甘薯丝裂原活化蛋白激酶MPK6对低温胁迫的响应

doi:10.3864/j.issn.0578-1752.2021.20.002

摘要/Abstract

摘要：

【目的】研究甘薯丝裂原活化蛋白激酶MPK6在抵御低温胁迫过程中的功能,为解析甘薯耐低温机制和遗传改良提供参考。【方法】采用农杆菌介导法,将35S::IbMPK6-GFP重组表达载体和对照载体质粒pDMC83转化甘薯愈伤组织,根据载体上特有的GFP序列设计引物对甘薯拟转基因材料进行分子鉴定,并通过qRT-PCR筛选表达量高的转基因株系。对非转基因植株（WT）和转基因株系进行低温胁迫和恢复处理,观察处理后及恢复后的表型变化;检测叶绿素荧光参数Fv/Fm、丙二醛（MDA）含量和过氧化氢（H₂O₂）含量等生理指标;利用二氨基联苯胺（DAB）和氮蓝四唑（NBT）染色法观察活性氧的积累情况;分析低温信号转导途径中关键转录因子基因IbCBF3和下游基因IbCOR27的表达水平。【结果】共获得12株IbMPK6过表达甘薯株系,筛选IbMPK6表达量最高的3个过表达株系（L3、L8和L11）用作低温胁迫分析材料。低温胁迫下WT的Fv/Fm为0.50,而转基因株系L3、L8和L11的Fv/Fm分别为0.79、0.79和0.80,与WT呈现极显著差异水平。温度恢复后,转基因植株中Fv/Fm恢复至低温处理前水平,而WT中Fv/Fm仅为0.70,极显著低于转基因植株。低温胁迫下,WT的丙二醛含量为0.05 μmol·g^-1,而转基因株系L3、L8和L11中的丙二醛含量分别为0.02、0.04和0.02 μmol·g ^-1,显著低于WT。温度恢复正常后,WT中的丙二醛含量为0.03 μmol·g ^-1,而转基因株系L3、L8和L11中的丙二醛含量均为0.01 μmol·g ^-1。DAB和NBT染色结果显示,低温胁迫下,WT叶片与转基因株系叶片相比,染色较深,说明WT比转基因植株积累了更多的过氧化氢和超氧阴离子。过氧化氢含量测定结果显示,低温胁迫下和温度恢复后,转基因植株中过氧化氢的含量均显著低于WT。qRT-PCR结果表明IbCBF3和IbCOR27受低温诱导表达,且在WT和转基因株系之间差异显著。【结论】过表达IbMPK6甘薯植株通过缓解光合系统和膜系统的损伤、减少活性氧的积累,提高了对低温胁迫的耐受性。IbMPK6通过调控低温通路中相关基因IbCBF3和IbCOR27的表达,参与甘薯低温信号转导途径。

关键词: 甘薯, IbMPK6, 转基因株系, 低温胁迫

Abstract:

【Objective】Studying the function of mitogen activated protein kinase (MAPK) IbMPK6 in respect of low temperature stress tolerance in sweetpotato, that will help us to understand the mechanism of adaption to low temperature stress and play a fundamental role in molecular breeding of sweetpotato. 【Method】Agrobacterium tumefaciens strain EHA105 harbored the plasmid 35S::IbMPK6-GFP were transformed intosweetpotato cv. Xushu29 embryogenic callus. Molecular examination and qRT-PCR were used to screen and select transgenic lines. For low temperature stress assay, selected transgenic lines were performed to observe the phenotype and determine the physiological indexes such as Fv/Fm, the content of malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) after low temperature treatment and recovery treatment. Diaminobenzidine (DAB) staining and nitro blue tetrazolium (NBT) staining analysis were performed to observe reactive oxygen species (ROS) accumulation. The expression level of the key transcription factor IbCBF3 and downstream gene IbCOR27 involved in low temperature signal transduction pathway were identified before and after low temperature treatment. 【Result】Twelve transgenic lines were generated and three transgenic lines (L3, L8 and L11) with a high expression level of IbMPK6 were selected for low temperature tolerance assay. Under low temperature stress, the level of Fv/Fm in transgenic lines L3, L8 and L11 was 0.79, 0.79 and 0.80, while that in WT was 0.05. After temperature recovery treatment, Fv/Fm in transgenic lines has recovered to former levels, whereas the level of Fv/Fm in WT was only 0.70, which was significantly lower than that in transgenic lines. MDA content of three transgenic (lines L3, L8 and L11) increased by 0.02, 0.04 and 0.02 μmol·g^-1, and it of WT increased by 0.05 μmol·g^-1 after low temperature stress treatment, respectively. After recovery treatment, MDA content in transgenic lines was 0.01 μmol·g^-1 on average, whereas it of WT was 0.03 μmol·g^-1. The results of DAB and NBT staining showed that the leaves of WT were stained deeper than those of transgenic lines, indicated that hydrogen peroxide and superoxide anion were accumulation less in transgenic lines than in WT. Furthermore, H₂O₂ level in WT was significantly higher than that in transgenic lines under low temperature stress condition and after recovery treatment. Low temperature regulated the expression level of IbCBF3 and IbCOR27 genes, but the expression level in transgenic lines was higher than that in WT. 【Conclusion】Overexpression of IbMPK6 in sweetpotato resulted in enhanced tolerance to low temperature stress, via alleviating the damage of membrane and photosynthetic system, and decreasing ROS accumulation. IbMPK6 involved in low temperature signaling transduction pathway by up-regulating the expression level of cold related genes IbCBF3 and IbCOR27.

Key words: sweetpotato, IbMPK6, transgenic lines, low temperature stress

靳容,刘明,赵鹏,张强强,张爱君,唐忠厚. 甘薯丝裂原活化蛋白激酶MPK6对低温胁迫的响应[J]. 中国农业科学, 2021, 54(20): 4265-4273.

JIN Rong,LIU Ming,ZHAO Peng,ZHANG QiangQiang,ZHANG AiJun,TANG ZhongHou. IbMKP6, A Mitogen-Activated Protein Kinase, Confers Low Temperature Tolerance in Sweetpotato[J]. Scientia Agricultura Sinica, 2021, 54(20): 4265-4273.

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引物名称 Gene name	正向引物序列 Forward primer sequence (5′-3′)	反向引物序列 Revers primer sequence (5′-3′)
IbMPK6-PCR	ATGGACGCTGGTTCGGCTCAG	TCACATTTGCAGCTCAAACTC
GFP6-PCR	AGGTTATTGGAAAATTAAGGGCC	AACCAGATCCGATTTTGGAGGATG
IbMPK6-RT	GCGGCAGATTCATCCAATAC	TATCGCTACGTGCTCATTCG
IbCBF3	TTCGCCACTGTCTTCTTC	TATCCTGGACTTCTTGTTG
IbCOR27	CCAATCTCAGCTTCCTTTGC	CATGAACCTCAGCATTGTCG
IbARF	CTTTGCCAAGAAGGAGATGC	TCTTGTCCTGACCACCAACA