马铃薯StCIPK11的克隆及响应干旱胁迫分析

doi:10.13560/j.cnki.biotech.bull.1985.2023-0062

摘要/Abstract

摘要：

明确马铃薯StCIPK11在响应干旱胁迫信号传导中的功能和作用机制，为深入研究StCIPK11响应马铃薯抗旱调控的分子机制提供理论依据。利用同源重组法和人工microRNA技术构建马铃薯StCIPK11过表达载体和干扰表达载体，通过根癌农杆菌介导法分别将其转入马铃薯栽培品种‘大西洋’中。RT-qPCR结果表明，过表达植株StCIPK11的表达量是非转基因植株（NT）的11.59和21.76倍，干扰表达植株StCIPK11干扰程度达到78%。经PEG模拟干旱胁迫，过表达植株叶片中丙二醛含量显著高于NT植株，脯氨酸含量、抗氧化酶（超氧化物歧化酶、过氧化物酶）活性均低于NT植株；StCIPK11干扰表达植株则表现出相反的趋势。StCIPK11参与了干旱胁迫应答过程，StCIPK11干扰表达可以降低马铃薯植株对水分胁迫的敏感性。

关键词: 马铃薯, StCIPK11, 干旱胁迫, 生理指标

Abstract:

This study is to clarify the function and mechanism of StCIPK11 in potato（Solanum tuberosum）in the signal transduction in responses to drought stress, aiming to provide theoretical basis for deeply understanding the molecular mechanism responding to drought stress. Homologous recombination and artificial microRNA technology were used to construct the overexpression vector and the interference vector of potato StCIPK11, and they were transferred into the potato cultivar ‘Atlantic’ via Agrobacterium. According to the RT-qPCR data, StCIPK11 was expressed 11.59 and 21.76 times more in the overexpression plants than in the non-transgenic plants, and the the interference degree of StCIPK11 in the interference plants reached 78%. Under drought stress via PEG, the proline content and antioxidant enzymes（superoxide dismutase and peroxidase）activities of the overexpression plants were lower than those of the non-transgenic plants, and the malondialdehyde content in their leaves was significantly higher than that of the non-transgenic plants. The StCIPK11-interfering plants, however, presented the opposite trend. StCIPK11 was involved in the drought stress response process, and StCIPK11 interference expression could lessen the sensitivity of potato plants to water stress.

Key words: potato, StCIPK11, drought stress, physiologic index

刘雯锦, 马瑞, 刘升燕, 杨江伟, 张宁, 司怀军. 马铃薯StCIPK11的克隆及响应干旱胁迫分析[J]. 生物技术通报, 2023, 39(9): 147-155.

LIU Wen-jin, MA Rui, LIU Sheng-yan, YANG Jiang-wei, ZHANG Ning, SI Huai-jun. Cloning of StCIPK11 Gene and Analysis of Its Response to Drought Stress in Solanum tuberosum[J]. Biotechnology Bulletin, 2023, 39(9): 147-155.

图/表 7

表 1 本研究所用的引物序列

Table 1 Primer sequences used in the study

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Purpose
StCIPK11-F	CGGGGGACGAGCTCGGTACCATGGCCTTATTCTCTTCTTCGG	PCR扩增 PCR amplification
StCIPK11-R	CCATGTCGACTCTAGATTATGGATCCACCTCTGTTGAAATC	PCR扩增 PCR amplification
StCIPK11-I	GATTCTATTTCCGCGCTAGCCTCTCTCTCTTTTGTATTCC	amiR-StCIPK11 PCR扩增 amiR-StCIPK11 for PCR amplification
StCIPK11-II	GAGAGGCTAGCGCGGAAATAGAATCAAAGAGAATCAATGA
StCIPK11-III	GAGAAGCTAGCGCGGTAATAGATTCACAGGTCGTGATATG
StCIPK11-IV	GAATCTATTACCGCGCTAGCTTCTCTACATATATATTCCT
StCIPK11-A	CTGCAAGGCGATTAAGTTGGGTAAC
StCIPK11-B	GCGGATAACAATTTCACACAGGAAACAG
Q- StCIPK11-F	TACGACACCCACACATCGTC	实时荧光定量PCR Quantitative real-time PCR
Q-StCIPK11-R	TTTGCGAACAATTCGCCTCC	实时荧光定量PCR Quantitative real-time PCR
StEf1a-F	CAAGGATGACCCAGCCAAG	内参基因Reference gene
StEf1a-R	TTCCTTACCTGAACGCCTGT	内参基因Reference gene
HYG-F	GTGATTTCATATGCGCGATTGCTG	HYG基因PCR扩增PCR amplification for HYG gene
HYG-R	ACGAGTGCTGGGGCGTCGGTTTCC	HYG基因PCR扩增PCR amplification for HYG gene
NPT II-F	GCTATGACTGGGCACAACAG	NPT II基因PCR扩增PCR amplification for NPT II gene
NPT II-R	ATACCGTAAAGCACGAGGAA	NPT II基因PCR扩增PCR amplification for NPT II gene

图1 StCIPK11的扩增产物条带及 amiR-StCIPK11前体片段扩增 A: StCIPK11扩增产物条带；B: a、b和 c小片段；C: d 片段；M: DNA marker DL2000；1-2：基因目的片段；3-4：d片段

Fig. 1 StCIPK11 amplified product bands and amiR-StCIPK11 precursor fragments amplification A: StCIPK11 amplification product bands. B: a, b and c are small fragments. C: d fragment. M: DNA marker DL2000. 1-2: Gene fragment. 3-4: d fragment

图2 StCIPK11蛋白序列及其他物种同源序列的多重比对黑色：完全相同序列；粉色：序列相似度75%以上；蓝色：序列相似度50%以上；白色：序列相似度小于30%

Fig. 2 Multiple alignments of homologous sequences between StCIPK11 protein and other species Black: Completely identitical sequence. Pink: Sequence identity above 75%. Blue: Sequence identity above 50%. White: Sequence identity below 30%

图 3 StCIPK11 蛋白的进化树分析

Fig. 3 Phylogenetic tree analysis of StCIPK11 protein

图4 重组质粒双酶切验证 A: p1300-StCIPK11双酶切验证；B: pBI121-amiR-StCIPK11双酶切验证；M: DNA marker DL2000；1-2: 酶切片段

Fig. 4 Double-digestion verification of recombinant plasmid A: Double-digestion verification of p1300-StCIPK11. B: Double-digestion verification of pBI121-amiR-StCIPK11. M: DNA marker DL2000. 1-2: Digestion fragment

图5 转基因植株的RT-qPCR检测 OE-1、OE-2：过表达植株；Ri-1、Ri-2：干扰表达植株；NT：非转基因植株；试验数据为3个生物学重复的平均值，数据为LSD法进行显著性分析。*：P< 0.05显著差异；**：P< 0.01极显著差异。下同

Fig. 5 RT-qPCR detection of the transgenic plants OE-1, OE-2: The overexpressed plants. Ri-1, Ri-2: The interfering expression plants. NT: Non-transgenic plants. Data are mean ±SD（n=3）. LSD post hoc test were used for significant analysis. *: P< 0.05; **: P< 0.01. The same below

图6 PEG6000处理下转基因植株和NT植株生理指标分析 A: MDA含量；B: Pro含量；C: SOD活性；D: POD活性

Fig. 6 Analysis of physiological indices of transgenic and NT potato plants under PEG6000 treatment A: MDA content; B: proline content; C: SOD activity; D: POD activity

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