茎瘤芥<i>BjuEAR1-1</i>的克隆与表达分析

doi:10.7668/hbnxb.20192689

摘要/Abstract

摘要：

茎瘤芥在生长发育过程中会受到非生物逆境胁迫的影响,导致产量降低。ABA作为植物逆境激素,在调控植株响应非生物逆境胁迫过程中具有重要作用。BjuEAR1-1是ABA信号通路中负调控因子EAR1的同源基因,其突变体ear1表现出对ABA超敏感的表型。因此,探究BjuEAR1-1的生物学功能,可为茎瘤芥抵抗不良环境的调控机制提供方向,也为有效利用基因资源培育耐逆稳产的茎瘤芥新品种提供重要的理论依据。以永安小叶为试验材料,采用PCR方法克隆BjuEAR1-1基因编码序列,采用酶切酶连方法构建植物过表达载体35S∷PTF101-GFP-BjuEAR1-1,采用烟草叶片瞬时转化技术对BjuEAR1-1的亚细胞定位情况进行分析,并对BjuEAR1-1基因启动子顺式元件进行分析,最后采用荧光定量PCR方法,检测BjuEAR1-1在不同非生物逆境胁迫处理下及不同组织器官的基因表达模式。烟草亚细胞定位结果表明,BjuEAR1-1定位于细胞核和细胞质中;BjuEAR1-1在茎瘤芥的根、茎、叶、花、种荚和膨大茎中均有表达,在根中表达量最高;BjuEAR1-1的表达水平显著受低温和ABA诱导,盐胁迫处理下BjuEAR1-1的表达水平与对照组没有显著性差异。BjuEAR1-1在细胞核和细胞质中发挥生物学功能,并且BjuEAR1-1调控茎瘤芥对于低温和ABA的响应。

关键词: 茎瘤芥, 非生物逆境胁迫, BjuEAR1-1, 基因克隆, 亚细胞定位, 基因表达

Abstract:

During the growth and development stages,tuber mustard is always affected by abiotic stresses.As a plant stress hormone,ABA plays an important role in regulating plant response to abiotic stress.BjuEAR1-1 was a homologue of negative regulator EAR1 in ABA signaling pathway,and its mutant ear1 exhibited an ABA sensitive phenotype.Therefore,to explore the biological function of BjuEAR1-1 will provide a direction for the regulation mechanism of resistance to adverse environment stresses,and also provide an important theoretical basis for the effective use of gene resources to cultivate new varieties of BjuEAR1-1 with resistance to adverse stresses and stable yield.The BjuEAR1-1 coding sequence was cloned by PCR method and the plant overexpression vector 35S∷PTF101-GFP-BjuEAR1-1 was constructed by enzyme digestion and ligation method.The subcellular localization of BjuEAR1-1 was analyzed by transient transformation technology of tobacco leaves.The cis-element of BjuEAR1-1 gene promoter was analyzed,and the gene expression patterns of BjuEAR1-1 under different abiotic stresses and different tissues and organs were detected by fluorescence quantitative PCR.The result of subcellular localization assay showed that BjuEAR1-1 was localized in the nucleus and cytoplasm of tobacco.BjuEAR1-1 was expressed in root,stem,leaf,flower,seed pod and swollen stem of tuber mustard,with the highest expression in root.The expression level of BjuEAR1-1 was significantly induced under low temperature and ABA stresses,and there was no significant difference under salt stress. BjuEAR1-1 was localized in the nucleus and cytoplasm and regulated the response of tuber mustard to low temperature and ABA stresses.

Key words: Tuber mustard, Abiotic stress, BjuEAR1-1, Gene clone, Subcellular localization, Gene expression

程春红, 游经番, 蔡兆明, 叶春宏, 陈丽. 茎瘤芥BjuEAR1-1的克隆与表达分析[J]. 华北农学报, 2022, 37(3): 37-43. doi: 10.7668/hbnxb.20192689.

CHENG Chunhong, YOU Jingfan, CAI Zhaoming, YE Chunhong, CHEN Li. Cloning and Expression Analysis of BjuEAR1-1 in Tuber Mustard[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(3): 37-43. doi: 10.7668/hbnxb.20192689.

http://www.hbnxb.net/CN/Y2022/V37/I3/37

图/表 6

图1 茎瘤芥BjuEAR1-1基因克隆及T载体连接 A.茎瘤芥BjuEAR1-1基因CDS序列扩增结果图:M.DL2000 DNA Marker;1-2.BjuEAR1-1基因CDS序列扩增。B.BjuEAR1-1-T转化子平板筛选图。C.BjuEAR1-1-T菌落PCR鉴定图:M.DL2000 DNA Marker;1-6.BjuEAR1-1-T菌落PCR鉴定结果。

Fig.1 Gene cloning and T vector ligation of BjuEAR1-1 in tuber mustard A.Amplification result of CDS sequence of BjuEAR1-1 in tuber mustard:M.DL2000 DNA Marker;1-2.Amplification of CDS sequence of BjuEAR1-1.B.Screening of BjuEAR1-1-T transformant plate.C.Identification of BjuEAR1-1-T colony by PCR:M.DL2000 DNA Marker;1-6.Identification of BjuEAR1-1-T colony by PCR.

图2 35S∷PTF101-GFP-BjuEAR1-1载体构建 A.BjuEAR1-1-T和35S∷PTF101-GFP双酶切后目的片段胶回收检测图:M.DL2000 DNA Marker;1-2.BjuEAR1-1-T双酶切后目的片段回收;3.35S∷PTF101-GFP双酶切后目的片段回收。B.35S∷PTF101-GFP-BjuEAR1-1菌落PCR检测:M.DL2000 DNA Marker;1-6.菌落PCR凝胶检测。C.35S∷PTF101-GFP-BjuEAR1-1酶切鉴定:M.DL2000 DNA Marker;1-4.35S∷PTF101-GFP-BjuEAR1-1质粒酶切鉴定。

Fig.2 Vector construction of 35S∷PTF101-GFP-BjuEAR1-1 A.Detection of target fragments after BjuEAR1-1-T and 35S∷PTF101-GFP double enzyme digestion:M.DL2000 DNA Marker;1-2.Target fragment recovery after BjuEAR1-1-T double digestion;3.Target fragment recovery after 35S∷PTF101-GFP double enzyme digestion.B.Detection of 35S∷PTF101-GFP-BjuEAR1-1 colony by PCR:M.DL2000 DNA Marker;1-6.Detection of colony by PCR.C.Enzyme digestion identification of 35S∷PTF101-GFP-BjuEAR1-1:M.DL2000 DNA Marker;1-4.35S∷PTF101-GFP-BjuEAR1-1 plasmid digestion identification.

图3 BjuEAR1-1蛋白亚细胞定位分析

Fig.3 Subcellular localization analysis of BjuEAR1-1

图4 BjuEAR1-1启动子顺式作用元件分析

Fig.4 The cis-acting elements analysis of BjuEAR1-1 promoter

图5 BjuEAR1-1在不同组织器官的时空表达模式分析不同小写字母表示各个处理之间在0.05显著水平上存在显著性差异。图6同。

Fig.5 Analysis of temporal and spatial expression patterns of BjuEAR1-1 in different tissues and organs The different normal letters indicate significant difference between treatments at the 0.05 level.The same as Fig.6.

图6 不同胁迫处理下BjuEAR1-1的表达分析

Fig.6 Expression analysis of BjuEAR1-1 under different stresses treatment

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