Identification and Expression Analysis of Genes Related to Longan miRNA Synthesis

doi:10.3969/j.issn.1000-2561.2021.06.001

Abstract

Abstract:

In order to understand the biological functions of genes related to the longan miRNA synthesis pathway, we identified and analyzed the miRNA synthesis pathway-related genes in the third-generation genomic data of longan. And longan transcriptomes in the early stage of somatic embryogenesis (SE) was used for miRNA synthesis pathway-related genes expression analysis. A total of 12 genes related to longan miRNA synthesis pathway were identified, namely DlDDL1, DlDDL2, DlDDL3, DlDCL1, DlSE, DlHYL1a, DlHYL1b, DlHYL1c, DlCBP20, DlCBP80, DlHEN1, DlHASTY. The number of amino acids encoded by these genes was mostly between 400-1000 aa, and the molecular weight of the protein was 23.92-220.25 kDa. Most proteins were acidic proteins, all the proteins were unstable proteins. Except DlHASTY which is hydrophobic protein, the others were hydrophilic proteins. Gene structure analysis showed that the number of introns of each member varied greatly from 2 to 23. All members contained conservative domains, and the number difference varied from 1 to 7. Analysis of cis-acting elements in the promoter regions of gens related to miRNA synthesis pathway showed that a large number of light-responsive elements and hormone-responsive elements were presented in the promoter region of these genes. Protein interaction prediction analysis showed that the longan miRNA synthesis pathway-related genes had strong interaction relationship. Analysis of expression during the early stage of SE in longan showed that each gene had a high level of transcription at the EC stage. This study indicates that the genes related to the longan miRNA synthesis pathway may participate in the early development of longan SE development and play an important role in the EC stage, as well as responding to multiple hormone responses and abiotic stresses. Moreover, it is suggested that miRNA synthesis pathway-related genes might have diversity and differences in biological functions.

Key words: longan, miRNA synthesis pathway-related genes, bioinformatics analysis, qPCR analysis

CLC Number:

S667.2

HUO Wen, CHEN Xiaohui, XU Xiaoping, SHEN Xu, LI Xiaofei, LAI Zhongxiong. Identification and Expression Analysis of Genes Related to Longan miRNA Synthesis[J]. Chinese Journal of Tropical Crops, 2021, 42(6): 1505-1513.

