Genetic Transformation and Chilling Resistance Analysis of Maize ZmDHN15 Gene in Tobacco

doi:10.13560/j.cnki.biotech.bull.1985.2022-1083

Abstract

Abstract:

Maize（Zea mays L.）originated in the subtropical zone, is a thermophilic crop, and is susceptible to low temperature stress. Dehydrin, group 2 members of the late embryogenesis abundant protein（LEA）, are a class of proteins that play an important role in plant abiotic stress. In this study, the ZmDHN15 gene was cloned and obtained, and the basic characteristics and tissue expression characteristics of the gene were analyzed using bioinformatics methods, real-time fluorescent quantitative PCR and other techniques, and the construction of plant overexpression vector and the genetic transformation of tobacco were carried out. T₂generation plants were used to verify their cold resistance. The results showed that the ZmDHN15 gene was 1 442 bp in length, encoded a total of 290 amino acids, with a molecular weight of 31.44 kD and a theoretical isoelectric point of 6.05. It was a hydrophilic non-transmembrane protein with a conserved domain unique to the dehydratin family. RT-qPCR analysis showed that the ZmDHN15 gene was highly expressed in maize leaves and increased under cold stress conditions.Nine T₂ generation transgenic tobacco plants were obtained. Compared with the wild type, the germination rate of transgenic tobacco increased by 1.40 times, the root length increased by 1.58 times, the wilting degree of its leaves was lower, the content of proline, malondialdehyde and peroxidase reduced by 41.17%, 28.47% and 23.33%, respectively. Soluble sugar content, oxide activity and superoxide dismutase activity increased by 58.97%, 47.85% and 47.53%, respectively, and the accumulation of H₂O₂ and O₂^- decreased by 34.78% and 47.00%, respectively. In conclusion, overexpression of ZmDHN15 gene can effectively improve the tolerance of tobacco plants to cold stress, which lays a foundation for further research on the function of ZmDHN15 gene in maize.

Key words: maize, ZmDHN15 gene, dehydrin, cold resistance, heterologous expression

CHEN Nan-nan, WANG Chun-lai, JIANG Zhen-zhong, JIAO Peng, GUAN Shu-yan, MA Yi-yong. Genetic Transformation and Chilling Resistance Analysis of Maize ZmDHN15 Gene in Tobacco[J]. Biotechnology Bulletin, 2023, 39(4): 259-267.

Figures/Tables 10

Table 1 Primer sequences

名称 Name	引物序列 Primer sequence（5'-3'）
ZmDHN15 for RT-qPCR-F	AAGCCAAAAGGCACTGAAGAAG
ZmDHN15 for RT-qPCR-R	ACAGAACAGATCAGCAGGCTAGCTA
Act-F	TTGAGGTAGGATGAGACT
Act-R	GGAGTGAAGCAGATGATT
ZmDHN15-F	AAGGCACTGAAGAAGCCAGTCA
ZmDHN15-R	GAAACCAAAGCAATTATTAACGCAT
ZmDHN15-3301-F	actcttgaccatggtagatctAAGGCACTGAAGAAG- CCAGTCA
ZmDHN15-3301-R	ggggaaattcgagctggtcaccGAAACCAAAGCAA- TTATTAACGCAT
Bar-F	TGACGCACAATCCCACTATCCT
Bar-R	GAAACCCACGTCATGCCAGT

Fig. 1 Phylogenetic tree of ZmDHN15 and other plant deh-ydrin proteins

Fig. 2 ZmDHN15 gene expression pattern analysis A: ZmDHN15 gene analysis of expression patterns in different parts. B: Analysis of the expression pattern of ZmDHN15 gene in leaves under intercooling treatment. * indicate significant differences among organizations, P < 0.05（*）, P < 0.01（**）

Fig. 3 Genetic transformation of tobacco A: Germination; B: co-culture; C: screening; D: differentiation; E: rooting; F: transplant; G: flowering; H: seed collection

Fig. 4 PCR validation of T2 generation tobacco M: DNA marker DL 2000; P: plasmid groups; N: negative control; CK: controlled plant ;1-9: positive plants

Fig. 5 RT-qPCR validation of transgenic tobacco of T2 ge-neration WT: Wild type, OE1-OE9: transgenic plants. * indicate significant differences from wild-type, P < 0.05（*）, P < 0.01（**）, and P < 0.001（***）. The same below

Fig. 6 Determination of germination rate and root length of transgenic tobacco plants under cold stress tre-atment A: Germination rate statistics; B: root length statistics. Control: Control group. Cold stress: Chilling stress at 4℃.The same below

Fig. 7 Phenotypic analysis A: Not under stress; B: cold stress for 24 h

Fig. 8 Determination of physiological and biochemical indexes A-C: Pro, MDA and soluble sugar contents. D-F:CAT, SOD and POD enzyme activities

Fig. 9 Qualitative and quantitative analysis of H2O2 and O2- in transgenic and wild-type tobacco leaves under cold stress A: NBT staining; B: DAB staining; C: H2O2 content; D: O2- content

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