通过马铃薯毛状根体系快速鉴定StHKT1基因的功能

doi:10.13560/j.cnki.biotech.bull.1985.2025-0658

摘要/Abstract

摘要：

目的建立一种简单、高效且无需组培的马铃薯遗传转化方法并鉴定StHKT1基因功能，为后续批量研究马铃薯基因功能及培育优质种质奠定基础。方法将蘸取发根农杆菌的马铃薯顶芽置于MS固体培养基上诱导毛状根，评估其诱导和转化效率；通过RT-qPCR检测毛状根中StHKT1基因的相对表达量。利用NaCl处理对照组和过表达StHKT1的复合体马铃薯植株并分析耐盐性。结果侵染顶芽约30 d后可获得具有大量毛状根的复合体植株，其中毛状根诱导和转化效率分别为100%和87.4%，复合体植株毛状根中StHKT1基因的相对表达量显著增加。在100 mmol/L NaCl处理下，转基因毛状根较对照组更长，复合体植株鲜重优于对照组；不同浓度NaCl诱导下，StHKT1基因的相对表达量显著上调，且转基因毛状根重量增幅大于对照组。最后，在200 mmol/L NaCl处理下，过表达StHKT1复合体马铃薯植株的生长状态优于对照组，丙二醛含量显著降低，叶绿素含量和SOD酶活性显著升高。结论利用马铃薯毛状根遗传转化体系可快速获得转基因复合体植株，且带有过表达StHKT1毛状根复合体马铃薯植株的耐盐性更强。

关键词: 毛状根转化, 发根农杆菌, 马铃薯, 复合体植株, StHKT1

Abstract:

Objective To establish a simple, high-efficiency and tissue culture-free transformation method and validatethefunction of StHKT1 gene, which will provide a theoretical basis for large-scale gene function studies and germplasm improvement in potato. Method Terminal buds of potato were scraped on the plate containing Agrobacterium rhizogenes and then planted on MS solid medium to induce hairy roots. The induction and transformation efficiency of hairy roots were evaluated. The expressions of the StHKT1 gene in hairy roots were detected by qRT-PCR. Both control plants and composite plants overexpressing StHKT1 gene were treated with NaCl to analyze the tolerance to salt. Result The composite plants with abundant hairy roots were successfully obtained after infecting terminal buds for 30 d. The induction and transformation efficiency of the hairy roots were 100% and 87.4% respectively, and the relative expression of the StHKT1 gene in the hairy roots of the composite plants significantly increased. Under 100 mmol/L NaCl stress, the composite plants showed better growth performance in root length and fresh weight compared to control plants. The StHKT1 gene was inducted by different concentration salt stress, and the weight of the transgenic hairy roots was significantly higher than that of controls. Finally, Under 200 mmol/L NaCl stress, the growth status of the composite potato plants remained superior to controls. Meanwhile, the MDA content decreased significantly in the composite plants, while the chlorophyll content and SOD activity increased significantly. Conclusion Composite plants can be rapidly obtained via Agrobacterium rhizogenes-mediated hairy root transformation in potato, and the hairy-root composite potato plants overexpressing StHKT1 show the enhanced tolerance to salt.

Key words: hairy root transformation, Agrobacterium rhizogenes, potato, composite plants, StHKT1

宋倩娜, 武诗云, 曹时瑾, 段永红, 冯瑞云. 通过马铃薯毛状根体系快速鉴定StHKT1基因的功能[J]. 生物技术通报, 2025, 41(10): 233-241.

SONG Qian-na, WU Shi-yun, CAO Shi-jin, DUAN Yong-hong, FENG Rui-yun. Rapid Identification of Gene StHKT1’s Function via Potato Hairy Root System[J]. Biotechnology Bulletin, 2025, 41(10): 233-241.

