水稻黄叶早衰突变体osyes1的生理特性和基因定位

doi:10.16819/j.1001-7216.2020.0301

摘要/Abstract

摘要：

【目的】叶片是水稻进行光合作用最重要的器官。叶色突变体是研究光合作用、叶绿素合成代谢和叶绿体发育的重要材料。【方法】利用⁶⁰Co辐射诱变中籼恢复系自选1号,获得黄叶早衰突变体osyes1。幼苗期对突变体和野生型进行外源H₂O₂处理。抽穗期对突变体和野生型叶片进行超氧化物歧化酶活性、过氧化氢酶活性、活性氧含量、丙二醛含量、可溶性蛋白含量、叶绿素含量、净光合速率测定以及透射电镜观察。成熟期考查突变体和野生型的主要农艺性状。以osyes1/02428的F₂群体中的隐性单株为作图群体,利用图位克隆方法定位OsYES1基因。【结果】osyes1的突变性状始于3~4叶期,水稻抽穗后,所有叶片均表现为黄叶衰老症状,致使突变体株高、穗长、每穗粒数及结实率极显著低于野生型对照。与野生型对照相比,突变体幼苗对外源H₂O₂更敏感。生理分析表明,孕穗期野生型倒3叶的叶绿素含量、过氧化物酶和过氧化氢酶活性极显著低于倒2叶和剑叶,而突变体的含量则极显著低于野生型且依次极显著降低;与野生型相比,突变体剑叶、倒2叶和倒3叶的丙二醛、H₂O₂和O₂^-含量极显著增加,而可溶性蛋白含量则相反。遗传分析表明,osyes1受一对隐性核基因控制,利用图位克隆技术将OsYES1基因定位于第7染色体短臂的RM21353与RM21384之间,物理距离为708 kb。【结论】由于osyes1叶片过早黄化衰老导致与产量相关的重要农艺性状显著下降。OsYES1基因定位在第7染色体两个SSR标记(RM21353和RM21384)之间的708 kb的物理区间内。

关键词: 水稻, osyes1, 黄叶早衰, 生理分析, 基因定位

Abstract:

【Objective】Leaf is a main photosynthetic organ and its color mutants of rice are ideal materials for the study of photosynthesis, chlorophyll biosynthesis and metabolism, and chloroplast development in plants. 【Method】The mutant osyes1 (Oryza sativa yellow-leaf and early senescence 1) was obtained through ⁶⁰Co γ-radiation treatment of indica cultivar Zixuan 1. The seedlings of osyes1 and its wild type were treated with exogenous H₂O₂. The SOD and CAT activities, ROS level, MDA content, soluble protein contents, chlorophyll contents, the net photosynthetic rate and chloroplast ultrastructure were analyzed for osyes1 and its wild type leaves at heading stage. The main agronomic traits of osyes1 and its wild type plants were analyzed under field conditions at maturation stage in Hangzhou. The recessive individuals in F₂ population derived from osyes1/02428 were used to locate the gene by the map cloning method.【Results】The yellow-leaf and early-senescence symptoms started at 3- or 4-leaf stage, and gradually spread to all of the leaves after heading. Due to the early-senescence of the leaves of the mutant osyes1, its major agronomic traits including plant height, panicle length, grain number per panicle and the seed setting rate were markedly reduced. Moreover, the mutant osyes1 exhibited hyper-sensitivity to exogenous H₂O₂. The physiological analysis indicated that the contents of chlorophyll and the activities POD and CAT in the third-top leaf was significantly lower than those in the flag leaf and the second-top leaf in wild-type (WT) plants, but all of them in the mutant plants were significantly lower than those in its WT. Compared with the WT plants, the contents of MDA, H₂O₂ and O₂^- followed a steady increasing trend in the flag, second-top and third-top leaves of the mutant, while their soluble protein levels were progressively dropping. Genetic analysis confirmed that osyes1 was controlled by a single recessive nuclear gene, which was mapped to a region of 708 kb flanked by two SSR markers (RM21353 and RM21384) on the short arm of chromosome 7. 【Conclusion】In this work, the main agricultural traits were significantly reduced in osyes1 for the yellow leaf and early senescence. OsYES1 was located in a 708 kb range between RM21353 and RM21384 by map-based cloning strategy.

Key words: rice, osyes1, yellow and early-senescence, physiological analysis, gene mapping

中图分类号:

Q755
S511.01

黄福灯, 赵超越, 吴鑫, 贺焕焕, 程方民, 李春寿, 潘刚. 水稻黄叶早衰突变体osyes1的生理特性和基因定位[J]. 中国水稻科学, 2020, 34(4): 307-315.

Fudeng HUANG, Chaoyue ZHAO, Xin WU, Huanhuan HE, Fangmin CHENG, Chunshou LI, Gang PAN. Physiological Characters and Gene Mapping of a Yellow Leaf and Early-senescence Mutant osyes1 in Rice[J]. Chinese Journal OF Rice Science, 2020, 34(4): 307-315.

