植物学报 ›› 2020, Vol. 55 ›› Issue (4): 407-420.DOI: 10.11983/CBB20009
收稿日期:
2020-01-15
接受日期:
2020-05-15
出版日期:
2020-07-01
发布日期:
2020-05-21
通讯作者:
韩榕
基金资助:
Fangfang He,Huize Chen,Jinlin Feng,Lin Gao,Jiao Niu,Rong Han()
Received:
2020-01-15
Accepted:
2020-05-15
Online:
2020-07-01
Published:
2020-05-21
Contact:
Rong Han
摘要: UV-B辐射对植物的影响体现在多个水平, 其会引起植物DNA损伤, 造成有丝分裂异常, 最终影响植物的生长发育及生理生化过程。RAD21.3是黏连蛋白复合物的一个亚基, 参与有丝分裂中染色体的分离。该研究以哥伦比亚生态型拟南芥(Arabidopsis thaliana)和atrad21.3突变体为材料, 设置对照(CK)及UV-B处理组, 对野生型(WT)、atrad21.3及过表达株系的根长、株高、抽薹时间和生理生化指标进行统计分析。利用碱性品红染色观察拟南芥根尖的有丝分裂现象, 并统计畸变率。SPSS分析结果表明, UV-B处理后, WT UV-B和atrad21.3 CK的抽薹时间、株高及各项生理生化指标与WT CK相比无显著差异, 但atrad21.3 UV-B与之相比差异显著。通过烟草(Nicotiana benthamiana)的瞬时表达和亚细胞定位观察, 发现RAD21.3集中在细胞核; 进一步观察分裂期细胞发现落后染色体、染色体桥和游离染色体等异常现象。统计结果表明, 与WT CK相比, WT UV-B和atrad21.3 CK的畸变率较高, 但atrad21.3 UV-B的畸变率更高, 表明RAD21.3可能响应UV-B辐射诱导的异常有丝分裂。
贺芳芳,陈慧泽,冯金林,高琳,牛娇,韩榕. 拟南芥黏连蛋白RAD21对增强UV-B辐射后细胞分裂的响应. 植物学报, 2020, 55(4): 407-420.
Fangfang He,Huize Chen,Jinlin Feng,Lin Gao,Jiao Niu,Rong Han. Response of Arabidopsis Cohesin RAD21 to Cell Division after Enhanced UV-B Radiation. Chinese Bulletin of Botany, 2020, 55(4): 407-420.
图1 atrad21.3的T-DNA插入示意图及鉴定 (A) atrad21.3基因结构以及T-DNA插入位点示意图; (B) atrad21.3 DNA水平鉴定(M: 分子量标记); (C) atrad21.3中RAD 21.3基因的转录本水平。WT: 野生型
Figure 1 T-DNA insertions and identification of atrad21.3 (A) Gene structure and T-DNA insertions of atrad21.3; (B) Identification of DNA level in atrad21.3(M: Marker); (C) Transcriptional level of RAD21.3 gene in atrad21.3. WT: Wild type
图2 野生型(WT)和atrad21.3的表型分析 (A) WT种子; (B) 第12天的WT莲座叶; (C) WT抽薹; (D) atrad21.3种子; (E) 第12天的atrad21.3莲座叶; (F) atrad21.3未抽薹; (G) WT和atrad21.3的株高. (A), (D) Bars=10 μm; (B), (C), (E)-(G) Bars=1.0 cm
Figure 2 Phenotypic analysis of wild type (WT) and atrad21.3 (A) Seed of WT; (B) Rosette leaf (day 12) of WT; (C) Bolting of WT; (D) Seed of atrad21.3; (E) Rosette leaf (day 12) of atrad21.3; (F) Bolting of atrad21.3; (G) Plant height of WT and atrad21.3. (A), (D) Bars=10 μm; (B), (C), (E)-(G) Bars=1.0 cm
图3 AtRAD21.3的克隆 (A) AtRAD21.3的扩增产物(1-6: 产物); (B) AtRAD21.3切胶回收电泳结果(1, 2: 切胶回收产物); (C) 菌液PCR结果(1-4, 6: 阳性单菌落; 5: 阴性对照); (D) 质粒双酶切验证(1-4: 双酶切结果)。M: 分子量标记
Figure 3 Cloning of AtRAD21.3 (A) PCR products of AtRAD21.3 (1-6: Products); (B) Gel cutting recovery result of AtRAD21.3 (1, 2: Gel cutting recovery products); (C) Bacterial PCR result (1-4, 6: Positive single colony; 5: Negative control products); (D) Dual-restriction digestion of plasmid (1-4: Dual-restriction result). M: DNA marker
图4 pSuper1300-RAD21.3-GFP在烟草叶片的瞬时表达 (A) pSuper1300-RAD21.3-GFP的GFP荧光信号图像; (B) pSuper1300-RAD21.3-GFP在烟草叶片的DAPI染色图像; (C) A和B叠加的图像。Bars=10 μm
Figure 4 Transient expression of pSuper1300-RAD21.3-GFP in leaves of Nicotiana benthamiana (A) GFP signal of pSuper1300-RAD21.3-GFP; (B) DAPI staining of pSuper1300-RAD21.3-GFP in leaves of Nicotiana benthamiana; (C) Merged image of A and B. Bars=10 μm
图6 UV-B辐射后AtRAD21基因表达量检测 (A) 半定量RT-PCR电泳图; (B) 灰度值分析。WT: 野生型。不同小写字母表示差异显著(Duncan法, P<0.05)。
Figure 6 Detection of UV-B radiation on AtRAD21 expres-sion quantity (A) Result of RT-PCR; (B) Gray value analysis. WT: Wild type. Different lowercase letters show significant differences (Duncan method, P<0.05).
