水稻叶片衰老基因LPS1的克隆与功能研究

doi:10.16819/j.1001-7216.2021.210303

摘要/Abstract

摘要：

【目的】早衰突变体是研究早衰机制的良好载体,对于探究早衰的遗传机理与作用机制及提高水稻的产量和品质具有重要作用。【方法】本研究利用EMS诱变获得了一个早衰突变体lps1,并对该突变体及其野生型进行表型观察、细胞学及组织化学分析、生理生化分析、遗传分析、基因定位和激素处理。【结果】lps1的叶片从3叶期开始发黄,成熟期株高、有效分蘖数、结实率、千粒重等极显著降低。电镜观察发现lps1叶表面光滑,硅质化突起和叶绿体数目减少、片层结构紊乱。生理生化分析表明lps1中有大量的活性氧积累,同时伴有蛋白质的降解、细胞膜的损伤以及大规模的细胞死亡。遗传分析表明该早衰表型受单隐性核基因控制,并且其在第5染色体上编码了一个泛素结合酶。亚细胞定位结果证明LPS1蛋白在细胞质与核中均有表达。外源激素处理发现,lps1对外源激素的处理更为敏感,且LPS1突变促进了ABA合成相关基因的表达。【结论】LPS1 突变使水稻ABA合成信号途径异常,进而引发H₂O₂等一系列与衰老相关生理指标的异常变动,导致lps1过早衰老,最终造成水稻产量严重降低。

关键词: 水稻, 早衰, 遗传分析, 亚细胞定位, 激素

Abstract:

【Objective】 As a good carrier, the premature aging mutant plays an important role in exploring the genetic mechanism and mechanism of action on premature aging, as well as improving grain yield and quality of rice.【Method】 In this study, a premature senescence mutant lps1 was obtained by EMS mutagenesis, and phenotypic observation, cytological and histochemical analysis, physiological and biochemical analysis, genetic analysis, gene localization and hormone treatment were performed on wild-type and mutants.【Result】 The leaves of lps1 began to turn yellow during the three-leaf stage, and the plant height, effective tiller number, seed-setting rate, and thousand-grain weight were extremely significantly reduced in the maturing stage. Electron microscopy observation found that the surface of lps1 leaves was smooth, the numbers of silicified protrusions and chloroplasts decreased, and the lamella structure was disordered. Physiological and biochemical analysis showed that a large amount of reactive oxygen species accumulated in mutant lps1, accompanied by protein degradation, cell membrane damage and large-scale cell death. Genetic analysis showed that the progeria phenotype was controlled by a single recessive nuclear gene, which encodes an ubiquitin conjugating enzyme on chromosome 5. The subcellular localization results prove that LPS1 protein is expressed in both the cytoplasm and the nucleus. Exogenous hormone treatment demonstrated that the lps1 was more sensitive to exogenous hormone treatment, and the LPS1 mutation promoted the expression of genes related to ABA synthesis.【Conclusion】 LPS1 mutation makes the rice ABA synthesis signal pathway abnormal, which in turn triggers abnormal changes in a series of aging-related physiological indicators such as H₂O₂, leading to premature senescence of lps1, and ultimately resulting in a serious decrease in rice yield.

Key words: rice, premature senescence, genetic analysis, subcellular localization, hormone

褚晓洁, 芦涛, 叶涵斐, 王盛, 林晗, 吴先美, 何瑞, 严钢, 王跃星, 李三峰, 路梅, 胡海涛, 杨窑龙, 饶玉春. 水稻叶片衰老基因LPS1的克隆与功能研究[J]. 中国水稻科学, 2021, 35(5): 427-438.

Xiaojie CHU, Tao LU, Hanfei YE, Sheng WANG, Han LIN, Xianmei WU, Rui HE, Gang YAN, Yuexing WANG, Sanfeng LI, Mei LU, Haitao HU, Yaolong YANG, Yuchun RAO. Cloning and Functional Analysis of Leaf Senescence Gene LPS1 in Oryza sativa[J]. Chinese Journal OF Rice Science, 2021, 35(5): 427-438.

图/表 12

表1 本研究所用引物及序列

Table 1 Primers and sequences used in this study.

