Cloning and Functional Analysis of miR398a from Chrysanthemum× grandiflora in Response to Salt Stress

doi:10.7525/j.issn.1673-5102.2022.06.008

Abstract

Abstract:

miRNAs played an important role in abiotic stress， and miR398a and pre-miR398a sequences were obtained from Small RNA database of Chrysanthemum×grandiflora. The sequence alignment showed that the cgr-miR398a was highly conserved with the identified miR398a sequences of other plants. Target genes prediction of cgr-miR398a showed that their annotation of targets include superoxide dismutase［Cu-Zn］（CSD2）， copper chaperone（CCS）， A20/AN1-like zinc finger family protein（SAP8）. The qPCR assay showed that the expression levels of miR398a was significantly decreased while target genes showed contrary trend during salt stress in Chrysanthemum×grandiflora. To explore the function of cgr-mir398a in response to salt stress， and an overexpression vector of cgr-mir398a was constructed and transformedinto Arabidopsis thaliana.The result showed that overexpression of the cgr-MIR398a in Arabidopsis could decrease seed germination under salt stress and salt tolerance at the adult seedling stage， therefore cgr-miR398a played a negative regulatory role in Arabidopsis in response to salt stress. The results provided a valuable reference for further exploring the functional mechanism of miR398a during salt stress in Chrysanthemum×grandiflora.

Key words: Chrysanthemum×grandiflora, cgr-miR398a, target gene, qPCR, salt stress

CLC Number:

S682.1+1

Liran YUE, Yingjie LIU, Chenxu LIU, Yunwei ZHOU. Cloning and Functional Analysis of miR398a from Chrysanthemum× grandiflora in Response to Salt Stress[J]. Bulletin of Botanical Research, 2022, 42(6): 986-996.

Figures/Tables 13

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引物名称 Primer name	引物序列 Primer sequence（5′→3′）
cgr-MIR398a-F	TACACTTCTCAACAAGGGCA
cgr-MIR398a-R	TAAGATAAATGTGAAGTGATTTGTAGAC
U6-F	GGAACGATACAGAGAAGATTAGC
U6-R	TGGAACGCTTCACGAATTTGCG
cgr-mir398a-F	GGAGTGACACTCAGAACACATG
cgr-MIR398aM-F	GGTACCTACACTTCTCAACAA
cgr-MIR398aM-R	GTCGACCGGTGTACCAATCAATTT
TRINITY_DN123102_c0_g2-F	GGCCTAATGTTGTGGTCGGA
TRINITY_DN123102_c0_g2-R	CACCACACCACAAGCAAGTC
TRINITY_DN96068_c0_g2-F	TGAAGCCTTGGAGCAGACAG
TRINITY_DN96068_c0_g2-R	GAACAATGACGATTGGCGCA
TRINITY_DN94774_c3_g5-F	GGACCCAAGAGATGCTCCAC
TRINITY_DN94774_c3_g5-R	CTTGGCAATGGCATCACGAC
CmEF1a-F	TTTTGGTATCTGGTCCTGGAG
CmEF1a-R	CCATTCAAGCGACAGACTCA
PBI121-GFP-F	TCATTTCATTTGGAGAGAACAC
PBI121-GFP-R	TTGCCAAATGTTTGAACGATC
At2g28190-F	ATGACACACGGAGCTCCAGAA
At2g28190-R	ATTGTTGTTTCTGCCACGCCA
At1g12520-F	GAGCCATGCCTCAGCTTCTTAC
At1g12520-R	TCACAGCATTAACACAACCCTCAC

植物组织 Plant tissue	靶基因 Target gene	功能注释 Annotation
叶和根 Leaf and root	TRINITY_DN118743_c0_g1	硫醇酶 Thiolase（PED1）
	TRINITY_DN121349_c0_g1	含CCT结构域的蛋白质 CCT domain-containing protein（APRR7）
	TRINITY_DN123102_c0_g2	叶绿体中铜-锌超氧化物歧化酶 Superoxide dismutase［Cu-Zn］，chloroplastic（CSD2）
	TRINITY_DN96068_c0_g2	铜伴侣蛋白 Copper chaperone for SOD1（CCS）
	TRINITY_DN97624_c8_g1	—
叶 Leaf	TRINITY_DN93471_c1_g2	未表征的蛋白质 Uncharacterized protein LOC107643668
	TRINITY_DN116098_c5_g1	叶绿体酶2 Chlorophyllase 2 （CLH2）
	TRINITY_DN97061_c0_g1	DNAJ热激家族蛋白 DNAJ heat shock family protein （DJA6）
	TRINITY_DN97655_c1_g2	过氧体（S）-2-羟基酸氧化酶球状体 Peroxisomal （S）-2-hydroxy-acid oxidase GLO1-like （GLO1）
	TRINITY_DN94774_c3_g5	类A20/AN1-l锌指家族蛋白 A20/AN1-like zinc finger family protein （SAP8）
	TRINITY_DN99012_c1_g2	假定蛋白Ccrd_005494 Hypothetical protein Ccrd_005494 （KAI2）
根 Root	TRINITY_DN105234_c5_g1	蛋白激酶样域 Protein kinase-like domain
	TRINITY_DN110642_c2_g2	糖苷水解酶，含催化域蛋白 Glycoside hydrolase， catalytic domain-containing protein （TAF15B）
	TRINITY_DN118599_c0_g1	Aux/IAA-ARF-二聚反应 Aux/IAA-ARF-dimerization（ARF9）
	TRINITY_DN122683_c2_g2	双功能多黏菌素抗性蛋白 Bifunctional polymyxin resistance protein
	TRINITY_DN124647_c1_g1	核苷酸结合相关 Nucleotide-binding， alpha-beta plait
	TRINITY_DN102787_c0_g1	假定蛋白CTI12_AA198490 Hypothetical protein CTI12_AA198490
	TRINITY_DN117385_c1_g3	含有PH、RCC1和FYVE结构域的蛋白质1样异构体X3 PH，RCC1 and FYVE domains-containing protein 1-like isoform X3 （UVR8）

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