水稻条纹叶和白穗基因SLWP的定位及变异分析

doi:10.16819/j.1001-7216.2018.7122

摘要/Abstract

摘要：

【目的】对叶绿体发育相关基因进行克隆和功能分析,为解析叶绿体功能奠定分子基础。【方法】用甲基磺酸乙酯(EMS)处理籼稻9311获得一个条纹叶和白穗突变体slwp,通过色素分析和农艺性状观察分析该突变体的表型,通过图位克隆方法分离该基因,进一步利用定量PCR分析相关基因的表达情况。【结果】突变体slwp从2叶期开始至抽穗期表现出条纹叶表型,抽穗后幼穗白化,光合色素含量明显低于野生型;株高降低、抽穗延迟、产量降低等表型。该突变性状为单隐性核基因控制,该基因定位于水稻第6染色体短臂C6-4和N14标记之间0.91 Mb区间内。基因组测序表明核糖核苷二磷酸还原酶小亚基基因(RNRS1)编码区第776位点发生单碱基替换,导致甘氨酸突变为天冬氨酸;该基因与已报导的水稻基因St1、Gws和St-wp为等位基因。通过对这4个等位基因的突变位点和表型进行分析,总结了该基因不同位点突变对植株表型的影响以及籼粳之间的差异。表达分析显示与叶绿素合成有关的基因受到不同程度调控,叶绿体发育第一和第二阶段基因上调表达,光合作用相关基因均下调表达。【结论】本研究分析了SLWP(RNRS1)基因不同位点的变异对水稻表型的影响,相关结果加深了对RNRS1基因功能的认识,有助于阐明叶绿体发育的分子机制。

关键词: 水稻, 突变体, 变异分析, 表达分析

Abstract:

【Objective】Cloning and function analysis of chloroplast development-associated genes will lay a molecular basis for clarifying chloroplast function. 【Method】A stripe leaf and white panicle mutant, slwp, was isolated from an ethylmethylsulfone (EMS) mutagenic population of indica rice cultivar 9311. The phenotypic characteristics of slwp were analyzed by pigments determination and agronomic traits observation. The SLWP gene was identified by map-based cloning method. The gene expression was analyzed by the quantitative real-time PCR.【Result】The slwp mutant showed stripe leaf phenotype from two-leaf stage to heading and white panicle after heading accompanied by obviously decreased pigment contents compared with wild type. The slwp mutant was also featured with delayed heading and decreased plant height and yield and so on. Genetic analysis demonstrated that the mutant phenotype was controlled by a recessive nuclear gene. The SLWP gene was fine-mapped to a 0.91 Mb interval between markers C6-4 and N14 on the short arm of rice chromosome 6. Sequence comparison displayed that a single nucleotide substitution (G776A) in the coding region of ribonucleoside-diphosphate reductase small chain(RNRS1), which led to the amino acid change from Gly to Asp. The SLWP was allelic to St1, Gws and St-wp. We analyzed the mutant sites and phenotypes of the four alleles and summarized the effect of mutation on plant phenotypes and the difference between indica and japonica. In addition, expression analysis revealed that genes involved in chlorophyll biosynthesis were differently regulated, genes involved in the first and second steps of chloroplast differentiation were up-regulated, and genes involved in photosynthesis were down-regulated. 【Conclusion】We analyzed the effect of mutant sites in SLWP(RNRS1) gene on rice phenotypes. It deepens understanding for RNRS1 function and help to elucidate the molecular mechanism of chloroplast development.

Key words: rice (Oryza sativa), mutant, mutation analysis, expression analysis

中图分类号:

周坤能, 夏加发, 马廷臣, 王元垒, 李泽福. 水稻条纹叶和白穗基因SLWP的定位及变异分析[J]. 中国水稻科学, 2018, 32(4): 325-334.

Kunneng ZHOU, Jiafa XIA, Tingchen MA, Yuanlei WANG, Zefu LI. Mapping and Mutation Analysis of Stripe Leaf and White Panicle Gene SLWP in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(4): 325-334.

