玉米ZmDHN15基因在烟草中的遗传转化及抗冷性分析

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

摘要/Abstract

摘要：

玉米起源于亚热带，是喜温作物，易受低温胁迫的影响，脱水素（dehydrin，DHN）作为胚胎发育晚期丰富蛋白（late embryogenesis abundant protein, LEA）Ⅱ家族成员，是一类在植物非生物胁迫中发挥重要功能的蛋白。本研究克隆获得ZmDHN15基因，使用生物信息学手段，实时荧光定量PCR等技术对该基因的基本特性、组织表达特性进行分析，并进行植物过表达载体的构建及烟草的遗传转化，对T₂代阳性植株进行抗冷性功能验证。结果表明，ZmDHN15基因全长1 442 bp，共编码290个氨基酸，分子量为31.44 kD，理论等电点为6.05，是亲水性非跨膜蛋白，具有脱水素家族特有保守结构域。RT-qPCR分析表明ZmDHN15基因的在玉米叶片中表达量较高且在冷胁迫条件下表达量增加；获得T₂代转基因烟草植株9 株；在冷胁迫下转基因烟草与野生型相比，萌发率提高1.40 倍，根长提高1.58 倍，其叶片萎蔫程度更低，脯氨酸含量、丙二醛含量和过氧化物酶活性分别降低41.17%、28.47%和23.33%，可溶性糖含量、氧化物酶活性和超氧化物歧化酶活性分别升高58.97%、47.85%和47.53%，H₂O₂ 和O₂^-积累量分别减少34.78%和47.00%。综上所述，过表达ZmDHN15 基因可以有效提高烟草植株对冷胁迫的耐受性，为进一步研究ZmDHN15基因在玉米中的功能奠定基础。

关键词: 玉米, ZmDHN15基因, 脱水素, 抗冷, 异源表达

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

陈楠楠, 王春来, 蒋振忠, 焦鹏, 关淑艳, 马义勇. 玉米ZmDHN15基因在烟草中的遗传转化及抗冷性分析[J]. 生物技术通报, 2023, 39(4): 259-267.

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.

图/表 10

表1 引物序列

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

图1 ZmDHN15蛋白和其他植物脱水素蛋白的系统进化树

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

图2 ZmDHN15 基因表达模式分析 A：ZmDHN15基因在不同部位表达模式分析；B：ZmDHN15基因在叶片中冷处理条件下表达模式分析；星号（*）表示各组织差异显著，P < 0.05（*）, P < 0.01（**）

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（**）

图3 烟草的遗传转化 A：萌发；B：共培养；C：筛选；D分化；E：生根；F：移栽；G：开花；H：收种

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

图4 T2代烟草的PCR验证 M：DNA marker DL 2000 ；P：质粒对照；N：阴性对照；CK：对照植株；1-9：阳性植株

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

图5 T2代转基因烟草的RT-qPCR验证 WT：野生型，OE1-OE9：转基因植株；星号（*）表示与野生型差异显著，P < 0.05（*）, P < 0.01（**）, P < 0.001（***）。下同

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

图6 冷胁迫处理下转基因烟草萌发率和根长的测定 A：萌发率统计；B：根长统计。Control：对照组；Cold stress：4℃低温胁迫。下同

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

图7 表型分析 A：未受胁迫； B：冷胁迫24 h

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

图8 生理生化指标测定 A-C：Pro、MDA、可溶性糖含量； D-F：CAT、SOD、POD酶活性

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

图9 在冷胁迫处理下转基因烟草与野生型烟草叶片H2O2和O2-的定性及定量分析 A：NBT染色；B：DAB染色；C：H2O2的含量； D：O2-的含量

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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