马铃薯StAS2-15基因的克隆及盐胁迫下功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1171

摘要/Abstract

摘要：

目的 ASYMMETRIC LEAVES2（AS2）基因家族是植物特有的转录家族，在植物生长发育和胁迫响应中发挥重要作用。研究AS2转录因子在马铃薯响应逆境胁迫特别是盐胁迫中的功能与机制，为培育抗盐马铃薯新品种提供理论和技术支撑。方法通过克隆野生型马铃薯品种底西芮（DES）叶片中的StAS2-15基因，并通过同源重组方法构建StAS2-15过表达载体，转化DES得到阳性植株，进行抗盐农艺性状分析及生理生化实验。结果盐胁迫表型实验表明，随着盐浓度的增加，过表达植株（OEs）和野生型（DES）的株高、鲜重、根数和根长显著降低，其中株高分别降低了2.42%-43.21%和28.77%-58.49%；同一盐浓度下，OEs的株高、鲜重、根数和根长较DES显著增加。不同盐浓度处理下，过表达StAS2-15基因阳性植株叶片中超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性和脯氨酸含量显著高于野生型，丙二醛（MDA）含量显著低于野生型。RT-qPCR结果显示，盐胁迫下马铃薯相关胁迫响应基因ABI3、MYB2、SnRK2s等在过表达植株中的表达量显著高于野生型。结论 StAS2-15基因的过表达使马铃薯体内一些抗氧化酶活性、渗透调节物质含量和其他盐胁迫响应基因的表达水平发生改变，且盐胁迫下植株生长状态呈现脱敏感表型，影响了马铃薯的生长发育。

关键词: 马铃薯, StAS2-15, 愈伤组织转化体系, 盐胁迫, RT-qPCR

Abstract:

Objective The ASYMMETRIC LEAVES2 (AS2) gene family is a plant-specific transcription family that plays an important role in plant growth and development and response to stress. The study of the function and mechanism of AS2 transcription factors in potato in response to adversity stress, especially salt stress, may provide theoretical and technical support for breeding new salt-resistant potato varieties. Method We cloned the StAS2-15 gene from the leaves of wild-type potato variety DES, constructed the StAS2-15 overexpression vector by homologous recombination, and transformed DES to obtain positive plants for salt-resistant agronomic traits and physiological and biochemical experiments. Result Salt stress phenotyping experiments showed that plant height, fresh weight, root number and root length of overexpressed plants (OEs) and wild type (DES) significantly decreased with increasing salt concentration, in which plant height decreased by 2.42%-43.21% and 28.77%-58.49%, respectively; and plant height, fresh weight, root number and root length of OEs significantly increased compared with those of DES at the same salt concentration. Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities and proline content in the leaves of positive plants overexpressing StAS2-15 gene were significantly higher and malondialdehyde (MDA) content was significantly lower than that of the wild type under different salt concentration treatments.The qRT-PCR results showed that the expressions of potato stress-related response genes ABI3, MYB2, and SnRK2s in overexpressed plants was significantly higher than that in wild type under salt stress. The RT-qPCR results showed that the expressions of potato stress-related response genes ABI3, MYB2, and SnRK2s in overexpressed plants was significantly higher than that in wild type under salt stress. Conclusion The overexpression of StAS2-15 gene alteres the expressions of some antioxidant enzymes, content of osmoregulatory substances and other salt stress responsive genes in potato, and the growth status of plants under salt stress shows a desensitized phenotype, which affects the growth and development of potato.

Key words: potato, StAS2-15, callus transformation system, salt stress, RT-qPCR

宋慧洋, 苏宝杰, 李京昊, 梅超, 宋倩娜, 崔福柱, 冯瑞云. 马铃薯StAS2-15基因的克隆及盐胁迫下功能分析[J]. 生物技术通报, 2025, 41(5): 119-128.

