龙眼<i>DlAGOMEL1</i>基因的克隆表达及亚细胞定位分析

陈荣珠; 王小平; 林美珍; 赖钟雄

doi:10.19303/j.issn.1008-0384.2021.10.008

龙眼DlAGOMEL1基因的克隆表达及亚细胞定位分析

doi: 10.19303/j.issn.1008-0384.2021.10.008

1.
漳州卫生职业学院药学系，福建　漳州　363000
2.
福建农林大学园艺植物生物工程研究所，福建　福州　350002

基金项目: 漳州市自然科学基金项目（ZZ2021J16）；漳州卫生职业学院高学历人才科研支持专项（ZWYGXL202101）；漳州卫生职业学院科技创新团队培育项目（Kjcx-3、Kjcx-5）；福建农林大学科技创新专项基金（CXZX2017189）

详细信息

作者简介:
陈荣珠（1986−），女，博士，讲师，研究方向：中药资源及药用植物生物技术（E-mail：349074689@qq.com）

通讯作者:
赖钟雄（1966−），男，博士，研究员，研究方向：园艺植物生物技术研究（E-mail：laizx01@163.com）

中图分类号: S 667.2
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出版历程
- 收稿日期: 2021-07-04
- 修回日期: 2021-08-11
- 网络出版日期: 2021-10-23
- 刊出日期: 2021-10-28

Cloning, Expression and Subcellular Localization of DlAGOMEL1 from Longan

1.
Department of pharmacy, Zhangzhou Health Vocational College, Zhangzhou, Fujian　363000, China
2.
Institute of Horticultural Biotechnology, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的探究DlAGOMEL1基因在龙眼体胚发生过程中的调控作用，并明确该基因在亚细胞中的定位，为深入研究DlAGOMEL1基因的功能提供理论依据。方法采用RT-PCR技术，基于龙眼基因组数据库，克隆DlAGOMEL1基因的CDS及该基因的5′端上游启动子序列，采用生物信息学方法分析该基因，通过实时荧光定量PCR技术检测其在龙眼体胚不同发育阶段的相对表达量，同时，构建该基因的瞬时融合表达载体，得到重组质粒，并将其瞬时转入洋葱内表皮细胞中，用荧光共聚焦显微镜观察细胞绿色荧光信号。结果获得DlAGOMEL1基因CDS全长序列及5′端上游启动子序列长度分别为2655 bp、1512 bp，该基因编码884个氨基酸；系统进化树分析表明，龙眼DlAGOMEL1蛋白与胡杨亲缘关系最近，与单子叶植物水稻、玉米等亲缘关系最远；实时荧光定量分析表明，该基因在龙眼体胚不同阶段均表达，在子叶胚时期（CE）的相对表达量较高，而在球形胚时期（GE）的相对表达量最低。亚细胞定位结果发现，DlAGOMEL1基因定位于细胞质中。结论 DlAGOMEL1基因在龙眼体胚阶段的相对表达量呈“V”字型，可能主要参与龙眼晚期体胚发生的转录调控过程，在细胞质中发挥主要的功能作用。
- 龙眼 /
- DlAGOMEL1基因 /
- 表达分析 /
- 亚细胞定位
Abstract: Objective Still unclear regulatory functions of DlAGOMEL1 on the nonembryonic callus and different embryogenic cultures of longan (Dimocarpus longan) were investigated. Method From the longan genome database, the full-length cDNA sequences (CDS) of DlAGOMEL1 and its promoter were obtained using RT-PCR. Bioinformatics of this gene was analyzed and the relative expressions of this gene in embryogenic cultures were detected by real-time quantitative PCR. The recombinant plasmid was constructed from the transient fusion expression vector, and the onion inner epidermal cells transformed into transient onion cells. Green fluorescence signal of the cells was searched under a fluorescence confocal microscope. Result The CDS of DlAGOMEL1 was 2,655 bp which encoded 884 amino acids and the promoter was 1,512 bp. The DlAGOMEL1 of longan and other plants were highly conserved as shown in a multiple sequences analysis. Phylogenetic tree of the protein indicated its close relation with Populars euphonium but not with monocotyledonous plants, such as rice and maize. The gene expressed differently in somatic embryo development stages—most strongly at cotyledon embryo stage and least at spherical embryo stage. The subcellular localization of DlAGOMEL1 was found in cytoplasm. Conclusion Since the relative expression of DlAGOMEL1 was shown to be high at the late stage of longan somatic embryo, the gene might be functionally active in the late stage of the development. The revealed subcellular localization of the gene seemed to predispose its involvement in the cytoplasm of longan.
- Dimocarpus longan /
- DlAGOMEL1 /
- gene expression /
- subcellular localization

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图 1 龙眼DlAGOMEL1基因CDS和启动子序列PCR扩增及融合表达载体的构建

注：（1）M1-DL 2 000 bp，M2-DL 15 kb；（2）A-DlAGOMEL1基因CDS电泳图；B-DlAGOMEL1基因5＇端上游启动子序列；C-融合表达载体的pCAMBIA1300-35s-DlAGOMEL1-GFP环状质粒。

Figure 1. Electrophoretograms of CDS of DlAGOMEL1 and promoter by PCR and fusion expression vector in longan

Note: (1) M1-DL 2 000 bp; M2:-DL 15 kb; (2) A-DlAGOMEL1 CDS; B- promoter of DlAGOMEL1; C- fusion expression vector of pCAMBIA1300-35s-DlAGOMEL1-eGFP circular plasmids.

