拟南芥GLABROUS 1两个新等位突变体的筛选和鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2020-0774

摘要/Abstract

摘要：

植物细胞命运决定机制的解析一直以来都是植物发育生物学研究的核心。模式植物拟南芥的表皮毛形成过程是研究植物细胞命运决定的优良模式系统。为了筛选和鉴定控制拟南芥表皮毛形成的新因子,我们进行了大规模的正向遗传筛选,获得了两株莲座叶表皮毛不能形成或数量显著减少的突变体f08-01和vat002-07。通过对突变基因的克隆和遗传互补实验,确定这两株突变体是GLABROUS 1（GL1）的新等位突变体。GL1是调控植物表皮毛发生的关键遗传因子,其表达模式在表皮毛形成过程中受到严格调控,但是具体的机制还不清楚。通过遗传互补实验,证实GL1的3'非编码区在表皮毛形成过程中至关重要,并且3'非编码区的序列影响GL1 mRNA的稳定性和转录激活。

关键词: 表皮毛, GL1, mRNA稳定性, 细胞命运决定, 转录后调控

Abstract:

The studies on plant developmental biology has always been centered on uncovering the underlying mechanisms of cell fate determination. Trichome in model plant Arabidopsis is an elegant system to study the molecular mechanisms of several aspects including cell fate determination of plant. To identify and screen the new genetic factors controlling the trichome initiation of Arabidopsi,we carried out the large-scale forward genetic screening,and obtained two mutants f08-01 and vat002-07 in which the trichome of rosette leaves could not be formed or the number was significantly reduced. After cloning and genetic complementation of the mutation genes,we confirmed that these two mutants represented the new alleles of GLABROUS 1（GL1）,which is known as a key regulator in trichome initiation. Previous studies suggested that the expression of GL1 itself might be tightly regulated during trichome development,but the underlying mechanisms are not clear yet. During our genetic complementation experiments,we found the 3' non-coding region of GL1 was vital for its function during trichome development. Further genetic and pharmacological analysis showed that the 3' non-coding sequence might be responsible for its mRNA stability and transcriptional activation.

Key words: trichome, GL1, mRNA-stability, cell fate determination, post-transcriptional regulation

郑叶子, 王丹, 潘咪, 王艳玲, 安丽君. 拟南芥GLABROUS 1两个新等位突变体的筛选和鉴定[J]. 生物技术通报, 2021, 37(2): 15-23.

ZHENG Ye-zi, WANG Dan, PAN Mi, WANG Yan-ling, AN Li-jun. Isolation and Characterization of Two New GLABROUS1 Alleles in Arabidopsis[J]. Biotechnology Bulletin, 2021, 37(2): 15-23.

图/表 5

表1 所用引物序列表

名称	序列（5'-3'）	用途
MFE16#1F	AAATCTACGCAACGCAATGG	F08-01突变基因的克隆
MFE16#1R	TCACCAAATCTGACTTCTGC
T21C14#1F	AAGCTTCCCATATCCTCATC
T21C14#1R	GACCTAACGAAACAACTATG
GL1F1	TGCTTATAGCCATGATTACAC	T-DNA 纯杂合的鉴定
GL1R1	GTTTGCACGACTAATACACT
LB	GAACAACACTCAACCCTATCTC
pGL13’ΔF	CGATAAGCTTTATACGACGACATGTCGTTAC	质粒构建
pGL13’ΔR	CATGGGATCCCCGGCAAAGATTACTACTG
pGL1F	CGATAAGCTTTATACGACGACATGTCGTTAC
pGL1R	CATGGGATCCAAGTACGTACTCCTAGATTG
35SGL1F	CATGGGATCCGAATCAGAGAATAAAAAAAGA
35SGL1R	CATGGGATCCAAGTACGTACTCCTAGATTG
pGL13’mF	CATGAAGCTTTATACGACGACATGTCGTTAC
pGL13’mR	CATGGGATCCGATTACTACTGTTTTGAAAAC
GL1qRT F	ATGAGAATAAGGAGAAGAGA	实时荧光定量PCR
GL1 qRT R	AAGGCAGTACTCAACATCAC
ACTIN2 F	TCAAAGACCAGCTCTTCCATCGAGA
ACTIN2 R	ACACACAAGCGCATCATAGAAACGA

图1 f08-01和vat002-07突变体的表皮毛表型 A-C：10 d龄 Col（A）、f08-01（B）和vat002 -07（C）第一对真叶的表皮毛表型,bar=1 mm。右边部分的图为对应的左边图中黄色方框部分的细节展示,bar= 250 μm;D-F：Col（D）、f08-01（E）、vat002 -07（F）主花序茎上的表皮毛表型;（G-H）莲座叶（G）和主花序茎（H）上表皮毛数量的统计分析。数值以平均值±标准差表示,***：P<0.0012.2 f08-01和vat002-07是GL1的两个新等位突变体

图2 F08-01和VAT002-07突变位点的克隆 A：f08-01突变基因的定位,MFE16#1和T21C14#1是用于连锁分析的两个分子标记,星号表示Chr III中GL1基因的位置,外显子、5'非翻译区和3'非翻译区分别用开放框和阴影框表示,实线表示内含子,bar=200 bp;B：TAIL-PCR产物的凝胶电泳,红色星号表示与GL1同源的片段;C：图B中红色星号标记的片段的测序结果,红色部分表示T-DNA序列对应的核苷酸,开放框代表起始密码子（ATG）;D：片段序列同源性比对;E：GL1基因结构模式图,不同形状图形代表的基因元件与图A中相同,GL1 F1,GL1R1以及LB分别指示为用于基因型鉴定的引物序列所在位置;F：vat002-07 BC2 4株独立植物中的GL1基因T-DNA插入的确定

图3 f08-01和vat002-07突变体的遗传互补 A：不同基因型植物中第一对莲座叶及主花序茎上的表皮毛表型i;B：fi08-01（gl1-44）,vat002-07（gl1-45）及不同回复株系中GL1基因的相对表达水平。数值以平均值±标准差表示,采用Student’s text进行显著性分析,***：P<0.001

图4 GL1的3'非编码区对其表达的影响 A：不同基因型植物第一对莲座叶的表皮毛表型,以GL1基因自身启动子或者35S启动子驱动GL1开放阅读框（ORF）表达不能回复gl1-45的表皮毛形成缺陷表型;B：将GL1基因3'非编码区的153 bp 序列中的E2F结合位点突变仍可以回复gl1-44的表型,图上部为GL1基因结构图,图中红色部分表示位于GL1基因3'非编码区的153 bp序列所在位置,黄色部分为GL1基因3'非编码区,其他图形所指示的基因元件与图2-E相同,下部为不同基因型植物莲座叶中的表皮毛表型;C：WT,PGL1∷GL1 gl1-45 和P35S∷GL1 gl1-45转基因植物中GL1的相对表达量;D：CHX处理后的PGL1∷GL1 gl1-45 和P35S∷GL1 gl1-45转基因植物中GL1的相对表达量。数据以均数±标准差,采用Student’s test进行显著性分析,***：P<0.001

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