马铃薯块茎变绿中StHY5对龙葵素合成的促进作用

doi:10.13560/j.cnki.biotech.bull.1985.2024-0514

摘要/Abstract

摘要：

【目的】马铃薯因采后贮藏、运输及货架摆放等因素，容易造成块茎“绿变”，导致块茎中SGAs大量累积，而HY5是植物中光信号的重要传递因子，研究其在块茎变绿中的作用，为马铃薯采后环节因光照导致的龙葵素积累提供分子基础。【方法】通过基因表达分析、转录组分析、靶向代谢物分析、亚细胞定位、酵母单杂交、双荧光素酶等试验，阐明StHY5在光照后块茎变绿中的作用。【结果】StHY5在薯皮和薯肉中均有表达，但表达模式不同；绿变处理后，薯肉中StHY5的表达水平和SGAs含量的变化一致，与此同时，龙葵素合成基因StSGT1/GAME1和StGAME4表达显著上调；酵母单杂交试验和双荧光素酶试验证明，StHY5可以直接结合StSGT1/GAME1和StGAME4的启动子并激活其表达。【结论】在马铃薯绿变过程中，StHY5通过直接上调StSGT1/GAME1和StGAME4的表达，从而促进SGAs的积累。

关键词: 马铃薯, StHY5, 光照诱导薯块绿变, 龙葵素积累

Abstract:

【Objective】Post-harvest storage, transportation, shelving, and other factors can lead to the “turning green” of potato, resulting in a significant accumulation of SGAs in tuber. HY5 plays a crucial role as a key mediator of light signals in plants. Investigating its involvement in the turning-green of tuber can establish a molecular foundation for understanding SGAs accumulation induced by light in post-harvest potato. 【Method】Through comprehensive gene expression analysis, transcriptome profiling, targeted metabolite analysis, subcellular localization studies, yeast single hybridization and double luciferase experiments, the involvement of StHY5 in tuber turning green following light exposure was preliminarily elucidated.【Result】The StHY5 gene was expressed in both the peel and flesh of potato, but with different expression patterns. During the treatment of potato greening, the expression of the StHY5 gene in potato meat correlated with changes in SGAs content, while the expression of SGAs synthesizing genes StSGT1/GAME1 and StGAME4 was significantly up-regulated. Yeast single hybridization and double luciferase experiments demonstrated that StHY5 directly up-regulated the expression of StSGT1/GAME1 and StGAME4.【Conclusion】During the process of potato greening, the StHY5 plays a role in promoting the accumulation of SGAs by directly up-regulating the expression of StSGT1/GAME1 and StGAME4. This finding provides a solid foundation for further investigation into the function of StHY5 in potato greening.

Key words: potato, StHY5 gene, tuber turning-green induced by light, SGAs accumulation

王超, 白如仟, 管俊梅, 罗稷林, 何雪姣, 迟绍轶, 马玲. 马铃薯块茎变绿中StHY5对龙葵素合成的促进作用[J]. 生物技术通报, 2024, 40(9): 113-122.

WANG Chao, BAI Ru-qian, GUAN Jun-mei, LUO Ji-lin, HE Xue-jiao, CHI Shao-yi, MA Ling. Promotion of StHY5 in the Synthesis of SGAs during Tuber Turning-green of Potato[J]. Biotechnology Bulletin, 2024, 40(9): 113-122.

图/表 10

表1 RT-qPCR所用引物

Table 1 Primers used in RT-qPCR

基因 Gene	正向引物序列Forward primer sequence（5'-3'）	反向引物序列Reverse primer sequence（5'-3'）
StActin	GGGATGGAGAAGTTTGGTGGTGG	CTTCGACCAAGGGATGGTGTAG
StHY5	AGATCTGGAAGCAAGGGTGAAG	CACCTGCTGTTGTGTTCTTCAG

表2 流动相梯度

Table 2 Eluent gradient

时间Time/min	A/%	B/%	流速Flow/（mL·min^-1）
0.00	95.00	5.00	0.800
5.00	60.00	40.00	0.800
6.00	0.00	100.00	0.800
8.00	0.00	100.00	0.800

图1 茄科中StHY5的系统发育树分析及同源序列比对 A：StHY5蛋白的系统发育树分析，番茄和马铃薯用红色字体标出；B：StHY5的同源序列比对，绿色标出的氨基酸序列区间（89-140 aa）为bZIP-HY5-like结构域

Fig. 1 Phylogenetic tree and homologous sequence alignment of StHY5 in Solanaceae A: Phylogenetic tree analysis and homologous sequence alignment of StHY5, Solanum lycopersicum and Solanum tuberosum are marked in red; B: homologous sequence alignment of StHY5, the amino acid sequence interval（89-140 aa）marked in green is the bZIP-HY5-like domain

