龙眼miR408与DlLAC12克隆及其在球形胚发生和非生物胁迫下的表达分析

doi:10.16420/j.issn.0513-353x.2021-0573

摘要/Abstract

摘要：

为探讨miR408及靶基因DlLAC12在龙眼球形体细胞胚诱导及不同非生物胁迫下的表达模式,采用miR-RACE PCR和Tail-PCR克隆获得pri-miR408 cDNA和转录起始位点、dlo-miR408 gDNA、靶基因DlLAC12 cDNA及启动子pro-MIR408序列。研究结果显示：dlo-pri-miR408 cDNA、gDNA全长均为706 bp,5′端转录起始位点为胞嘧啶（C）。DlLAC12 cDNA全长为1 725 bp,pro-MIR408全长为1 532 bp。pro-MIR408序列上存在ABA、GA₃、JA等激素信号传导顺式作用元件和响应光、低温胁迫等相关元件。qRT-PCR结果显示,dlo-miR408-3p随外源添加的蔗糖浓度、Cu²⁺浓度及培养温度的变化呈现动态表达;而dlo-miR408-5p1对蔗糖不敏感,在铜离子失衡和低温时下调表达。dlo-miR408-3p与DlLAC12负调控模式能响应高浓度ABA（≥ 500 μmol · L^-1）处理,而不同浓度GA₃处理下二者表达模式一致。dlo-miR408-3p与DlLAC12在球形胚诱导不同天数呈现负调控,说明在球形胚诱导过程发挥作用。试验表明,dlo-miR408与靶基因DlLAC12可参与龙眼体胚发生与非生物胁迫响应。

关键词: 龙眼, 体胚发生, miR408, DlLAC12, 非生物胁迫

Abstract:

To investigate the expression patterns of miR408 and its target DlLAC12 during longan globular embryo（GE）development and different abiotic stresses. The miR-RACE PCR and Tail-PCR were used to clone the cDNA and gDNA of dlo-miR408 and cDNA of DlLAC12,and the transcriptional start site（TSS）,and promoter of MIR408（pro-MIR408）were ascertained. Characteristics of dlo-miR408 sequence,homologous and cis-acting elements of the promoter were bioinformatically analyzed. The transcriptional expression levels of the members of dlo-miR408 family at various abio-stress conditions were verified with qRT-PCR,and the expression patterns of dlo-miR408-3p with its target gene DlLAC12 were further analyzed at early somatic embryogenesis stages with various plant growth regulator treatments. pro-MIR408 sequence contains hormone signal transducing cis-acting elements such as ABA,GA₃ and JA,and light and low temperature stress cis-elements. The results showed that the length of pri-miR408 cDNA,gDNA were 706 bp. The TSS was“C”. The length of DlLAC12 cDNA was 1 725 bp. The length of pro-MIR408 was 1 532 bp. dlo-miR408-3p showed dynamic expressions in responding to sucrose,Cu²⁺ and temperature treatments by means of qRT-PCR,whereas dlo-miR408-5p1 showed downregulation in the imbalanced Cu²⁺and low temperature conditions,and maintained stable expression to sucrose changes. Moreover,dlo-miR408-3p and DlLAC12 responded to high concentration of ABA（≥ 500 μmol · L^-1）with negative regulations,and GA₃ treatment made them same in the expression pattern. The negative regulation at the late globular embryo（GE）developmental stage played an important role in the GE morphogenesis. The present work showed that dlo-miR408 and DlLAC12 were involved in the longan somatic embryogenesis and abiotic stress.

Key words: longan, somatic embryogenesis, miR408, DlLAC12, abiotic stress

中图分类号:

S667.2

徐小萍, 曹清影, 蔡柔荻, 官庆栩, 张梓浩, 陈裕坤, 徐涵, 林玉玲, 赖钟雄. 龙眼miR408与DlLAC12克隆及其在球形胚发生和非生物胁迫下的表达分析[J]. 园艺学报, 2022, 49(9): 1866-1882.

XU Xiaoping, CAO Qingying, CAI Roudi, GUAN Qingxu, ZHANG Zihao, CHEN Yukun, XU HAN, LIN Yuling, LAI Zhongxiong. Gene Cloning and Expression Analysis of miR408 and Its Target DlLAC12 in Globular Embryo Development and Abiotic Stress in Dimocarpus longan[J]. Acta Horticulturae Sinica, 2022, 49(9): 1866-1882.

