ZmJAZ与ZmMYC2的BiFC互作研究

doi:10.13560/j.cnki.biotech.bull.1985.2021-0181

摘要/Abstract

摘要：

JAZ（jasmonate ZIM-domain）蛋白是茉莉酸（jasmonic acid,JA）信号途径中关键的负调控因子,明确JAZ蛋白和MYC2之间的结合关系对整个JA信号通路至关重要。通过qRT-PCR筛选了在籽粒中较特异表达的ZmJAZ4、生殖器官中高表达的ZmJAZ8以及组成型表达的ZmJAZ12,利用玉米叶肉细胞原生质体瞬时表达体系,通过亚细胞定位和双分子荧光互作（bimolecular fluorescence complementation）研究了这些JAZ蛋白的定位以及是否可以与ZmMYC2相互作用。研究结果发现ZmJAZ4、ZmJAZ8和ZmJAZ12均定位于细胞核,同时BiFC结果显示这些JAZ蛋白都可与ZmMYC2在细胞核中相互作用,证实了ZmJAZ家族蛋白可与JA信号通路关键转录因子ZmMYC2结合的功能,提示它们可通过与ZmMYC2的结合影响其转录调控活性。

关键词: 茉莉酸, JAZ蛋白, MYC2转录因子, 双分子荧光互作（BiFC）

Abstract:

JAZ（Jasmonate ZIM-domain）proteins are negative regulators in the JA signaling pathway,clarifying the interaction between JAZ protein and MYC2 is crucial for understanding the JA signaling pathway. qRT-PCR was applied to examine the expressions of ZmJAZ genes,and discovered seed preferentially expressed ZmJAZ4,reproductive organ predominantly expressed ZmJAZ8,and constitutively expressed ZmJAZ12. Then,the subcellular localizations of these AZ proteins and ZmMYC2 were investigated using protoplasts of maize mesophyll cells transiently expressing vector. Besides,the interactions between these JAZ proteins and ZmMYC2 were analyzed by bimolecular fluorescence complementation（BiFC）. The results showed that ZmJAZ4,ZmJAZ8,and ZmJAZ12 were located in cell nucleus,and BiFC assay revealed that these JAZ proteins interacted with ZmMYC2 in cell nucleus,confirming that ZmJAZ family proteins had function of binding key transcription factor ZmMYC2 in JA signal pathway,suggesting that ZmMYC2 can regulate its transcriptional activity by binding to ZmMYC2.

Key words: jasmonic acids, JAZ protein, MYC2 transcription factor, bimolecular fluorescence complementation

吕迪, 陈茹梅, 周晓今. ZmJAZ与ZmMYC2的BiFC互作研究[J]. 生物技术通报, 2022, 38(1): 77-85.

LV Di, CHEN Ru-mei, ZHOU Xiao-jin. Interactions Between ZmJAZ and ZmMYC2 Using Bimolecular Fluorescence Complementation Assay[J]. Biotechnology Bulletin, 2022, 38(1): 77-85.

图/表 7

表1 实时荧光定量PCR所用引物

Table 1 Primers used in qRT-PCR

基因名称 Gene name	引物名称 Primer name	引物序列 Primer sequence（5'-3'）
ZmActin	ZmActin-qF	ATGTTTCCTGGGATTGCCGAT
ZmActin	ZmActin-qR	CCAGTTTCGTCATACTCTCCCTTG
ZmJAZ4	ZmJAZ4-qF	CGCTCAGGCTCTGAAACAGTGAAACC
ZmJAZ4	ZmJAZ4-qR	CGACGACAGACACAGTGCCTAAGAAT
ZmJAZ8	ZmJAZ8-qF	TTGAGAAACGCCGTGACAGGGT
ZmJAZ8	ZmJAZ8-qR	GTCAGCCTTCACCGATGGGACT
ZmJAZ12	ZmJAZ12-qF	CTGATGCTAAGAAGCCTACTCGC
ZmJAZ12	ZmJAZ12-qR	GCGTCTGAAGGAGAAGTTTGGTA

