水稻斑马叶突变体zl7的鉴定与基因的精细定位

doi:10.16819/j.1001-7216.2023.220314

中国水稻科学 ›› 2023, Vol. 37 ›› Issue (2): 113-124.DOI: 10.16819/j.1001-7216.2023.220314

• 研究报告 • 下一篇

水稻斑马叶突变体zl7的鉴定与基因的精细定位

廉院训¹^,²^,^#, 韦子芸¹^,²^,^#, 张强², 李清², 任德勇², 胡江², 朱丽², 高振宇², 张光恒², 郭龙彪², 曾大力², 钱前²^,^*(), 沈兰²^,^*()

¹浙江师范大学化学与生命科学学院, 浙江金华 321004
²中国水稻研究所水稻生物学国家重点实验室, 杭州310006

收稿日期:2022-03-28 修回日期:2022-05-09 出版日期:2023-03-10 发布日期:2023-03-10
通讯作者: 钱前,沈兰
作者简介:第一联系人：^#共同第一作者
基金资助:
国家自然科学基金资助项目(31971872);国家自然科学基金资助项目(32171987);水稻生物学国家重点实验室自主课题(2020ZZKT10205);中国水稻研究所所级重点研发项目(CNRRI-2020-01);浙江省重点研发计划资助项目(2021C02056)

Identification and Gene Mapping of a Zebra Leaf Mutant zl7 in Rice

LIAN Yuanxun¹^,²^,^#, WEI Ziyun¹^,²^,^#, ZHANG Qiang², LI Qing², REN Deyong², HU Jiang², ZHU Li², GAO Zhenyu², ZHANG Guangheng², GUO Longbiao², ZENG Dali², QIAN Qian²^,^*(), SHEN Lan²^,^*()

¹College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China
²State Key Laboratory of Rice Biology China National Rice Research Institute, Hangzhou 310006, China

Received:2022-03-28 Revised:2022-05-09 Online:2023-03-10 Published:2023-03-10
Contact: QIAN Qian, SHEN Lan
About author:First author contact:^#These authors contributed equally to this work

摘要/Abstract

摘要：

【目的】斑马叶突变体作为水稻叶色突变体的重要种质资源，是研究植物光合作用机制和高光效育种的理想材料，对于解析光合作用机理和提高水稻产量具有重要意义。【方法】用甲基磺酸乙酯（EMS）诱变粳稻品种春江06建立突变体库，从突变体库中筛选到1份苗期为斑马叶的突变体，该突变体被命名为zl7 (zebra leaf 7)。在常规大田种植条件下分别比较突变体与野生型在苗期、抽穗期和成熟期叶色表型和产量性状差异，通过透射电镜实验分析叶片叶绿体发育情况，利用图位克隆方法克隆候选基因，利用荧光定量PCR 方法分析参与叶绿素合成和叶绿体发育相关基因的表达水平。【结果】从苗期开始，突变体zl7表现出典型的斑马叶，叶绿素含量降低，直到抽穗期，斑马叶表型消失，叶片逐渐复绿，叶绿素含量无明显差异。光合速率测定和电镜观察结果显示，突变体zl7的光合速率、气孔导度下降，叶绿体发育异常。与野生型相比，突变体的株高、分蘖、穗长、一次枝梗、二次枝梗和每穗粒数均显著降低，而粒长、粒宽和千粒重均略有增加。荧光定量PCR结果表明突变体中参与叶绿素降解相关基因的表达量显著升高，而参与叶绿素合成和叶绿体发育相关基因的表达量显著降低。遗传分析表明，该突变体受一对隐性核基因调控。通过图位克隆将该基因定位在第7染色体，测序发现突变体的目标基因ZL7编码区发生单碱基替换，导致一个氨基酸由丝氨酸变为天冬酰胺。【结论】ZL7 (Zebra Leaf 7)突变导致叶绿体发育异常，水稻叶片出现斑马叶表型，该基因在叶绿素合成及叶绿体发育中起重要作用。

关键词: 水稻, 叶绿体, 遗传分析, 图位克隆

Abstract:

