水稻OsWAK16通过调节抗氧化酶活性调控种子抗老化能力(长英文摘要）

doi:10.11983/CBB24038

植物学报 ›› 2025, Vol. 60 ›› Issue (1): 17-32.DOI: 10.11983/CBB24038 cstr: 32102.14.CBB24038

水稻OsWAK16通过调节抗氧化酶活性调控种子抗老化能力(长英文摘要）

田建红¹^,^†, 刘燕¹^,^†, 尹梦琪¹, 王静¹, 陈婷¹, 汪燕¹, 姜孝成¹^,²^,^*()

¹湖南师范大学生命科学学院, 长沙 410081
²作物不育资源创新与利用湖南省重点实验室, 长沙 410081

收稿日期:2024-03-09 接受日期:2024-07-14 出版日期:2025-01-10 发布日期:2024-07-23
通讯作者: * 姜孝成, 博士, 湖南师范大学二级教授, 博士生导师。主要从事水稻种子活力调控及其分子机制研究, 主持和承担国家自然科学基金、国家科技部基础条件平台建设、湖南省自然科学基金等科研项目。以第一作者或通讯作者身份在Journal of Integrative Plant Biology、BMC Plant Biology等权威期刊上发表论文60余篇。兼任中国植物学会种子科学与技术专业委员会委员, 湖南省植物学会常务理事, 湖南省植物生理与分子生物学学会常务理事, 《生命科学研究》编辑委员会委员。担任Plant Biotechnology Journal、BMC Plant Biology、Molecular Breeding等国际期刊审稿人。E-mail: jxclc@hunnu.edu.cn
作者简介:^†共同第一作者
基金资助:
国家自然科学基金(32072125);湖南省发育生物学工程与新产品协同创新中心(20134486)

OsWAK16 Regulates Seed Anti-aging Ability by Modulating Antioxidant Enzyme Activity in Rice

Jianhong Tian¹^,^†, Yan Liu¹^,^†, Mengqi Yin¹, Jing Wang¹, Ting Chen¹, Yan Wang¹, Xiaocheng Jiang¹^,²^,^*()

¹School of Life Sciences, Hunan Normal University, Changsha 410081, China
²Hunan Province Key Laboratory of Crop Sterile Germplasm Resource Innovation and Application, Changsha 410081, China

Received:2024-03-09 Accepted:2024-07-14 Online:2025-01-10 Published:2024-07-23
Contact: * E-mail: jxclc@hunnu.edu.cn
About author:^†These authors contributed equally to this paper

摘要/Abstract

摘要： 细胞壁相关蛋白激酶(WAK)家族成员在水稻(Oryza sativa)基因组中已注释了大约130个WAK基因, 它们在水稻生长发育和应激响应中发挥重要作用。该文探讨了水稻WAK16-RLK的编码基因OsWAK16对水稻种子活力和抗老化能力的调控作用及其生理机制。结果表明, OsWAK16敲除突变体的种子活力在种子未老化和人工老化12天时均显著低于野生型种子, 过表达种子的活力则显著高于野生型种子, 说明OsWAK16正调控种子活力和抗老化能力。同时, 相比野生型种子, 未老化和人工老化12天的OsWAK16敲除突变体种子中丙二醛含量以及种子浸泡液电导率显著增加, 抗氧化酶活性则显著下降; 过表达种子中的变化则相反。此外, 野生型、OsWAK16突变和过表达种子在人工老化处理后, OsWAK16的差异表达也引起OsPER1A、OsbZIP23、OsPIMT1、OsSdr4、OsMSRB5和OsHSP18.2等种子活力相关基因表达的协同变化。因此, 推测OsWAK16可能与其它种子活力相关基因协同作用, 行使清除细胞中活性氧的功能, 从而调控种子活力和抗老化能力。

关键词: OsWAK16, 种子活力, 人工老化, 丙二醛, 抗氧化酶

Abstract: INTRODUCTION: The cell wall-associated kinase (WAK) family has annotated approximately 130 WAK genes in the genome of rice (Oryza sativa), which play an important role in rice growth and development and stress responses.

RATIONALE: Here, we investigated the regulation and physiological mechanism of OsWAK16, an encoding gene of the cell wall-associated kinase WAK16-RLK, on rice seed vigor and anti-aging ability.

RESULTS: The results showed that before and after artificial aging, the seed vigor of OsWAK16 knock out mutants and overexpression lines was significantly lower and higher than that of wild-type seeds, respectively, indicating that OsWAK16 positively regulates the anti-aging ability of seeds. Physiological and biochemical analyses indicated that compared with wild-type seeds before and after artificial aging treatment, malondialdehyde (MDA) content and electrical conductivity (EC) of seed soaking solution of OsWAK16 knock out mutant seeds were significantly increased, while antioxidant enzyme activity was significantly decreased. The reverse was true in overexpression seeds. In addition, the differential expression of OsWAK16 in three types of seeds, whether artificially aged or not, also caused synergistic changes in the expression of other seed vigor-related genes OsPER1A, OsbZIP23, OsPIMT1, OsSdr4, OsMSRB5 and OsHSP18.2.