Figures/Tables 10

References 29

[1]	Hutvagner G. A microRNA in a multiple-turnover RNAi enzyme complex[J]. Science, 297(5589):2056-2060. DOI URL
[2]	Zeng Y, Yi R, Cullen B R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(17):9779-9784.
[3]	Doench J G, Petersen C P, Sharp P A. siRNAs can function as miRNAs[J]. Genes and Development, 2003, 17(4):438-442. DOI URL
[4]	付蓉. 玉米自交系盐胁迫响应miRNA及其靶基因的鉴定研究[D]. 南通: 南通大学, 2016.
[5]	刘元龙. 参与柑橘果实发育的miRNA和靶基因的挖掘与分析[D]. 武汉: 华中农业大学, 2015.
[6]	林玉玲, 赖钟雄. 龙眼胚性愈伤组织miR398b前体克隆及其在龙眼体胚发生过程中的表达分析[J]. 热带作物学报, 2012, 33(11):2012-2017.
[7]	Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymerase II[J]. Embo Journal, 2004, 23 (20):4051-4060. DOI URL
[8]	Kurihara Y, Watanabe Y. Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions[J]. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(34):12753-12758.
[9]	Yu B, Yang Z, Li J, et al. Methylation as a crucial step in plant microRNA biogenesis[J]. Science, 2005, 307(5711):932-935. DOI URL
[10]	Yu B, Bi L, Zheng B, et al. The FHA domain proteins DAWDLE in Arabidopsis and SNIP₁ in humans act in small RNA biogenesis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(29):10073-10078.
[11]	李冬梅. 龙眼体细胞胚胎成熟机理的研究[D]. 福州: 福建农林大学, 2003:3-5.
[12]	林玉玲, 赖钟雄. 利用Solexa测序技术鉴定龙眼体胚发生过程中的miRNAs[EB/OL].( 2011-02-10) [2020-05-25]. http://www.paper.edu.cn/releasepaper/content/201102-64.
[13]	Lin Y, Lai Z, Lin L, et al. Endogenous target mimics, microRNA167, and its targets ARF₆ and ARF₈ during somatic embryo development in Dimocarpus longan Lour[J]. Molecular Breeding, 2015, 35(12):1-15. DOI URL
[14]	赖钟雄. 龙眼生物技术研究[M]. 福州: 福建科学技术出版社, 2003:18-22.
[15]	Chen C J, Xia R, Chen H, et al. TBtools, a toolkit for biologists integrating various biological data handling tools with a user-friendly interface[J/OL]. BioRxiv, 2018: 289660 [2020-05-25]. http://dx.doi.org/10.1101/289660.
[16]	Lin Y L, Lai Z X. Reference gene selection for qPCR analysis during somatic embryogenesis in longan tree[J]. Plant Science, 2010, 178(4):359-365. DOI URL
[17]	Morris E R, Chevalier D, Walker J C. DAWDLE, a forkhead- associated domain gene, regulates multiple aspects of plant development[J]. Plant Physiology, 2006, 141(3):932-941. DOI URL
[18]	Hiraguri A, Itoh R, Kondo N, et al. Specific interactions between Dicer-like proteins and HYL1/DRB-family dsRNA- binding proteins in Arabidopsis thaliana[J]. Plant Molecular Biology, 2005, 57(2):173-188. DOI URL
[19]	Dong Z, Han M H, Fedoroff N. The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of pri-miRNA by DCL1[J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(29):9970-9975.
[20]	Zhou C, Chen R J, Gao X L, et al. Heterologous expression of a rice RNA-recognition motif gene OsCBP20 in escherichia coli confers abiotic stress tolerance[J]. Plant Omics, 2014, 7(1):28-34.
[21]	Kim S, Yang J Y, Xu J, et al. Two cap-binding proteins CBP20 and CBP80 are involved in processing primary MicroRNAs[J]. Plant Cell Physiology, 2008, 49(11):1634-1644. DOI URL
[22]	Chen X, Liu J, Cheng Y, et al. HEN₁ functions pleiotropically in Arabidopsis development and Acts in C function in the flower[J]. Development, 2002, 129(5):1085-1094. DOI URL
[23]	Bollman M K. HASTY, the Arabidopsis ortholog of exportin 5/MSN5, regulates phase change and morphogenesis[J]. Development, 2003, 130(8):1493-1504. PMID
[24]	Finkelstein R. Abscisic acid synthesis and response[J]. Arabidopsis Book, 2013, 11:e0166. DOI URL
[25]	Gómez-Cadenas A, Zentella R, Walker-Simmons M K, et al. Gibberellin/abscisic acid antagonism in barley aleurone cells: site of action of the protein kinase PKABA1 in relation to gibberellin signaling molecules[J]. Plant Cell, 2001, 13(3):667-679. PMID
[26]	Chen M, Du X, Zhu Y, et al. Seed Fatty Acid Reducer Acts downstream of gibberellin signalling pathway to lower seed fatty acid storage in Arabidopsis[J]. Plant Cell Environment, 2012, 35(12):2155-2169. DOI URL
[27]	朱华国, 张献龙, 金双侠, 等. 两种常用激素组合下棉花体细胞胚胎发生过程的组织学观察[J]. 棉花学报, 2012, 24(2):159-166.
[28]	魏帅杰. 拟南芥HEN1调控胚珠发育的分子机理研究[D]. 泰安: 山东农业大学, 2019.
[29]	陈晓慧. 龙眼体胚发生过程中 DlDCLs的克隆与功能分析 [D]. 福州: 福建农林大学, 2018.

名称 Name	序列（5°-3°） Sequence (5°-3°)	退火温度 T_m/℃	用途 Annotation
DlDDL1	F: ACGGTTGATGAGAGTGCGAG	58	qRT-PCR
	R: GCTCACCAATTTCACGCTCT	58
DlDDL2	F: CGATTTCGTGCTTGATCATC	58	qRT-PCR
	R: AGTTCAACGGGAGTGTCTTTG
DlDDL3	F: TCCAGTTACTGCTGTGGATGC	58	qRT-PCR
	R: ACTATCGTCGGCATTGCTCA
DlDCL1	F: GCGTTTAGAGTTTGTTGGTGATG	58	qRT-PCR
	R: TCTGTCTTTCAAGGGCACTGG
DLSE	F: CCATGGAGTGGATTATTATGGG	58	qRT-PCR
	R: CAGCAGTCGCATCAATCTTC
DlHYL1a	F: ATGCTAGCCGCTTTAGGTGTA	58	qRT-PCR
	R: CCTGTCCAGTGTAAACCTCACC
DlHYL1b	F: GGCTGCTGAAGTTGCTCTTATG	58	qRT-PCR
	R: TTCATGCTGGAATGGACAAC
DlHYL1c	F: CCAGATCATGACCCTCGCT	58	qRT-PCR
	R: GAAGTGGAGAAAAGCAAGCATG
DlCBP20	F: CTACTGAGGAGCAAATTTACGAGC	58	qRT-PCR
	R: CGAATAGGACGGTCATCAAGA
DlCBP80	F: GCAAAGTCCTACAGCCTGGT	58	qRT-PCR
	R: GTTACATAAAAGTCGCCACGTG
DlHEN1	F: TTGCGAGCAACAGAACCTC	58	qRT-PCR
	R: AAACTTCCGGAGCCACATC
DlHASTY	F: ATCCAGCAAGGGTCCTGTTC	58	qRT-PCR
	R: TGCCTGCCAACTTCACTTAGT