图2 马铃薯StHY5的时空表达分析（A）、原位杂交（B）和亚细胞定位分析（C）不同小写字母表示在0.05水平差异显著。下同

Fig. 2 Temporal and spatial expression analysis (A), in-situ hybridization (B) and subcellular localization analysis (C) of StHY5 in potato Different lowercases indicate significant differences at the 0.05 level. The same below

表3 马铃薯StHY5启动子顺式作用元件分析

Table 3 Cis-acting elements of StHY5 promoter in potato

名称 Name	序列 Sequence	数量 Account	功能 Function
MBS	CAACTG	1	参与干旱诱导的MYB结合位点MYB binding site involved in drought-inducibility
LTR	CCGAAA	1	参与低温响应的顺式作用元件Cis-acting element involved in low-temperature responsiveness
GC-motif	CCCCCG	1	参与缺氧特异性诱导的增强剂Enhancer-like element involved in anoxic specific inducibility
P-box	CCTTTTG	1	赤霉素反应元件Gibberellin-responsive element
GT1-motif	GGTTAA	1	光敏元件Light-sensitive element
TCCC-motif	TCTCCCT	1	光响应元件的一部分Part of a light responsive element
TCA-element	CCATCTTTTT/TCAGAAGAGG	3	参与水杨酸反应的顺式作用元件Cis-acting element involved in salicylic acid responsiveness
ABRE	ACGTG/GCAACGTGTC	3	参与脱落酸反应的顺式作用元件Cis-acting element involved in the abscisic acid responsiveness
ARE	AAACCA	2	厌氧诱导所需的顺式作用元件Cis-acting regulatory element essential for the anaerobic induction
G-Box	CACGTT	3	参与光反应的顺式作用元件Cis-acting regulatory element involved in light responsiveness
CGTCA-motif	CGTCA	5	茉莉酸甲酯响应元件Cis-acting regulatory element involved in the MeJA-responsiveness
TGACG-motif	TGACG	5	茉莉酸甲酯响应元件Cis-acting regulatory element involved in the MeJA-responsiveness

图3 绿变处理中SGAs含量及StHY5表达量 A-B：CIP183薯皮、薯肉的SGAs含量；C-D：CIP183薯皮、薯肉的StHY5表达量；E-F：CIP150薯皮、薯肉的SGAs含量；G-H：CIP150薯皮、薯肉的StHY5表达量

Fig. 3 SGAs content and StHY5 expression in green turning treatment A-B: CIP183 SGAs intensity of potato peel and flesh. C-D: StHY5 expression of CIP183 potato peel and flesh. E-F: CIP150 SGAs intensity of potato peel and flesh.G-H: StHY5 expression in CIP150 potato peel and flesh

图4 马铃薯块茎CIP150薯肉绿变前后的转录组分析 A：火山图分析；B：KEGG富集分析；C：GO富集分析

Fig. 4 Transcriptome analysis of potato tuber CIP150 before and after flesh turning green A: Volcanic map analysis. B: KEGG enrichment analysis. C: GO enrichment analysis

图5 马铃薯块茎CIP150薯肉绿变前后SGAs合成基因的差异表达 StAACT至St7-DR2为胆固醇合成途径，StGAME7至StSGT3为龙葵素合成途径，箭头指示的基因顺序即为龙葵素合成通路基因顺序

Fig. 5 Differential expression of SGAs synthetic genes in potato tuber CIP150 before and after flesh turning green StAACT to St7-DR2 is the synthesis pathway of cholesterol, StGAME7 to StSGT3 is the synthesis pathway of SGAs, the arrow indicates the sequence of genes involved in the SGAs synthesis pathway

图6 StHY5与StSGT1/GAME1和StGAME4启动子间的酵母单杂交分析 L、W、H分别代表亮氨酸、色氨酸和组氨酸，10-1、10-2、10-3分别代表酵母菌OD600分别调至0.1、0.01、0.001时的点板浓度

Fig. 6 Yeast single hybridization analysis between StHY5 and StSGT1/GAME1 and StGAME4 promoters L, W and H indicate Leu, Trp and His respectively, and 10-1, 10-2 and 10-3 indicate the point plate concentration of yeast OD600 when it is adjusted to 0.1, 0.01 and 0.001, respectively

图7 StHY5与StSGT1/GAME1和StGAME4启动子间的双荧光素酶分析

Fig. 7 Double luciferase analysis between StHY5 and StS-GT1/GAME1 and StGAME4 promoters **P<0.01, ***P<0.001

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