生物技术通报 ›› 2023, Vol. 39 ›› Issue (4): 259-267.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1083
陈楠楠1,3(), 王春来1,3, 蒋振忠1,3, 焦鹏1,3, 关淑艳2,3(), 马义勇2,3()
收稿日期:
2022-09-01
出版日期:
2023-04-26
发布日期:
2023-05-16
通讯作者:
关淑艳,女,博士,教授,研究方向:作物遗传育种与基因工程;E-mail: guanshuyan@jlau.edu.cn;作者简介:
陈楠楠,女,硕士研究生,研究方向:作物重要性状形成的分子机制;E-mail: 2356014208@qq.com
基金资助:
CHEN Nan-nan1,3(), WANG Chun-lai1,3, JIANG Zhen-zhong1,3, JIAO Peng1,3, GUAN Shu-yan2,3(), MA Yi-yong2,3()
Received:
2022-09-01
Published:
2023-04-26
Online:
2023-05-16
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。
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.
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 |
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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