异源过表达大豆GmNF-YB24提高转基因烟草抗旱性

doi:10.13560/j.cnki.biotech.bull.1985.2025-0046

摘要/Abstract

摘要：

目的核因子（NF-Y）基因参与植物抗旱性的调控。对大豆GmNF-YB24的抗旱功能及抗旱机制进行解析，为后续NF-YB基因在高抗大豆遗传育种中的应用提供理论依据。方法利用RT-qPCR检测GmNF-YB24在盐、干旱和低温胁迫下的表达。克隆GmNF-YB24，构建植物表达载体并转化烟草。对GmNF-YB24转基因烟草的抗旱性进行鉴定。结果盐、干旱和低温胁迫均能诱导GmNF-YB24的表达，且GmNF-YB24的表达对干旱胁迫的响应最明显。GmNF-YB24开放阅读框全长516 bp，编码的蛋白含有171个氨基酸残基。GmNF-YB24蛋白序列中含有1个组蛋白折叠基序（HFM）。构建pRI101-GmNF-YB24植物表达载体并转化烟草。共获得5株GmNF-YB24转基因烟草植株（OE1-OE5）。干旱及复水处理后，GmNF-YB24转基因烟草的表现优于野生型烟草。干旱胁迫下，与野生型烟草相比，GmNF-YB24转基因烟草的脯氨酸和可溶性糖含量增加，相对电解质渗透率和丙二醛含量降低，抗氧化酶活性提高，减少了过氧化物的积累。GmNF-YB24转基因烟草中胁迫相关基因NtOsmotin和NtERD10B的表达量高于野生型烟草。结论 GmNF-YB24在烟草中的异源过表达提高了转基因烟草的抗旱性。

关键词: 大豆, 核因子Y, NF-YB, 转基因烟草, 抗旱性

Abstract:

Objective Nuclear factor (NF-Y) genes are involved in the regulation of drought resistance in plants. The function of resistance to drought and mechanism of resistance to drought of soybean GmNF-YB24 were analyzed, which may provide theoretical basis for the subsequent application of NF-YB genes in the genetic breeding of soybean with high resistance. Method The expression of GmNF-YB24 under salt, drought and cold stresses was detected by RT-qPCR. GmNF-YB24 was cloned and its plant expression vector was constructed to transformed tobacco. The resistance of GmNF-YB24 transgenic tobacco to drought was evaluated. Result Salt, drought and cold stresses induced the expression of GmNF-YB24, and the expression of GmNF-YB24 was the most obvious in response to drought stress. The open reading frame of GmNF-YB24 was 516 bp, and the encoded protein contained 171 amino acid residues. The GmNF-YB24 protein contained a histone-fold motif (HFM). The plant expression vector pRI101-GmNF-YB24 was constructed and transformed into tobacco. Five GmNF-YB24 transgenic tobacco plants (OE1-OE5) were obtained. After drought and rewater treatment, the performance of GmNF-YB24 transgenic tobacco was superior to that of wild-type tobacco. Under drought stress, compared with wild-type tobacco, GmNF-YB24 transgenic tobacco had more proline and soluble carbohydrate, lower relative electrolyte leakage and malondialdehyde content, and antioxidant enzyme activity increased and accumulation of peroxide reduced. The expressions of stress-related genes NtOsmotin and NtERD10B in GmNF-YB24 transgenic tobacco were higher than those in wild-type tobacco. Conclusion Heterologous overexpression of GmNF-YB24 in tobacco improves the resistance of transgenic tobacco to drought.

Key words: soybean, nuclear factor Y, NF-YB, transgenic tobacco, resistance to drought

翟莹, 计俊杰, 陈炯辛, 于海伟, 李珊珊, 赵艳, 马天意. 异源过表达大豆GmNF-YB24提高转基因烟草抗旱性[J]. 生物技术通报, 2025, 41(8): 137-145.

ZHAI Ying, JI Jun-jie, CHEN Jiong-xin, YU Hai-wei, LI Shan-shan, ZHAO Yan, MA Tian-yi. Heterologous Overexpression of Soybean GmNF-YB24 Improves the Resistance of Transgenic Tobacco to Drought[J]. Biotechnology Bulletin, 2025, 41(8): 137-145.