引物名称 Primer name	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）	用途 Use
M1	CACACATTCTAAATTTGGAAAAAGG	TACGTGGCTACTTGGCGTTC	精细定位Fine mapping
M2	GCTGAAGAGCCTCCTCGAA	GGTATCATGAGAGCGAGTCTGA	精细定位Fine mapping
M3	ACCCATGACCATGAGACGAT	GGGTACTAGCCGTGCTTATCC	精细定位Fine mapping
M4	AGCCATTAGGGGCTTAGGAA	CCCCTGAGTGATATGCTTGG	精细定位Fine mapping
M5	TAAATTACATCGGCCGGAGA	CCCACCAAAGAAATCTCCAA	精细定位Fine mapping
M6	ATTTGGGGGAAAGTTTGCTT	AATAGTATGCGTGCGCTGTG	精细定位Fine mapping
LPS1	TCCTAAGAAGGGCTCGGAAA	CTGCCTCAACCACACTTACA	载体构建Vector construction
LPS1-GFP	GCCCAGATCAACTAGTATGGAT CTATATGCAATTGAC	TCGAGACGTCTCTAGACGGGCTGC AGGGGATGCCGG	载体构建Vector construction
Q-YGL1	AACCTTACCGTCCTATTCCTT	CCATACATCTAACAGAGCACC	qRT-PCR
Q-CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC	qRT-PCR
Q-NYC3	TCTATCTAGGTGCCAAAGGC	ATTCTGGCACCTGCTGTTTC	qRT-PCR
Q-DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTC	qRT-PCR
Q-RLS1	CTTGGGCTGTTGATGCAGC	CTTCAACACCCGCCTCGC	qRT-PCR
Q-OsABA1	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsABA2	AGCAAACCTGAAAGGTGTGGA	AAAGCCACCATCCACCATGA	qRT-PCR
Q-OsABA3	GGGCAAGATTTTGTTCGGCA	AAGGGTACACTTGTTGCCCC	qRT-PCR
Q-OsNCED1	ACCATGAAGTCCATGAGGCT	TCTCGTAGTCTTGGTCTTGG	qRT-PCR
Q-OsNCED2	ATGGAAACGAGGATAGTGGT	CTTATTGTTGTGCGAGAAGT	qRT-PCR
Q-OsNCED3	CTCCCAAACCATCCAAACCG	TGAGCATATCCTGGCGTCGT	qRT-PCR
Q-OsNCED5	TCCGAGCTCCTCGTCGTGAA	AGGTGTTTTGGAATGAACCA	qRT-PCR
Q-OsZEP	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsZDS	CACGTGTTCTTCGGGTGTTA	ATGTAACGGAGCTCCCACAG	qRT-PCR
Q-OsABA80x1	AAGCTGGCAAAACCAACATC	CCGTGCTAATACGGAATCCA	qRT-PCR
Q-OsABA80x2	CTACTGCTGATGGTGGCTGA	CCCATGGCCTTTGCTTTAT	qRT-PCR
Q-OsABA80x3	AGTACAGCCCATTCCCTGTG	ACGCCTAATCAAACCATTGC	qRT-PCR
Actin	CAGGCCGTCCTCTCTCTGTA	AAGGATAGCATGGGGGAGAG	qRT-PCR

表1 本研究所用引物及序列

Table 1 Primers and sequences used in this study.

