马铃薯StDREBs基因的克隆及其表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0509

摘要/Abstract

摘要：

【目的】探究马铃薯中OsDREB1C同源基因StDREBs在低氮环境下的表达特性，以评估其是否具备提高马铃薯氮利用效率的潜力，为后续基因功能验证及作物改良提供理论依据。【方法】通过同源克隆获得OsDREB1C在马铃薯（Solanum tuberosum L.）中的同源基因，发现马铃薯中共有4个OsDREB1C的同源基因，对其进行生物信息学、亚细胞定位、低氮条件下的表达量分析。【结果】马铃薯中的StDREBs蛋白含有AP2保守结构域，蛋白产物均定位在细胞核中；StDREBs基因在根、茎、叶中均表达，在根中具有较高的表达量，在茎中其次，在叶片中的相对表达量最低。缺氮处理下，不同组织中的StDREBs基因表达量随着处理时间增加而上升，其中1/10 N处理18 h时，StDREBs基因的表达量达到峰值，表明StDREBs在马铃薯中参与对低氮环境的响应。【结论】马铃薯中OsDREB1C同源基因（StDREBs）在马铃薯低氮胁迫响应中起关键作用。

关键词: StDREBs, 马铃薯, 氮胁迫响应, 光诱导

Abstract:

【Objective】This study is aimed to investigate the expression characteristics of OsDREB1C homologous genes（StDREBs）in potato（Solanum tuberosum L.）under low nitrogen conditions, evaluating their potential to enhance nitrogen use efficiency in potato, thereby providing a theoretical basis for subsequent gene functional validation and crop improvement. 【Method】The homologous gene of OsDREB1C in potato was obtained by homologous cloning. Four homologous genes of OsDREB1C were found in the potato. Their expressions were analyzed via bioinformatics, subcellular localization and low nitrogen conditions. 【Result】 StDREBs protein in potato contained AP2 conserved domain, and the protein products were located in the nucleus. StDREBs gene was expressed in the roots, stems and leaves, with a higher expression in the roots, followed by in the stems, and the lowest relative expression in the leaves. Under nitrogen deficiency treatment, the expressions of StDREBs gene in different tissues increased with the increasing of treatment time, and the expression of StDREBs gene reached its peak when 1/10N was treated for 18 h, indicating that StDREBs participated in the response to low-nitrogen environment in potato. 【Conclusion】 OsDREB1C homologs（StDREBs）play a key role in potato under low-nitrogen conditions.

Key words: StDREBs, potato, response to nitrogen stress, photo-induction

张小妹, 周南伶, 张赛行, 王超, 沈玉龙, 管俊梅, 马玲. 马铃薯StDREBs基因的克隆及其表达分析[J]. 生物技术通报, 2024, 40(9): 42-50.

ZHANG Xiao-mei, ZHOU Nan-ling, ZHANG Sai-hang, WANG Chao, SHEN Yu-long, GUAN Jun-mei, MA Ling. Cloning and Expression Analysis of StDREBs Gene in Solanum tuberosum L.[J]. Biotechnology Bulletin, 2024, 40(9): 42-50.

