Cloning and Functional Analysis of Gynostemma pentaphyllum GpMIR156a and GpMIR166b

doi:10.13560/j.cnki.biotech.bull.1985.2021-1072

Abstract

Abstract:

This study aims to deeply understand the role of two important miRNAs involved in the Gynostemma pentaphyllum aerial stem-to-rhizome transition,GpmiR156a and GpmiR166b,in regulating plant growth and development. GpMIR156a and GpMIR166b genes were obtained by homologous cloning and analyzed by bioinformatics. The effects of GpMIR156a and GpMIR166boverexpression on plant phenotype were investigated by constructing their overexpression vectors and then transforming Arabidopsis thaliana. The overexpression of GpMIR156a increased the number of leaves and branches of A. thaliana seedlings,and accelerated seed germination and root elongation,indicating that GpmiR156a promoted vegetative organ development and seed germination,but inhibited juvenile-to-adult transition. However,the overexpression of GpMIR166b led to the premature and dwarf plants,i.e.,increase in the number of trichomes on the inflorescence stems,as well as delayed seed germination but promoted root elongation. These findings indicated that GpmiR166b affected the normal development of vegetative organs and delayed seed germination to a certain extent,whereas promoted juvenile-to-adult transition. The results of this study broaden the understanding of miR156a and miR166b in regulating plant growth and development.

Key words: Gynostemma pentaphyllum, GpmiR156a, GpmiR166b, bioinformatics analysis, functional analysis

YU Qiu-lin, MA Jing-yi, ZHAO Pan, SUN Peng-fang, HE Yu-mei, LIU Shi-biao, GUO Hui-hong. Cloning and Functional Analysis of Gynostemma pentaphyllum GpMIR156a and GpMIR166b[J]. Biotechnology Bulletin, 2022, 38(7): 186-193.

Figures/Tables 9

Table 1 Primer information of G. pentaphyllu GpmiR156a and GpmiR166b

名称Name	序列Sequence（5'-3'）
GpmiR156a-F	TCTTYACWCAACWTCTCACCCA
GpmiR156a-R	GAGARAYCWGCATAWCTCAATMACC
GpmiR166b-F	CAGATACATGTACGAGGWGTTCA
GpmiR166b-R	GCTTCATCATYAYACCAATCTGC
GpmiR156a-Ft	TGGAGAGAACACGGGGGACTCTAGA TCTTYACWCAACWTCTCACCCA
GpmiR156a-Rt	TAACATAAGGGACTGACCACCCGGG GAGARAYCWGCATAWCTCAATMACC
GpmiR166b-Ft	TGGAGAGAACACGGGGGACTCTAGA CAGATA- CATGTACGAGGWGTTCA
GpmiR166b-Rt	TAACATAAGGGACTGACCACCCGGG GCTTCA- TCATYAYACCAATCTGC
p121-F	CGTCTTCAAAGCAAGTGGATT
p121-R	CCAACGCTGATCAATTCCAC

Fig. 1 Genomic DNA electrophoresis of G. pentaphyllumM：Marker. 1,2：Genomic DNA of G. pentaphyllum

Fig. 2 Precursor sequences of G. pentaphyllu GpmiR156a and GpmiR166b A：GpmiR156a precursor sequence. B：GpmiR166b precursor sequence. Underlines indicate mature sequences of GpmiR156a and GpmiR166b,respectively

Fig. 3 Secondary structures of G. pentaphyllu GpmiR156a and GpmiR166b A：Secondary structure of GpmiR156a. B：Secondary structure of GpmiR166b. The color change in the figure refers to the base pairing probability and the red ones refer to the highest probability

Fig. 4 Phylogenetic analysis of G. pentaphyllu GpmiR156a and GpmiR166b A：Phylogenetic analysis of GpmiR156a. B：Phylogenetic analysis of GpmiR166b. Arrows indicate the position of GpmiR156a and GpmiR166b,respectively

