绞股蓝GpMIR156a和GpMIR166b的克隆与功能分析

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

摘要/Abstract

摘要：

本研究旨在深入了解药用植物绞股蓝地上茎向根状茎转变过程中两个重要的miRNAs（GpmiR156a和GpmiR166b）在调控植物生长发育方面的作用。采用同源克隆的方法获得GpMIR156a和GpMIR166b基因,进行了生物信息学分析。通过构建植物过表达载体并成功转化拟南芥,研究GpMIR156a和GpMIR166b的过表达对植株表型的影响。GpMIR156a过表达导致拟南芥幼苗叶片数量和分枝增多,并加速了种子萌发、促进根的伸长,表明GpmiR156a能够促进营养器官发育和种子萌发,但抑制童期向成熟期发育的转变。然而,GpMIR166b过表达导致拟南芥植株矮小及早熟性状即花序茎上毛状体数量增多,并延缓种子的萌发,但促进根伸长,表明GpmiR166b在一定程度上影响营养器官的发育和延缓种子萌发,但能促进童期向成熟期发育的转变。研究结果拓宽了miR156a和miR166b在调控植物生长发育方面的认识。

关键词: 绞股蓝, GpmiR156a, GpmiR166b, 生物信息学分析, 功能分析

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

于秋琳, 马婧怡, 赵盼, 孙鹏芳, 何玉美, 刘世彪, 郭惠红. 绞股蓝GpMIR156a和GpMIR166b的克隆与功能分析[J]. 生物技术通报, 2022, 38(7): 186-193.

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.

图/表 9

表1 绞股蓝GpmiR156a和GpmiR166b的引物信息

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

图1 绞股蓝基因组DNA电泳图 M：Marker;1,2：绞股蓝基因组DNA

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

图2 绞股蓝GpmiR156a和GpmiR166b的前体序列 A：GpmiR156a前体序列;B：GpmiR166b前体序列;下划线指示GpmiR156a和GpmiR166b的成熟体

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

图3 绞股蓝GpmiR156a和GpmiR166b的二级结构 A：GpmiR156a的二级结构;B：GpmiR166b的二级结构;图中颜色的变化代表碱基配对概率,红色代表概率最大

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

图4 绞股蓝GpmiR156a和GpmiR166b的进化分析 A：GpmiR156a的进化分析;B：GpmiR166b的进化分析;Gynostemma pentaphyllum：绞股蓝;Arabidopsis thaliana：拟南芥;Brassica rapa：芜菁;Zea mays：玉米;Glycine max：大豆;Solanum lycopersicum：番茄;Brachypodium distachyon：二穗短柄草;Cucumis melo：甜瓜;Citrullus lanatus：西瓜;Momordica charantia：苦瓜;Cucurbita pepo：西葫芦;Cucurbita maxima：笋瓜;Cucurbita moschata：南瓜;Vitis vinifera：葡萄;Solanum tuberosum：马铃薯;箭头指示GpmiR156a和GpmiR166b的位置

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

表2 绞股蓝GpmiR156a和GpmiR166b的靶基因预测

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

图5 转基因植株的检测与表型分析 A：GpmiR156a和GpmiR166b转基因株系的部分PCR鉴定结果;M：marker;1：阴性对照;2：GpmiR156a的阳性对照;3-12：GpmiR156a;13：GpmiR166b的阳性对照;14-24：GpmiR166b;B：GpmiR156a和GpmiR166b转基因株系的GUS染色鉴定;1：野生型拟南芥;2-7：GpmiR156a转基因植株;8-13：GpmiR166b转基因植株;Bar=5 mm;C：Kan抗性筛选;a：GpmiR156a筛选培养基;b：GpmiR166b筛选培养基;红色箭头指示Kan抗性筛选出的转基因拟南芥;D：野生型与转基因拟南芥的表型;a,d：野生型拟南芥;b,e：GpmiR156a转基因拟南芥;c,f：GpmiR166b转基因拟南芥;Bar=1 cm

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

图6 转基因拟南芥与野生型拟南芥的叶片及分枝数量 A：过表达GpmiR156a拟南芥叶片数量;B：过表达GpmiR156a拟南芥分枝数量;C：过表达GpmiR166b拟南芥叶片数量;GpmiR156a-1,GpmiR156a-2,GpmiR156a-3为3个转基因株系;GpmiR166b-1,GpmiR166b-2,GpmiR166b-3为3个转基因株系;分枝数量指除主枝以外的数目。n=10。小写字母a,b表示野生型和转基因株系之间的差异显著性（P<0.05）。下同

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

图7 过表达GpmiR156a、GpmiR166b拟南芥与野生型拟南芥种子的萌发与根长 A：过表达GpmiR156a拟南芥种子的萌发;B：过表达GpmiR166b拟南芥种子的萌发;C：过表达GpmiR156a拟南芥的根长;D：过表达GpmiR166b拟南芥的根长;GpmiR156a-1,GpmiR156a-2,GpmiR156a-3为3个转基因株系;GpmiR166b-1,GpmiR166b-2,GpmiR166b-3为3个转基因株系;A,B：n=36;C,D：n=5

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

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