图/表 11

表1 RT-qPCR引物序列

Table 1 Primer sequences for RT-qPCR

基因（GenBank登录号）

Gene （GenBank accession no.）

引物序列

Primer sequences （5′‒3′）

GmNF-YB24 （XM003546151）

F-AGACAGGTTCCTTCCGATAGCG

R-TTATGAAGCTGATGAACTCCGACAC

Gmβ-Tubulin （GMU12286）

F-GGAAGGCT TTCTTGCATTGGTA

R-AGTGGCATCCTGGTACTGC

NtActin （AB158612）

F-TTGCTGGTCGTGATCTTACTGATTG

R-CAGTCTCCAACTCTTGCTCATAGTC

NtOsmotin （M29279）

F-CTCCTTGCCTTGGTGACTT

R-ACCGCCTATGGGTGTCG

NtERD10B （AB049336）

F-TCCCATTCGTCAAACCG

R-CCCACCAAGTATGCCAGT

NtP5CS （HM854026）

F-GACACGGACTGATGGAAGATTAG

R-TTCATAGCCTTGCGAGTTAAGC

图1 GmNF-YB24在非生物胁迫下的表达不同字母表示差异显著（P<0.05）。下同

Fig. 1 Expression of GmNF-YB24 under abiotic stressDifferent letters indicate significant difference (P<0.05). The same below

图2 GmNF-YB24基因克隆M：DL2000分子量标记物；1：GmNF-YB24 PCR扩增产物

Fig. 2 Cloning of GmNF-YB24M: DL2000 marker; 1: PCR amplified products of GmNF-YB24

图3 GmNF-YB24基因核苷酸及其编码蛋白氨基酸序列阴影代表HFM结构域；*代表终止密码子

Fig. 3 Nucleotide and protein amino acid sequences of GmNF-YB24Shadow refers to the HFM domain; * refers to the termination codon

图4 GmNF-YB24植物表达载体构建和农杆菌遗转化M：DL2000分子量标记物；1‒2：pRI101-GmNF-YB24质粒双酶切；3‒4：农杆菌菌液PCR

Fig. 4 Construction of GmNF-YB24 plant expression vector and A. tumefaciens transformationM: DL2000 marker. 1-2: Double enzyme digestion of pRI101-GmNF-YB24 plasmid. 3‒4: PCR of Agrobacterium tumefaciens

图5 GmNF-YB24转基因烟草鉴定A：GmNF-YB24转基因烟草基因组DNA PCR检测；B：GmNF-YB24转基因烟草RT-qPCR检测；M：DL2000分子量标记物；+：pRI101-GmNF-YB24质粒；WT：野生型烟草；1‒5：GmNF-YB24转基因烟草；OE1-OE5：GmNF-YB24转基因烟草。下同

Fig. 5 Identification of GmNF-YB24 transgenic tobaccoA: Genomic DNA PCR detection of GmNF-YB24 transgenic tobacco; B: RT-qPCR detection of GmNF-YB24 transgenic tobacco; M: DL2000 marker for molecular weight; +: pRI101-GmNF-YB24 plasmid; WT: wild-type tobacco; 1-5: GmNF-YB24 transgenic tobacco; OE1-OE5: GmNF-YB24 transgenic tobacco. The same below

图6 GmNF-YB24转基因烟草在干旱及复水处理下的表型A：停止浇水前；B：停止浇水10 d；C：停止浇水20 d；D：复水3 d

Fig. 6 Phenotype of GmNF-YB24 transgenic tobacco under drought and rewater treatmentA: Before stop watering; B: stop watering for 10 d; C: stop watering for 20 d; D: rewater for 3 d

图7 GmNF-YB24转基因烟草干旱胁迫下脯氨酸含量、可溶性糖含量、相对电解质渗透率和丙二醛含量

Fig. 7 Proline content, soluble carbohydrate content, relative electrolyte leakage,and malondialdehyde content of GmNF-YB24 transgenic tobacco under drought stress

图8 GmNF-YB24转基因烟草干旱胁迫下SOD活力和POD活力

Fig. 8 SOD activity and POD activity of GmNF-YB24 transgenic tobacco under drought stress

图9 GmNF-YB24转基因烟草干旱胁迫下叶片DAB染色

Fig. 9 Leaf DAB staining of GmNF-YB24 transgenic tobacco under drought stress

图10 GmNF-YB24转基因烟草胁迫相关基因的表达

Fig. 10 Expressions of stress-related genes in GmNF-YB24 transgenic tobacco

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