图/表 10

参考文献 24

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引物用途与名称 Usage and name	正向引物 Forward sequence(5′-3′)	反向引物 Reverse sequence(5′-3′)
定位和测序引物 Primers for mapping and sequencing
C6-4	CAGTTAACACCAATCCAATCCA	CCAAATGGGCAGTAGTTTGAA
C6-5	GCTTCTCCCGGAGTATGTCA	TGGTCTGAAAAGTGCCAAAA
C6-6	CCCCAAGCTTGTTTGTCTCT	TTGGGTCTTTGAGCTTCCTC
N4	TCAAGTTGCTAAACCTTATCTG	AGATGAACTGTGCTAAAAGATG
N12	GTAACTTAAAAGCCAATGTTGA	GAGTACTACCATCCATCCCTGT
N14	TCTGTGGACGTAGTAGGTTGA	CCTTCCTTAGGTCTGGCTC
slwp-g	ACAACCCCAAATCCCCATCCA	ACCACGCGCATGTATTACTACT
定量PCR引物 Primers for quantitative RT-PCR
PORA	ATCACCAAGGGCTACGTCTC	GAGTTGTTGTTCCAGCTCCA
HEMA1	CACCAGTCTGAATCATAT	CTACCACTTCTCTAATCC
YGL1	TGGACAGTTGAAGATGTT	GAATAGGACGGTAAGGTT
CHLI	AGTAACCTTGGTGCTGTG	AATCCATCAACATTCAACTCTG
CHLH	CTATACATTCGCCACACT	TATCACACAACTCCCAAG
CHLD	GGAAAGAGAGGGCATTAG	CAATACGATCAAGTAAGTGTT
FtsZ	GTTGGTGTTTCTTCCAGCAA	CCTCAATAGACGACCCGATT
RpoTp2	AAGTCTGGCTTACGCTGGTT	AGGATCCTCAGCATTCATCC
rpoA	AAATCGTTGATACGGCACAA	ATTCACATTTCGAACAGGCA
rpoB	GCATTGTTGGAACTGGATTG	GCCGATGGGTAACTAAAGGA
rbcL	GTTGAAAGGGATAAGTTGA	AATGGTTGTGAGTTTACG
rbcS	TCATCAGCTTCATCGCCTAC	ACTGGGAACACACGAAACAA
psbA	AAGTTTCTCTGATGGTATG	ATAGCACTGAATAGGGAA
psaA	GAGATACCACTTCCTCAT	ACTAAGAAATTCTGCGTATT
psaB	TTGGTATTGCTACCGCACAT	CCGGACGTCCATAGAAAGAT
psbB	TCATATTGCTGCGGGTACAT	AGTTGCTGACCCATACCACA
psbC	TACAACCTTGGCAAGAACGA	TACGCCACCCACAGAATTTA
SLWP	AGTAATACATGCGCGTGGTG	ACACGGGCAGCTGATACTAA
RNRS2	AGATGTACAACGTCGCCAAC	GACATTACGGACGCCTTCTG
Ubq	GCTCCGTGGCGGTATCAT	CGGCAGTTGACAGCCCTAG

引物用途与名称 Usage and name	正向引物 Forward sequence(5′-3′)	反向引物 Reverse sequence(5′-3′)
定位和测序引物 Primers for mapping and sequencing
C6-4	CAGTTAACACCAATCCAATCCA	CCAAATGGGCAGTAGTTTGAA
C6-5	GCTTCTCCCGGAGTATGTCA	TGGTCTGAAAAGTGCCAAAA
C6-6	CCCCAAGCTTGTTTGTCTCT	TTGGGTCTTTGAGCTTCCTC
N4	TCAAGTTGCTAAACCTTATCTG	AGATGAACTGTGCTAAAAGATG
N12	GTAACTTAAAAGCCAATGTTGA	GAGTACTACCATCCATCCCTGT
N14	TCTGTGGACGTAGTAGGTTGA	CCTTCCTTAGGTCTGGCTC
slwp-g	ACAACCCCAAATCCCCATCCA	ACCACGCGCATGTATTACTACT
定量PCR引物 Primers for quantitative RT-PCR
PORA	ATCACCAAGGGCTACGTCTC	GAGTTGTTGTTCCAGCTCCA
HEMA1	CACCAGTCTGAATCATAT	CTACCACTTCTCTAATCC
YGL1	TGGACAGTTGAAGATGTT	GAATAGGACGGTAAGGTT
CHLI	AGTAACCTTGGTGCTGTG	AATCCATCAACATTCAACTCTG
CHLH	CTATACATTCGCCACACT	TATCACACAACTCCCAAG
CHLD	GGAAAGAGAGGGCATTAG	CAATACGATCAAGTAAGTGTT
FtsZ	GTTGGTGTTTCTTCCAGCAA	CCTCAATAGACGACCCGATT
RpoTp2	AAGTCTGGCTTACGCTGGTT	AGGATCCTCAGCATTCATCC
rpoA	AAATCGTTGATACGGCACAA	ATTCACATTTCGAACAGGCA
rpoB	GCATTGTTGGAACTGGATTG	GCCGATGGGTAACTAAAGGA
rbcL	GTTGAAAGGGATAAGTTGA	AATGGTTGTGAGTTTACG
rbcS	TCATCAGCTTCATCGCCTAC	ACTGGGAACACACGAAACAA
psbA	AAGTTTCTCTGATGGTATG	ATAGCACTGAATAGGGAA
psaA	GAGATACCACTTCCTCAT	ACTAAGAAATTCTGCGTATT
psaB	TTGGTATTGCTACCGCACAT	CCGGACGTCCATAGAAAGAT
psbB	TCATATTGCTGCGGGTACAT	AGTTGCTGACCCATACCACA
psbC	TACAACCTTGGCAAGAACGA	TACGCCACCCACAGAATTTA
SLWP	AGTAATACATGCGCGTGGTG	ACACGGGCAGCTGATACTAA
RNRS2	AGATGTACAACGTCGCCAAC	GACATTACGGACGCCTTCTG
Ubq	GCTCCGTGGCGGTATCAT	CGGCAGTTGACAGCCCTAG

