植物学报 ›› 2020, Vol. 55 ›› Issue (6): 677-692.DOI: 10.11983/CBB20048
宋凝曦1,2, 谢寅峰2, 李霞1,2,3,*
收稿日期:
2020-03-21
接受日期:
2020-08-26
出版日期:
2020-11-01
发布日期:
2020-11-11
通讯作者:
李霞
作者简介:
*E-mail: jspplx@jaas.ac.cn基金资助:
Ningxi Song1,2, Yingfeng Xie2, Xia Li1,2,3,*
Received:
2020-03-21
Accepted:
2020-08-26
Online:
2020-11-01
Published:
2020-11-11
Contact:
Xia Li
摘要: 为探究干旱胁迫下表观遗传机制对高表达玉米(Zea mays) C4型PEPC转基因水稻(Oryza sativa)种子萌发的影响, 以转C4型PEPC水稻(PC)和野生型水稻Kitaake (WT)为试材, 采用10% (m/v)聚乙二醇6000 (PEG6000)模拟干旱条件, 通过单独和联合施用PEG6000、DNA甲基化抑制剂5-氮杂胞苷(5azaC)和可变剪接抑制剂大环内酯类(PB)进行种子发芽实验, 测定种子活力、萌发过程中可溶性糖和可溶性蛋白含量、α-淀粉酶活性以及PEPC、糖信号相关基因和部分剪接因子基因的表达。结果表明, 0.25 µmol·L-1PB处理对2种供试水稻在干旱条件下种子萌发均表现出显著抑制作用, 使干旱条件下种子萌发过程中可溶性总糖、蔗糖、葡萄糖和果糖含量以及可溶性蛋白含量均有所下降, PB也抑制糖信号-蔗糖非发酵1 (SNF1)相关蛋白激酶(SnRKs)家族和剪接因子丝氨酸/精氨酸富集蛋白家族(SR proteins)相关基因的表达以及α-淀粉酶的活性, 但对PC的抑制作用小于WT。5 µmol·L-15azaC处理对干旱条件下种子萌发的效果与可变剪接抑制剂相反。5 µmol·L -1 5azaC联合PEG6000干旱处理部分减缓了干旱对水稻种子发芽率的抑制作用, 使供试材料发芽率升高, 表明DNA甲基化和可变剪接机制参与了水稻芽期干旱耐性, 其中对PC的作用更大。
宋凝曦, 谢寅峰, 李霞. 干旱胁迫下表观遗传机制对转C4型PEPC基因水稻种子萌发的影响. 植物学报, 2020, 55(6): 677-692.
Ningxi Song, Yingfeng Xie, Xia Li. Effects of Epigenetic Mechanisms on C4 Phosphoenolpyruvate Carboxylase Transgenic Rice (Oryza sativa) Seed Germination Under Drought Stress. Chinese Bulletin of Botany, 2020, 55(6): 677-692.
Gene | Forward primer (5'-3') | Reverse primer (5'-3') |
---|---|---|
Actin | CCCTCAAACATCGGTATGGA | TTGATCTTCATGCTGCTTGG |
OsK1a | AACCAGAGGTAACAGGCAGG | AACCAGAGGTAACAGGCAGG |
OsK24 | CGTGTTGGCTTCAGTGAAT | CCTTCTCTATCTAAGGGCCG |
OsK35 | TTGTGTTGGCTTCAGTGAAA | CCTTCGCTGTCTAAGGACTG |
C4-PEPC | CCCACTATCCTTCGCAAGAC | CTAGCCAGTGTTCTGCATGCCGG |
Osppc2a | CTGGTTGAGATGGTTTTCGC | GGTGTGAATTCAGGCACTTC |
SAPK8 | ATAGATGATAATGTCCAGCG | GTTCCTACAGTGGATTTTGG |
SAPK9 | CACAGCAACGCCGTCTCC | CACACTTCCACCGCTACCAA |
SAPK10 | TGCTGATGTGTGGTCGTGTG | TGCTGGTATGGTCGCCTCT |
SR40 | CAATCTGGGGACTGCTTTC | TCCTGCTTGGGCTTTTACT |
SR33 | ATATTGCCTGCTACCCGAAAG | CAGAGCAGCACCCAGTTTATTAC |
OsAmy1A | TTTCGGTCCTCATCGTCCTCC | TCCACGACTCCCAGTTGAATC |
OsAmy1C | TGGTATCGATCAGAAACCGGC | GTCCGACCTTCGTGATGACC |
OsAmy3C | AAGCATTCCACCACAATGAGC | AGGAAGTTGTACCACCCACC |
OsAmy3E | TCACCCTGTGTTGTGTCGTT | AAAGTTGTACCACCCGCCTT |
表1 引物序列
Table 1 Primers used in this study
Gene | Forward primer (5'-3') | Reverse primer (5'-3') |
---|---|---|
Actin | CCCTCAAACATCGGTATGGA | TTGATCTTCATGCTGCTTGG |
OsK1a | AACCAGAGGTAACAGGCAGG | AACCAGAGGTAACAGGCAGG |
OsK24 | CGTGTTGGCTTCAGTGAAT | CCTTCTCTATCTAAGGGCCG |
OsK35 | TTGTGTTGGCTTCAGTGAAA | CCTTCGCTGTCTAAGGACTG |
C4-PEPC | CCCACTATCCTTCGCAAGAC | CTAGCCAGTGTTCTGCATGCCGG |
Osppc2a | CTGGTTGAGATGGTTTTCGC | GGTGTGAATTCAGGCACTTC |
SAPK8 | ATAGATGATAATGTCCAGCG | GTTCCTACAGTGGATTTTGG |
SAPK9 | CACAGCAACGCCGTCTCC | CACACTTCCACCGCTACCAA |
SAPK10 | TGCTGATGTGTGGTCGTGTG | TGCTGGTATGGTCGCCTCT |
SR40 | CAATCTGGGGACTGCTTTC | TCCTGCTTGGGCTTTTACT |
SR33 | ATATTGCCTGCTACCCGAAAG | CAGAGCAGCACCCAGTTTATTAC |
OsAmy1A | TTTCGGTCCTCATCGTCCTCC | TCCACGACTCCCAGTTGAATC |
OsAmy1C | TGGTATCGATCAGAAACCGGC | GTCCGACCTTCGTGATGACC |
OsAmy3C | AAGCATTCCACCACAATGAGC | AGGAAGTTGTACCACCCACC |
