Cloning and Functional Analysis of the StAS2-15 Gene in Potato under Salt Stress

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

Abstract

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

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.

Figures/Tables 7

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

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

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

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

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

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

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

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