Gene Cloning and Expression Analysis of miR408 and Its Target DlLAC12 in Globular Embryo Development and Abiotic Stress in Dimocarpus longan

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

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (9): 1866-1882.doi: 10.16420/j.issn.0513-353x.2021-0573

• Research Papers • Previous Articles Next Articles

Gene Cloning and Expression Analysis of miR408 and Its Target DlLAC12 in Globular Embryo Development and Abiotic Stress in Dimocarpus longan

XU Xiaoping¹, CAO Qingying¹, CAI Roudi¹, GUAN Qingxu¹, ZHANG Zihao¹, CHEN Yukun¹, XU HAN¹^,², LIN Yuling¹, LAI Zhongxiong¹^,^*()

¹College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China
²Institut de la Recherche Interdisciplinaire de Toulouse,IRIT-ARI,Toulouse 31300,France

Received:2021-10-01 Revised:2022-06-16 Online:2022-09-25 Published:2022-10-08
Contact: LAI Zhongxiong E-mail:laizx01@163.com

Abstract

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

CLC Number:

S667.2

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.

Figures/Tables 11

References 41

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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

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

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

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

Gene Cloning and Expression Analysis of miR408 and Its Target DlLAC12 in Globular Embryo Development and Abiotic Stress in Dimocarpus longan

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