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图 2 龙眼DlAGOMEL1蛋白三级结构预测

Figure 2. Predicted tertiary structure of DlAGOMEL1

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图 3 龙眼DlAGOMEL1蛋白保守结构域预测

Figure 3. Predicted functional domains of DlAGOMEL1

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图 4 AGOMEL1基因编码氨基酸多序列比对

Figure 4. Multiple sequence alignment of AGOMEL1

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图 5 AGOMEL1蛋白系统发育进化树

Figure 5. Phylogenetic analysis on DIAGOMEL1 from longan and other plants

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图 6 龙眼体胚发生不同阶段DlAGOMEL1的相对表达量

注：（1）NEC：非胚性愈伤组织；EC：胚性愈伤组织；ICpEC：不完全胚性紧实结构；GE：球形胚；CE：子叶胚。（2）图中不同小写字母表示差异显著（P＜0.05）。

Figure 6. Relative expressions of DlAGOMEL1 at different somatic embryogenesis stages of longan

Note: (1) NEC: Non-embryonic callus; EC: Embryonic callus; ICpEC: Incomplete embryogenic compact structure; GE: Globular embryo; CE: Cotyledon embryo. (2) Different lowercase letters indicate significant differences (P＜0.05).

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图 7 pCAMBIA1300-35s-DlAGOMEL1-GFP蛋白在洋葱内表皮细胞的亚细胞定位

注：A～C：导入DlLAGOMEL1重组质粒的洋葱内表皮细胞；D～F：导入pCAMBIA1300-35s-GFP空载体的洋葱内表皮细胞；A、D：荧光激发图；B、E：叠加图；C、F：明场图。

Figure 7. Subcellular localization of pCAMBIA1300-35s-DlAGOMEL1-GFP protein in onion

Note: A–C: subcellular locations of endoepidermal cells of onion scale leaf introduced into DlAGOMEL1 recombinant plasmid; D–F: subcellular locations of endoepidermal epidermis cells of onion scale leaf introduced into pcambia1300-35s-GFP empty vector; A and D: flourescent field; B and E: merged field; C and F: bright field.

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表 1 引物序列

Table 1. Primer sequence

引物名称 Primer name	引物序列　　　　　　　　 Sequence （5′-3′）	用途　　　　　　 Purpose	退火温度 Annealing temperature/℃
DlAGOMEL1-CF	AATATCGACTTCACGCGTCTG	全长验证 Full length validation	61
DlAGOMEL1-CQ	GCAAGCAACCACAATGAAGC	全长验证 Full length validation	61
pro-AGOMEL1-F	ACTTGTTAGAGACCGACCTAC	启动子克隆 Promoter cloning	54
pro-AGOMEL1-R	ATACGAGAATCTTGACAATAACC	启动子克隆 Promoter cloning	54
DlAGOMEL1-1300-F	CAGTGGTCTCACAACATGACTCGATGCATGATAAG	载体构建 Vector construction	61
DlAGOMEL1-1300-R	CAGTGGTCTCATACATTCCTGGTGAATAGCATCCA
GFP-F	AACATACGGAAAACTTACCCT	荧光信号验证 Fluorescence Signal Verification	55
GFP-R	GTGCAACTCGCTGATCATTAT	荧光信号验证 Fluorescence Signal Verification	55
DlAGOMEL1-QF	GGTCTCACTCTCCAAACTCAG	实时荧光定量 PCR Real-time quantitative PCR	58
DlAGOMEL1-QR	TTGAAGAAGCAGCTGTCTCC	实时荧光定量 PCR Real-time quantitative PCR	58
注：加粗CAGT为保护碱基，下划线为酶切位点。 Note: bold CAGT are the protective bases, and underlined are the restriction sites.

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表 2 龙眼AGOMEL1基因启动子序列顺式作用元件

Table 2. Cis-acting elements of DlAGOMEL1 promoter

顺式元件 Cis element	序列 Sequences	方向 Strand matrix	位置 Position/bp	功能 Function
ABRE	ACGTG/AACCCGG/CACGTG/ACGTG	+/+/−/+	616/798/1153/1154	脱落酸反应Abscisic acid responsiveness
Box 4	ATTAAT	−	948	光响应Light responsiveness
G-Box	CACGTT/CACGTG/GCCACGTGGA	−/−/+	615/1153/1151	光响应Light responsiveness
GATA-motif	GATAGGA/GATAGGG	+/+	690/1107	光响应Light responsiveness
TCT-motif	TCTTAC	+	212	光响应Light responsiveness
CAT-box	GCCACT	+	1285	分生组织表达Meristem expression
LTR	CCGAAA	+	1353	低温响应Low-temperature responsivenes
MBS	CAACTG	+	1407	参与干旱诱导的MYB结合位点 MYB binding site involved in drought-inducibility
注: +: 正向; −: 负向。 Note: +: positive strand; −: negative strand.

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