Table 2 Prediction of G. pentaphyllu GpmiR156a and GpmiR166b target genes

GpmiR156a靶基因预测GpmiR156a target gene prediction		GpmiR166b靶基因预测GpmiR166b target gene prediction
编号Serial No.	名称Name	编号Serial No.	名称Name
AT5G43270.3	SPL2 \| squamosa promoter binding protein-like 2	AT1G30490.1	PHV,ATHB9
AT2G33810.1	SPL3 \| squamosa promoter binding protein-like 3	AT1G30490.1	PHV,ATHB9
AT1G53160.1	SPL4 \| squamosa promoter binding protein-like 4	AT1G52150.3	ATHB-15,ATHB15,CNA
AT3G57920.1	SPL5 \| squamosa promoter binding protein-like 5	AT1G52150.3	ATHB-15,ATHB15,CNA
AT2G42200.1	SPL9,AtSPL9 \| squamosa promoter binding protein-like 9	AT2G34710.1	PHB,ATHB14,ATHB-14
AT1G27370.3	SPL10 \| squamosa promoter binding protein-like 10	AT2G34710.1	PHB,ATHB14,ATHB-14
AT1G27360.4	SPL11 \| squamosa promoter-like 11	AT4G32880.1	ATHB-8,ATHB8,HB-8 \| homeobox gene 8
AT5G50570.2	SPL13A,SPL13 \| Squamosa promoter-binding protein-like（SBP domain）transcription factor family protein	AT4G32880.1	ATHB-8,ATHB8,HB-8 \| homeobox gene 8
AT5G50670.1	SPL13B,SPL13 \| Squamosa promoter-binding protein-like（SBP domain）transcription factor family protein	AT5G60690.1	REV,IFL
AT3G57920.1	SPL15 \| squamosa promoter binding protein-like 15	AT5G60690.1	REV,IFL

Fig. 5 Detection and phenotype analysis of positive transg-enic plants A：Partial PCR identification results of GpmiR156a and GpmiR166b transgenic lines. M：Marker. 1：Negative control. 2：Positive control of GpmiR156a. 3-12：GpmiR156a. 13：Positive control of GpmiR166b. 14-24：GpmiR166b. B：Identification of GpmiR156a and GpmiR166b transgenic lines by GUS staining. 1：Wild-type（WT）A. thaliana. 2-7：GpmiR156a transgenic plants. 8-13：GpmiR166b transgenic plants. Bar=5 mm. C：Kan resistance screening. a：GpmiR156a screening medium. b：GpmiR166b screening medium. Red arrows indicate the transgenic A. thaliana selected for Kan resistance. D：Phenotype of WT and transgenic A. thaliana. a,d：WT Arabidopsis thaliana. b,e：GpmiR156a transgenic A. thaliana. c,f：GpmiR166b transgenic A. thaliana. Bar=1 cm

Fig. 6 Number of rosette leaves and branches in transgenic A. thaliana and WT A：Number of rosette leaves in the transgenic A. thaliana overexpressing GpmiR156a. B：Number of branches in the transgenic A. thaliana overexpressing GpmiR156a. C：Number of rosette leaves in the transgenic A. thaliana overexpressing GpmiR166b. GpmiR156a-1,GpmiR156a-2,and GpmiR156a-3 are three separate lines of transgenic A. thaliana. GpmiR166b-1,GpmiR166b-2,and GpmiR166b-3 are three separate lines of transgenic A. thaliana. Number of branches refers to the branches other than the main branches. n=10. Lowercase letters a and b refer to the significant difference between wild type and transgenic plants. The same below

Fig. 7 Seed germination and root length of overexpressing GpmiR156a and GpmiR166b and wild-type A. thal-iana A：Seed germination of transgenic Arabidopsis overexpressing GpmiR156a. B：Seed germination of transgenic Arabidopsis overexpressing GpmiR166b. C：Root length of transgenic Arabidopsisoverexpressing GpmiR156a. D：Root length of transgenic Arabidopsis overexpressing GpmiR166b. GpmiR156a-1,GpmiR156a-2,and GpmiR156a-3 are three separate lines of transgenic Arabidopsis. GpmiR166b-1,GpmiR166b-2,and GpmiR166b-3 are three separate lines of transgenic Arabidopsis. A,B：n=36;C,D：n=5

