水稻黄绿叶突变体w390的遗传分析和基因定位

doi:10．3969/j．issn．1001G7216．2015．03．003

中国水稻科学 ›› 2015, Vol. 29 ›› Issue (3): 241-249.DOI: 10．3969/j．issn．1001G7216．2015．03．003

水稻黄绿叶突变体w390的遗传分析和基因定位

董青^1,², 张迎信^1,², 张振华^1,², 周全^1,², 秦亚芝^1,^2,³, 王宏^1,², 程式华^1,², 曹立勇^1,^2,^*(), 沈希宏^1,^2,^*()

¹中国水稻研究所/国家水稻改良中心, 杭州 311401
2.浙江省超级稻研究重点实验室, 杭州 311401
3.四川农业大学水稻研究所/西南作物基因资源与遗传改良教育部重点实验室, 成都 611130

收稿日期:2014-11-27 修回日期:2014-12-24 出版日期:2015-05-10 发布日期:2015-05-10
通讯作者: 曹立勇,沈希宏
基金资助:
国家科技支撑计划资助项目(2011BAD35B02);农业部水稻生物学与遗传育种学科群项目;浙江省公益技术应用研究项目(2013C32003);中国农业科学院科技创新工程资助项目(CAAS-ASTIP-2013-CNRRI)

Genetic Analysis and Gene Mapping of a Yellow-green Leaf Mutant w390 in Rice (Oryza sativa L.)

Qing DONG^1,², Ying-xin ZHANG^1,², Zhen-hua ZHANG^1,², Quan ZHOU^1,², Ya-zhi QIN^1,^2,³, Hong WANG^1,², Shi-hua CHENG^1,², Li-yong CAO^1,^2,^*(), Xi-hong SHEN^1,^2,^*()

¹China National Rice Research Institute/National Center for Rice Improvement, Hangzhou 311401, China
2.Key Laboratory for Zhejiang Super Rice Research, Hangzhou 311401, China
3.Rice Research Institute/Key Laboratory of Southwest Crop Genetic Resources and Improvement, Ministry of Education, Sichuan Agricultural University, Chengdu 611130, China

Received:2014-11-27 Revised:2014-12-24 Online:2015-05-10 Published:2015-05-10
Contact: Li-yong CAO, Xi-hong SHEN

摘要/Abstract

摘要：

通过⁶⁰Co-γ辐射诱变籼稻中恢8015获得了一个在全生育期叶片均呈黄绿色的突变体w390。与野生型相比,突变体中检测不到叶绿素b的存在,且叶绿素a和类胡萝卜素的含量分别降低了50.6%和44.8%;主要农艺性状调查结果显示,突变体的株高、单株有效穗数、每穗总粒数、每穗实粒数较野生型分别降低了12.0%、22.3%、18.5%和27.6%;透射电镜结果显示:突变体的类囊体数量明显减少,基粒垛叠方向异常;遗传分析表明该突变性状受一对隐性核基因控制。利用突变体与粳稻日本晴杂交构建的F₂群体,将突变基因定位至水稻第10染色体长臂约71.8 kb的区域内。对该区间包含的15个ORFs进行序列分析,发现突变体中编码叶绿素酸酯氧化酶1(chlorophyllide a oxygenase 1)的基因OsCAO1的第8外显子发生了两个单碱基突变,导致第394和396位的亮氨酸和甘氨酸分别突变为组氨酸和谷氨酸,推测该突变基因是一个OsCAO1功能丧失的新等位基因。

关键词: 水稻, 突变体, 黄绿叶, 基因定位

Abstract:

A yellow-green leaf mutant, designated as w390, was isolated from the progeny of ⁶⁰Co-γ-treated indica cultivar R8015. The mutant exhibited a stable yellow-green leaf phenotype during the whole life cycle. The chlorophyll b was not detected in w390, and the chlorophyll a and carotenoids contents of w390 were reduced by 50.6% and 44.8%, compared with the wild-type. The plant height, the number of panicle per plant, the number of spikelets per panicle, the number of grains per panicle of w390 were reduced by 12.0%, 22.3%,18.5% and 27.6%, respectively. The transmission electron microscope assay demonstrated that the number of thylakoids decreased and granas were poorly stacked in the mutant, resulting in underdevelopment of chloroplasts. Genetic analysis showed that the yellow-green leaf phenotype was controlled by a single recessive gene. Using an F₂ mapping population derived from the cross of w390 mutant and Nipponbare, the gene was delimitated to a region of 71.8-kb on the long arm of chromosome 10, which contained 15 ORFs. Sequence analysis revealed that the mutant carried two nucleotide substitutions in the eighth exon of OsCAO1 gene, which led to the substitution of leucine and glycine for histidine and glutamic acid, respectively. This implied that W390 might be a novel allele of OsCAO1 gene.

Key words: Oryza sativa L., mutant, yellow-green leaf, gene mapping

中图分类号:

董青, 张迎信, 张振华, 周全, 秦亚芝, 王宏, 程式华, 曹立勇, 沈希宏. 水稻黄绿叶突变体w390的遗传分析和基因定位[J]. 中国水稻科学, 2015, 29(3): 241-249.

Qing DONG, Ying-xin ZHANG, Zhen-hua ZHANG, Quan ZHOU, Ya-zhi QIN, Hong WANG, Shi-hua CHENG, Li-yong CAO, Xi-hong SHEN. Genetic Analysis and Gene Mapping of a Yellow-green Leaf Mutant w390 in Rice (Oryza sativa L.)[J]. Chinese Journal OF Rice Science, 2015, 29(3): 241-249.

