利用荧光标记高通量鉴定减数分裂重组抑制突变体

doi:10.11983/CBB18229

摘要/Abstract

摘要：

正向遗传学突变体筛选被广泛用于揭示减数分裂中涉及的遗传基因, 如调控减数分裂II型交叉形成途径的重组抑制基因。该研究利用拟南芥(Arabidopsis thaliana)花粉荧光标记系进行EMS突变体的正向遗传学筛选, 鉴定拟南芥野生型Col遗传背景下的重组抑制突变体, 共获得18个重组率显著提高3倍以上的重组抑制突变体, 其中包括显性和隐性遗传突变。研究表明, 基于荧光标记高通量鉴定重组抑制突变体是可行的, 可为植物减数分裂重组调控分子机制研究提供新方法和突变材料。

关键词: 减数分裂, 重组抑制突变体, 荧光标记, 高通量可视分析法

Abstract:

The forward genetic approach has become a widespread methodology to reveal genetic factors involved in meiosis, such as the crossover negative regulators, which limit the class II crossover formation. Here we developed a forward genetics mutant screen to identify anti-CO mutants under the Col wild-type background of Arabidopsis thaliana. We isolated 18 mutant lines showing more than three-fold increase in male meiotic recombination frequency as compared with the wild type, including dominant and recessive mutants. Thus, the EMS screen based on fluorescent reporters allows for high-throughput identification of meiotic anti-CO mutants and provides a novel approach and genetic materials to study the molecular mechanism of meiotic recombination regulation.

Key words: meiosis, anti-CO mutants, fluorescent marker, high-throughput visual assay

李帆,阮继伟. 利用荧光标记高通量鉴定减数分裂重组抑制突变体. 植物学报, 2019, 54(4): 522-530.

Fan Li,Jiwei Ruan. High-throughput Identification of Meiotic Anti-CO Mutants by Fluorescent Reporters. Chinese Bulletin of Botany, 2019, 54(4): 522-530.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB18229

https://www.chinbullbotany.com/CN/Y2019/V54/I4/522

图/表 6

图1 拟南芥花粉荧光标记系FTL-I2b示意图 (A) 拟南芥花粉荧光标记系FTL-I2b四分体花粉荧光显微图, 其包含1个DsRed和1个eYFP标记, 能在不同的荧光激发下分别表达红荧光和绿荧光(Bars=50 μm); (B) 拟南芥花粉荧光标记系FTL-I2b在染色体上的位置示意图(Bar=1 Mb), 红色(下)和绿色(上)椭圆分别代表对应的荧光标记DsRed和eYFP, 并构建出1个I2b标记区间(1.45 Mb)。BF: 明场

Figure 1 A schematic illustration of Arabidopsis fluorescent tagged line FTL-I2b (A) Fluorescence micrograph of a tetrad pollen of FTL-I2b, which contains a DsRed and an eYFP fluorescent marker and expresses red fluorescence and green fluorescence under different fluorescence excitations respectively (Bars=50 μm); (B) The genomic location of FTL-I2b fluorescent markers on the chromosome (Bar=1 Mb), the DsRed and eYFP fluorescent markers are indicated by filled circles colored by red (down) and green (up), respectively, which constructing a I2b interval (1.45 Mb). BF: Bright field

图2 拟南芥FTL-I2b半合子荧光标记(I2b/++) (A) FTL-I2b与Col杂交示意图, F1代经过杂交后具有半合子荧光标记, 通过减数分裂形成重组和未重组的配子; (B) 杂交F1代花粉半合子荧光标记显微图, 图中黄色花粉(GR)为未重组配子, 单红色(R)和单绿色(G)花粉为重组配子(Bars=50 μm); (C) 利用流式细胞仪统计花粉中荧光标记可计算该标记区间I2b的重组率, 计算公式为重组率(RF)=(G+R)/(2GR+G+R)×100。

