中国农业科学 ›› 2016, Vol. 49 ›› Issue (4): 609-620.doi: 10.3864/j.issn.0578-1752.2016.04.001

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

水稻OsABC1K3突变体鉴定及其对强光胁迫的响应

高清松1,徐梦彬2,袁彩勇3,纪剑辉1,周 勇2,梁国华2   

  1. 1淮阴师范学院生命科学学院/江苏省区域现代农业与环境保护协同创新中心/江苏省环洪泽湖生态农业生物技术重点实验室,江苏淮安 223300
    2扬州大学农学院/江苏省作物遗传生理重点实验室,江苏扬州 225009
    3江苏徐淮地区淮阴农业科学研究所,江苏淮安 223301
  • 收稿日期:2015-09-14 出版日期:2016-02-16 发布日期:2016-02-16
  • 通讯作者: 周勇,Tel:0514-87937619;E-mail:zhouyong@yzu.edu.cn。梁国华,Tel:0514-87972138;E-mail:ricegb@yzu.edu.cn
  • 作者简介:高清松,E-mail:qsgao@hytc.edu.cn。徐梦彬,E-mail:454007514@qq.com。高清松和徐梦彬为同等贡献作者。
  • 基金资助:
    国家自然科学基金(31471458)、江苏省科技厅重点研发计划(BE2015341)

Rice OsABC1K3 Gene Mutant and Its Response to High Light Stress

GAO Qing-song1, XU Meng-bin2, YUAN Cai-yong3, JI Jian-hui1, ZHOU Yong2, LIANG Guo-hua2   

  1. 1 College of Life Sciences, Huaiyin Normal University/Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection/Jiangsu Key Laboratory for Eco-Agricultural Biotechnology around Hongze Lake, Huai’an 223300, Jiangsu
    2 College of Agriculture, Yangzhou University/Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Yangzhou 225009, Jiangsu
    3 Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu, Huai’an 223301, Jiangsu
  • Received:2015-09-14 Online:2016-02-16 Published:2016-02-16

摘要: 【目的】强光胁迫能够抑制植物光合作用,严重时造成光合器官破坏,影响作物生长和产量。以T-DNA插入突变体为材料,研究水稻ABC1(activity of bc1 complex)激酶基因OsABC1K3响应强光胁迫的生理功能。【方法】根据水稻基因组注释数据库预测的OsABC1K3不同转录本共有序列设计引物,采用3′ RACE对OsABC1K3可变剪接进行验证;从水稻T-DNA插入序列数据库中获得该基因的T-DNA插入突变体osabc1k3,通过加代繁殖和PCR检测取得纯合突变体,调查突变体株高、结实率、种子大小等农艺性状,采用Arnon法测定叶片色素含量,利用LI-6400便携式光合测定系统测定抽穗期旗叶光合速率,采用透射电镜分析叶绿体超微结构;将生长于300 μmol·m-2·s-1光照强度下的野生型和突变体幼苗转移至800 μmol·m-2·s-1光照强度进行强光处理,观察植株表型,分析叶绿体超微结构和叶绿素含量变化;用含20 μmol·L-1甲基紫精(MV)和0.1%吐温20的水溶液喷洒野生型和突变体幼苗叶片表面,以单独用0.1%吐温20喷洒的幼苗作为对照,观察叶片表型,测定处理后超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;采用实时荧光定量PCR分析突变体淀粉合成、叶绿体异戊二烯基脂合成及叶绿体ABC1激酶基因的表达。【结果】OsABC1K3仅检测到一个转录本LOC_Os05g25840.3osabc1k3突变体株高和结实率下降,种子变小,其中,粒宽下降了13.4%,粒厚下降了6.8%,千粒重下降了18.2%,而粒长与野生型无显著差异。突变体叶片叶绿素含量下降,而叶绿素a/b和类胡萝卜素含量高于对照。突变体叶绿体形态正常,但缺乏淀粉粒。光合活性分析表明,突变体叶片光合速率与野生型无显著差异。强光处理7 d后,突变体叶片发生黄化;9 d后,部分植株枯死。强光处理后突变体叶绿体类囊体结构紊乱,出现大量的空泡。进一步对叶绿素含量进行测定表明,突变体叶片叶绿素衰减程度显著高于对照。然而,MV处理后突变体叶片坏死程度、SOD活性及MDA含量与野生型无显著差异。此外,OsABC1K3突变影响了叶片淀粉合成、叶绿体异戊二烯基脂合成及叶绿体ABC1激酶基因的表达。【结论】OsABC1K3可能不参与水稻氧化胁迫的防御,而通过遗传控制的信号途径调控强光胁迫响应。