表2 PCR扩增所用引物

Table 2 Primers used for gene cloning

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	扩增产物 Product
ZmMYC2-F	ATGAACCTGTGGACGGAC	ZmMYC2-ORF
ZmMYC2-R	TTACCTGCCCATGGCAGACCCG
ZmJAZ4-F	ATGGCCGCCTCCGGGAACAACA	ZmJAZ4-ORF
ZmJAZ4-R	TCAGAGCCTGAGCGCGAGCCACGA
ZmJAZ8-F	ATGGATCTGTTGGAGCGGAATATT	ZmJAZ8-ORF
ZmJAZ8-R	TTACTGGGCCTTGCCCTCCGCTGT
ZmJAZ12-F	ATGGCGGCGTCCGCGAGGCCCG	ZmJAZ12-ORF
ZmJAZ12-R	CTAGCTCAGGTTCAGGTTGGACTT
ZmMYC2-GF	TCATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGACGACAA	ZmMYC2-GFP
ZmMYC2-GR	CCTCGCCCTTGCTCACCATTCTAGACCTGCCCATGGCAGACCCGGGCT
ZmJAZ4-GF	ATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAACA	ZmJAZ4-GFP
ZmJAZ4-GR	CCTCGCCCTTGCTCACCATTCTAGAGAGCCTGAGCGCGAGCCAC
ZmJAZ8-GF	ATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAATA	ZmJAZ8- GFP
ZmJAZ8-GR	CCTCGCCCTTGCTCACCATTCTAGACTGGGCCTTGCCCTCCGCT
ZmJAZ12-GF	ATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC	ZmJAZ12-GFP
ZmJAZ12-GR	CCTCGCCCTTGCTCACCATCTAGAGCTCAGGTTCAGGTTGGACT
ZmMYC2-NF	ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC	ZmMYC2-YFP^N
ZmMYC2-NR	CTTTTGCTCCATTCTAGACCTGCCCATGGCAGACCC
ZmMYC2-NF	ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC	ZmMYC2-YFP^C
ZmMYC2-CR	GTATGGGTACATTCTAGACCTGCCCATGGCAGACCC
ZmJAZ4-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA	ZmJAZ4-YFP^N
ZmJAZ4-NR	AACTTTTGCTCCATTCTAGAGAGCCTGAGCGCGAGCCACGAGG
ZmJAZ4-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA	ZmJAZ4-YFP^C
ZmJAZ4-CR	CATCGTATGGGTACATTCTAGAGAGCCTGAGCGCGAGCCACGAGG
ZmJAZ8-NF	CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT	ZmJAZ8-YFP^N
ZmJAZ8-NR	AACTTTTGCTCCATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC
ZmJAZ8-NF	CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT	ZmJAZ8-YF^PC
ZmJAZ8-CR	CATCGTATGGGTACATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC
ZmJAZ12-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC	ZmJAZ12-YFP^N
ZmJAZ12-NR	AACTTTTGCTCCATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC
ZmJAZ12-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC	ZmJAZ12-YFP^C
ZmJAZ12-CR	CATCGTATGGGTACATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC

表2 PCR扩增所用引物

Table 2 Primers used for gene cloning

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	扩增产物 Product
ZmMYC2-F	ATGAACCTGTGGACGGAC	ZmMYC2-ORF
ZmMYC2-R	TTACCTGCCCATGGCAGACCCG
ZmJAZ4-F	ATGGCCGCCTCCGGGAACAACA	ZmJAZ4-ORF
ZmJAZ4-R	TCAGAGCCTGAGCGCGAGCCACGA
ZmJAZ8-F	ATGGATCTGTTGGAGCGGAATATT	ZmJAZ8-ORF
ZmJAZ8-R	TTACTGGGCCTTGCCCTCCGCTGT
ZmJAZ12-F	ATGGCGGCGTCCGCGAGGCCCG	ZmJAZ12-ORF
ZmJAZ12-R	CTAGCTCAGGTTCAGGTTGGACTT
ZmMYC2-GF	TCATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGACGACAA	ZmMYC2-GFP
ZmMYC2-GR	CCTCGCCCTTGCTCACCATTCTAGACCTGCCCATGGCAGACCCGGGCT
ZmJAZ4-GF	ATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAACA	ZmJAZ4-GFP
ZmJAZ4-GR	CCTCGCCCTTGCTCACCATTCTAGAGAGCCTGAGCGCGAGCCAC
ZmJAZ8-GF	ATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAATA	ZmJAZ8- GFP
ZmJAZ8-GR	CCTCGCCCTTGCTCACCATTCTAGACTGGGCCTTGCCCTCCGCT
ZmJAZ12-GF	ATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC	ZmJAZ12-GFP
ZmJAZ12-GR	CCTCGCCCTTGCTCACCATCTAGAGCTCAGGTTCAGGTTGGACT
ZmMYC2-NF	ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC	ZmMYC2-YFP^N
ZmMYC2-NR	CTTTTGCTCCATTCTAGACCTGCCCATGGCAGACCC
ZmMYC2-NF	ATTTGGAGAGGACCTCGAGATGAACCTGTGGACGGAC	ZmMYC2-YFP^C
ZmMYC2-CR	GTATGGGTACATTCTAGACCTGCCCATGGCAGACCC
ZmJAZ4-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA	ZmJAZ4-YFP^N
ZmJAZ4-NR	AACTTTTGCTCCATTCTAGAGAGCCTGAGCGCGAGCCACGAGG
ZmJAZ4-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCCGCCTCCGGGAACAA	ZmJAZ4-YFP^C
ZmJAZ4-CR	CATCGTATGGGTACATTCTAGAGAGCCTGAGCGCGAGCCACGAGG
ZmJAZ8-NF	CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT	ZmJAZ8-YFP^N
ZmJAZ8-NR	AACTTTTGCTCCATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC
ZmJAZ8-NF	CATTTCATTTGGAGAGGACCTCGAGATGGATCTGTTGGAGCGGAAT	ZmJAZ8-YF^PC
ZmJAZ8-CR	CATCGTATGGGTACATTCTAGACTGGGCCTTGCCCTCCGCTGTCAC
ZmJAZ12-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC	ZmJAZ12-YFP^N
ZmJAZ12-NR	AACTTTTGCTCCATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC
ZmJAZ12-NF	CATTTCATTTGGAGAGGACCTCGAGATGGCGGCGTCCGCGAGGCC	ZmJAZ12-YFP^C
ZmJAZ12-CR	CATCGTATGGGTACATTCTAGAGCTCAGGTTCAGGTTGGACTTGAC

图1 ZmJAZ4、ZmJAZ8和ZmJAZ12基因的相对表达量 Root：根;Stem：茎;Leaf：叶片;Tassel：未散粉的雄花;Ear：未授粉的雌穗;En：胚乳;Em：胚;DAP（Days after pollination）：授粉后天数;数据以平均值±标准差表示

Fig.1 Relative expressions of ZmJAZ4,ZmJAZ8,and ZmJAZ12 En：Endosperm. Em：Embryo. DAP：Day after pollination. The data are in means ± SD

图2 亚细胞定位载体和酶切鉴定电泳图 A：亚细胞定位所用载体构建的示意图,包括对照GFP载体和细胞核定位marker载体。B：亚细胞定位载体的Xho I和Xba I酶切检测结果,其中M1为100 bp DNA Ladder;M2为1 kb DNA Ladder;泳道1为pZmMYC2-GFP;泳道2为pZmJAZ4-GFP;泳道3为pZmJAZ8-GFP;泳道4为pZmJAZ12-GFP