Figure 2 The Arabidopsis hemizygous fluorescent markers (I2b/++) of FTL-I2b (A) A schematic illustration of FTL-I2b cross with Col, the hybrid F1 contains a hemizygous fluorescent markers after hybridization, the recombined and unrecombined gametes are formed through meiosis; (B) Fluorescence micrograph of pollen in hybrid F1 generation, in which yellow pollen (GR) is unrecombined gamete, and monochromatic red (R) and green (G) pollen is recombined gametes (Bars=50 μm); (C) The recombination frequency (RF) of I2b interval can be calculated by counting the fluorescent marker pattern in the gametes via flow cytometry, the formula calculating recombination frequency=(G+R)/(2GR+G+R)×100.

图3 不同时间的EMS (75 mmol∙L-1)处理下M1代拟南芥种子的萌发率种子萌发率为3次重复的平均发芽率, 不同小写字母表示差异显著(P<0.05)。

Figure 3 The germination rate of M1 Arabidopsis seeds by different treat time using 75 mmol∙L-1 EMS The average germination rate was derived from three repeats, different lowercase letters indicate significant differences (P<0.05).

图4 拟南芥减数分裂重组抑制突变体正向遗传学筛选示意图突变体正向遗传学的筛选建立在拟南芥野生型(WT)遗传背景基础上, 利用花粉荧光标记系FTL-I2b半合子荧光标记(I2b/++)进行高通量重组率检测, 筛选获得重组率提高的突变体。图中FTL-I2b中的DsRed和eYFP荧光标记分别由R和G表示。m代表基因的显性或隐性突变。RF: 重组率

Figure 4 A schematic illustration of Arabidopsis meiotic anti-CO mutants forward genetics screen The forward genetics mutants screen was used the FTL-I2b containing linked hemizygous fluorescent reporters (I2b/++) on the genetic background of Arabidopsis wild type (WT). High-throughput detecting the recombination frequency by flow cytometry could identify the mutants with increased recombination rate. The DsRed and eYFP fluorescent reporters of FTL-I2b are indicated by R and G, respectively. The m refers to a dominant or recessive mutation. RF: Recombination frequency

图5 拟南芥野生型与大花粉突变体花粉显微图 (A) 拟南芥野生型(WT)花粉大小为(21.82±0.98) µm (n=10); (B) 大花粉突变体1 (big pollen 1)的大花粉率为100%, 花粉大小为(38.36±1.37) µm (n=10); (C) 大花粉突变体2 (big pollen 2)的大花粉率为50%, 花粉大小为(36.00±2.70) µm (n=10)。Bars=50 µm

Figure 5 The pollen micrograph of Arabidopsis wild type and big pollen mutants (A) The pollen size of Arabidopsis wild type (WT) is (21.82±0.98) µm (n=10); (B) The large pollen rate of big pollen 1 mutant is 100%, pollen size is (38.36±1.37) µm (n=10); (C) The large pollen rate of big pollen 2 mutant is 50%, pollen size is (36.00±2.70) µm (n=10). Bars=50 µm

图6 拟南芥野生型与重组抑制突变体 (A) 拟南芥野生型(WT)、显性重组抑制突变体(drs)和隐性重组抑制突变体(rrs1)植株表型(Bars=1 cm); (B) 拟南芥野生型(WT)、显性重组抑制突变体(drs)和隐性重组抑制突变体(rrs1)花粉荧光标记显微图(Bars=50 μm)。

Figure 6 Arabidopsis wild type and recombination suppressor mutants (A) The plant phenotype of Arabidopsis wild type (WT), dominant recombination suppressor mutant (drs) and recessive recombination suppressor mutant (rrs1) (Bars=1 cm); (B) The fluorescent pollen micrograph of Arabidopsis wild type (WT), dominant recombination suppressor mutant (drs) and recessive recombination suppressor mutant (rrs1) (Bars=50 μm)

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