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作物学报 ›› 2022, Vol. 48 ›› Issue (5): 1191-1198.doi: 10.3724/SP.J.1006.2022.13027

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

干旱锻炼对B73自交后代当代干旱胁迫记忆基因表达及其启动子区DNA甲基化的影响

王霞*(), 尹晓雨, 于晓明, 刘晓丹   

  1. 吉林农业科技学院,吉林吉林 132101
  • 收稿日期:2021-03-24 接受日期:2021-09-09 出版日期:2022-05-12 网络出版日期:2021-10-14
  • 通讯作者: 王霞
  • 基金资助:
    吉林省教育厅“十三五”科学技术项目(JJKH20190972KJ);吉林农业科技学院青年基金项目(吉农院合字[2018]第1001号)资助

Effects of drought hardening on contemporary expression of drought stress memory genes and DNA methylation in promoter of B73 inbred progeny

WANG Xia*(), YIN Xiao-Yu, Yu Xiao-Ming, LIU Xiao-Dan   

  1. College of Jilin Agriculture Science and Technology, Jilin 132101, Jilin, China
  • Received:2021-03-24 Accepted:2021-09-09 Published:2022-05-12 Published online:2021-10-14
  • Contact: WANG Xia
  • Supported by:
    “13th five-year plan” Science and Technology Project of Jilin Province(JJKH20190972KJ);Science Technology Foundation for Young Scientists of College of Jilin Agriculture Science and Technology (Ji Nong Yuan He Zi [2018] 1001).

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摘要：

植物可能会记住已有的逆境刺激,且这种记忆可以跨代遗传。为研究G0代干旱锻炼对玉米B73 G1代当代干旱胁迫记忆基因表达的影响及其启动子区DNA甲基化的影响,实验用20% PEG-6000模拟干旱条件,选取在玉米和拟南芥已知的当代均具有干旱胁迫记忆特性的6个基因为研究对象,利用qRT-PCR技术分析这些基因的表达水平变化,并进一步利用重亚硫酸盐测序PCR技术分析在2个世代中表达差异最显著基因启动子区的DNA甲基化率变化规律。结果表明：干旱胁迫上调了6个当代胁迫记忆基因的表达水平,均呈+/+模式,同当代多次干旱胁迫时表达水平变化趋势一致,且G1代表达水平显著高于G0代;干旱胁迫降低了GRMZM2G088396基因启动子区的甲基化水平,检测区1两个世代DNA甲基化率的降低主要由CHG和CHH甲基化水平降低造成,检测区2 DNA甲基化率的降低主要是由CG和CHH甲基化水平降低造成,G1代2个检测区总胞嘧啶甲基化率均显著低于G0代,说明G0代干旱锻炼使G1代GRMZM2G088396基因启动子区的DNA甲基化修饰产生了可遗传的变异,它可能直接参与GRMZM2G088396基因的表达。

关键词: 干旱锻炼, B73, 干旱胁迫记忆基因, 跨代记忆, qRT-PCR, 重亚硫酸盐测序PCR技术

Abstract:

Plants may remember the existing stresses, and this memory can be passed down across generations. To study the effects of drought hardening in G0 generation on the contemporary drought stress memory genes’ expression and DNA methylation in promoter region in G1 generation, 20% PEG-6000 was used to simulate drought condition, qRT-PCR was used to analyze the expression level changes of six genes with drought-stressed memory function in maize and Arabidopsis. The changes of DNA methylation rates in the promoter regions of the most differentially expressed gene was analyzed by bisulfite sequencing technology. The results showed that the expression of six genes were up-regulated under drought stress in a +/+ mode, and the expression level of G1 generation was significantly higher than that of G0 generation. Drought stress reduced the DNA methylation’ level in the promoter region of GRMZM2G088396 gene. The reduction of methylation in the detection region 1 of two generations was mainly caused by the reduction of CHG and CHH methylation level, the decrease of methylation in the detection region 2 was mainly caused by the decrease of CG and CHH methylation level. The total cytosine methylation rate of G1 generation was significantly lower than that of G0 generation, suggesting that drought exercise in G0 generation resulted in heritable variation of DNA methylation in the promoter region of GRMZM2G088396 gene in G1 generation, which may be directly involved in the expression of GRMZM2G088396 gene.

Key words: drought hardening, B73, drought stress memory gene, transgenerational memory, qRT-PCR, bisulfite sequencing PCR

表1

qRT-PCR及BSP引物"

引物名称
Primer name		 引物序列
Primer sequence (5′-3′)		 引物名称
Primer name		 引物序列
Primer sequence (5′-3′)
GAPDH-F CCCTTCATCACCACGGACTAC GRMZM2G059836-R ACGCTCGCAACCTTCGCAGTC
GAPDH-R TCCCACCACGGTTCTTCCAA GRMZM2G088396-F TGAGAAGGGGCAAGAATACCA
GRMZM5G851862-F TGTTTTCTGCTGGGAGTGACA GRMZM2G088396-R CTCGTCTCCAAGCACTGTCCT
GRMZM5G851862-R GAACCTTCGGGAATCACGTAG GRMZM2G079440-F GCACTTGCGAGTGGCTTTACTTG
GRMZM2G126505-F CCACCGCAACAAGCAGAGT GRMZM2G079440-R ACCGCTGGAGGTAATATCGACAC
GRMZM2G126505-R CGGTTTTTCGCGTTCCTGG GRMZM2G088396-BSP1-F TTTATAGTAGTAAAAGAGGTATTTTTAAT
GRMZM2G028535-F CAAAGGCGAAAAGATCGGTAC GRMZM2G088396-BSP1-R TCCCTATACTAAATTTTAAACTCTAC
GRMZM2G028535-R TTGCGTTCCTCTGATGACAAC GRMZM2G088396-BSP2-F TAGTATAGGGAATTAAATAATTGTTA
GRMZM2G059836-F TTGCGTTCCTCTGATGACAAC GRMZM2G088396-BSP2-R CATCCCTAACACCACTAAAAAATATC

图1

干旱胁迫处理B73 G0和G1代表型"

表2

干旱胁迫记忆基因信息"

基因编号Gene ID 基因描述Gene description 位置Position 染色体Chr.
GRMZM5G851862 cytochrome P450 family 76 subfamily C polypeptide 7 21,883,592-21,886,533 5
GRMZM2G126505 abscisic acid 8’-hydroxylase2 162,654,597-162,657,413 4
GRMZM2G028535 Deltal-pyrroline-5-carboxylate synthetase 173,415,159-173,423,316 8
GRMZM2G059836 Farnesylated protein 2 669,554-670,477 6
GRMZM2G088396 4-hydroxyphenylpyruvate dioxygenase 1 86,084,655-86,086,755 5
GRMZM2G079440 dehydrin DHN1 141,323,718-141,325,549 6

图2

G0、G1代干旱胁迫响应基因表达水平比较 不同字母表示P < 0.05差异显著。"

图3

GRMZM2G088396基因启动子区CG位点分布预测 蓝色区域为CG富集区。"

图4

GRMZM2G088396启动子区BSP扩增电泳图 M：100 bp DNA marker;1~4：G0代胁迫0、1、3、5 h;5~8：G1代胁迫0、1、3、5 h。"

图5

GRMZM2G088396基因启动子区CG、CHG和CHH位点分布图"

图6

干旱锻炼对G1代GRMZM2G088396基因启动子区DNA甲基化率的影响 不同字母表示差异显著,P < 0.05;DNA甲基化率是甲基化胞嘧啶占所有胞嘧啶的比例。"

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