References 25

[1]	Razmovski-Naumovski V, Huang THW, Tran VH, et al. Chemistry and pharmacology of Gynostemma pentaphyllum[J]. Phytochem Rev, 2005, 4(2/3):197-219. doi: 10.1007/s11101-005-3754-4 URL
[2]	Yang Q, Liu SB, Han XN, et al. Integrated transcriptome and miRNA analysis uncovers molecular regulators of aerial stem-to-rhizome transition in the medical herb Gynostemma pentaphyllum[J]. BMC Genomics, 2019, 20(1):865. doi: 10.1186/s12864-019-6250-8 pmid: 31730459
[3]	Axtell MJ, Bowman JL. Evolution of plant microRNAs and their targets[J]. Trends Plant Sci, 2008, 13(7):343-349. doi: 10.1016/j.tplants.2008.03.009 URL
[4]	Ma JY, Zhao P, Liu SB, et al. The control of developmental phase transitions by microRNAs and their targets in seed plants[J]. Int J Mol Sci, 2020, 21(6):1971. doi: 10.3390/ijms21061971 URL
[5]	Schwab R, Palatnik JF, Riester M, et al. Specific effects of microRNAs on the plant transcriptome[J]. Dev Cell, 2005, 8(4):517-527. doi: 10.1016/j.devcel.2005.01.018 URL
[6]	Franco-Zorrilla JM, Valli A, Todesco M, et al. Target mimicry provides a new mechanism for regulation of microRNA activity[J]. Nat Genet, 2007, 39(8):1033-1037. pmid: 17643101
[7]	He J, Xu ML, Willmann MR, et al. Threshold-dependent repression of SPL gene expression by miR156/miR157 controls vegetative phase change in Arabidopsis thaliana[J]. PLoS Genet, 2018, 14(4):e1007337.
[8]	Wang J, Gao XY, Li L, et al. Overexpression of Osta-siR2141 caused abnormal polarity establishment and retarded growth in rice[J]. J Exp Bot, 2010, 61(6):1885-1895. doi: 10.1093/jxb/erp378 URL
[9]	Williams L, Grigg SP, Xie MT, et al. Regulation of Arabidopsis shoot apical meristem and lateral organ formation by microRNA miR166g and its AtHD-ZIP target genes[J]. Development, 2005, 132(16):3657-3668. pmid: 16033795
[10]	Jung JH, Park CM. MIR166/165 genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in Arabidopsis[J]. Planta, 2007, 225(6):1327-1338. doi: 10.1007/s00425-006-0439-1 URL
[11]	Wang JW, Park MY, Wang LJ, et al. miRNA control of vegetative phase change in trees[J]. PLoS Genet, 2011, 7(2):e1002012.
[12]	Xiong JS, Bai YB, Ma CJ, et al. Molecular cloning and characterization of SQUAMOSA-promoter binding protein-like gene FvSPL10 from woodland strawberry(Fragaria vesca)[J]. Plants(Basel), 2019, 8(9):342.
[13]	Guo Q, Li L, Zhao K, et al. Genome-wide analysis of poplar SQUAMOSA-promoter-binding protein(SBP)family under salt stress[J]. Forests, 2021, 12(4):413. doi: 10.3390/f12040413 URL
[14]	Xu ML, Hu TQ, Zhao JF, et al. Developmental functions of mir156-regulated Squamosa promoter binding protein-like(spl)genes in Arabidopsis thaliana[J]. PLoS Genet, 2016, 12(8):e1006263.
[15]	Gao RM, Gruber MY, Amyot L, et al. SPL13 regulates shoot branching and flowering time in Medicago sativa[J]. Plant Mol Biol, 2018, 96(1/2):119-133. doi: 10.1007/s11103-017-0683-8 URL
[16]	Miao CB, Wang Z, Zhang L, et al. The grain yield modulator miR156 regulates seed dormancy through the gibberellin pathway in rice[J]. Nat Commun, 2019, 10(1):3822. doi: 10.1038/s41467-019-11830-5 URL
[17]	Zhang L, Ding H, Jiang HL, et al. Regulation of cadmium tolerance and accumulation by miR156 in Arabidopsis[J]. Chemosphere, 2020, 242:125168. doi: 10.1016/j.chemosphere.2019.125168 URL
[18]	Barrera-Rojas CH, Rocha GHB, Polverari L, et al. miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses[J]. J Exp Bot, 2020, 71(3):934-950. doi: 10.1093/jxb/erz475 pmid: 31642910
[19]	Willmann MR, Poethig RS. The effect of the floral repressor FLC on the timing and progression of vegetative phase change in Arabidopsis[J]. Development, 2011, 138(4):677-685. doi: 10.1242/dev.057448 pmid: 21228003
[20]	Tang XR, Bian SM, Tang MJ, et al. microRNA-mediated repression of the seed maturation program during vegetative development in Arabidopsis[J]. PLoS Genet, 2012, 8(11):e1003091.
[21]	Li ZX, Zhang LF, Li WF, et al. MIR166a affects the germination of somatic embryos in larixleptolepis by modulating IAA biosynthesis and signaling genes[J]. J Plant Growth Regul, 2017, 36(4):889-896. doi: 10.1007/s00344-017-9693-7 URL
[22]	Barik S, SarkarDas S, Singh A, et al. Phylogenetic analysis reveals conservation and diversification of micro RNA166 genes among diverse plant species[J]. Genomics, 2014, 103(1):114-121. doi: 10.1016/j.ygeno.2013.11.004 URL
[23]	Chaves I, Lin YC, Pinto-Ricardo C, et al. miRNA profiling in leaf and cork tissues of Quercus suber reveals novel miRNAs and tissue-specific expression patterns[J]. Tree Genet Genomes, 2014, 10(3):721-737. doi: 10.1007/s11295-014-0717-1 URL
[24]	Li ZX, Qi LW. Over-expression of LaMIR166a promotes organs development in Nicotiana benthamiana[J]. Russ J Plant Physiol, 2019, 66(5):718-724. doi: 10.1134/S1021443719050133 URL
[25]	Gautam V, Singh A, Yadav S, et al. Conserved LBL1-ta-siRNA and miR165/166-RLD1/2 modules regulate root development in maize[J]. Development, 2021, 148(1):dev190033.