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作物学报 ›› 2024, Vol. 50 ›› Issue (1): 67-75.doi: 10.3724/SP.J.1006.2024.32012

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小肽Ospep5对水稻耐镉性的影响

李明月1,3(), 张文婷1,2, 李阳1,2, 张保龙1,2, 杨立明3, 王金彦1,2,*()   

  1. 1江苏省农业科学院种质资源与生物技术研究所, 江苏南京 210014
    2生物育种钟山实验室, 江苏南京 210014
    3南京林业大学生物与环境学院, 江苏南京 210037
  • 收稿日期:2023-04-01 接受日期:2023-06-29 出版日期:2024-01-12 网络出版日期:2023-07-13
  • 通讯作者: *王金彦, E-mail: wangjy@jaas.ac.cn
  • 作者简介:E-mail: 15236535936@qq.com
  • 基金资助:
    江苏省农业科技自主创新资金项目(CX(22)3124)

Effects of small peptide Ospep5 on cadmium tolerance in rice

LI Ming-Yue1,3(), ZHANG Wen-Ting1,2, LI Yang1,2, ZHANG Bao-Long1,2, YANG Li-Ming3, WANG Jin-Yan1,2,*()   

  1. 1Institute of Germplasm Resources and Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China
    2Zhongshan Biological Breeding Laboratory, Nanjing 210014, Jiangsu, China
    3College of Biology and Environment, Nanjing Forestry University, Nanjing 210037, Jiangsu, China
  • Received:2023-04-01 Accepted:2023-06-29 Published:2024-01-12 Published online:2023-07-13
  • Contact: *E-mail: wangjy@jaas.ac.cn
  • Supported by:
    Jiangsu Agricultural Science and Technology Innovation Fund(CX(22)3124)

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摘要：

镉(Cd)胁迫是植物面临的主要重金属胁迫之一, 对植物的生长和发育产生严重的影响。尽管已有研究表明植物小肽具有缓解胁迫的作用, 但水稻中镉毒害的研究很少。在前期研究中, 通过翻译组、转录组和蛋白质组鉴定了一系列水稻小肽, 其中发现Ospep5可以显著地提高水稻的耐盐性。本研究以日本晴野生型、日本晴过表达Ospep5-OX和日本晴CRISPR/Cas9突变体ospep5-3作为水稻试验材料, 研究Ospep5对镉胁迫下水稻幼苗生长的影响。试验结果表明: 500 μmol L-1 CdCl2处理显著抑制了水稻幼苗的形态生长和叶绿素含量, 同时超氧化物歧化酶(SOD)、脯氨酸(Pro)含量、丙二醛(MDA)含量和镉离子含量显著提高。与单独的镉胁迫相比, 外源施加Ospep5后, 可以有效缓解镉胁迫对水稻幼苗的形态生长的抑制, 显著提高超氧化物歧化酶(SOD)活力, 同时显著降低丙二醛(MDA)含量、脯氨酸(Pro)含量和镉离子含量, 并且能够促进耐镉基因(OsHMA2、OsHMA3、OsCAL1)的表达。总之, Ospep5通过调节水稻幼苗各种生理生化反应以及调控耐镉基因表达的方式, 最终提高水稻幼苗对镉胁迫的耐受性。

关键词: 水稻, 镉胁迫, Ospep5, 幼苗生长

Abstract:

Cadmium (Cd) stress is one of the major heavy metal stresses which causes serious impacts on plant growth and development. Although studies have shown that plant small peptides have a mitigating effect on stress, research on their tolerance to cadmium toxicity in rice is limited. In previous studies, a few peptides were identified through translatome, transcriptome, and proteome analysis, among which Ospep5 was found to significantly improve salt tolerance in rice. In this study, the effects of Ospep5 on the growth of rice seedlings under cadmium stress were investigated using the japonica cultivar Nipponbare, Ospep5 overexpressing line Ospep5-OX, and the CRISPR/Cas9 mutant ospep5-3 as the experimental materials. The results showed that 500 μmol L-1 CdCl2 significantly inhibited the morphological growth and chlorophyll content of rice seedlings, while the activities of superoxide dismutase (SOD), contents of proline (Pro), malondialdehyde (MDA), and cadmium ion content were significantly increased. Compared to cadmium stress alone, exogenous application of synthetic Ospep5 effectively alleviated the inhibition of cadmium stress on the morphological growth of rice seedlings. Furthermore, SOD activity was significantly increased, and MDA content, Pro content, and cadmium ion content were significantly reduced. And the relative expression level of cadmium tolerance genes (OsHMA2, OsHMA3, OsCAL1) were upregulated. In conclusion, Ospep5 improves the tolerance of rice seedlings to cadmium stress by regulating various physiological and biochemical reactions and the expression of cadmium tolerance genes.