【Objective】 Zebra leaf mutants, as important germplasm resources of rice leaf color mutants, are ideal materials for studying the mechanism of plant photosynthesis and high photosynthetic efficiency breeding, and play an important role in analyzing the mechanism of photosynthesis and improving rice yield. 【Method】 A zebra leaf mutant, designated as zebra leaf 7(zl7), was isolated from an ethyl methanesulfonate (EMS)-mutagenized population of Chunjiang 06 (CJ06). Under conventional paddy field management, the differences in leaf color phenotype and yield characters between the mutant and its wild type at seedling stage, heading stage and maturity stage were compared respectively. The development of leaf chloroplast were analyzed with a transmission electron microscope. Candidate genes were cloned by map-based cloning method, and the expression levels of genes involved in chlorophyll synthesis and chloroplast development were analyzed by fluorescence quantitative PCR. 【Result】From seedling stage, the mutant zl7 showed typical zebra leaves with decreased chlorophyll contents. During the heading stage, the zebra leaf phenotype disappeared and the leaves gradually turned green, and there was no significant difference in chlorophyll contents. The measurement of photosynthetic rate and the observation with an electron microscope showed that the photosynthetic rate, stomatal conductance and chloroplast development of the mutant zl7 were abnormal. Compared with the wild type, the plant height, the number of tillers, panicle length, primary rachis branch number, secondary rachis branch number and grains per panicle of the mutant decreased significantly, while the grain length, grain width and 1000-grain weight increased slightly. The results of fluorescence quantitative PCR showed that the expression levels of genes involved in chlorophyll degradation increased significantly, while the expression levels of genes involved in chlorophyll synthesis and chloroplast development decreased significantly. Genetic analysis showed that the mutant was regulated by a pair of recessive nuclear genes. The gene was mapped on chromosome 7 by map-based cloning, and the sequencing results showed that the coding region of ZL7 in zl7 has a single-base substitution mutation, causing an amino acid change from serine to asparagine. 【Conclusion】ZL7 mutation leads to abnormal chloroplast development, consequently resulting in zebra leaf phenotype, which plays an important role in rice chlorophyll synthesis and chloroplast development.

Key words: rice, chloroplast, genetic analysis, map cloning

廉院训, 韦子芸, 张强, 李清, 任德勇, 胡江, 朱丽, 高振宇, 张光恒, 郭龙彪, 曾大力, 钱前, 沈兰. 水稻斑马叶突变体zl7的鉴定与基因的精细定位[J]. 中国水稻科学, 2023, 37(2): 113-124.

LIAN Yuanxun, WEI Ziyun, ZHANG Qiang, LI Qing, REN Deyong, HU Jiang, ZHU Li, GAO Zhenyu, ZHANG Guangheng, GUO Longbiao, ZENG Dali, QIAN Qian, SHEN Lan. Identification and Gene Mapping of a Zebra Leaf Mutant zl7 in Rice[J]. Chinese Journal OF Rice Science, 2023, 37(2): 113-124.

图/表 12

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标记 Marker	正向引物序列 Forward primer sequence	反向引物序列 Reverse primer sequence
M1	GTCCATGCATCCATCTCTAG	ACGGAAGGAATACGTCTGTA
M2	TGTGGACAACCTCAACTGAAAGC	CATAATCACCAACATCGGAGAAGC
M3	TGTTGAGCTAGAAGAGAGGGG	TGAACACAAAAGGATGCGCT
M4	CCAAGTCTTAAGCTACCCCT	CGCAGGGCTTAATAGAATAC
M5	GATAGAGCGAGTGAGCAAAC	CCTACCAATTCAACTCCAAC
M6	TGGATTGAGGATCAGGATAG	TCTGGAATTTTCCCTAATGA
M7	GAAATCAGTCAGAAAGACCG	CCATCTTCTCACTGTGGAGT
M8	GCCCTAATTGCTCCAGGTCT	AATTCTAGCAGTGTTCCATTGTG
M9	TTCTTCCATGTAGCAAGCATT	CCTACTGCCTGCCAAATCTAT

标记 Marker	正向引物序列 Forward primer sequence	反向引物序列 Reverse primer sequence
M1	GTCCATGCATCCATCTCTAG	ACGGAAGGAATACGTCTGTA
M2	TGTGGACAACCTCAACTGAAAGC	CATAATCACCAACATCGGAGAAGC
M3	TGTTGAGCTAGAAGAGAGGGG	TGAACACAAAAGGATGCGCT
M4	CCAAGTCTTAAGCTACCCCT	CGCAGGGCTTAATAGAATAC
M5	GATAGAGCGAGTGAGCAAAC	CCTACCAATTCAACTCCAAC
M6	TGGATTGAGGATCAGGATAG	TCTGGAATTTTCCCTAATGA
M7	GAAATCAGTCAGAAAGACCG	CCATCTTCTCACTGTGGAGT
M8	GCCCTAATTGCTCCAGGTCT	AATTCTAGCAGTGTTCCATTGTG
M9	TTCTTCCATGTAGCAAGCATT	CCTACTGCCTGCCAAATCTAT