图/表 9

图1 突变体osyes1及其野生型的表型 A-幼苗期; B-开花期; C-开花期叶片; D-成熟期; F, 2, 3, 4分别代表剑叶、倒2叶、倒3叶和倒4叶; 标尺=10 cm。E和F-野生型叶肉细胞;G和H-突变体叶肉细胞。cl-叶绿体;nu-细胞核;sg-淀粉粒。标尺=1 μm。

Fig. 1. Phenotype of osyes1 and its WT plants at different growth stages. A, Seedling stage; B, Heading stage; C, Leaves at heading stage; D, Maturity stage; F, 2, 3 and 4 mean flag leaf, the 2nd, the 3rd and the 4th leaf from top, respectively; Bar=10 cm. Chloroplast ultrastructure of the wild-type (E, F) and osyes1(G, H). cl, Chloroplast; nu, Nucleus; sg, Starch granule; Bar=1 μm.

表1 突变体及其野生型的主要农艺性状

Table 1 Main agronomic traits of osyes1 and its wild type (WT) plants.

性状Trait	2018		2019
性状Trait	野生型WT	突变体osyes1	野生型WT	突变体osyes1
株高 Plant height / cm	104.83±2.04	94.01±4.52^**	108.15±4.35	95.27±4.74^**
穗长 Panicle length / cm	24.04±1.64	19.72±1.28^**	24.78±1.43	19.13±1.76^**
有效穗数 Effective panicle number	8.14±1.14	9.17±2.11	8.53±1.78	9.15±1.87
每穗粒数 Grain number per panicle	174.97±8.88	119.03±10.25^**	175.24±7.99	118.48±8.94^**
结实率 Seed-setting rate / %	85.27±6.68	50.63±3.93^**	85.84±4.98	48.72±4.34^**
千粒重 1000-grain weight / g	22.63±0.48	21.66±2.10	22.87±0.75	21.69±1.71

图2 H2O2胁迫下osyes1突变体及其野生型(WT)的表型 **在0.01水平上差异显著。

Fig. 2. Phenotype of osyes1 mutant and its wild type (WT) plants under H2O2 exposure. ** Significantly different at P<0.01 (t-test).

图3 孕穗期突变体osyes1及其野生型叶片的叶绿素含量及其净光合速率**在0.01水平上差异显著(t-test)。

Fig. 3. Chlorophyll (chl) levels and net photosynthetic rate of leaves in osyes1 and its WT plants at the booting stage. **Significantly different at P<0.01 (t-test).

图4 孕穗期突变体osyes1及其野生型叶片中的H2O2和O2-含量及抗氧化酶活性*在0.05水平上差异显著, **在0.01水平上差异显著(t-test)。

Fig. 4. H2O2 and O2- levels, and the activities of antioxidases in osyes1 and its WT plants at booting stage. *Significantly different at P<0.05, **Significantly different at P<0.01(t-test).

图5 孕穗期突变体osyes1及其野生型叶片的MDA和可溶性蛋白含量**在0.01水平上差异显著。

Fig. 5. MDA and soluble protein contents of leaves in osyes1 and its WT plants at booting stage. **Significantly different at P<0.01 (t-test).

表2 突变体osyes1的遗传分析

Table 2 Genetic analysis of the mutant osyes1.

组合 Cross	野生型表型单株数 No. of WT-type plants	突变表型单株数 No. of mutant-type plants	植株总数 Total number of plants	χ²_3:1	P值 P value
osyes1/自选1号 osyes1/Zixuan 1	596	191	687	0.22	0.64
osyes1/浙恢7954 osyes1/Zhehui 7954	814	279	1093	0.16	0.69

图6 水稻黄叶早衰基因OsYES1在第7染色体上的分子定位

Fig. 6. Molecular mapping of OsYES1 gene on the short arm of chromosome 7.

表3 定位区间内在叶中表达的基因功能注释

Table 3 Functional annotations of genes expressed in leaves within the target interval.

基因登录号	功能注释
GeneBank accession No.	Annotation
LOC_Os07g17130	含蛋白质的FYVE锌指结构域 FYVE zinc finger domain containing protein
LOC_Os07g17184	表达蛋白 Expressed protein
LOC_Os07g17220	抗病蛋白 Disease resistance protein
LOC_Os07g17230	WRKY123
LOC_Os07g17250	抗病RPP13样蛋白1 Disease resistance RPP13-like protein 1
LOC_Os07g17280	Ser/Thr蛋白磷酸酶家族蛋白 Ser/Thr protein phosphatase family protein
LOC_Os07g17330	B12D蛋白 B12D protein
LOC_Os07g17350	转座子蛋白 Transposon protein
LOC_Os07g17390	光系统Ⅱ的外周蛋白 PsbP
LOC_Os07g17400	环状锌指蛋白 RING-type zinc finger protein
LOC_Os07g17560	表达蛋白 Expressed protein
LOC_Os07g17689	表达蛋白 Expressed protein
LOC_Os07g17970	含AMP结合域的蛋白质 AMP-binding domain containing protein
LOC_Os07g18050	RNA结合基序蛋白 RNA-binding motif protein
LOC_Os07g18070	表达蛋白 Expressed protein

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