Key words: rice, cadmium stress, Ospep5, seedling growth

表1

引物信息"

基因名称
Gene ID		 正向引物
Forward sequence (5'-3')		 反向引物
Reverse sequence (5'-3')
Ospep5-OE gttacttctgcactaggtaccATGCTTGATCCAACGCACAT tcttagaattcccggggatccTCATGATGGTTTCTCGTAGC
Ospep5-Crispr TGTGCATGTTCGTCCAGCCTCACG AAACCGTGAGGCTGGACGAACATG
Ospep5-RT ATGCTTGATCCAACGCACATG TCATGATGGTTTCTCGTAGCTG
OsHMA2 CGCTGAATCAGGCAAGGTTG AATGCTCGAACAGCGACACG
OsHMA3 GTCCTTCTTCGAGTGGCTCT CGATGAGCATGAGGACGTTG
OsCAL1 GCTTCTGCAAGAAGGTCTGC GCCAAGAACAGAAGCTCGTC
Actin TGGTCGTACCACAGGTATTGTGTT AAGGTCGAGACGAAGGATAGCAT

图1

Ospep5的基因编辑突变体和过表达株系分析 A: Ospep5的靶向突变位点和Sanger测序图; B: Ospep5的基因表达量变化; 图中不同小写字母表示处理间在0.05概率水平差异显著(ANOVA分析)。"

图2

Ospep5对镉胁迫下水稻幼苗生长的表型影响 A: 野生型日本晴在不同处理下的表型; B: CRISPR/Cas9基因编辑突变体ospep5-3植株在不同处理下的表型; C: 在500 μmol L-1 CdCl2处理下野生型, 突变体ospep5-3, 过表达Ospep5-OX的表型; 标尺为5 cm。CK: 营养液培养; Cd: 含有500 μmol L-1 CdCl2的营养液培养; Cd+S5: 含有500 μmol L-1 CdCl2的营养液并同时向地上部分喷施300 nmol L-1 Ospep5; R: 日本晴; -OX: 过表达株系Ospep5-OX; -3: 基因编辑突变体ospep5-3。"

表2

Ospep5对镉胁迫下水稻幼苗的鲜重、株高和根长的影响"

材料
Material		 处理
Treatment		 指标Indicator
鲜重 Fresh weight (g) 株高 Plant height (cm) 根长 Root length (cm)
日本晴Nipponbare CK 0.77±0.11 a 36.40±0.60 a 20.18±1.65 a
Cd 0.18±0.04 cd 13.93±1.28 ef 14.08±1.91 c
Cd+S5 0.38±0.04 b 19.90±1.51 d 16.48±0.94 b
Ospep5-3 CK 0.52±0.03 b 29.88±4.05 b 12.71±0.77 cd
Cd 0.16±0.01 d 12.61±0.89 f 9.35±0.64 e
Cd+S5 0.24±0.02 c 15.16±0.62 e 12.01±1.91 d
Ospep5-OX CK 0.50±0.09 b 25.35±0.76 c 16.50±1.00 b
Cd 0.21±0.04 cd 14.35±0.72 ef 14.05±1.28 c

图3

Ospep5对镉胁迫下水稻幼苗生理指标的影响 A: 叶绿素含量; B: 丙二醛(malondialdehyde, MDA)含量; C: 脯氨酸(proline, Pro)含量; D: 超氧化物歧化酶(superoxide dismutase, SOD)活性的影响。横坐标与表2处理相同。不同小写字母表示处理间在0.05概率水平差异显著(ANOVA分析)。"

图4

Ospep5对镉胁迫下水稻幼苗镉含量的影响 A: 地上部分镉离子含量; B: 地下部分镉离子含量。CK、Cd和Cd+S5同表2。不同小写字母表示处理间在0.05概率水平差异显著(ANOVA分析)。"

图5

Ospep5对镉胁迫下水稻幼苗相关基因的qRT-PCR的分析 A: Ospep5的基因表达量变化; B: OsHMA2的基因表达量变化; C: OsHMA3的基因表达量变化; D: OsCAL1的基因表达量变化。CK、Cd和Cd+S5同表2。不同小写字母表示处理间在0.05概率水平差异显著(ANOVA分析)。"

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