应答元件Part of a light responsive element	+	-	-
CAAT-box	增强子元件cis-Acting element playing an enhancing role	+	+	+
CAT-box	分生组织表达有关的应答元件cis-Acting element related to meristem expression	-	-	+
CCAAT-box	MYBHv1结合位点MYBHv1 binding site	+	-	-
CGTCA-motif	茉莉酸甲酯应答元件cis-Acting element involved in the MeJA-responsiveness	+	+	+
circadian	昼夜节律应答元件cis-Acting element involved in circadian control	-	+	-
ERE	乙烯应答元件Ethylene responsive element	+	+	-
GA-motif	部分光应答元件Part of a light responsive element	+	-	-
GARE-motif	赤霉素应答元件Gibberellin-responsive element	-	-	+
GATA-motif	部分光应答元件Part of a light responsive element	+	-	+
G-Box	光应答元件cis-Acting element involved in light responsiveness	+	+	+
GCN4_motif	胚乳表达的应答元件cis-Acting element involved in endosperm expression	+	-	-
I-box	部分光应答元件Part of a light responsive element	+	-	-
MBS	干旱诱导中MYB 结合位点MYB binding site involved in drought-inducibility	+	-	-
MYB	MYB结合位点MYB binding site	+	+	+
MYC	MYC结合位点MYC binding site	+	+	+
O2-site	玉米醇溶蛋白代谢的应答元件cis-Acting element involved in zein metabolism regulation	+	-	+
P-box	赤霉素应答元件cis-Acting element involved in gibberellin-responsive	+	+	-
TATA-box	核心启动子元件Core promoter elements	+	+	+
TCA-element	水杨酸应答元件cis-Acting element involved in salicylic acid responsiveness	+	-	-
TGACG-motif	茉莉酸甲酯应答元件cis-Acting element involved in the MeJA-responsiveness	-	+	+

顺式作用元件 cis-acting elements	功能 Function	野菊 Chrysanthemum indicum	拟南芥 Arabidopsis thaliana	水稻 Oryza sativa
A-box	顺式作用调节元件cis-Acting regulatory element	+	-	-
ABRE	脱落酸应答元件cis-Acting element involved in ABA responsiveness	+	+	+
ACE	光应答元件cis-Acting element involved in light responsiveness	-	-	+
AE-box	部分光应答元件Part of a light responsive element	-	-	+
ARE	厌氧诱导应答元件cis-Acting element essential for the anaerobic induction	+	+	-
Box 4	部分光应答元件Part of a light responsive element	+	+	-
BoxⅡ	部分光应答元件Part of a light responsive element	+	-	-
CAAT-box	增强子元件cis-Acting element playing an enhancing role	+	+	+
CAT-box	分生组织表达有关的应答元件cis-Acting element related to meristem expression	-	-	+
CCAAT-box	MYBHv1结合位点MYBHv1 binding site	+	-	-
CGTCA-motif	茉莉酸甲酯应答元件cis-Acting element involved in the MeJA-responsiveness	+	+	+
circadian	昼夜节律应答元件cis-Acting element involved in circadian control	-	+	-
ERE	乙烯应答元件Ethylene responsive element	+	+	-
GA-motif	部分光应答元件Part of a light responsive element	+	-	-
GARE-motif	赤霉素应答元件Gibberellin-responsive element	-	-	+
GATA-motif	部分光应答元件Part of a light responsive element	+	-	+
G-Box	光应答元件cis-Acting element involved in light responsiveness	+	+	+
GCN4_motif	胚乳表达的应答元件cis-Acting element involved in endosperm expression	+	-	-
I-box	部分光应答元件Part of a light responsive element	+	-	-
MBS	干旱诱导中MYB 结合位点MYB binding site involved in drought-inducibility	+	-	-
MYB	MYB结合位点MYB binding site	+	+	+
MYC	MYC结合位点MYC binding site	+	+	+
O2-site	玉米醇溶蛋白代谢的应答元件cis-Acting element involved in zein metabolism regulation	+	-	+
P-box	赤霉素应答元件cis-Acting element involved in gibberellin-responsive	+	+	-
TATA-box	核心启动子元件Core promoter elements	+	+	+
TCA-element	水杨酸应答元件cis-Acting element involved in salicylic acid responsiveness	+	-	-
TGACG-motif	茉莉酸甲酯应答元件cis-Acting element involved in the MeJA-responsiveness	-	+	+