引物名称 Primer name	正向引物 Forward primer（5′-3′）	反向引物 Reverse primer（5′-3′）	用途 Use
M1	CACACATTCTAAATTTGGAAAAAGG	TACGTGGCTACTTGGCGTTC	精细定位Fine mapping
M2	GCTGAAGAGCCTCCTCGAA	GGTATCATGAGAGCGAGTCTGA	精细定位Fine mapping
M3	ACCCATGACCATGAGACGAT	GGGTACTAGCCGTGCTTATCC	精细定位Fine mapping
M4	AGCCATTAGGGGCTTAGGAA	CCCCTGAGTGATATGCTTGG	精细定位Fine mapping
M5	TAAATTACATCGGCCGGAGA	CCCACCAAAGAAATCTCCAA	精细定位Fine mapping
M6	ATTTGGGGGAAAGTTTGCTT	AATAGTATGCGTGCGCTGTG	精细定位Fine mapping
LPS1	TCCTAAGAAGGGCTCGGAAA	CTGCCTCAACCACACTTACA	载体构建Vector construction
LPS1-GFP	GCCCAGATCAACTAGTATGGAT CTATATGCAATTGAC	TCGAGACGTCTCTAGACGGGCTGC AGGGGATGCCGG	载体构建Vector construction
Q-YGL1	AACCTTACCGTCCTATTCCTT	CCATACATCTAACAGAGCACC	qRT-PCR
Q-CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC	qRT-PCR
Q-NYC3	TCTATCTAGGTGCCAAAGGC	ATTCTGGCACCTGCTGTTTC	qRT-PCR
Q-DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTC	qRT-PCR
Q-RLS1	CTTGGGCTGTTGATGCAGC	CTTCAACACCCGCCTCGC	qRT-PCR
Q-OsABA1	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsABA2	AGCAAACCTGAAAGGTGTGGA	AAAGCCACCATCCACCATGA	qRT-PCR
Q-OsABA3	GGGCAAGATTTTGTTCGGCA	AAGGGTACACTTGTTGCCCC	qRT-PCR
Q-OsNCED1	ACCATGAAGTCCATGAGGCT	TCTCGTAGTCTTGGTCTTGG	qRT-PCR
Q-OsNCED2	ATGGAAACGAGGATAGTGGT	CTTATTGTTGTGCGAGAAGT	qRT-PCR
Q-OsNCED3	CTCCCAAACCATCCAAACCG	TGAGCATATCCTGGCGTCGT	qRT-PCR
Q-OsNCED5	TCCGAGCTCCTCGTCGTGAA	AGGTGTTTTGGAATGAACCA	qRT-PCR
Q-OsZEP	GGATGCCATTGAGTTTGGTT	TGGCTGACTGAAGTCTCTCG	qRT-PCR
Q-OsZDS	CACGTGTTCTTCGGGTGTTA	ATGTAACGGAGCTCCCACAG	qRT-PCR
Q-OsABA80x1	AAGCTGGCAAAACCAACATC	CCGTGCTAATACGGAATCCA	qRT-PCR
Q-OsABA80x2	CTACTGCTGATGGTGGCTGA	CCCATGGCCTTTGCTTTAT	qRT-PCR
Q-OsABA80x3	AGTACAGCCCATTCCCTGTG	ACGCCTAATCAAACCATTGC	qRT-PCR
Actin	CAGGCCGTCCTCTCTCTGTA	AAGGATAGCATGGGGGAGAG	qRT-PCR

图1 水稻早衰突变体lps1及其野生型的表型及农艺性状A-3叶期植株表型(标尺=3 cm);B-3叶期叶片表型(标尺=1 cm);C-分蘖期表型(标尺=6 cm);D-成熟期表型(标尺=8 cm);E-籽粒表型(标尺=10 cm); F~I-野生型与lps1的农艺性状(株高、分蘖、结实率、千粒重);J~L, 粒厚、粒长和粒宽。*和**分别表示野生型与lps1在0.05和0.01水平上差异显著。

Fig. 1. Phenotype and agronomic traits of the wild type and lps1. A, Plant phenotype(Bar=3 cm); B, Leaf phenotype at the three-leaf stage(Bar=1 cm); C, Tillering stage(Bar=6 cm); D, Maturity stage(Bar=8 cm); E, Seed phenotype of wild type and lps1(Bar=10 cm); F-I, Agronomic traits of wild type and lps1; J-L, Statistics of seed thickness, length and width of the wild type and lps1. * and ** indicate that the wild-type and mutant lps1 are significantly different at the levels of 0.05 and 0.01, respectively.

图2 水稻早衰突变体lps1及其野生型分蘖期叶绿素含量及相关基因表达分析A-分蘖期野生型与lps1不同部位叶表型,标尺为2 cm;B~D-野生型与lps1叶绿素a、叶绿素b和类胡萝卜素含量测定;E-叶绿素合成与代谢相关基因表达水平。L1―倒1叶;L2―倒2叶;L3―倒3叶。*和**分别表示野生型与lps1在0.05和0.01水平上差异显著

Fig. 2. Chlorophyll contents and related gene expression analysis at the tillering stage.A, Leaf phenotype of the wild type and lps1 in the tillering stage, bar=2 cm; B-D, Chlorophyll a, chlorophyll b and carotenoid contents of the wild type and lps1; E, Chlorophyll synthesis and metabolism-related gene expression level. L1, First leaf from the top; L2, Second leaf from the top; L3, Third leaf from the top. * and ** indicate that the wild type and lps1 are significantly different at the levels of 0.05 and 0.01, respectively.

