桑树MnDREB6E的克隆及耐盐抗旱性分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-0705

摘要/Abstract

摘要：

目的解析桑树DREB转录因子家族成员MnDREB6E的生物学功能，阐明其在非生物胁迫响应中的作用机制，为木本植物抗逆分子育种提供基因资源。方法基于桑树盐胁迫转录组数据克隆MnDREB6E，通过生物信息学分析其序列特征及系统进化关系；利用酵母单杂交技术分析其顺式作用元件；构建过表达及抑制表达载体，采用农杆菌瞬时转化技术获得转基因植株，测定盐/干旱胁迫下生理生化指标及相关基因表达水平。结果 MnDREB6E开放阅读框（ORF）全长1 173 bp，编码390个氨基酸，具有典型AP2/ERF结构域，归属DREB A6亚组；MnDREB6E亚细胞定位预测在细胞核，可特异性结合GCC-box/DRE顺式元件；盐/干旱胁迫下，过表达植株的超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、过氧化物酶（POD）、谷胱甘肽S-转移酶（GST）活性显著提高，抗坏血酸（AsA）、还原型谷胱甘肽（GSH）和脯氨酸含量增加，超氧阴离子（O₂^•-）、过氧化氢（H₂O₂）、羟自由基（•OH）水平降低，相对电导率和丙二醛含量下降，相关抗氧化酶基因表达显著上调。结论 MnDREB6E通过协同调控抗氧化代谢与渗透保护通路，正向增强桑树对盐/干旱胁迫耐受性。

关键词: 桑树, DREB转录因子, 干旱胁迫, 盐胁迫, 瞬时表达

Abstract:

Objective To elucidate the biological function of MnDREB6E, a member of the DREB transcription factor family in mulberry, and clarify its mechanism of action in response to abiotic stresses, thereby providing critical genetic resources for molecular breeding of stress-tolerant woody plants. Method MnDREB6E was cloned from salt-stressed mulberry using transcriptomic data. Bioinformatics tools were employed to analyze its sequence characteristics and phylogenetic relationships. Cis-acting elements were validated using yeast one-hybrid assays. Overexpression and RNAi-mediated suppression vectors were constructed and transiently transformed into mulberry via Agrobacterium-mediated transformation. Physiological and biochemical parameters as well as the antioxidant gene expression were measured under salt/drought stress. Result MnDREB6E contains a 1 173 bp ORF encoding 390 amino acids with a conserved AP2/ERF domain, classifying it into the DREB A6 subgroup. Its subcellular localization was predicted to be in the nucleus and specifically bound to GCC-box/DRE cis-elements. Under salt/drought stress, the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione S-transferase (GST) in the overexpressed plants were significantly enhanced. The contents of ascorbic acid (AsA), reduced glutathione (GSH), and proline increased, while the levels of reactive oxygen species (ROS), including superoxide anion (O₂^•-), hydrogen peroxide (H₂O₂), and hydroxyl radical (•OH) decreased. Additionally, the relative electrolyte leakage and malondialdehyde (MDA) content were reduced, and the expressions of antioxidant-related genes was significantly upregulated. Conclusion MnDREB6E coordinately regulates antioxidant metabolism and osmoprotection pathways, enhancing mulberry tolerance to salt/drought stress.

Key words: mulberry, DREB transcription factor, drought stress, salt stress, transient expression

董亚茹, 朱红, 王照红, 赵东晓, 刘惠芬. 桑树MnDREB6E的克隆及耐盐抗旱性分析[J]. 生物技术通报, 2026, 42(2): 306-316.

DONG Ya-ru, ZHU Hong, WANG Zhao-hong, ZHAO Dong-xiao, LIU Hui-fen. Cloning and Salt-drought Tolerance Analysis of MnDREB6E Gene in Mulberry[J]. Biotechnology Bulletin, 2026, 42(2): 306-316.

图/表 12

表1 基因克隆和载体构建所用引物

Table 1 Primers used in gene cloning and vector construction

引物名称 Primer name	序列 Sequence （5′‒3′）	用途 Application
MnDREB6E-F	ATGGAAGATCAGTTTCCCAAGATG	基因克隆 Gene cloning
MnDREB6E-R	TCATGATGTATCAGAGACCAG	基因克隆 Gene cloning
pROKⅡ-MnDREB6E-F	CTCTAGAGGATCCCCATGGAAGATCAGTTTCCCAAG	载体构建 Vector construction
pROKⅡ-MnDREB6E-R	TCGAGCTCGGTACCCTCATGATGTATCAGAGACCAG
pFGC5941-MnDREB6E-cis-F	ACAATTACCATGGGGCGTCATCGGTTTCATCACCGGG
pFGC5941-MnDREB6E-cis-R	AAATCATCGATTGGGCCACTCTTTTCTTGTGGGGTTG
pFGC5941-MnDREB6E-anti-F	AGTTAATTAAGACCCCGTCATCGGTTTCATCACCGGG
pFGC5941-MnDREB6E-anti-R	CTAGGGACTAGTCCCCCACTCTTTTCTTGTGGGGTTG
pGADT7-Rec2-F	ATGAACATGGAGGCCAGTG
pGADT7-Rec2-R	GATGGATCCCGTATCGATG
pHIS2-F	GCCTTCGTTTATCTTGCCTGCTC
pHIS2-R	CGATCGGTGCGGGCCTCTTC