基因 Gene	基因登录号 Gene ID	定量正向引物序列 Forward primer sequence	定量反向引物序列 Reverse primer sequence	功能 Function
NYC1	LOC_Os01g12710	CATGCAACACCAACAAAAGG	GACCATTCCAGGAGAAGCAG	叶绿素b还原酶基因
NOL	LOC_Os03g45194	CCACGAAAGGTATAGGATATG	TCAAGTCAGTCACCGCAGAT	叶绿素b还原酶基因
NYC3	LOC_Os06g24730	TCTATCTAGGTGCCAAAGGC	ATTCTGGCACCTGCTGTTTC	α/β折叠水解酶家族蛋白
PAO	LOC_Os03g05310	AAGCCTCCGATGTTACCGAA	CGAGGGTTTCCAGAATTTGA	脱镁叶绿酸a加氧酶，叶绿体前体
SGR	LOC_Os09g36200	GCAATGTCGCCAAATGACG	GCTCACCACACTCATTCCCTAAAG	镁离子去螯合酶
RCCR1	LOC_Os10g25020	GGATCGACGATTGATTTCATG	GTCGAGGCGTTCAGAAAGAT	红色叶绿素分解代谢还原酶
RCCR2	LOC_Os10g25040	TGGCGAGGGACAGGAAGGT	GGATGTGGTGGCGAGAGAAAC	红色叶绿素分解代谢还原酶
LchP2	LOC_Os09g17740	GAAGAAGATCAAGAACGGCC	TTGCCGGGGACGAAGTTGGT	捕光叶绿素a/b结合蛋白基因
PsbA	LOC_Os12g19580	AGAGACGCGAAAGTACAAGC	AAGTTGCGGTCAATAAGGTA	光合反应中心蛋白
RpoC2	LOC_Os04g16830	ATGCATCGCAGGTACACCAA	CCCTCGCGTAAATTGCTTTG	DNA定向RNA聚合酶亚单位β
Rps15	LOC_Os12g10580	AGATACGGAGACTTGCTTCA	GCTCCCTAATATCCAACTGACT	核酮糖二磷酸羧化酶大链前体
V1	LOC_Os03g45400	AGAATCAGCGCGAGAAGAGAACCT	TACACCAGCTTTGGAGGAGCTGAA	质体蛋白
V2	LOC_Os03g20460	AGCAGATCCGTGATTACATGGCGA	TGCCTCTTCACTCTCTGCAACCAA	鸟苷酸激酶
YGL8	LOC_Os01g17170	TGGATCTAACATGACACGCACCCA	ACTGTAACGGCATTCTTCTCCGGT	镁原卟啉IX单酯环化酶的催化亚基
CAO1	LOC_Os10g41780	TTGGCTCAGTTAATGAGGGCAGAATCC	GGATGCGCACGTTGAGCATCTTTGTGG	叶绿素a加氧酶
PORA	LOC_Os04g58200	ATGGCTCTCCAAGTTCAG	TGGCTCACGCTAAGGAAC	NADPH:原叶绿素酸酯氧化还原酶A
PORB	LOC_Os10g35370	CCGCAAGGAGGGAGCGGTG	CCCTCTTGGTGCTAAGGCCG	原叶绿素酸酯氧化还原酶B
CHLH	LOC_Os03g20700	GCACGGGAACTTGGCGTTTCATTA	ACATGTCCTGGAGCTGCTTCTCAT	镁离子螯合酶H亚基
CHLD	LOC_Os03g59640	TAGCACAGCTGTCAGAGTGGGTTT	TTGCCAGCCACCTCAAGTATCTCA	镁离子螯合酶D亚基
CHLI	LOC_Os03g36540	AGGGATGCTGAACTCAGGGTGAAA	AAGTAGGACTCACGGAACGCCTTT	镁离子螯合酶I亚基
DVR	LOC_Os03g22780	AGCCCAGGTTCATCAAGGT	TGATCACCCTCTCGAAGAACT	联乙烯还原酶基因
OsCHLM	LOC_Os06g04150	GCTTCATCTCCACGCAGTTCTACT	GCAATGACGAATCGAAGACGCACA	镁原卟啉O-甲基转移酶
YGL1	LOC_Os05g28200	CCAGCCACTGATGAAAGCAGCAAT	AGAGCGCTAATACACTCGCGAACA	叶绿素合成酶
OsHEMA1	LOC_Os10g35840	GATGCAATCACTGCTGGAAAGCGT	CCATCTTGCCAGCACCAATCAACA	莽草酸/奎宁酸5-脱氢酶
OsHEML	LOC_Os08g41990	AGAACAAAGGGCAGATTGCTGCTG	TGTTTCGTCAAGTCACGGAGAGCA	转氨酶
OsHEMB	LOC_Os06g49110	TGGCATTGTCAGGGAAGATGGAGT	CCAAAGCAGCACGTATTGCTCCAA	δ-氨基乙酰丙酸脱水酶，叶绿体前体
GUN4	LOC_Os11g16550	AAGGGAAGGAGAGGCCAAAGTTCA	ACCATGACCAGCATCTCTGCATCA	镁离子螯合酶H亚基结合蛋白