名称 Name	序列（5°-3°） Sequence (5°-3°)	退火温度 T_m/℃	用途 Annotation
DlDDL1	F: ACGGTTGATGAGAGTGCGAG	58	qRT-PCR
	R: GCTCACCAATTTCACGCTCT	58
DlDDL2	F: CGATTTCGTGCTTGATCATC	58	qRT-PCR
	R: AGTTCAACGGGAGTGTCTTTG
DlDDL3	F: TCCAGTTACTGCTGTGGATGC	58	qRT-PCR
	R: ACTATCGTCGGCATTGCTCA
DlDCL1	F: GCGTTTAGAGTTTGTTGGTGATG	58	qRT-PCR
	R: TCTGTCTTTCAAGGGCACTGG
DLSE	F: CCATGGAGTGGATTATTATGGG	58	qRT-PCR
	R: CAGCAGTCGCATCAATCTTC
DlHYL1a	F: ATGCTAGCCGCTTTAGGTGTA	58	qRT-PCR
	R: CCTGTCCAGTGTAAACCTCACC
DlHYL1b	F: GGCTGCTGAAGTTGCTCTTATG	58	qRT-PCR
	R: TTCATGCTGGAATGGACAAC
DlHYL1c	F: CCAGATCATGACCCTCGCT	58	qRT-PCR
	R: GAAGTGGAGAAAAGCAAGCATG
DlCBP20	F: CTACTGAGGAGCAAATTTACGAGC	58	qRT-PCR
	R: CGAATAGGACGGTCATCAAGA
DlCBP80	F: GCAAAGTCCTACAGCCTGGT	58	qRT-PCR
	R: GTTACATAAAAGTCGCCACGTG
DlHEN1	F: TTGCGAGCAACAGAACCTC	58	qRT-PCR
	R: AAACTTCCGGAGCCACATC
DlHASTY	F: ATCCAGCAAGGGTCCTGTTC	58	qRT-PCR
	R: TGCCTGCCAACTTCACTTAGT

基因ID Gene ID	基因名称 Gene name	氨基酸数目 Amino acid number	分子量 Molecular weight/kDa	等电点 pI	不稳定系数 Instability index	亲水性指数 GRAVY
Dlo015725	DlDDL1	516	59.53	10.32	65.66	-1.529
Dlo021086	DlDDL2	405	43.85	6.36	45.20	-0.638
Dlo030112	DlDDL3	680	76.82	5.05	47.06	-0.870
Dlo011843	DlDCL1	516	59.53	10.32	65.66	-1.529
Dlo025444	DlSE	405	43.85	6.36	45.20	-0.638
Dlo004247	DlHYL1a	680	76.82	5.05	47.06	-0.870
Dlo013793	DlHYL1b	1968	220.25	5.75	80.95	-0.446
Dlo025470	DlHYL1c	835	94.65	8.70	63.05	-0.954
Dlo027356	DlCBP20	206	23.92	7.71	75.24	-0.343
Dlo025028	DlCBP80	871	99.51	5.95	97.72	-0.131
Dlo022071	DlHEN1	974	108.53	5.48	86.39	-0.220
Dlo003920	DlHASTY	1228	136.13	5.87	103.43	0.049

基因ID Gene ID	基因名称 Gene name	氨基酸数目 Amino acid number	分子量 Molecular weight/kDa	等电点 pI	不稳定系数 Instability index	亲水性指数 GRAVY
Dlo015725	DlDDL1	516	59.53	10.32	65.66	-1.529
Dlo021086	DlDDL2	405	43.85	6.36	45.20	-0.638
Dlo030112	DlDDL3	680	76.82	5.05	47.06	-0.870
Dlo011843	DlDCL1	516	59.53	10.32	65.66	-1.529
Dlo025444	DlSE	405	43.85	6.36	45.20	-0.638
Dlo004247	DlHYL1a	680	76.82	5.05	47.06	-0.870
Dlo013793	DlHYL1b	1968	220.25	5.75	80.95	-0.446
Dlo025470	DlHYL1c	835	94.65	8.70	63.05	-0.954
Dlo027356	DlCBP20	206	23.92	7.71	75.24	-0.343
Dlo025028	DlCBP80	871	99.51	5.95	97.72	-0.131
Dlo022071	DlHEN1	974	108.53	5.48	86.39	-0.220
Dlo003920	DlHASTY	1228	136.13	5.87	103.43	0.049