桑树MnERF2的表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0016

摘要/Abstract

摘要：

乙烯响应因子（ethylene responsive factors，ERFs）是植物特异性转录因子，参与多种生物过程，特别是非生物胁迫耐受。研究干旱胁迫下MnERF2的表达情况，为研究桑树AP2/ERF转录因子的抗逆分子机制提供基础理论数据。从桑树中鉴定了一个ERF转录因子亚家族成员，命名为MnERF2，对其进行生物信息学分析，并通过瞬时转化分别获得过表达MnERF2和RNA干扰（RNAi）沉默MnERF2桑树植株，利用功能增益和功能损失方法分析其抗旱功能。结果显示，MnERF2开放阅读框为1 050 bp，编码349个氨基酸，编码蛋白质分子量为39.48 kD，理论等电点为5.6，具有一个典型的AP2/ERF保守结构域。过表达MnERF2显著提高桑树对干旱胁迫的耐受性，相比之下，MnERF2的沉默显著降低了对干旱胁迫的耐受性。进一步试验表明，MnERF2增强SOD、POD、CAT、GST活性，促进ASA、GSH、脯氨酸含量的升高，降低O₂^·-、H₂O₂、·OH、MDA的含量和电解质渗透率。MnERF2通过提高脯氨酸的生物合成来调节渗透势，通过提高抗氧化酶活性和抗氧化物质含量来提高活性氧的清除能力，从而提高桑树干旱胁迫的耐受性。

关键词: 桑树, AP2/ERF转录因子, 干旱胁迫, 瞬时表达

Abstract:

Ethylene responsive factors（ERFs）is a plant-specific transcription factor that involves in various biological processes，especially abiotic stress tolerance. The expression of MnERF2 under drought stress was studied to provide basic theoretical data for understanding the stress resistance molecular mechanism of AP2/ERF transcription factor in mulberry. A member of the ERF transcription factor subfamily，named MnERF2，was identified from mulberry，and analyzed by bioinformatics. Transiently transfected mulberry plants with transiently overexpressed MnERF2 and RNAi-silenced MnERF2 were generated for its drought resistance via gain- and loss-of-function analysis. The results showed that the open reading frame of MnERF2 was 1 050 bp，encoding 349 amino acids. The relative molecular weight of MnERF2 protein was 39.48 kD，with isoelectric point 5.6 and a typical AP2/ERF conserved domain. Overexpression of MnERF2 significantly elevated the tolerance of mulberry to drought stress. In contrast，silencing of MnERF2 significantly decreased the tolerance to drought stress. Further experiments demonstrated that MnERF2 enhanced the activities of SOD，POD，CAT and GST，increased the contents of ASA，GSH and proline，while decreased the contents of O₂^·-，H₂O₂，·OH，MDA and electrolyte permeability. MnERF2 regulated osmotic potential by increasing proline contents，and enhanced the ability of ROS scavenging by increasing antioxidant enzyme activity and antioxidant substance content，thus improved drought stress tolerance in mulberry.

Key words: mulberry, AP2/ERF transcription factor, drought stress, transient expression

董亚茹, 赵东晓, 耿兵, 李云芝, 王照红. 桑树MnERF2的表达分析[J]. 生物技术通报, 2022, 38(11): 112-121.

DONG Ya-ru, ZHAO Dong-xiao, GENG Bing, LI Yun-zhi, WANG Zhao-hong. Expression Analysis of MnERF2 in Mulberry[J]. Biotechnology Bulletin, 2022, 38(11): 112-121.

图/表 9

表1 试验所用引物

Table 1 Primers used in this study

引物名称 Primer name	序列 Sequence（5'-3'）	用途 Application
MnERF2-F	ATGGCGACCATAAACGAAGTC	基因克隆 Gene cloning
MnERF2-R	TCACACAACCATTAGTTGTGG	基因克隆 Gene cloning
MnERF2-F	TCCCGTTGGAAGCCGGGA	实时荧光定量PCR Quantitative real-time PCR
MnERF2-R	CGACAGAGGCGGGACGTT
MnRPL15-F	GGCTATGTGATTTACCGTGTT
MnRPL15-R	TTGGTCCAGTATGAGTTGAGAA
β-actin-F	AGCAACTGGGATGACATGGAGA
β-actin-R	CGACCACTGGCGTAAAGGGA

图1 MnERF2与拟南芥AP2/ERF家族蛋白的系统进化关系

Fig. 1 Phylogenetic relationships of MnERF2 with Arabid-opsis thaliana AP2/ERF family protein

图2 MnERF2与来自拟南芥B-3组的其他ERF序列比对 *表示完全一致的残基；：表示性质特别相近的残基；.表示性质微弱相近的残基，箭头分别表示AP2/ERF结构域的第14位和第19位

Fig. 2 Multiple sequence alignments of MnERF2 with other group B-3 ERFs from A. thaliana * indicates positions which have a single，fully conserved residue.：indicates that one of the following ‘strong’ groups is fully conserved.. indicates that one of the following ‘weaker’ groups is fully conserved，and arrow indicates the amino acids at the position of 9 and 14 of the AP2/ERF domain respectively

图3 MnERF2瞬时转化桑树植株RT-qPCR检测不同小写字母表示处理间差异显著（P<0.05）。下同

Fig. 3 RT-qPCR detection of mulberry transiently transfo-rmed with MnERF2 Different lowercase letters indicate significant difference at 0.05 level among treatments. The same below

图4 干旱协迫下OE、RNAi及CK桑树植株电解质渗透率及MDA含量分析

Fig. 4 Analysis of electrolyte permeability and MDA contents of mulberry plant OE，RNAi and CK under drought stress

图5 干旱胁迫下OE、RNAi及CK桑树植株O2·-、H2O2和·OH含量的变化

Fig. 5 Changes of O2·-，H2O2，and ·OH contents of mulberry plant transiently OE，RNAi and CK under drought stress

图6 干旱胁迫下OE、RNAi及CK桑树植株NBT、DAB和Evans Blue染色比较

Fig. 6 NBT，DAB，and Evans Blue staining comparisons between mulberry plant OE，RNAi and CK under drought stress

图7 干旱胁迫下OE、RNAi及CK桑树植株SOD、POD、CAT和GST活性的变化

Fig. 7 Changes of SOD，POD，CAT and GST activities of mulberry plant OE，RNAi and CK under drought stress

图8 干旱胁迫下OE、RNAi及CK桑树植株AsA、GSH和脯氨酸含量的变化

Fig. 8 Changes of AsA，GSH and proline contents of mu-lberry plant OE，RNAi and CK under drought stress

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