OsAmy3E | TCACCCTGTGTTGTGTCGTT | AAAGTTGTACCACCCGCCTT |
图1 不同处理对转基因水稻(PC)和野生型水稻(WT)种子活力的影响 (A) 发芽势; (B) 发芽率; (C) 发芽指数; (D) 活力指数; (E) 种子发芽图片。不同小写字母表示差异显著(P<0.05)。Bars=0.5 cm
Figure 1 Effects of different treatments on seed vigor of transgenic rice (PC) and wild type rice (WT) (A) Germination; (B) Germination rate; (C) Germination index; (D) Vigor index; (E) Images of germinating seeds at different time. Different lowercase letters indicate significant differences (P<0.05). Bars=0.5 cm
图2 不同处理下转基因水稻(PC)和野生型水稻(WT)萌发种子根长(A)、芽长(B)和根芽比(C)的变化 不同小写字母表示差异显著(P<0.05)。
Figure 2 Effects of different treatments on root length (A), shoot length (B) and root/bud ratio (C) of transgenic rice (PC) and wild type rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
图3 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中可溶性总糖含量(A)和可溶性蛋白含量(B)的影响 不同小写字母表示差异显著(P<0.05)。
Figure 3 Effects of different treatments on total soluble sugar content (A) and soluble protein content (B) of transgenic rice (PC) and wild type rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
图4 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中糖组分含量的影响 (A), (D) 分别为发芽24和48小时的蔗糖含量; (B), (E) 分别为发芽24和48小时的葡萄糖含量; (C), (F) 分别为发芽24和48小时的果糖含量。不同小写字母表示差异显著(P<0.05)。
Figure 4 Effects of different treatments on sugar content of transgenic rice (PC) and wild type rice (WT) germinating seeds (A), (D) Sucrose content at 24 h and 48 h after germination, respectively; (B), (E) Glucose content at 24 h and 48 h after germination, respectively; (C), (F) Fructose content at 24 h and 48 h after germination, respectively. Different lowercase letters indicate significant differences (P<0.05).
图5 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中SnRK相关基因表达的影响 不同小写字母表示差异显著(P<0.05)。
Figure 5 Effects of different treatments on the expression of SnRK related genes in transgenic rice (PC) and wild type rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
图6 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中剪接因子相关基因表达的影响 不同小写字母表示差异显著(P<0.05)。
Figure 6 Effects of different treatments on expression of splicing factor related genes in transgenic rice (PC) and wild type rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
图7 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中PEPC相关基因表达的影响 不同小写字母表示差异显著(P<0.05)。
Figure 7 Effects of different treatments on expression of PEPC-related genes in transgenic rice (PC) and wild type rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
图8 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中α-淀粉酶相关基因表达的影响 不同小写字母表示差异显著(P<0.05)。
Figure 8 Effects of different treatments on expression of α-amylase related genes in transgenic rice (PC) and type wild rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
图9 不同处理对转基因水稻(PC)和野生型水稻(WT)种子萌发过程中α-淀粉酶活性的影响 不同小写字母表示差异显著(P<0.05)。
Figure 9 Effects of different treatments on α-amylase activity in transgenic rice (PC) and wild type rice (WT) germinating seeds Different lowercase letters indicate significant differences (P<0.05).
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