异源表达大豆转录因子GmNF-YA19提高转基因烟草抗旱性

doi:10.13560/j.cnki.biotech.bull.1985.2022-1285

摘要/Abstract

摘要：

核因子（NF-Y）广泛存在于真核生物中，是一类能够在植物非生物胁迫反应中发挥重要调控作用的转录因子。探究大豆GmNF-YA19的抗旱性及作用机制，为GmNF-YA19在抗旱植物育种中的应用奠定基础。运用实时荧光定量PCR（RT-qPCR）对GmNF-YA19在非生物胁迫下的表达量进行检测，克隆GmNF-YA19，构建GmNF-YA19植物表达载体并转化烟草，对转基因烟草的抗旱性进行鉴定。RT-qPCR结果显示GmNF-YA19在大豆中能够响应干旱、高盐、低温及外源ABA，且干旱胁迫下GmNF-YA19的表达量升高最显著。以大豆叶片cDNA为模板，通过PCR克隆GmNF-YA19。序列分析结果显示，GmNF-YA19编码一个含有213个氨基酸的蛋白质，预测分子量22.99 kD，预测等电点9.40。GmNF-YA19蛋白序列包含一个保守的CBF结构域。蛋白系统进化分析表明，GmNF-YA19蛋白与OsNF-YA7蛋白、AtNF-YA4蛋白和AtNF-YA7蛋白亲缘关系较近。共获得4棵转基因烟草植株。在干旱胁迫下，GmNF-YA19的异源表达增强了转基因烟草的抗旱性。与野生型烟草相比，GmNF-YA19转基因烟草中3个抗逆相关基因（NtABA2、NtNCED3-2和NtP5CS）的表达量显著升高。GmNF-YA19的异源表达增强了转基因烟草的抗旱性。

关键词: 大豆, 核因子, NF-YA, 非生物胁迫, 抗旱性

Abstract:

Nuclear factor（NF-Y）is a kind of transcription factor widely existing in eukaryotes, and can play an important role in the regulation of abiotic stress response in plants. Exploring the drought resistance and mechanism of soybean GmNF-YA19 will lay a foundation for the application of GmNF-YA19 in drought resistant breeding. Real-time fluorescence quantitative PCR（RT-qPCR）was used to detect the expression of GmNF-YA19 under abiotic stress. GmNF-YA19 was cloned. Its plant expression vector was constructed and transformed into tobacco, and the drought resistance of transgenic tobacco was identified. RT-qPCR results showed that GmNF-YA19 responded to drought, salt, cold and exogenous ABA in soybean, and the expression of GmNF-YA19 increased most significantly under drought stress. GmNF-YA19 was cloned by PCR using soybean leaf cDNA as template. Sequence analysis revealed that GmNF-YA19 encoded a protein containing 213 amino acids, with predicted molecular weight of 22.99 kD and predicted isoelectric point of 9.40. GmNF-YA19 protein sequence contained a conserved CBF domain. Phylogenetic analysis demonstrated that GmNF-YA19 protein was closely related to OsNF-YA7, AtNF-YA4 and AtNF-YA7 proteins. Four transgenic tobacco plants were obtained. The heterologous expression of GmNF-YA19 enhanced the drought resistance of transgenic tobacco plant under drought stress. Compared with wild-type tobacco, the expressions of three stress-related genes（NtABA2, NtNCED3-2 and NtP5CS）in GmNF-YA19 transgenic tobacco significantly increased.

Key words: soybean, nuclear factor, NF-YA, abiotic stress, drought resistance

翟莹, 李铭杨, 张军, 赵旭, 于海伟, 李珊珊, 赵艳, 张梅娟, 孙天国. 异源表达大豆转录因子GmNF-YA19提高转基因烟草抗旱性[J]. 生物技术通报, 2023, 39(5): 224-232.

ZHAI Ying, LI Ming-yang, ZHANG Jun, ZHAO Xu, YU Hai-wei, LI Shan-shan, ZHAO Yan, ZHANG Mei-juan, SUN Tian-guo. Heterologous Expression of Soybean Transcription Factor GmNF-YA19 Improves Drought Resistance of Transgenic Tobacco[J]. Biotechnology Bulletin, 2023, 39(5): 224-232.

