龙眼pri-miR319a编码短肽活性的研究

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

摘要/Abstract

摘要：

为研究龙眼miRNA编码短肽(miRNA encode regulatory peptide,miPEP)的潜在活性,以miR319为对象,从7个龙眼品种中克隆得到pri-miR319a 序列并预测其潜在编码miPEP,并通过遗传转化和人工合成miPEP的方法验证miPEP319a的活性。结果显示,7个龙眼品种中pri-miR319a序列存在10个核苷酸的差异,并具有3个潜在ORF可以编码miPEP,其中1个核苷酸突变位点导致miPEP319a氨基酸序列的改变。进一步通过原位转化法获得转化miPEP的过表达载体及相应突变表达载体的龙眼幼芽,并结合人工合成miPEP319处理龙眼胚性愈伤组织,进一步验证龙眼miPEP的生物学活性。结果显示,pri-miR319a编码的3个潜在miPEP仅miPEP319a-2具有生物学活性并促进下游miR319a的表达。最后采用烟草叶片的瞬时转化法验证龙眼miPEP319a在烟草当中的活性及对miR319a的影响。结果表明龙眼miPEP319a具有物种特异性,在烟草叶片中miR319a的表达模式无差异。该研究结果提示龙眼pri-miR319序列在不同品种中具有多态性,且其编码的3个潜在miPEP仅miPEP319a-2具有生物学活性,并具有物种特异性,同时也暗示龙眼miPEP319a-2可以参与到龙眼的生长发育过程中。

关键词: 龙眼, miR319a, miRNA编码短肽, 基因克隆, 遗传转化

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

中图分类号:

S667.2

苏立遥, 王培育, 蒋梦琦, 黄倏祺, 薛晓东, 刘梦雨, 肖学宸, 赖春旺, 张梓浩, 陈裕坤, 赖钟雄, 林玉玲. 龙眼pri-miR319a编码短肽活性的研究[J]. 园艺学报, 2021, 48(5): 908-920.

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.

图/表 10

表1 本试验中所用引物

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

图1 龙眼pCAMBIA1301-miPEP(A)和pCAMBIA1301-Mutant-miPEP(B)过表达载体示意图

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

图2 7个龙眼品种中pri-miR319a序列全长验证 HHZ:红核子;FY:福眼;QLBH:泉龙白核;QL104:泉龙104;SEY:十二月;SJM:四季蜜;YTB:油潭本。箭头表示突变后的短肽序列。

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.

图3 龙眼转基因植株的获得 A:实生苗;B、B’:去除顶芽及真叶;C、C’:农杆菌侵染伤口;D:原位转化幼苗伤口保湿;E:原位转化幼苗发芽情况;F:原位转化幼苗。

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.

图4 转化miPEP319a-1/-2/-3及其相应的突变载体龙眼植株抗性芽PCR检测 M：Marker 2000；CK：质粒阳性对照；1 ~ 20：转基因抗性芽。

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.

图5 转化miPEP319a-1/-2/-3及其相应的突变载体龙眼植株抗性芽GUS表达量分析

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

图6 龙眼转miPEP319a-1/-2/-3(T)及其突变载体(M)抗性芽中miR319a表达模式

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)

图7 人工合成miPEP319a-1/-2/-3处理对miR319a表达的影响

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

图8 过表达miPEP319a-1/-2/-3和Mutant-miPEP319a-1/-2/-3载体侵染烟草叶片GUS染色

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

图9 过表达miPEP319a-1/-2/-3(T)和Mutant-miPEP319a-1/-2/-3(M)烟草叶片中miR319a表达模式分析

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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