植物学报 ›› 2019, Vol. 54 ›› Issue (4): 522-530.DOI: 10.11983/CBB18229
收稿日期:
2018-10-30
接受日期:
2019-02-11
出版日期:
2019-07-01
发布日期:
2020-01-08
通讯作者:
阮继伟
基金资助:
Received:
2018-10-30
Accepted:
2019-02-11
Online:
2019-07-01
Published:
2020-01-08
Contact:
Jiwei Ruan
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。
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.
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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