图/表 8

图1 StHKTI蛋白进化树构建与结构域分析A：系统进化树；B：StHKT1蛋白结构域；C：StHKT1蛋白PA、PB、PC和PD孔域处的氨基酸序列对比

Fig. 1 Phylogenetic trees constructing and domain analysis of StHKT1 proteinA: Phylogenetic trees. B: Domain of StHKT1 protein. C: Sequence alignment of p-loop (PA, PB, PC and PD) of StHKT1 protein

图2 马铃薯毛状根转化及复合体植株获得A：待转化马铃薯组培苗；B：顶芽外植体；C：MSU440农杆菌侵染；D：共培养2 d后的外植体；E：10 d后诱导获得毛状根；F：筛选GFP毛状根；G：获得复合体马铃薯植株；H：复合体植株继续培养

Fig. 2 Genetic transformation of potato hairy roots and obtaining of composite plantsA: Ideal potato seedings for transformation. B: Explants on terminal bud. C: Infected by MSU440 Agrobacterium rhizogenes. D: Explants co-cultured for 2 d. E: Hairy roots of induction 10 d after inoculation. F: Selection of GFP-positive hairy roots. G: Potato plants with composite. H: The composite plants cultured continuously

表1 毛状根诱导效率和转化效率的评估

Table 1 Evaluation on the induction efficiency and transformation efficiency of hairy roots

转化

Transformation

毛状根诱导效率

Induction efficiency of hairy roots

毛状根群体

Hairy root clones

转化效率

Transformation efficiency

图3 复合体马铃薯植株中StHKT1基因相对表达量检测A：PCR检测StHKT1基因的整合；P：质粒；W：野生型；K：蒸馏水；B： RT-qPCR检测StHKT1基因的相对表达量。不同的小写字母表示P<0.05。下同

Fig. 3 Detection of relative expressions of StHKT1 in the composite potato plantsA: PCR detection of StHKT1 geneintegration. P: Plasmid; W: wild type; K: ddH2O. B: Detecting relative expression of StHKT1 by RT-qPCR. The different lowercase letters indicate P<0.05. The same below

图4 马铃薯复合体植株毛状根耐盐性及StHKT1表达模式分析A：不同盐浓度处理下，毛状根增加百分比；B：不同盐浓度处理下，毛状根中StHKT1基因的相对表达量；VC和OE为3株独立复合体植株毛状根的混样

Fig. 4 Analysis of tolerance to salt and StHKT1 expression pattern of hairy roots in the composite potato plantsA: Increase ratio of hairy roots under different NaCl concentrations. B: Relative expression of StHKT1 in hairy roots under different NaCl concentrations. VC and OE contain mixed samples of hairy roots from three independent composite plants

表2 盐胁迫下马铃薯复合体植株的生长指标

Table 2 Growth indicators of the composite potato plants under salt stress

样本 Sample	NaCl浓度 Concentrations of NaCl （mmol/L）	根长 Length of root （cm）	根数 No. of roots	根鲜重 Fresh weight of root （g）	茎长 Length of shoot （cm）	植株鲜重 Fresh weight of plant （g）
VC	0	9.833±0.763a	3.000±1.000b	0.021±0.008a	4.567±0.551a	0.059±0.001a
VC	100	5.300±0.650bc	3.000±1.000b	0.006±0.001b	1.967±0.642b	0.039±0.002bc
OE	0	9.600±2.970a	5.000±0.001a	0.025±0.021a	5.100±0.529a	0.080±0.012a
OE	100	6.900±0.360b	4.000±1.000b	0.007±0.002b	2.967±0.586b	0.059±0.008b

图5 StHKT1过表达复合体马铃薯植株盐胁迫下的表型分析A-B：盐胁迫处理2周后复合体植株生长状况；C：盐胁迫处理下复合体植株毛状根的生长状况

Fig. 5 Phenotypic analysis of StHKT1-overexpressed composite potato plants under salt stressA-B: Growth status of composite plants treated with salt for two weeks. C: Growth status of hairy roots of composite treated with salt for two weeks

图6 盐胁迫下StHKT1过表达复合体马铃薯植株生理指标测定A：盐胁迫下毛状根中StHKT1基因的相对表达量；B：叶片中叶绿素含量；C、E：叶片和毛状根中SOD酶活性；D、F：叶片和毛状根中MDA含量。OE-1、OE-2和OE-3代表3株独立植株的毛状根；VC和OE包含3株复合体植株叶片和毛状根的混样

Fig. 6 Physiological characteristics of overexpressing StHKT1 composite potato plants under salt stressA: Relative expression of StHKT1 in the hairy roots under salt stress. B: Chlorophyll content in leaf. C, E: SOD activity in leaf and hairy roots. D, F: MDA content of leaf and hairy roots. OE-1, OE-2 and OE-3 refer to three independent hairy roots from composite plants; VC and OE contain mixed samples of leaves and hairy roots from three independent composite plants

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