Heterologous Expression of Soybean Transcription Factor GmNF-YA19 Improves Drought Resistance of Transgenic Tobacco

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

Abstract

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

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.

Figures/Tables 9

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

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

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

Fig. 3 Phylogenetic tree of plant NF-YA proteins

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

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

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

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

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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