过表达长穗偃麦草EeHKT1；4基因增强拟南芥抗旱耐盐性分析

doi:10.11686/cyxb2021304

摘要/Abstract

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。

关键词: 长穗偃麦草, EeHKT1, 4, 转基因, DAB染色, NBT染色

Abstract:

Plant high-affinity potassium transporter （HKT） genes encode K⁺ or Na⁺ transport or K⁺-Na⁺ co-transport plasma membrane channel proteins， which play an important role in the process of plant stress resistance. This research investigated the function of EeHKT1；4 in Elytrigia elongata （GenBank： KF956112.1）. An EeHKT1；4 overexpression plant expression vector was constructed and transformed into Arabidopsis thaliana， and the drought and salt tolerance of transgenic Arabidopsis plants were evaluated and analyzed. No difference was found in the primary root length between wild-type （WT） and transgenic lines under normal growth conditions， but NaCl and mannitol treatments inhibited the growth of WT and transgenic plant roots， and inhibited the root length of transgenic lines more than the root length of WT under the same drought and salt treatment conditions. There was no significant difference between WT and transgenic lines under normal growth conditions， but under NaCl and mannitol treatment， WT showed leaf atrophy and yellowing， while only some plants in transgenic lines showed leaf atrophy， and the survival rate of transgenic lines was higher than WT under the same stress conditions. Nitroblue tetrazolium and diaminobenzidine staining results showed that the leaf staining in WT and transgenic lines was relatively weak under normal growth conditions， and the intensity of staining in all leaves gradually deepened with increase in NaCl and mannitol concentration. Also， the intensity of staining in the WT was higher than transgenic lines under the same stress. With the expression level under normal growth conditions as the control， AtSOS1 gene were incrementally up-regulated in WT and transgenic plants with increase in NaCl concentration， and the expression levels in the transgenic plants were higher than that in WT. The expression of the gene AtNHX1 was up-regulated under NaCl treatment and the expression level in transgenic plants was lower than in the WT. Except for a transgenic line ‘L5’， no change in AtNHX1 gene expression associated with increase in NaCl concentration was detected in WT or transgenic lines. The genes AtRD29B and AtP5CS1 were both up-regulated under mannitol treatment， and their expression levels in transgenic plants were higher than in WT plants. In summary， EeHKT1；4 overexpression reduced the accumulation of superoxide anion and H₂O₂ in A. thaliana under adversity stress， induced the up-regulated expression of stress-resistant genes， and enhanced the drought and salt tolerance of A. thaliana.

Key words: Elytrigia elongata, EeHKT1, 4, transgenic, diaminobenzidine staining, nitroblue tetrazoliun staining

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.

图/表 6

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	CTAGTCTAGAATGCAACTCCCAAGTCATAACA
	EeHKT1；4-Knp I	CGGGGTACCCTAACTAAGCTTCCAGGTCCTGC
	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

图3 NaCl与甘露醇处理下转基因与野生型拟南芥表型分析

Fig.3 Phenotype analysis of transgene and wild-type Arabidopsis under NaCl and mannitol treatment

图4 NaCl与甘露醇处理下转基因株系与野生型拟南芥的DAB和NBT染色

Fig.4 DAB and NBT staining for the transgenic and wild-type plants under NaCl and mannitol treatment

图5 NaCl与甘露醇处理下拟南芥抗性相关基因的表达分析

Fig.5 Expression analysis of Arabidopsis resistance-related genes under NaCl and mannitol treatments

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