Fig.2 Vectors for subcellular localization and enzyme digestion identification A：Schematic diagram of vectors for subcellular localization,including control GFP vector and cell nucleus localization marker vector. B：Enzyme digestion identification of Xho I + Xba I in subcellular localization vectors. M1：100 bp DNA ladder. M2：1000 bp DNA ladder. Lane 1：pZmMYC2-GFP,lane 2：pZmJAZ4-GFP,lane 3：pZmJAZ8-GFP,and lane 4：pZmJAZ12-GFP

图3 BiFC载体构建示意图和酶切鉴定电泳图 A：BiFC所用载体构建的示意图。B：各BiFC载体的Xho I和Xba I酶切检测结果,其中M1为100 bp DNA Ladder;M2为全式金公司1 kb DNA Ladder;泳道1为pZmMYC2-YFPN;泳道2为pZmMYC2-YFPC;泳道3为pZmJAZ4-YFPN;泳道4为pZmJAZ4-YFPC;泳道5为pZmJAZ8-YFPN;泳道6为pZmJAZ8-YFPC;泳道7为pZmJAZ12-YFPN;泳道8为pZmJAZ12-YFPC

Fig.3 Vectors for BiFC assay and enzyme digestion identification A：Schematic diagram of vectors for BiFC assay. B：Enzyme digestion identification of BiFC vectors by Xho I + Xba I. M1：100 bp DNA ladder. M2：1 000 bp DNA ladder. Lane 1：pZmMYC2-YFPN,lane 2：pZmMYC2-YFPC,lane 3：pZmJAZ4-YFPN,lane 4：pZmJAZ4-YFPC,lane 5：pZmJAZ8-YFPN,lane 6：pZmJAZ8-YFPC,lane 7：pZmJAZ12-YFPN,and lane 8：pZmJAZ12-YFPC

图4 ZmJAZ4、ZmJAZ8、ZmJAZ12和ZmMYC2的亚细胞定位将ZmJAZ4、ZmJAZ8、ZmJAZ12和ZmMYC2构建到GFP的N端。GFP示目标蛋白荧光通道;DsRed示核定位标记载体荧光通道;Chl示叶绿体自发荧光通道;Bright示明场通道;Merge示GFP、DsRed、Chl和明场的叠加通道。Bar=10 μm

Fig. 4 Subcellular localization of ZmJAZ4,ZmJAZ8,ZmJAZ12 and ZmMYC2 The ORFs of ZmJAZ4、ZmJAZ8、ZmJAZ12 and ZmMYC2 were fused at the N terminal of GFP. GFP indicates the target protein fluorescence channel. DsRed indicates fluorescence channel for a nuclear localization maker. Chl indicates the chloroplast spontaneous fluorescence channel. Bright indicates the bright field. The Merge indicates channel combined the GFP,DsRed,Chl and the bright field. Scale bar = 10 μm

图5 ZmMYC2与ZmJAZ4、ZmJAZ8和ZmJAZ12的BiFC互作验证将ZmMYC2和待验证的3个基因分别构建到YFP的N端及C端进行双向验证。YFP示BiFC互作激发荧光通道;DsRed示核定位信号载体荧光通道;Chl示叶绿体自发荧光通道;Bright示明场通道;Merge示YFP、DsRed、Chl和明场的叠加通道。Bar=10 μm

Fig. 5 BiFC assay to verify interactions between ZmJAZs with ZmMYC2 ZmMYC2 and 3 to-be-verified genes were constructed into the N-terminal and C-terminal of YFP for BiFC assay. YFP indicates fluorescence channel triggered by BiFC interaction. The DsRed indicates fluorescence channel for nuclear localization maker. Chl indicates the chloroplast spontaneous fluorescence channel. Bright indicates the bright field. Merge indicates channel combined the GFP,DsRed,Chl and the bright field. Scale bar = 10 μm

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