图/表 11

表1 基因克隆及qRT-PCR所用引物

Table 1 Primers used in gene cloning and qRT-PCR

用途 Purpose	引物名称 Primer name	引物序列 Primer sequence	退火温度/℃ Temperature	延伸时间/s Time
miR408 qPCR	dlo-miR408-3p-qF	ATGCACTGCCTCTTCCCTGGC	60	30
miR408 qPCR	dlo-miR408-5p1-qF	ACGGGGAACAGGCAGAGCATG	60	30
miRNA qRT-PCR	Univer-primer	GATCGCCCTTCTACGTCGTAT	60	30
pre-miR408 qPCR	pre-miR408-qF	AGAGCAGCAAGAGACAAAGACG	60	30
	pre-miR408-qR	GAAAGAGGAGAAGAAGGAGCC	60	30
pri-miR408 qPCR	pri-miR408-qF	GGCTTCACCCATGCACTGC	60	30
	pri-miR408-qR	TCACTCACCCAGTAATCAGTTGC	60	30
靶基因定量Target gene qPCR	DlLAC12-qF	GCTTCTCCTAGCTTCTACATTGTCC	60	30
	DlLAC12-qR	CAAAGTTGTCAACAAAGCCAG	60	30
内参基因Reference gene/miRNA	DlFSD-qF	GGTCAGATGGTGAAGCCGTAGAG	60	30
	DlFSD-qR	GTCTATGCCACCGATACAACAAACCC	60	30
	dlo-miR156a^*-qF	TGCTCACTTCTCCTCTGTCAG	60	30
pri-miR408 5RACE	pri-miR408-5R1	CAGGGAAGAGGCAGTGCATGG	56.7	30
	pri-miR408-5R2	GCATCCATGCTCTGCCTGTTC	54.8	45
pri-miR408 3RACE	pri-miR408-3R1	ACAGGCAGAGCATGGATGCAACTA	55	45
	pri-miR408-3R2	GGCTTCACCCATGCACTGC	54	45
pri-miR408 cDNA	pri-miR408-F	TTGTGGAAGGAAACGTGG	56	45
	pri-miR408-R	TGAACCTGCATTTCTGTCTACAC	56	45
pro-MIR408	pro-MIR408-F	CGAGTGAGATGAATCCGAAACAAAG	65	90
	pro-MIR408-R	CGAGTGAGATGAATCCGAAACAAAG	65	90
DlLAC12 cDNA	DlLAC12-F	ATGGAGCTTCTCAAAAGCAC	56	120
	DlLAC12-R	CACCAGATTTGCCTATATGCTAA	56	120

图1 pri-miR408及其靶标DlLAC12克隆和成熟体序列（蓝色）及前体序列（划线部分） M：Marker;胞嘧啶“C”为转录起始位点。

Fig. 1 Gene cloning of pri-miR408 and DlLAC12,miR408（blue）and pre-miR408 sequence（underlined） M：Marker;“C”is the transcription start site.

图2 龙眼dlo-miR408家族4个成熟体多序列比对

Fig. 2 Multiple alignment of dlo-miR408 family members

图3 dlo-pre-miR408序列二级结构箭头代表miRNA 5′端第1个碱基

Fig. 3 The secondary structure of dlo-pre-miR408 sequence stem-loop structure The arrows point to the first base of 5′ of miRNA.

图4 龙眼dlo-miR408（A）、dlo-MIR408（B）与植物多物种聚类分析 32种植物的miR408,Dlo：龙眼Dimocarpus longan;stu：马铃薯Solanum tuberosum;nta：烟草Nicotiana tabacum;dpr：紫花洋地黄Digitalis purpurea;gma：大豆Glycine max;bdi：二穗短柄草Brachypodium distachyon;aly：琴叶拟南芥Arabidopsis lyrata;vvi：葡萄Vitis vinifera;pta：火炬松Pinus taeda;sof：甘蔗Saccharum officinarum;ath：拟南芥Arabidopsis thaliana;osa：水稻Oryza sativa;ppt：小立碗藓Physcomitrella patens;sbi：高粱sorghum bicolor;ahy：花生Arachis hypogaea;ssp：甘蔗Saccharum ssp;mes：木薯Manihot esculenta;mdm：苹果Malus domestica;cpa：番木瓜Carica papaya;ptc：毛果杨Populus trichocarpa;tae：小麦Triticum aestivum;rco：蓖麻Ricinus communis;vun：豇豆Vigna unguiculata;cme：香瓜Cucumis melo;ata：山羊草Aegilops tauschii;zma：玉米Zea mays;csi：甜橙Citrus sinensis;mtr：蒺藜苜蓿Medicago truncatula;cca：刺菜蓟Cynara cardunculus;bra：芜菁Brassica rapa;lja：百脉根Lotus japonicus;hbr：橡胶树Hevea brasiliensis.miRNA前体聚类分析共选取33种植物,Dlo：龙眼Dimocarpus longan;rco：蓖麻Ricinus communis;cpa：番木瓜Carica papaya;hbr：橡胶树Hevea brasiliensis;mes：木薯Manihot esculenta;ptc：毛果杨Populus trichocarpa;vvi,葡萄Vitis vinifera;mtr：蒺藜苜蓿Medicago truncatula;mdm：苹果Malus domestica;aqc：耧斗菜Aquilegia caerulea;stu：马铃薯Solanum tuberosum;cca：刺菜蓟Cynara cardunculus;pta：火炬松Pinus taeda;ppt：小立碗藓Physcomitrella patens;smo：江南卷柏Selaginella moellendorffii;sof：甘蔗Saccharum officinarum;gma,大豆Glycine max;ssp：甘蔗Saccharum ssp;dpr：毛地黄Digitalis purpurea;cme：香瓜Cucumis melo;zma：玉米Zea mays;ata：山羊草Aegilops tauschii;tae,小麦Triticum aestivum;bdi：二穗短柄草Brachypodium distachyon;osa：水稻Oryza sativa;sbi：高粱sorghum bicolor;bra：芜菁Brassica rapa;ath：拟南芥Arabidopsis thaliana;Aly：琴叶拟南芥Arabidopsis lyrata;csi：甜橙Citrus sinensis;vun：豇豆Vigna unguiculata;Lus：亚麻Linum usitatissimum;lja：百脉根Lotus japonicus.