图/表 9

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试剂和仪器Reagent and instrument	用途Fuctinon	来源Source
pRI101-AN-35S-EGFP 载体	构建载体	实验室提供
内切酶 EcoR I	构建载体	南京诺唯赞生物科技股份有限公司
内切酶 Kpn I	构建载体	南京诺唯赞生物科技股份有限公司
同源重组酶	构建载体	南京诺唯赞生物科技股份有限公司
PCR 高保真酶（PrimeSTAR^® GXL DNA Polymerase）	构建载体	宝日医生物技术（北京）有限公司
琼脂糖凝胶 DNA 回收试剂盒（TIANgel Purification Kit）	构建载体	天根生化科技（北京）有限公司
大肠杆菌 DH5α	构建载体	上海唯地生物技术有限公司
农杆菌 GV3101（pSoup-p19）	构建载体	上海唯地生物技术有限公司
核定位染料 DAPI 溶液	亚细胞定位	北京索莱宝科技有限公司
TRIzol^® reagent	总RNA提取	维百奥（北京）生物科技有限公司
氯仿（三氯甲烷）	总RNA提取	实验室提供
异丙醇	总RNA提取	天津市致远化学试剂有限公司
无水乙醇	总RNA提取	天津市致远化学试剂有限公司
cDNA 合成试剂盒	反转录合成	北京全式金生物技术股份有限公司
实时荧光定量试剂盒	定量分析	北京全式金生物技术股份有限公司
BIORADPCR 仪	构建载体	实验室提供
化学发光检测仪	构建载体	实验室提供
Zeiss 倒置共聚焦显微镜	亚细胞定位	实验室提供
Applied Biosystems 实时荧光定量PCR 仪	定量分析	实验室提供

试剂和仪器Reagent and instrument	用途Fuctinon	来源Source
pRI101-AN-35S-EGFP 载体	构建载体	实验室提供
内切酶 EcoR I	构建载体	南京诺唯赞生物科技股份有限公司
内切酶 Kpn I	构建载体	南京诺唯赞生物科技股份有限公司
同源重组酶	构建载体	南京诺唯赞生物科技股份有限公司
PCR 高保真酶（PrimeSTAR^® GXL DNA Polymerase）	构建载体	宝日医生物技术（北京）有限公司
琼脂糖凝胶 DNA 回收试剂盒（TIANgel Purification Kit）	构建载体	天根生化科技（北京）有限公司
大肠杆菌 DH5α	构建载体	上海唯地生物技术有限公司
农杆菌 GV3101（pSoup-p19）	构建载体	上海唯地生物技术有限公司
核定位染料 DAPI 溶液	亚细胞定位	北京索莱宝科技有限公司
TRIzol^® reagent	总RNA提取	维百奥（北京）生物科技有限公司
氯仿（三氯甲烷）	总RNA提取	实验室提供
异丙醇	总RNA提取	天津市致远化学试剂有限公司
无水乙醇	总RNA提取	天津市致远化学试剂有限公司
cDNA 合成试剂盒	反转录合成	北京全式金生物技术股份有限公司
实时荧光定量试剂盒	定量分析	北京全式金生物技术股份有限公司
BIORADPCR 仪	构建载体	实验室提供
化学发光检测仪	构建载体	实验室提供
Zeiss 倒置共聚焦显微镜	亚细胞定位	实验室提供
Applied Biosystems 实时荧光定量PCR 仪	定量分析	实验室提供

引物Primer	序列Sequence（5'-3'）
720-CDS-F	ATGATGAAGTTATTCTTAGC
720-CDS-R	TTATATAGAATAATTCCATA
730-CDS-F	ATGTTTCCAAGCTATTATTC
730-CDS-R	TTAGATTGAATAATTCCATA
740-CDS-F	ATGAATATCTTTGAAACCTA
740-CDS-R	TTAGATAGAATAATTCCATA
750-CDS-F	ATGGATATCTTTCGAAGCTA
750-CDS-R	TTAGATAGAATAGCTCCATA
pRI101-720-F	agagttgttgattcagaattcATGATGAAGTTATTCTTAGCTTCAAATAAC
pRI101-720-R	ctgtacaagcccgggggtaccTATAGAATAATTCCATAAAGGCATATAAGC
pRI101-730-F	agagttgttgattcagaattcATGTTTCCAAGCTATTATTCGGAGC
pRI101-730-R	ctgtacaagcccgggggtaccTTAGATTGAATAATTCCATAAAGACATGTAA
pRI101-740-F	agagttgttgattcagaattcATGAATATCTTTGAAACCTATTATTCAGACC
pRI101-740-R	ctgtacaagcccgggggtaccTTAGATAGAATAATTCCATAAAGTCATGTAAGC
pRI101-750-F	agagttgttgattcagaattcATGGATATCTTTCGAAGCTATTATTCG
pRI101-750-R	ctgtacaagcccgggggtaccTTAGATAGAATAGCTCCATAAAGGCATAT
720qPCR-F	CCAACAGTAGAAATGGCAGCTAGAG
720qPCR-R	CTTCCGACTGATCTGATGATGATGG
730qPCR-F	GAACTTTGCTGACTCTGTTTGGAG
730qPCR-R	CCTCATTCATAGAGAGGGCCTTTTC
740qPCR-F	TAAAGATATTCAAAAGGCGGCCGC
740qPCR-R	CTCTGGCGCTTCTTCGTTCATAAA
750qPCR-F	GCTATAGCATTAAGAGGCCGTTCT
750qPCR-R	CATCCGGAGTACTAGATTGATCTCC
Actin-F	GGGATGGAGAAGTTTGGTGGTGG
Actin-R	CTTCGACCAAGGGATGGTGTAGC

