Expression Analysis of MnERF2 in Mulberry

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

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

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

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

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

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