Fig. 4 The phylogenetic tree construction of dlo-miR408（A）and dlo-MIR408（B）nucleotide sequence in multiple plant species

图5 pro-MIR408序列克隆

Fig. 5 The pro-MIR408 sequence cloning

图6 pro-MIR408顺式作用元件分析

Fig. 6 The pro-MIR408 sequence information analysis

表2 pro-MIR408顺式作用元件分析

Table 2 The cis-acting element analysis of pro-MIR408

顺式作用元件 Cis-acting element	位点数 Number of site	正、负链 Positive and negative chain	矩阵分数 Matrix fraction	核心序列 Core sequence	注释 Annotation
ABRE	1	+	9	GCAACGTGTC	参与脱落酸反应性 Participates in abscisic acid reactivity
ARE	1	-	6	AAACCA	厌氧诱导 Anaerocabically
Box 4	2	+/-	6	ATTAAT	光响应有关保守DNA模块 Light response related to conserved DNA modules
CAT-box	2	-/+	6	GCCACT	分生组织表达相关 Meristem expression
CGTCA-motif	1	+	5	CGTCA	参与MeJA反应 Participate in MeJA responsing
GARE-motif	1	-	7	TCTGTTG	赤霉素应答 Gibberellin response
GT1-motif	1	+	6	GGTTAA	光响应元件 Light response element
LTR	2	+/-	6	CCGAAA	参与低温反应 Participate in low temperature
MYB	4	+/-/+/-	6	CAACAG、CAACCA、TAACCA	MYB结合位点 MYB binding site
MYB-like sequence	1	-	6	TAACCA
MYC	1	-	6	CATTTG	MYC结合位点MYC binding site
Myb-binding site	2	+	6	CAACAG
Myc	1	-	7	TCTCTTA
TATC-box	2	-	7	TATCCCA	赤霉素反应Gibberellin response
TCCC-motif	1	-	7	TCTCCCT	光响应Light response
TGACG-motif	1	-	5	TGACG	参与MeJA反应Participate in MeJA response

图7 dlo-miR408在不同浓度蔗糖、铜离子和不同温度胁迫下的表达模式 “*”代表与对照相比在0.05水平具有显著性差异,“**”代表在0.01水平下具有显著性差异,每个处理设置3个独立的生物学重复。

Fig. 7 Expression patterns of dlo-miR408 during different concentrations of sucrose,Cu2+ and different temperatures “*”represents significant difference at 0.05 level,“**”represents significant difference at 0.01 level. Every treatment sets up three independent biological replicates.

图8 dlo-miR408与DlLAC12在龙眼球形胚不同诱导阶段的表达模式 ** 代表P < 0.01水平下具有极显著性差异。每个处理设置3个独立的生物学重复。

Fig. 8 Different expression patterns of dlo-miR408 and DlLAC12 during different days of GE induction in longan ** represents a very significant difference at P < 0.01 level. Every treatment sets up three independent biological replicates.

图9 dlo-miR408与靶基因DlLAC12在不同浓度ABA和GA3处理24 h的定量表达分析 * 代表P < 0.05水平下具有显著性差异,** 代表P < 0.01水平下具有极显著性差异。

Fig. 9 The different expression analysis of dlo-miR408-3p and DlLAC12 during different concentration of ABA and GA3 * represents significant difference at P < 0.05. ** represents a very significant difference at P < 0.01 level.

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