图3 水稻野生型（A~C）和早衰突变体lps1（D~F）叶片下表皮的扫描电镜（SEM）观察

Fig. 3. Scanning electron microscope (SEM) observations of the epidermis of the wild type (A-C) and lps1(D-F).

图4 水稻野生型（A~C）和早衰突变体lps1（D~F）叶片的透射电镜（TEM）观察N-细胞核; C-叶绿体; Thy-类囊体; S-淀粉颗粒; Og-嗜锇颗粒。

Fig. 4. Transmission electron microscopy (TEM) observation of the wild type (A-C) and lps1(D-F) leaves. N, Nucleus; C, Chloroplast; Thy, Thylakoid; S, Starch granules; Og, Osmophilic granules.

图5 水稻早衰突变体lps1及其野生型生理生化检测A-DAB染色; 标尺为2 cm;B-NBT染色, 标尺为2 cm;C-衰老相关生理指标（过氧化氢、丙二醛、可溶性蛋白含量、过氧化氢酶、过氧化物酶、超氧化物歧化酶活性）的测定。*和**分别表示野生型与lps1在0.05和0.01水平上差异显著。

Fig. 5. Physiological and biochemical detection of the wild type and lps1.A, DAB staining; bar=2 cm; B, NBT staining, bar=2 cm; C, Determination of aging-related physiological indicators (H2O2, MDA, SP, CAT, POD, SOD). * and ** indicate that the wild-type and lps1 are significantly different at the levels of 0.05 and 0.01, respectively.

图6 野生型(A、B)和lps1(C、D)叶片的TUNEL处理 A-D,标尺为100 μm。蓝色荧光代表正常细胞,绿色荧光代表凋亡细胞。

Fig. 6. TUNEL treatment results of the wild type (A and B) and lps1 (C and D) leaves. A-D, bar=100 μm. Blue fluorescence represents normal cells, green fluorescence represents apoptotic cells.

表2 F2代分离群体统计结果

Table 2 Statistical results of F2 segregating population.

杂交组合 Combination	F₁表型 Phenotype of F₁	F₂表型 Phenotype of F₂			χ²_(3:1)
杂交组合 Combination	F₁表型 Phenotype of F₁	正常表型 Normal	突变表型 Mutant	总株数 Total	χ²_(3:1)
lps1/浙辐802 lps1/Zhefu 802	正常表型 Normal	253	82	335	0.049
浙辐802/lps1 Zhefu 802/lps1	正常表型 Normal	233	76	309	0.027

图7 LPS1基因的图位克隆

Fig. 7. Map-based cloning of LPS1 gene.

图8 LPS1蛋白的亚细胞定位

Fig. 8. Subcellular localization of LPS1 protein.

图9 外源激素处理对野生型和lps1幼苗的影响 A-外源激素处理对野生型WT(左)和lps1(右)表型的影响,标尺为3 cm;B-激素处理后地上部分长度;C-激素处理后地下部分长度。*和**分别表示野生型与lps1在0.05和0.01水平上差异显著。

Fig. 9. Effect of exogenous hormone treatment on the wild type and lps1 seedlings. A, Effect of exogenous hormone treatment on the phenotype of the wild type(left) and lps1(right), bar=3 cm; B, Length of the aerial part after hormone treatment; C, Length of the underground part after hormone treatment. * and ** indicate significant difference between the wild type and lps1 at 0.05 and 0.01 level, respectively.

图10 ABA合成与降解相关基因表达水平 A-处理前野生型（WT）和lps1中ABA相关基因表达水平;B-处理后野生型（WT）和lps1中ABA相关基因表达水平;*和**分别表示野生型与lps1在0.05和0.01水平上差异显著。

Fig. 10. ABA synthesis and degradation related gene expression levels. A, ABA-related gene expression levels in the wild type and lps1 before treatment; B, ABA-related gene expression levels in the wild type and lps1 after treatment; * and ** indicate significant difference between the wild type and lps1 at 0.05 and 0.01 level, respectively.

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