图/表 9

表1 RT-qPCR引物序列

Table 1 Primer sequences for RT-qPCR

基因（GenBank登录号）Genes（GenBank accession number）	正向引物Forward primer（5'-3'）	反向引物Reverse primer（5'-3'）
Gmβ-tubulin（GMU12286）	GGAAGGCTTTCTTGCATTGGTA	AGTGGCATCCTGGTACTGC
GmNF-YA19（NM001253093）	AGCCATTCTTGTCGAATCATACTG	CCTGTGGTCCATAAGCAACTATTG
NtActin（AB158612）	TTGCTGGTCGTGATCTTACTGATTG	CAGTCTCCAACTCTTGCTCATAGTC
NtABA2（EU123520）	TCTCCAAACTTTCCTACTCGCTTTC	CACTGCTTGACGCTCTTCCTTC
NtNCED3-1（JX101472）	CCCAAATGTGTTCAAGGCGTTTAC	TGCCGTCACCGTCAAAGAAATG
NtNCED3-2（JX101473）	ATATGGGAAGAAATCGCCTGATGTC	ACAACTTGCTGGTCGGGAATTAC
NtCAT1（U93244）	CCTCGTGGTTTTGCTGTC	GGGATTTAGGATTTGGCTT
NtP5CS（HM854026）	GACACGGACTGATGGAAGATTAG	TTCATAGCCTTGCGAGTTAAGC

图1 GmNF-YA19在干旱、高盐、低温和ABA处理下的表达量不同字母表示差异显著性（P <0.05）。下同

Fig. 1 Expressions of GmNF-YA19 under drought, salt, cold and ABA treatments Different letters indicate significant difference（P <0.05）. The same below

图2 GmNF-YA19核苷酸及蛋白质氨基酸序列阴影为CBF结构域；下划线为NF-YB/NF-YC结合子域；黑体为DNA结合子域；*为终止密码子

Fig. 2 Nucleotide and protein amino acid sequences of GmNF-YA19 The shadow is the CBF domain. The underline is NF-YB/NF-YC binding subdomain. The bold one is the DNA binding subdomain. * is the termination codon

图3 植物NF-YA蛋白的系统进化树

Fig. 3 Phylogenetic tree of plant NF-YA proteins

图4 GmNF-YA19植物表达载体构建和农杆菌转化 M：DL2000分子量标记物；1：pRI101-GmNF-YA19质粒双酶切；2：农杆菌菌液PCR

Fig. 4 Construction of GmNF-YA19 plant expression vector and Agrobacterium tumefaciens transformation M: DL2000 marker. 1: Double enzyme digestion of pRI101-GmNF-YA19 plasmid.2: PCR of Agrobacterium tumefaciens

图5 GmNF-YA19 转基因烟草基因组DNA PCR检测（A）和RT-qPCR检测（B） M：DL2000分子量标记物；+：pRI101-GmNF-YA19质粒；WT：野生型烟草；OE1-OE4：GmNF-YA19转基因烟草

Fig. 5 Genomic DNA PCR detection (A) and RT-qPCR detection (B) of GmNF-YA19 transgenic tobacco M: DL2000 marker. +: pRI101-GmNF-YA19 plasmid. WT: Wild-type tobacco. OE1-OE4: GmNF-YA19 transgenic tobacco plants

图6 GmNF-YA19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18 d；D：复水3 d；WT：野生型烟草；OE1-OE3：GmNF-YA19转基因烟草

Fig. 6 Phenotype of GmNF-YA19 transgenic tobacco plants under drought and rehydration treatment A: Before stop watering. B: Stop watering for 10 d. C: Stop watering for 18 d. D: Rehydration 3 d. WT: Wild-type tobacco. OE1-OE3: GmNF-YA19 transgenic tobacco plants

图7 GmNF-YA19转基因烟草干旱胁迫下电解质渗透率、丙二醛含量、可溶性糖含量和脯氨酸含量 WT：野生型烟草；OE1-OE3：GmNF-YA19转基因烟草

Fig. 7 Electrolyte leakage, malondialdehyde contents, soluble carbohydrate contents and proline contents in GmNF-YA19 transgenic tobacco plants under drought stress WT: Wild-type tobacco; OE1-OE3: GmNF-YA19 transgenic tobacco plants

图8 GmNF-YA19转基因烟草抗逆相关基因表达量 WT：野生型烟草；OE1：GmNF-YA19转基因烟草

Fig. 8 Expression of stress-related genes in GmNF-YA19 transgenic tobacco plants WT: Wild-type tobacco. OE1: GmNF-YA19 transgenic tobacco plants

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