草业学报 ›› 2022, Vol. 31 ›› Issue (8): 188-198.DOI: 10.11686/cyxb2021304
• 研究论文 • 上一篇
收稿日期:
2021-08-10
修回日期:
2021-11-10
出版日期:
2022-08-20
发布日期:
2022-07-01
通讯作者:
孟林
作者简介:
E-mail: menglin9599@sina.com基金资助:
Yong ZHANG(), Xiao-xia TIAN, Ming-li ZHENG, Pei-chun MAO, Lin MENG()
Received:
2021-08-10
Revised:
2021-11-10
Online:
2022-08-20
Published:
2022-07-01
Contact:
Lin MENG
摘要:
植物高亲和性K+转运蛋白基因(HKT)编码K+、Na+转运或K+-Na+共转运质膜通道蛋白,在植物抗逆过程中发挥重要作用。为了研究长穗偃麦草EeHKT1;4(GenBank: KF956112.1)的功能作用,构建了EeHKT1;4过表达植物表达载体转化拟南芥,进行拟南芥转基因植株的抗旱耐盐性评价分析。结果显示,正常生长条件下野生型(WT)与转基因株系的主根长度无差异,NaCl与甘露醇处理下WT和转基因株系根的生长受到抑制,转基因株系根长度均大于同等胁迫条件下(WT)的根长;正常生长条件下WT与转基因株系表型无显著差异,但在NaCl与甘露醇处理下WT表现出叶片萎缩和植株枯黄,转基因株系仅部分植株表现出叶片萎缩,同一胁迫条件下转基因株系的植株存活率皆高于WT。硝基氮蓝四唑(NBT)与二氨基联苯胺(DAB)染色结果显示,正常生长条件下WT与转基因株系叶片染色相对较浅,随着NaCl与甘露醇浓度提高,所有叶片染色程度逐渐加深且同等胁迫下WT染色程度高于转基因株系。以正常生长条件下基因的表达量为对照,随着NaCl浓度的增加,AtSOS1基因在WT和转基因植株中逐渐上调且在转基因中的表达量高于WT;AtNHX1基因在NaCl处理下上调表达且转基因植株中表达量低于WT,除转基因株系L5外并未检测到WT和转基因株系自身因NaCl浓度的提高AtNHX1基因表达量发生改变;在甘露醇处理下,AtRD29B和AtP5CS1基因均上调表达且转基因植株中表达量高于WT。综上所述,EeHKT1;4过表达降低了逆境胁迫下拟南芥中超氧阴离子和H2O2的积累,诱导抗逆基因上调表达,增强拟南芥抗旱耐盐性。
张勇, 田小霞, 郑明利, 毛培春, 孟林. 过表达长穗偃麦草EeHKT1;4基因增强拟南芥抗旱耐盐性分析[J]. 草业学报, 2022, 31(8): 188-198.
Yong ZHANG, Xiao-xia TIAN, Ming-li ZHENG, Pei-chun MAO, Lin MENG. Analysis of drought and salt resistance of EeHKT1;4 gene from Elytrigia elongata in Arabidopsis[J]. Acta Prataculturae Sinica, 2022, 31(8): 188-198.
功能Function | 引物名称Primer name | 序列Sequences (5'-3') |
---|---|---|
载体构建 Construction of expression vector | EeHKT1;4-Xba I | CTAG |
EeHKT1;4-Knp I | CGG | |
EeHKT1;4-F | ATGCAACTCCCAAGTCATAACA | |
EeHKT1;4-R | CTAACTAAGCTTCCAGGTCCTGC | |
qPCR | qEeHKT1;4-F | CTACTGATCGGCTGCAACAGCAGC |
qEeHKT1;4-R | ATGGAGAGGGAGATTGTAGCAGAG | |
qAtSOS1-F | TCGTTTCAGCCAAATCAGAAAGT | |
qAtSOS1-R | GCTACATAGTTCGGAGTTCCACA | |
qAtNHX1-F | GACTCCTTCATGCGACCCG | |
qAtNHX1-R | CCACGTTACCCTCAAGCCTTAC | |
qAtP5CS1-F | TACACAGGCCCTCCAAGTGA | |
qAtP5CS1-R | CTTGATTTGTCGCCGAATGT | |
qAtRD29B-F | GGAGAGAGCAGAGAGGCTCA | |
qAtRD29B-R | CCGTTGACCACCGAGATAGT | |
qAtACTIN -F | AGCACTTGCACCAAGCAGCATG | |
qAtACTIN -R | ACGATTCCTGGACCTGCCTCATC |
表1 本研究所用的引物序列及用途
Table 1 The primer sequence and application used in this study
功能Function | 引物名称Primer name | 序列Sequences (5'-3') |
---|---|---|
载体构建 Construction of expression vector | EeHKT1;4-Xba I | CTAG |
EeHKT1;4-Knp I | CGG | |
EeHKT1;4-F | ATGCAACTCCCAAGTCATAACA | |
EeHKT1;4-R | CTAACTAAGCTTCCAGGTCCTGC | |
qPCR | qEeHKT1;4-F | CTACTGATCGGCTGCAACAGCAGC |
qEeHKT1;4-R | ATGGAGAGGGAGATTGTAGCAGAG | |
qAtSOS1-F | TCGTTTCAGCCAAATCAGAAAGT | |
qAtSOS1-R | GCTACATAGTTCGGAGTTCCACA | |
qAtNHX1-F | GACTCCTTCATGCGACCCG | |
qAtNHX1-R | CCACGTTACCCTCAAGCCTTAC | |
qAtP5CS1-F | TACACAGGCCCTCCAAGTGA | |
qAtP5CS1-R | CTTGATTTGTCGCCGAATGT | |
qAtRD29B-F | GGAGAGAGCAGAGAGGCTCA | |
qAtRD29B-R | CCGTTGACCACCGAGATAGT | |
qAtACTIN -F | AGCACTTGCACCAAGCAGCATG | |
qAtACTIN -R | ACGATTCCTGGACCTGCCTCATC |
图1 转基因拟南芥植株的获得与鉴定A: EeHKT1;4植物表达载体示意图A diagram showing EeHKT1;4 overexpression vector; B: 转基因植株潮霉素筛选Hygromycin screening of transgenic plants; C: 转基因植株PCR鉴定PCR identification of transgenic plants; D: 转基因拟南芥转录水平鉴定Transcription level identification of transgenic Arabidopsis; DL2000: DL2000 Marker; L1~L5: 不同转基因株系Different transgenic lines; +: 阳性对照Positive control; -: 阴性对照Negative control; WT: 野生型Wild type.下同The same below.
Fig.1 Obtaining and identification of transgenic Arabidopsis
图2 NaCl与甘露醇处理下根的表型及根长统计不同小写字母表示在0.05水平上差异显著,下同。 Different lowercase letters indicate significant differences at the 0.05 level, the same below.
Fig.2 Root phenotype and root length statistics under NaCl and mannitol treatment
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