CONCLUSION: Therefore, it is speculated that OsWAK16 may work synergistically with other seed vigor-related genes to clear reactive oxygen species in cells, thereby regulating seed vigor and anti-aging capacity.

Key words: OsWAK16, seed vigor, artificial aging, malondialdehyde, antioxidant enzyme

田建红, 刘燕, 尹梦琪, 王静, 陈婷, 汪燕, 姜孝成. 水稻OsWAK16通过调节抗氧化酶活性调控种子抗老化能力(长英文摘要）. 植物学报, 2025, 60(1): 17-32.

Jianhong Tian, Yan Liu, Mengqi Yin, Jing Wang, Ting Chen, Yan Wang, Xiaocheng Jiang. OsWAK16 Regulates Seed Anti-aging Ability by Modulating Antioxidant Enzyme Activity in Rice. Chinese Bulletin of Botany, 2025, 60(1): 17-32.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB24038

https://www.chinbullbotany.com/CN/Y2025/V60/I1/17

图/表 8

表1 实验所用引物

Table 1 The primers used in the experiments

Primer name	Sequence (5′-3′)	Purpose
gRT1	CCGTTACAAGGCCCTTCTGT	CRISPR/Cas9 vector construction
OsU6aT1	ACAGAAGGGCCTTGTAACGGC
gRT2	ATTACAACTGGTATTAGTTG
OsU6bT2	CAACTAATACCAGTTGTAATC
PB-L	GCGCGCGGTCTCGCTCGACTAGTATGG
PB-R	GCGCGCGGTCTCTACCGACGCGTATCC
OsWAK16-cas9-F1	GCCAGGTACTGATACCTAC
OsWAK16-cas9-R1	CTCAAGAAAAGATCAGTCGC
OsWAK16-cas9-F2	TTTACATGCACTAGTTGCCC
OsWAK16-cas9-R2	GCGCTTTCATCTGAGATTAG
OsWAK16-pHB-F	ATGAGGTCGAGCTTTGTGGC	Overexpression vector construction
OsWAK16-pHB-R	CTAGCGTGGCAAACTGACTGAG	Overexpression vector construction
OsWAK16-1301-F	CCAATGAGTACATTTCTGCTATAGACAG	GUS vector construction
OsWAK16-1301-R	CTTTGCGCCAGCCGAGAC	GUS vector construction
OsActin1-F	CAATGTGCCAGCTATGTATGTCGCC	Quantitative internal control
OsActin1-R	TTCCCGTTCAGCAGTGGTAGTGAAG	Quantitative internal control
qRT-OsWAK16-F	GATGGCAACTTTACTACAA	OsWAK16 expression analysis
qRT-OsWAK16-R	TGTGATAATACTCTGGGTC	OsWAK16 expression analysis
OsPER1A-F	GACCCGGACGAGAAGGATTC	Gene expression analysis
OsPER1A-R	ACCACCTCATCCATGTTCCG
OsbZIP23-F	CTGGGAAATGGGCTGGTCT
OsbZIP23-R	CCATCTTGCCGAAGCCATT
OsPIMT1-F	CACCGACTGTGGTCAAGC
OsPIMT1-R	AGCACCAGGAGGCACAAA
OsSdr4-F	AAGACGGCGGAGGAGGTGGA
OsSdr4-R	CATGGACGGATGACCACTTGC
OsMSRB5-F	GCCATAAACCGAACACCG
OsMSRB5-R	GCTCATCAGTAGGCGTCTTG
OsHSP18.2-F	GCTCAAGTCCTCCGACATCAAG
OsHSP18.2-R	TCCGCAGGTACTTGCACGAC

图1 OsWAK16的生物信息学分析 (A) OsWAK16基因结构; (B) OsWAK16蛋白结构; (C) OsWAK16与水稻其它WAK同源基因序列构建的系统进化树(蓝色字体为OsWAK16)。

Figure 1 Bioinformatics analysis of OsWAK16 (A) OsWAK16 gene structure; (B) OsWAK16 protein structure; (C) Phylogenetic tree of OsWAK16 and its homologous genes from Oryza sativa (OsWAK16 is highlighted in blue).

图2 OsWAK16在水稻不同组织及不同活力种子中的表达模式 (A) OsWAK16在Kasalath (WT)不同组织中的表达水平; (B) OsWAK16启动子驱动的GUS表达分析(a: 幼苗; b: 根; c: 叶; d: 穗; e: 种子); (C) 老化处理不同时间WT种子的萌发率; (D) 老化处理不同时间WT种子中OsWAK16的表达水平。(B) Bars in a-d=1 cm; bar in e=0.1 cm。数值为平均值±标准差(n=3)。根与其它组织之间的差异显著性(A)以及未老化种子与老化不同天数种子之间的差异显著性(C, D)用Student’s t-test计算(* P<0.05, ** P<0.01)。

Figure 2 Expression patterns of OsWAK16 in different tissues and seeds of rice with different vigor (A) Expression level of OsWAK16 in different tissues of Kasalath (WT); (B) Analysis of OsWAK16 promoter-driven GUS expression (a: Seedling; b: Root; c: Leaf; d: Spikelet; e: Seed); (C) Germination rate of WT seeds at different time of artificial aging; (D) Expression of OsWAK16 in WT seeds at different time of artificial aging. (B) Bars in a-d=1 cm; bar in e=0.1 cm. Data represent means±SD (n=3). Significant differences between root and other tissues (A), and significant differences between unaged seeds and seeds artificially aged for different days (C, D) were determined using Student’s t-test (* P<0.05, ** P<0.01).