表2 实时荧光定量PCR引物序列

Table 2 Primer sequences for RT-qPCR

基因 Gene	正向引物 Forward primer （5′‒3′）	反向引物 Reverse primer （5′‒3′）
MnDREB6E	CCAAATCAAAAGAACAAGCCT	ATTCTACTCAGCTGAACAGCT
MnRPL15	GGCTATGTGATTTACCGTGTT	TTGGTCCAGTATGAGTTGAGAA
β-actin	AGCAACTGGGATGACATGGAGA	CGACCACTGGCGTAAAGGGA
sodc	GCAGCACCAAAGCCACTCT	CCGTCGTCTTGTTGGGTCA
sod1	GACGCTGATGAGAAAGGT	TTAGACCTTGCCGGTGAC
mpod12	CCCCACGAACATCACGGTTGCCACTA	GCACGTATCTCACCTTGGCTGCCTGT
pod4	TCCCCTCTCGACAGCACC	AGTTTACCACCACGCAAT
cat1	CGTCACGCTGAGAGGTAC	TCAAATGCTTGGCCTCAC
gst10	GCCCTAGGTGACAAAGAT	CTACTCAATGCCATTCAT

图1 MnDREB6E的克隆M：DNA marker DL 2 000；1：阴性对照；2：MnDREB6E条带

Fig. 1 Cloning of MnDREB6E geneM: DNA marker DL 2 000; 1: negative control; 2: MnDREB6E gene band

图2 MnDREB6E与拟南芥A6组蛋白序列比对绿色线条代表保守的DNA结合结构域（AP2/ERF结构域），3个红色方框、1个蓝色方框和▲符号分别表示3个β折叠片层、1个α螺旋结构以及V14位点

Fig. 2 Sequence alignment of MnDREB6E with protein sequences from group A6 of Arabidopsis thalianaThe green line indicates the conserved DNA-binding domain (AP2/ERF domain), 3 red boxes, 1 blue box and ▲ respectively indicate 3 β-sheets, 1 α-helix and V14

图3 MnDREB6与拟南芥、水稻、玉米、胡杨、棉花、大豆DREB家族蛋白的系统进化关系

Fig. 3 Phylogenetic relationships between MnDREB6 and DREB family proteins from A. thaliana, Oryza sativa, Zea mays, Populus euphratica, Gossypium hirsutum, and Glycine max

图4 MnDREB6E结合GCC-box和DRE顺式元件分析阳性对照、阴性对照和试验菌体分别稀释5、50、500和5 000倍，TDO（SD/-His/-Leu/-Trp）固体培养基含有60 mmol/L 3-AT；1是阳性对照，2是阴性对照，3和7分别是GCC-box和DRE核心序列，4-6和8-9分别是GCC-box和DRE的核心突变序列

Fig. 4 Analyses of MnDREB6E binding to GCC-box and DRE motifsThe positive control, negative control, and experimental bacterial cells were diluted 5, 50, 500, and 5 000 fold respectively. The TDO (SD/-His/-Leu/-Trp) solid medium contained 60 mmol/L 3-AT; 1 refers to the positive control, 2 refers to the negative control, 3 and 7 correspond to the GCC-box and DRE core sequences respectively, while 4‒6 and 8‒9 refer to core mutant sequences of GCC-box and DRE motifs

图5 MnDREB6E瞬时转化桑树植株的RT-qPCR检测A和B分别为盐和干旱胁迫处理下瞬时转化桑树植株MnDREB6E表达量。*表示在P<0.05水平上差异显著。下同

Fig. 5 RT-qPCR detection of mulberry transiently transformed with MnDREB6EA and B are the expressions of the MnDREB6E gene in transiently transformed mulberry plants under salt and drought stress treatments, respectively. * indicates significantly different at the P<0.05 level. The same below

图6 逆境胁迫下Control、OE及RNAi桑树植株中相对电导率及MDA含量分析A：盐胁迫处理；B：干旱胁迫处理

Fig. 6 Analysis of electrolyte permeability and MDA content in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

图7 逆境胁迫下Control、OE及RNAi桑树植株中O2•-、H2O2和•OH含量的变化A：盐胁迫处理；B：干旱胁迫处理

Fig. 7 Changes in the contents of O2•-, H2O2, and •OH in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

图8 逆境胁迫下Control、OE及RNAi桑树植株中SOD、POD、CAT和GST活性的变化A：盐胁迫处理；B：干旱胁迫处理

Fig. 8 Changes in the activities of SOD, POD, CAT and GST in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

图9 逆境胁迫下Control、OE及RNAi桑树植株中AsA、GSH和脯氨酸含量的变化A：盐胁迫处理；B：干旱胁迫处理

Fig. 9 Changes in the contents of AsA, GSH and proline in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment. B: Drought stress treatment

图10 逆境胁迫下Control、OE及RNAi桑树植株中氧化酶基因表达分析A：盐胁迫处理（48 h）；B：干旱胁迫处理（48 h）

Fig. 10 Analysis of oxidase gene expression in Control, OE and RNAi mulberry plants under stress conditionsA: Salt stress treatment (48 h). B: Drought stress treatment (48 h)

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