关键词: 水稻, ABC1, T-DNA插入突变体, 强光胁迫, 淀粉合成

Abstract: 【Objective】 High light (HL) stress inhibits plant photosynthesis. Severe HL stress will destroy photosynthetic potential, affecting crop growth and production. The objective of this study was to investigate the physiological function of rice ABC1 (activity of bc1 complex) kinase gene OsABC1K3 in HL stress response pathway. 【Method】 To verify the alternative splicing forms of OsABC1K3, 3′ RACE was performed using primers designed from common sequences of different OsABC1K3 transcripts predicted in the Rice Genome Annotation Project Database. Subsequently, a T-DNA insertion mutant of the gene, osabc1k3, was obtained from the Rice T-DNA Insertion Sequence Database. The homozygous mutants were identified by adding-generation propagation and PCR. Then, the mutant agronomic traits such as plant height, seed setting rate and seed size were examined. The leaf pigment contents were determined according to the method of Arnon. The leaf photosynthetic rate and chloroplast ultrastructure were analyzed using a LI-6400 portable photosynthesis measurement system and transmission electron microscopy, respectively. To perform HL treatment, wild type and mutant seedlings grown at 300 μmol·m-2·s-1 light intensity were transferred into 800 μmol·m-2·s-1. The plant phenotype was photographed, and the variation of chloroplast ultrastructure and chlorophyll content after treatment was analyzed. To perform methyl viologen (MV) treatment, wild type and mutant seedlings were sprayed with 20 μmol·L-1 MV in 0.1% Tween 20 (V/V). Control plants were sprayed with water and 0.1% Tween 20. The leaf phenotype after treatment was photographed, and the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured. Finally, the expression of starch biosynthesis, chloroplast prenyllipid biosynthesis and chloroplast ABC1 kinase genes in mutants was analyzed using quantitative real-time PCR. 【Result】 Only one transcript, LOC_Os05g25840.3, was detected for the OsABC1K3 gene. The plant height, seed setting rate and seed size of osabc1k3 mutants were reduced compared with wild type. The grain width, grain thickness and 1 000-grain weight were reduced by 13.4%, 6.8% and 18.2%, respectively. However, the grain length was not significantly different from that of wild type. The chlorophyll content of mutant leaves was lower than wild type, while the chlorophyll a/b ratio and carotenoid content were higher. In addition, the mutant chloroplasts were morphologically normal, but lacking starch granules. Photosynthetic activity analysis revealed that the photosynthetic rate of mutants did not differ significantly from that of wild type. After 7 days of HL stress, the mutant leaves etiolated. After 9 days, some of the plants died. The chloroplast thylakoid structure of mutants was disrupted after treatment, presenting extensive vacuolation. Further determination of chlorophyll content revealed that the chlorophyll attenuation of mutants was more severe than wild type. However, the necrosis degree, SOD activity and MDA content of mutant leaves were not significantly different from wild type after MV treatment. Furthermore, OsABC1K3 mutation affected the expression of leaf starch biosynthesis, chloroplast prenyllipid biosynthesis and chloroplast ABC1 kinase genes. 【Conclusion】 OsABC1K3 might participate in HL stress response through genetically controlled signaling pathways, rather than direct oxidative stress defense.

Key words: rice, ABC1, T-DNA insertion mutant, high light stress, starch synthesis