引物Primer	序列Sequence（5'-3'）
720-CDS-F	ATGATGAAGTTATTCTTAGC
720-CDS-R	TTATATAGAATAATTCCATA
730-CDS-F	ATGTTTCCAAGCTATTATTC
730-CDS-R	TTAGATTGAATAATTCCATA
740-CDS-F	ATGAATATCTTTGAAACCTA
740-CDS-R	TTAGATAGAATAATTCCATA
750-CDS-F	ATGGATATCTTTCGAAGCTA
750-CDS-R	TTAGATAGAATAGCTCCATA
pRI101-720-F	agagttgttgattcagaattcATGATGAAGTTATTCTTAGCTTCAAATAAC
pRI101-720-R	ctgtacaagcccgggggtaccTATAGAATAATTCCATAAAGGCATATAAGC
pRI101-730-F	agagttgttgattcagaattcATGTTTCCAAGCTATTATTCGGAGC
pRI101-730-R	ctgtacaagcccgggggtaccTTAGATTGAATAATTCCATAAAGACATGTAA
pRI101-740-F	agagttgttgattcagaattcATGAATATCTTTGAAACCTATTATTCAGACC
pRI101-740-R	ctgtacaagcccgggggtaccTTAGATAGAATAATTCCATAAAGTCATGTAAGC
pRI101-750-F	agagttgttgattcagaattcATGGATATCTTTCGAAGCTATTATTCG
pRI101-750-R	ctgtacaagcccgggggtaccTTAGATAGAATAGCTCCATAAAGGCATAT
720qPCR-F	CCAACAGTAGAAATGGCAGCTAGAG
720qPCR-R	CTTCCGACTGATCTGATGATGATGG
730qPCR-F	GAACTTTGCTGACTCTGTTTGGAG
730qPCR-R	CCTCATTCATAGAGAGGGCCTTTTC
740qPCR-F	TAAAGATATTCAAAAGGCGGCCGC
740qPCR-R	CTCTGGCGCTTCTTCGTTCATAAA
750qPCR-F	GCTATAGCATTAAGAGGCCGTTCT
750qPCR-R	CATCCGGAGTACTAGATTGATCTCC
Actin-F	GGGATGGAGAAGTTTGGTGGTGG
Actin-R	CTTCGACCAAGGGATGGTGTAGC

蛋白质 Protein	氨基酸数量Amount of amino acids	分子质量Molecular mass/Da	原子总数 Total atoms	不稳定系数Coefficient of instability	pI	脂溶性指数Fat solubility index	亲水指数Hydrophilic index	亚细胞定位Subcellular localization
StDREB720	199	22 478	3 125	65.51	6.44	69.70	-0.37	细胞核
StDREB730	236	26 319	3 628	61.92	4.98	67.50	-0.48	细胞核、细胞质
StDREB740	258	28 601	3 909	70.35	4.55	57.67	-0.56	细胞核、细胞质
StDREB750	217	24 264	3 334	55.26	5.20	57.65	-0.60	细胞核、细胞质