The Activity Verification of pri-miR319a Encode Regulatory Peptide of Dimocarpus longan

doi:10.16420/j.issn.0513-353x.2020-0579

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (5): 908-920.doi: 10.16420/j.issn.0513-353x.2020-0579

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The Activity Verification of pri-miR319a Encode Regulatory Peptide of Dimocarpus longan

SU Liyao¹, WANG Peiyu², JIANG Mengqi¹, HUANG Shuqi¹, XUE Xiaodong¹, LIU Mengyu¹, XIAO Xuechen¹, LAI Chunwang¹, ZHANG Zihao¹, CHEN Yukun¹, LAI Zhongxiong¹, LIN Yuling¹^,^*()

¹Institute of Horticultural Biotechnology,Fujian Agriculture and Forestry University,Fuzhou 350002,China
²Sanming Academy of Agricultural Sciences,Sanming,Fujian 365000,China

Received:2020-09-15 Revised:2021-04-06 Online:2021-05-25 Published:2021-06-07
Contact: LIN Yuling E-mail:buliang84@163.com

Abstract

Abstract:

To study the potential activity of pri-miR319a encoding regulatory peptide of Dimocarpus longan,the pri-miR319a full-length sequences were cloned from seven longan varieties and the positions of potential miPEP(miRNA encode regulatory peptide)in longan pri-miR319a sequences were further analyzed. Then,the activity of miPEP319a was verified by genetic transformation and artificial synthesis of miPEP319a. The results showed that among the seven longan varieties,pri-miR319a had 10 nucleotide differences and three potential ORFs that could encode miPEPs,and one nucleotide mutation site resulted in the change of miPEP319a amino acid sequence. The biological activity of longan miPEP was further verified by thein plantatransformation technology and the synthetic miPEP319 treated embryogenic callus of longan. The results indicated that only the miPEP319a-2 of the three potential miPEPs was biological activity and promoted the expression of miR319a. Finally,the vectors were transiently expressed in tobacco leaves by Agrobacterium-mediated transformation method to verify the activity of longan miPEP319a in tobacco. The results showed that there was no significant difference in the expression of miR319a in tobacco leaves,which indicated that miPEP319a was specie-specific. The results suggested that longan miPEP319a-2 had biological activity and species-specific,and also suggested that longan miPEP319a-2 might participate in the growth and development of longan.

Key words: Dimocarpus longan, miR319a, miRNA encode regulatory peptide, gene clone, genetic transformation

CLC Number:

S667.2

SU Liyao, WANG Peiyu, JIANG Mengqi, HUANG Shuqi, XUE Xiaodong, LIU Mengyu, XIAO Xuechen, LAI Chunwang, ZHANG Zihao, CHEN Yukun, LAI Zhongxiong, LIN Yuling. The Activity Verification of pri-miR319a Encode Regulatory Peptide of Dimocarpus longan[J]. Acta Horticulturae Sinica, 2021, 48(5): 908-920.

Figures/Tables 10

Table 1 The primer used in this study

用途 Annotation	引物名称 Name of primer	序列(5′-3′) Sequence
初级体克隆 Clone	FL-319a-F	ATGAGATATTGCTTTGGTTAACAGG
	FL-319a-R	GTAGCATAGTTTAGTAGTGTGACTG
载体构建	miPEP319a-1F	GGGGTACCCTCCATTTGCAGATCCATGAG
Plasmid construction	miPEP319a-1R	CGGGATCCAGTATCACTAGCTCTAGCCTGTTAACC
	Mutant-miPEP319a-1F	GGGGTACCCTCCATTTGCAGATCCATTAGAT
	miPEP319a-2F	GGGGTACCAGATTTCATGGTCTATATATGTC
	miPEP319a-2R	CGGGATCCGCTCCCATCACAAGTCACAAC
	Mutant-miPEP319a-2F	GGGGTACCAGATTTCATTGTCTATATATTTC
	miPEP319a-3F	GGGGTACCTGGGGTTGTGACTTGTGATG
	miPEP319a-3R	CGGGATCCTGCAAGACAGATGGACAGATCTAG
	Mutant-miPEP319a-3F	GGGGTACCTGGGGTTGTGACTTGTGATTGGAG
定量及克隆	GUS-F	CCGTCCTGTAGAAACCCCAACC
Clone and qRT-PCR	GUS-R	TCCCGGCAATAACATACGGCGT
	Fe-SOD-F	GGTCAGATGGTGAAGCCGTAGAG
	Fe-SOD-R	GTCTATGCCACCGATACAACAAACCC
miRNA定量	miR319a	AGGGAGCTCCCTTCAGTCCAA
miRNA qRT-PCR	Ath5.8S*	ACGTCTGCCTGGGTGTCACAA
	Uniprimer	GATCGCCCTTCTACGTCGTAT

Fig. 1 The schematic diagram of pCAMBIA1301-miPEP(A)and pCAMBIA1301-Mutant-miPEP(B)vectors

Fig. 2 Full length of pri-miR319a in seven longan species HHZ:Honghezi;FY:Fuyan;QLBH:Quanlong Baihe;QL104:Quanlong 104;SEY:Shieryue;SJM:Sijimi;YTB:Youtanben. The arrow represents the mutant small peptide sequence.

Fig. 3 The process of obtaining transgenic gene longan plant A:Longan seedling;B,B’:Pruning terminal bud and euphylla;C,C’:Agrobacteriuminfection;D:Moisturize of longan seedling wound;E:The situation of transgenic gene bud;F:Transgenic gene plant.

Fig. 4 PCR verification of transferred miPEP319a-1/-2/-3 vectors and their mutant vectors longan buds M:Marker 2000;CK:Positive control with plasmid;1-20:Transgenic resistant shoots.

Fig. 5 The expression of GUS gene in transferred pCAMBIA1301-miPEP319a-1/-2/-3 and pCAMBIA1301-Mutant-miPEP319a vectors longan buds

Fig. 6 The relative expression of miR319a in longan buds that transferred pCAMBIA1301-miPEP319a-1/ miPEP319a-2/miPEP319a-3(T)and their mutant vectors(M)

Fig. 7 Relative expression of miR319a treated after synthetic miPEP319a-1,miPEP319a-2 and miPEP319a-3

Fig. 8 GUS staining of tobacco leaves that transferred pCAMBIA1301-miPEP319a-1/ miPEP319a-2 miPEP319a-3 and their mutant vector

Fig. 9 Relative expression of miR156a in tobacco leaves that transferred pCAMBIA1301-miPEP319a-1,miPEP319a-2,miPEP319a-3(T)and their mutant vectors(M)

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The Activity Verification of pri-miR319a Encode Regulatory Peptide of Dimocarpus longan

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