图7 UV-B处理对拟南芥种子萌发率及幼苗根长、株高和抽薹时间的影响 (A), (E) UV-B处理对拟南芥种子萌发率的影响; (B), (F) UV-B处理对拟南芥幼苗根长的影响; (C), (H) UV-B处理对拟南芥抽薹时间的影响; (D), (G) UV-B处理对拟南芥株高的影响。WT: 野生型。不同小写字母表示差异显著(Duncan法, P<0.05)。
Figure 7 The seed germination rate, root length, plant height and bolting time of Arabidopsis thaliana with UV-B treatment (A), (E) The seed germination rate of Arabidopsis thaliana with UV-B treatment; (B), (F) The root length of Arabidopsis thaliana seedling with UV-B treatment; (C), (H) The bolting time of Arabidopsis thaliana with UV-B treatment; (D), (G) The plant height of Arabidopsis thaliana with UV-B treatment. WT: Wild type. Different lowercase letters show significant differences (Duncan method, P<0.05).
图8 UV-B辐射对拟南芥植株生理生化指标的影响 (A) UV-B辐射对拟南芥丙二醛(MDA)浓度的影响; (B) UV-B辐射对拟南芥超氧化物歧化酶(SOD)活性的影响; (C) UV-B辐射对拟南芥叶绿素含量的影响; (D) UV-B辐射对拟南芥叶片可溶性糖含量的影响。WT: 野生型。不同小写字母(或*)表示差异显著(Duncan法, P<0.05)。
Figure 8 The influence of physiological and biochemical in Arabidopsis thaliana with UV-B treatment (A) Effect of UV-B radiation on Arabidopsis thaliana malondialdehyde (MDA) concentration; (B) Effect of UV-B radiation on Arabidopsis thaliana superoxide dismutase (SOD) activity; (C) Effect of UV-B radiation on chlorophyll content in Arabidopsis thaliana; (D) Effect of UV-B radiation on content of soluble sugar of Arabidopsis thaliana. WT: Wild type. Different lowercase letters (or * ) show significant differences (Duncan method, P<0.05).
图9 拟南芥细胞周期相关基因的相对表达量 WT: 野生型。不同小写字母表示差异显著(Duncan法, P<0.05)。
Figure 9 Relative expression of cell cycle related genes in Arabidopsis thaliana WT: Wild type. Different lowercase letters show significant differences (Duncan method, P<0.05).
图10 拟南芥根尖正常有丝分裂各时期图 (A) 间期; (B), (C) 前期; (D) 中期; (E) 中后期; (F) 后期; (G) 末期。Bars=20 μm
Figure 10 Normal phase types of mitosis in root tip of Arabidopsis thaliana (A) Interphase; (B), (C) Prophase; (D) Metaphase; (E) Meta-anaphase; (F) Anaphase; (G) Telephase. Bars=20 μm
图11 拟南芥根尖染色体畸变的不同类型 (A) 前期不定向(箭头); (B) 前期散乱(箭头); (C) 游离染色体(箭头); (D) 游离染色体(箭头); (E) 后期不对称(箭头); (F) 染色体桥(箭头); (G) 落后染色体(箭头); (H) 落后染色体(箭头)。Bars=20 μm
Figure 11 Different types of chromosome aberration in root tip of Arabidopsis thaliana (A) Unorientation at prophase (arrow); (B) Cofusion at prophase (arrow); (C) Fragments chromosomes (arrow); (D) Fragments chromosomes (arrow); (E) Asymmetric at anaphase (arrow); (F) Chromosome bridge (arrow); (G) Lagging chromosome (arrow); (H) Lagging chromosome (arrow). Bars=20 μm
Treatment | Total of observing cells | Total of dividing cells | Total of aberration cells | Percentage of dividing cells (%) | Percentage of chromosomal aberration (%) |
---|---|---|---|---|---|
WT CK | 5390 | 195 | 8 | 3.6±0.202 a | 0.148±0.102 c |
WT UV-B | 5376 | 166 | 25 | 3.08±0.258 c | 0.465±0.056 b |
atrad21.3 CK | 6998 | 215 | 32 | 3.07±0.195 c | 0.457±0.085 b |
atrad21.3 UV-B | 7044 | 194 | 45 | 2.75±0.182 d | 0.638±0.105 a |
35S:RAD21.3 CK | 5603 | 186 | 7 | 3.3±0.132 ab | 0.125±0.136 c |
35S:RAD21.3 UV-B | 5536 | 193 | 21 | 3.48±0.129 ab | 0.379±0.125 bc |
表1 UV-B辐射对拟南芥有丝分裂的影响
Table1 Effects of the UV-B radiation on mitosis of Arabidopsis thaliana
Treatment | Total of observing cells | Total of dividing cells | Total of aberration cells | Percentage of dividing cells (%) | Percentage of chromosomal aberration (%) |
---|---|---|---|---|---|
WT CK | 5390 | 195 | 8 | 3.6±0.202 a | 0.148±0.102 c |
WT UV-B | 5376 | 166 | 25 | 3.08±0.258 c | 0.465±0.056 b |
atrad21.3 CK | 6998 | 215 | 32 | 3.07±0.195 c | 0.457±0.085 b |
atrad21.3 UV-B | 7044 | 194 | 45 | 2.75±0.182 d | 0.638±0.105 a |
35S:RAD21.3 CK | 5603 | 186 | 7 | 3.3±0.132 ab | 0.125±0.136 c |
35S:RAD21.3 UV-B | 5536 | 193 | 21 | 3.48±0.129 ab | 0.379±0.125 bc |
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