基因 Gene	基因登录号 Gene ID	定量正向引物序列 Forward primer sequence	定量反向引物序列 Reverse primer sequence	功能 Function
NYC1	LOC_Os01g12710	CATGCAACACCAACAAAAGG	GACCATTCCAGGAGAAGCAG	叶绿素b还原酶基因
NOL	LOC_Os03g45194	CCACGAAAGGTATAGGATATG	TCAAGTCAGTCACCGCAGAT	叶绿素b还原酶基因
NYC3	LOC_Os06g24730	TCTATCTAGGTGCCAAAGGC	ATTCTGGCACCTGCTGTTTC	α/β折叠水解酶家族蛋白
PAO	LOC_Os03g05310	AAGCCTCCGATGTTACCGAA	CGAGGGTTTCCAGAATTTGA	脱镁叶绿酸a加氧酶，叶绿体前体
SGR	LOC_Os09g36200	GCAATGTCGCCAAATGACG	GCTCACCACACTCATTCCCTAAAG	镁离子去螯合酶
RCCR1	LOC_Os10g25020	GGATCGACGATTGATTTCATG	GTCGAGGCGTTCAGAAAGAT	红色叶绿素分解代谢还原酶
RCCR2	LOC_Os10g25040	TGGCGAGGGACAGGAAGGT	GGATGTGGTGGCGAGAGAAAC	红色叶绿素分解代谢还原酶
LchP2	LOC_Os09g17740	GAAGAAGATCAAGAACGGCC	TTGCCGGGGACGAAGTTGGT	捕光叶绿素a/b结合蛋白基因
PsbA	LOC_Os12g19580	AGAGACGCGAAAGTACAAGC	AAGTTGCGGTCAATAAGGTA	光合反应中心蛋白
RpoC2	LOC_Os04g16830	ATGCATCGCAGGTACACCAA	CCCTCGCGTAAATTGCTTTG	DNA定向RNA聚合酶亚单位β
Rps15	LOC_Os12g10580	AGATACGGAGACTTGCTTCA	GCTCCCTAATATCCAACTGACT	核酮糖二磷酸羧化酶大链前体
V1	LOC_Os03g45400	AGAATCAGCGCGAGAAGAGAACCT	TACACCAGCTTTGGAGGAGCTGAA	质体蛋白
V2	LOC_Os03g20460	AGCAGATCCGTGATTACATGGCGA	TGCCTCTTCACTCTCTGCAACCAA	鸟苷酸激酶
YGL8	LOC_Os01g17170	TGGATCTAACATGACACGCACCCA	ACTGTAACGGCATTCTTCTCCGGT	镁原卟啉IX单酯环化酶的催化亚基
CAO1	LOC_Os10g41780	TTGGCTCAGTTAATGAGGGCAGAATCC	GGATGCGCACGTTGAGCATCTTTGTGG	叶绿素a加氧酶
PORA	LOC_Os04g58200	ATGGCTCTCCAAGTTCAG	TGGCTCACGCTAAGGAAC	NADPH:原叶绿素酸酯氧化还原酶A
PORB	LOC_Os10g35370	CCGCAAGGAGGGAGCGGTG	CCCTCTTGGTGCTAAGGCCG	原叶绿素酸酯氧化还原酶B
CHLH	LOC_Os03g20700	GCACGGGAACTTGGCGTTTCATTA	ACATGTCCTGGAGCTGCTTCTCAT	镁离子螯合酶H亚基
CHLD	LOC_Os03g59640	TAGCACAGCTGTCAGAGTGGGTTT	TTGCCAGCCACCTCAAGTATCTCA	镁离子螯合酶D亚基
CHLI	LOC_Os03g36540	AGGGATGCTGAACTCAGGGTGAAA	AAGTAGGACTCACGGAACGCCTTT	镁离子螯合酶I亚基
DVR	LOC_Os03g22780	AGCCCAGGTTCATCAAGGT	TGATCACCCTCTCGAAGAACT	联乙烯还原酶基因
OsCHLM	LOC_Os06g04150	GCTTCATCTCCACGCAGTTCTACT	GCAATGACGAATCGAAGACGCACA	镁原卟啉O-甲基转移酶
YGL1	LOC_Os05g28200	CCAGCCACTGATGAAAGCAGCAAT	AGAGCGCTAATACACTCGCGAACA	叶绿素合成酶
OsHEMA1	LOC_Os10g35840	GATGCAATCACTGCTGGAAAGCGT	CCATCTTGCCAGCACCAATCAACA	莽草酸/奎宁酸5-脱氢酶
OsHEML	LOC_Os08g41990	AGAACAAAGGGCAGATTGCTGCTG	TGTTTCGTCAAGTCACGGAGAGCA	转氨酶
OsHEMB	LOC_Os06g49110	TGGCATTGTCAGGGAAGATGGAGT	CCAAAGCAGCACGTATTGCTCCAA	δ-氨基乙酰丙酸脱水酶，叶绿体前体
GUN4	LOC_Os11g16550	AAGGGAAGGAGAGGCCAAAGTTCA	ACCATGACCAGCATCTCTGCATCA	镁离子螯合酶H亚基结合蛋白