图/表 9

表1 突变体和野生型叶片的光合色素含量比较

Table 1 Comparison of photosynthetic pigment contents in leaves between the mutant and its wild-type parent.mg/g

光合色素含量 Photosynthetic pigment content	中恢8015 R8015	w390	P
叶绿素a Chlorophyll a	2.65±0.03	1.34±0.05	<0.0001
叶绿素b Chlorophyll b	0.74±0.05	0.00	<0.0001
类胡萝卜素 Carotenoids	0.66±0.04	0.36±0.01	<0.0003
总含量 Total content	4.05±0.07	1.70±0.06	<0.0001

图1 野生型和突变体的表型

Fig. 1. Plant phenotype of the mutant and its wild-type parent.

表2 突变体和野生型的主要农艺性状比较

Table 2 Comparison of main agronomic traits between the mutant and its wild-type parent.

性状 Trait	中恢8015 R8015	w390	P
株高 Plant height/cm	112.3±5.2	98.9±5.5	0.0003
单株有效穗数 No. of panicles per plant	8.1±1.4	6.3±1.8	0.0227
每穗总粒数 No. of spikelets per panicle	118.0±9.1	96.2±7.3	<0.0001
每穗实粒数 No. of grains per panicle	71.1±6.7	51.5±5.9	<0.0001
千粒重 1000-grain weight /g	33.5±0.4	31.9±0.5	0.3067
单株产量 Grain yield per plant /g	19.3±2.8	10.3±1.4	<0.0001

图2 野生型和突变体的叶绿体超显微结构比较（A和B-野生型的叶肉细胞和叶绿体结构; C和D-突变体的叶肉细胞和叶绿体结构; N-细胞核;CP-叶绿体; G-基粒。）

Fig. 2. Comparison of ultrastructures of chloroplasts in the mesophyll cells between the mutant and its wild-type parent.（A and B, Mesophyll cell and chloroplast of wild-type, respectively; C and D, Mesophyll cell and chloroplast of mutant, respectively. N, Nucleus; CP, Chloroplast; G, Grana.）

表3 精细定位和测序所用的分子标记

Table 3 Markers used for fine mapping and sequencing.

引物名称 Primer name	上游引物 Forward primer (5'-3')	下游引物 Reverse primer (5'-3')	退火温度 Anealing temperature/℃
D2	CAGCCTCTCTAATTGACTCTC	CCAAGTAGAATGGCCAAATGT	55
D3	GCATCAAACTATAATAACTGACT	AGGAAAGTAAACAAGGCCTTA	55
D4	ACACATATGTAGAGTATTATCCG	GCAAGCTGTCTACACGGTT	55
D8	AACTCTATTGTTTATGGTGG	TAATTCCGAATTTCAGCTCT	55
CAO1-1	CATGCCTACTTGTGTCACT	ACCGCTCATGTGTACCATC	55
CAO1-2	GGCGTATTCCAAACCTATTCG	TTAGAATAAAATACGGTGTGCT	55
CAO1-3	ATTTCCTACTACCCGAAGCTG	GACTATAGTATGCGGTTACCTT	55
CAO1-4	GATTACAATTGCATCCCGTGA	ATGCCATCCACAAAGATGCTC	55
CAO1-5	CAGAAGAAGTCCATGCTCCA	CCGGTTCGATATCCAGTATTGC	55
CAO1-6	AGATGATACTCTAGTTTCCGACA	AATTAGACAAAACCACCCTCG	60

图3 突变体基因的定位与图位克隆（A-将突变体基因定位至第10染色体RM228和RM6673; B-突变体基因被界定在InDel标记D3和D4之间约71.8 kb的基因组区域内; C-序列分析发现突变体在LOC_Os10g41760的第8外显子存在两个单碱基突变,导致第394和396位的亮氨酸和甘氨酸分别突变为组氨酸和谷氨酸。）

Fig. 3. Fine mapping and positional cloning of the mutant gene.（A, The mutant locus was mapped on the chromosome 10 between RM228 and RM6673. B, The locus was narrowed to a 71.8 kb genomic region between InDel marker D3 and D4. C, Sequence analysis revealed that the mutant carried two nucleotide substitutions in the eighth exon of LOC_Os10g41760, which led to the substitution of leucine and glycine for histidine and glutamic acid, respectively.）

图4 野生型和突变体CAO1蛋白三维预测结构的比较（A-野生型; B-突变体; C-野生型与突变体的叠合。红色与深蓝色分别代表突变体位点在野生型与突变体中所在位置,紫色和浅蓝色分别代表野生型和突变体的PaO结构域。）

Fig. 4. Comparison of 3D protein structure model of CAO1 between wild-type and the mutant.（A, Wild-type; B, Mutant; C, Superposition of the mutant and its wild-type. The red and dark blue lines represent the structure haboring the mutations in the mutant and its wild-type, respectively. The purple and light blue regions represent the pheophorbide a oxygenase(PaO) domain in the mutant and its wild-type, respectively.）

图5 野生型和突变体CAO1蛋白三维模型的拉氏构象（A-野生型; B-突变体。）

Fig. 5. Ramachandran plots of 3D protein structure models of CAO1 in the mutant and its wild-type.（A, Wild-type; B, Mutant.）

图6 实时荧光定量PCR 分析野生型与突变体中叶色相关基因的表达

Fig. 6. Expression analysis of genes associated with leaf color by real-time PCR in the mutant and its wild-type.

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水稻黄绿叶突变体w390的遗传分析和基因定位

Genetic Analysis and Gene Mapping of a Yellow-green Leaf Mutant w390 in Rice (Oryza sativa L.)

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