SONG Hui-yang, SU Bao-jie, LI Jing-hao, MEI Chao, SONG Qian-na, CUI Fu-zhu, FENG Rui-yun. Cloning and Functional Analysis of the StAS2-15 Gene in Potato under Salt Stress[J]. Biotechnology Bulletin, 2025, 41(5): 119-128.

图/表 7

表1 荧光定量引物序列

Table 1 Primer sequences for fluorescence quantification

引物名称 Primer name	引物序列 Sequence（5'-3'）
Actin-F	GGGATGGAGAAGTTTGGTGGTGG
Actin-R	CTTCGACCAAGGGATGGTGTAGC
StAS2-15-F	CGTCTCCGAGATCCAGTCTA
StAS2-15-R	GGTACTAGCAATGCCTCC
ABF4-F	CTTCTGCACATACAAGGATATTCAA
ABF4-R	TGCCTAGCCAAAGGGAAATCA
MYB2-F	ATGACATGGACCTCCGACGA
MYB2-R	CGTTCGCTTTAGACCAGCAC
SnRK2s-F	TTGCTGTAGAGAAGGTGGGT
SnRK2s-R	GTCCTACTGTCACTGCCGTC
DREB2A-F	CACAGAAGAAAAGAGAAATGGCT
DREB2A-R	TCCACTATAATCCAACGGCAGA
ABI3-F	TGCTGACATGAAGTGTGGCA
ABI3-R	TTTTGCCCTCCGACTTTGGT
ABI5-F	TAAAACAGGCCCTGGCGGAG
ABI5-R	TTGCGCCTTTGTTTGAGCTT

图1 马铃薯StAS2-15过表达载体的构建和基因表达量分析A：1代表含目的基因StAS2-15的质粒；2代表Hind Ⅲ-Sac Ⅰ酶切质粒；M代表DNA marker。B：DES代表野生型；P代表含目的基因StAS2-15的质粒；1-3代表过表达阳性植株。C：OEs代表过表达阳性植株；显著性差异通过Duncan新复极差法确定；不同字母表示差异显著（P<0.05）；误差线表示3个独立生物学实验获得的标准差；以Actin为内参对目的基因相对表达量进行标准化计算，下同

Fig. 1 Construction of potato StAS2-15 overexpression vector and gene expression analysisA: 1 refers to the plasmid containing the gene of interest StAS2-15; 2 refers to HindIII-Sac I digest plasmid; M refers to DNA marker. B: DES refers to wild type; P refers to the plasmid containing the gene of interest StAS2-15; 1-3 refers to overexpression-positive plants. C: OEs refer to overexpressed positive plants. Significant differences were determined by Duncan's new complex polarity difference method. Different letters showed significant differences (P<0.05). Error bars indicate standard deviations obtained from 3 independent biological experiments; Actin was used as the internal reference gene to standardize the relative expression of the target gene. The same below

图2 马铃薯StAS2-15过表达阳性植株筛选流程A：茎段预培养；B：农杆菌侵染；C：愈伤组织形成；D：愈伤组织出苗；E：移苗入培养瓶

Fig. 2 Screening protocol of potato StAS2-15 overexpressing positive plantA: Pre-culture of stem segments. B: Agrobacterium infection. C: Callus formation. D: callus emergence. E: Transfer seedlings into culture flasks

图3 盐胁迫20 d后组培苗生理指标测定

Fig. 3 Physiological indices of tissue culture seedlings after 20 d of salt stress

图4 盐胁迫20 d后组培苗生长状态DES：野生型；OEs：过表达阳性植株

Fig. 4 Growth state of tissue culture seedlings after 20 d of salt stressDES: Wild type. OEs: Overexpressed positive plants

图5 不同氯化钠浓度下植株中抗氧化指标的变化

Fig. 5 Variations in antioxidant indexes in plants under different sodium chloride concentrations

图6 马铃薯 StAS2-15过表达阳性植株中盐胁迫相关基因的表达量分析

Fig. 6 Expression analysis of salt stress-related genes in potato StAS2-15 overexpressing positive plants

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