关键词: 马铃薯, 高温胁迫, 结薯因子StSP6A, 转基因, 试管块茎

Abstract:

【Objective】High temperatures, as a major abiotic stress factor, affect potato tuber formation. Exploring the mechanisms underlying potato tuber formation under high-temperature stress holds significant theoretical value for elucidating the intrinsic mechanisms by which environmental factors regulate tuber development.【Method】Using in vitro seedlings from 103 potato germplasm accessions as experimental material, the seedlings were subjected to either optimal temperature (17 ℃) or high temperature (28 ℃) in plant growth chamber for 150 days. After collecting and analyzing the phenotypic traits of the seedlings, representative germplasm accessions were selected for transcriptomic analysis, which was validated using real-time quantitative PCR (RT-qPCR). Furthermore, following the construction of an SP6A overexpression vector, genetic transformation was performed using Longshu 10. After subjecting the transgenic material to high-temperature treatment, phenotypic evaluation and molecular-level analysis were conducted.【Result】After treatment at 17 ℃ and 28 ℃, 103 accessions were classified into four temperature response patterns: widespread tuber-forming type (GroupⅠ, 46 accessions; tubers formed at both temperatures), heat-sensitive type (GroupⅡ, 33 accessions; tubers formed only at 17 ℃), high temperatures /high sugar induced sensitive type (GroupⅢ, 11 accessions; tubers formed only at 28 ℃), and non-tuber-forming (GroupⅣ, 13 accessions; no tubers formed at either temperature). Furthermore, a variance analysis indicated that high-temperature stress shortened the number of days to tuber formation, reduced plant height, and significantly decreased the weight and number of test-tube tubers. At the same time, the coefficients of variation for those indicators all increased significantly. Transcriptome analysis revealed that Longshu 11 (which tuberizes only at 17 ℃) and Lily (which tuberizes only at 28 ℃) had 3 398 and 2 197 differentially expressed genes, respectively. with the differentially expressed genes in Longshu 11 focused on molecular functions such as catalytic activity, as well as metabolic processes and pathways, while those in Lily were enriched in cell wall remodeling and carbohydrate metabolism pathways. A combined transcriptomic analysis revealed that the two groups shared 730 differentially expressed genes, which were classified into eight categories based on their expression patterns. Among the 14 key differentially expressed genes, the tuber-specific gene SP6A is the primary factor responsible for the differences in tuber formation in in vitro tubers under high-temperature stress. An increased number of microtubers was observed in the overexpression plants (SP6A-OE) under 17 ℃ treatment. Under 28 ℃ heat treatment, although the formation of in vitro tubers was inhibited in both the SP6A-OE and control, the SP6A-OE line still produced more in vitro tubers. Western blot results indicated that the expression level of the SP6A protein in SP6A-OE plants was lower at 28 ℃ than at 17 ℃. Meanwhile, RT-qPCR results showed that SP6A expression was suppressed at high temperatures, whereas the expression levels of both SP5G and HSFA6b were significantly elevated at 28 ℃.【Conclusion】Under high-temperature stress, different potato germplasm exhibit varying tuber-forming habits. Overexpression of StSP6A alleviates the inhibitory effect caused by heat stress on axillary tuber formation, thereby enhancing the ability of in-vitro seedlings to form tubers under high-temperature conditions.

Key words: potato, high temperature stress, SELF-PRUNING 6A (StSP6A), genetic modification, in vitro tubers