图3 OsWAK16转基因水稻株系的构建 (A) CRISPR/Cas9的OsWAK16靶位点(靶位点1和靶位点2)及突变体oswak16-1和oswak16-2的碱基序列和对应的氨基酸序列变化; (B) 野生型(WT)和OsWAK16过表达株系OE-1、OE-2和OE-3叶片中OsWAK16的相对表达量, 数值为平均值±标准差(n=3), WT与过表达株系间的差异显著性用Student’s t-test计算(** P<0.01)。

Figure 3 Construction of transgenic rice lines of OsWAK16 (A) Target sites (target site 1 and target site 2) selected by CRISPR/Cas9 and changes of the nucleotide sequences as well as the corresponding amino acid sequences of mutants oswak16-1 and oswak16-2; (B) Relative expression levels of OsWAK16 in leaves of wild type (WT) and overexpression lines OE-1, OE-2, and OE-3, data represent means±SD (n=3), significant differences between WT and overexpression lines were determined using Student’s t-test (** P<0.01).

图4 Kasalath (WT)、oswak16突变体和OsWAK16过表达株系种子的萌发表型及其相关活力指标 (A) 未老化和人工老化12天种子的萌发表型(bar=1 cm); (B) 幼苗芽长; (C) 幼苗根长; (D) 萌发率; (E) 萌发势; (F) 萌发指数; (G) OsWAK16相对表达量。数值为平均值±标准差(n=3), WT与其它基因型之间的差异显著性用Student’s t-test计算(* P<0.05, ** P<0.01)。

Figure 4 Seed germination phenotypes and the related indexes of rice Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines (A) Germination phenotypes of unaged seeds and seeds with 12 days of artificial aging (bar=1 cm); (B) Seedling shoot length; (C) Seedling root length; (D) Germination rate; (E) Germination potential; (F) Germination index; (G) The relative expression level of OsWAK16. Data represent means±SD (n=3), significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

图5 Kasalath (WT)、oswak16突变体和OsWAK16过表达株系种子老化处理前后丙二醛(MDA)含量(A)和种子浸泡液的相对电导率(EC) (B)比较数值为平均值±标准差(n=3), WT与其它基因型之间的差异显著性用Student’s t-test计算(* P<0.05, ** P<0.01)。

Figure 5 Comparison of malondialdehyde (MDA) content (A) and relative conductivity (EC) (B) of seeds of Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines before and after artificial aging Data represent means±SD (n=3); significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

图6 Kasalath (WT)、oswak16突变体和OsWAK16过表达株系种子人工老化前后抗氧化酶活性以及H2O2和O2-.含量比较 (A) 超氧化物歧化酶(SOD); (B) 过氧化物酶(POD); (C) 过氧化氢酶(CAT); (D) O2-. ; (E) H2O2。数值为平均值±标准差(n=3), WT与其它基因型之间的差异显著性用Student’s t-test计算(* P<0.05, ** P<0.01)。

Figure 6 Comparison of antioxidase activities and H2O2 and O2-. contents in seeds before and after artificial aging of Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines (A) Superoxide dismutase (SOD); (B) Peroxidase (POD); (C) Catalase (CAT); (D) O2-. ; (E) H2O2. Data represent means±SD (n=3); significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

图7 Kasalath (WT)、oswak16突变体和OsWAK16过表达株系种子中种子活力相关基因表达量分析 (A) OsPER1A; (B) OsbZIP23; (C) OsPIMT1; (D) OsSdr4; (E) OsMSRB5; (F) OsHSP18.2。数值为平均值±标准差(n=3), WT与其它基因型之间的差异显著性用Student’s t-test计算(* P<0.05, ** P<0.01)。

Figure 7 Analysis of seed vigor-related genes expression in seeds before and after artificial aging of Kasalath (WT), oswak16 mutants and OsWAK16 overexpression lines (A) OsPER1A; (B) OsbZIP23; (C) OsPIMT1; (D) OsSdr4; (E) OsMSRB5; (F) OsHSP18.2. Data represent means±SD (n=3), significant differences between WT and other genotypes were determined using Student’s t-test (* P<0.05, ** P<0.01).

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水稻OsWAK16通过调节抗氧化酶活性调控种子抗老化能力(长英文摘要）

OsWAK16 Regulates Seed Anti-aging Ability by Modulating Antioxidant Enzyme Activity in Rice

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