农艺性状 Agronomic trait	野生型 Wild type	突变体 Mutant zl7
株高 Plant height/cm	91.2±4.0	79.5±2.7^**
分蘖 Tillering number	10.0±1.2	8.0±0.8^*
穗长 Panicle length/cm	22.5±1.3	20.2±0.8^*
一次枝梗 Primary rachis branches	17.0±0.5	14.0±1.1^*
二次枝梗 Secondary rachis branches	28.0±1.9	23.0±1.6^*
每穗粒数 No. of grains per panicle	152.0±6.0	126.0±4.7^**
结实率Seed setting rate/%	93.4±0.0	93.7±0.0
千粒重 1000-grain weight/g	23.2±0.6	24.9±0.4
粒长 Grain length/mm	7.35±0.40	7.67±0.63
粒宽 Grain width/mm	3.08±0.23	3.17±0.24

水稻斑马叶突变体zl7的鉴定与基因的精细定位

Identification and Gene Mapping of a Zebra Leaf Mutant zl7 in Rice

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图/表 12

参考文献 42

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Metrics

材料 Material	光合作用速率 P_n/ (μmol·m⁻² s⁻¹)	气孔导度 G_s / (mol·m⁻² s⁻¹)	胞间CO₂浓度 C_i / (μmol·mol⁻¹)	蒸腾速率 T_r/ (mol·m⁻² s⁻¹)
野生型WT	26.41±0.34	0.06±0.01	442.34±24.64	2.94±0.08
Mutant-g	21.67±0.51 ^**	0.05±0.01	246.63±29.41^**	2.83±0.32
Mutant-y	13.21±0.47 ^**	0.04±0.01	133.69±7.49^**	2.34±0.17^*

材料 Material	光合作用速率 P_n/ (μmol·m⁻²·s⁻¹)	气孔导度 G_s / (mol·m⁻²·s⁻¹)	胞间CO₂浓度 C_i / (μmol·mol⁻¹)	蒸腾速率 T_r/ (mol·m⁻²·s⁻¹)
WT	16.06±0.56	0.15±0.02	204.24±17.42	3.29±0.30
Mutant-g	18.34±0.64**	0.28±0.02*	278.66±9.48**	4.79±0.26**
Mutant-y	9.93±1.41**	0.13±0.03	262.71±15.41**	2.71±0.58

杂交组合 Hybrid combination	F₁		F₂		χ² (3:1)
杂交组合 Hybrid combination	正常表型 Wild-type phenotype	突变表型 zl7 phenotype	正常表型 Wild-type phenotype	突变表型 zl7 phenotype	χ² (3:1)
zl7/93-11	13	0	2065	655	1.225
93-11/ zl7	8	0	822	271	0.025

预测基因 Predictive gene	基因功能注释 Gene function annotation
ORF1	转座子蛋白
ORF2	表达蛋白
ORF3	ζ-胡萝卜素脱氢酶
ORF4	表达蛋白
ORF5	细胞色素b-c1复合亚单位7
ORF6	表达蛋白
ORF7	核苷酸结合蛋白
ORF8	羧基末端肽酶
ORF9	表达蛋白
ORF10	肽酶，M24家族蛋白
ORF11	细胞周期蛋白相关蛋白
ORF12	表达蛋白

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