农艺性状 Agronomic trait	野生型 Wild type	突变体 swlp
株高Plant height / cm	124.7±3.4	98.2±4.8^**
播始历期Days from sowing to heading / d	98.4±1.2	108.3±1.0^**
穗长Panicle length / cm	25.5±1.1	22.1±1.3^**
有效穗数Number of effective panicles per plant	7.1±1.4	6.6±1.7
每穗粒数Grain number per panicle	245.1±32.6	145.0±21.0^**
结实率Seed-setting rate / %	88.2±1.3	87.9±1.6
千粒重1000-grain weight / g	30.8±0.4	27.9±0.6^**
理论产量Theoretical yield per plot / kg	10.74±0.23	5.34±0.35^**
实际产量Actual yield per plot / kg	9.16±0.26	4.43±0.47^**

农艺性状 Agronomic trait	野生型 Wild type	突变体 swlp
株高Plant height / cm	124.7±3.4	98.2±4.8^**
播始历期Days from sowing to heading / d	98.4±1.2	108.3±1.0^**
穗长Panicle length / cm	25.5±1.1	22.1±1.3^**
有效穗数Number of effective panicles per plant	7.1±1.4	6.6±1.7
每穗粒数Grain number per panicle	245.1±32.6	145.0±21.0^**
结实率Seed-setting rate / %	88.2±1.3	87.9±1.6
千粒重1000-grain weight / g	30.8±0.4	27.9±0.6^**
理论产量Theoretical yield per plot / kg	10.74±0.23	5.34±0.35^**
实际产量Actual yield per plot / kg	9.16±0.26	4.43±0.47^**

突变体 Mutant	野生型 Wild type	氨基酸突变位点 Mutant site of amino acid	叶片表型 Leaf phenotype	穗部表型 Panicle phenotype	主要农艺性状变化 Agronomic trait change compared with its wild type
st1	FL176	p. Lys40Arg	4或5叶期至分蘖期出现条纹;抽穗后表型恢复Display stripe from 4- or 5-leaf stage to tillering, then revert to normal after heading	无 None	无 None
gws	日本晴 Nipponbare	104原有+插入14个氨基酸 104 original+Inserted 14 amino acid residues	苗期至抽穗期叶片均出现条纹Display stripe from the seedling to heading stage	无 None	株高、穗长、有效分蘖数、结实率、千粒重、单株生物量均降低Decreased plant height, panicle length, tiller number, seed-setting rate, 1000-grain weight, and plant biomass per plant
st-wp	9311	p. Glu103Val	苗期至抽穗期叶片均出现条纹Display stripe from the seedling to heading stage	白穗 White panicle	株高、穗长、每穗粒数、结实率、千粒重降低,抽穗期延长,有效穗数不变 Decreased plant height, panicle length, grain number per panicle, seed-setting rate, and 1000-grain weight; prolonged heading date; similar tiller number
slwp	9311	p. Gly259Asp	苗期至抽穗期叶片出现条纹 Display stripe from the seedling to heading stage	白穗 White panicle	株高、穗长、每穗粒数、千粒重、理论产量、实际产量下降,抽穗期延长,有效穗数和结实率不变 Decreased plant height, panicle length, grain number per panicle, 1000-grain weight, theoretical yield, and actual yield; prolonged heading date, similar tiller number and seed-setting rate