一个由可变剪接造成的水稻开颖不育突变体ohms1 的鉴定及基因定位

doi:10.3969/j.issn.1001G7216.2015.05.002

中国水稻科学 ›› 2015, Vol. 29 ›› Issue (5): 457-466.DOI: 10.3969/j.issn.1001G7216.2015.05.002

一个由可变剪接造成的水稻开颖不育突变体ohms1 的鉴定及基因定位

孙廉平^1,², 张迎信², 张沛沛², 杨正福², 占小登², 沈希宏², 张振华², 胡霞², 轩丹丹², 吴玮勋², 曹立勇^2,^*(), 程式华^1,^2,^*()

中国水稻研究所/国家水稻改良中心/浙江省超级稻研究重点实验室, 杭州 310006

收稿日期:2015-02-04 修回日期:2015-05-06 出版日期:2015-09-10 发布日期:2015-09-10
通讯作者: 曹立勇,程式华
作者简介:
^*通讯录作者：E-mail:caoliyong@cass.cn;shcheng@mail.hz.zj.cn
基金资助:
基金项目: 国家科技支撑计划资助项目(2011BAD35B02)国家转基因重大专项(2011ZX08001-002)浙江省自然科学基金资助项目(LQ14C130003)中国农业科学院创新工程超级稻育种创新团队、水稻杂种优势研究机理研究创新团队(CAAS-ASTIP-2013-CNRRI)

Characterization and Gene Mapping of an Open Hull Male Sterile Mutant ohms1 Caused by Alternative Splicing in Rice

Lian-ping SUN^1,², Ying-xin ZHANG², Pei-pei ZHANG², Zheng-fu YANG², Xiao-deng ZHAN², Xi-hong SHEN², Zhen-hua ZHANG², Xia HU², Dan-dan XUAN², Wei-xun WU², Li-yong CAO^2,^*(), Shi-hua CHENG^1,^2,^*()

¹College of Agronomy, Shenyang Agricultural University, Shenyang 110866, China
²National Center for Rice Improvement/Key Laboratory for Zhejiang Super Rice Research,China National Rice Research Institute, Hangzhou 310006, China

Received:2015-02-04 Revised:2015-05-06 Online:2015-09-10 Published:2015-09-10
Contact: Li-yong CAO, Shi-hua CHENG
About author:
^*Corresponding author：E-mailcaoliyong@cass.cn;shcheng@mail.hz.zj.cn

摘要/Abstract

摘要：

在⁶⁰Co-γ射线辐射诱变籼稻中恢8015的突变体库内发现了一个花器官发育突变体,暂命名为开颖不育突变体ohms1(open hull and male sterile 1)。ohms1突变体表现为颖花开裂,在雄蕊和柱头之间形成类似内外稃的结构,使得突变体的颖花形成类似“三齿稃”状的三个颖壳,小穗完全不育,花粉育性为60%~70%,但自交不结实。遗传分析和基因定位结果表明,ohms1受一对隐性单基因控制,位于第3染色体短臂KY2和KY29标记之间,物理距离约42 kb,该区域包含4个开放阅读框ORFs。进一步序列分析发现,突变体中一个编码MADS盒的基因LOC_Os03g11614的第5内含子末位碱基由A突变为G。酶切实验和cDNA测序证实,该基因的第5内含子未被剪切,致使该基因的第6外显子所编码的14个氨基酸完整缺失,但并未造成该蛋白MADS结构域的改变或移码。qRT-PCR结果显示,突变体中OsMADS1 基因的表达水平显著降低,水稻开花调控因子和内外稃发育调控基因的表达量也发生了显著变化。说明该基因对水稻花器官发育尤其是内外稃发育和小花原基的分化具有重要作用。

关键词: 水稻, 开颖不育, 基因定位, 可变剪接

Abstract:

A rice floral organ development mutant was obtained from the ⁶⁰Co-γ-treated indica restorer line Zhonghui 8015 and termed as open hull and male sterile 1 (ohms1). The mutant was characterized as open hull and lemma- and palea-like structure between anthers and stigma that made the spikelet of the mutant showed three “triodia-like ” flower glumes. The mutant was self-sterile but 60%-70% pollen were fertile. Genetic analysis and gene mapping showed ohms1 was controlled by a single ressessive gene and the mutant gene was fine mapped to a region of 42 kb on the short arm of chromosome 3 between markers KY2 and KY29. Sequence analysis of the four open reading frames(ORFs) in this region revealed that LOC_Os03g11614 was probably corresponding to ohms1, which encoded a MADS-box gene allelic with OsMADS1, and had a single nucleotide transformation (A to G) at the bottom of the fifth intron. Enzyme digestion and cDNA sequencing furtherly indicated that the variable splicing was responsible for the delection of the sixth exon of ohms1,but no structure changes in MADS domain or amino acid frameshift occurred. Additionally,real-time fluorescence quantitative PCR analysis showed that the expression level of OHMS1 decreased significantly and the expression level of rice flowering factors and floral glume development-related genes changed significantly. All those results demonstrated that OHMS1 may play an important role in rice floral organ development,particularly in floral glume development and floret primordium differentiation.

Key words: rice, open hull male sterile, gene mapping, alternative splicing

孙廉平, 张迎信, 张沛沛, 杨正福, 占小登, 沈希宏, 张振华, 胡霞, 轩丹丹, 吴玮勋, 曹立勇, 程式华. 一个由可变剪接造成的水稻开颖不育突变体ohms1 的鉴定及基因定位[J]. 中国水稻科学, 2015, 29(5): 457-466.

Lian-ping SUN, Ying-xin ZHANG, Pei-pei ZHANG, Zheng-fu YANG, Xiao-deng ZHAN, Xi-hong SHEN, Zhen-hua ZHANG, Xia HU, Dan-dan XUAN, Wei-xun WU, Li-yong CAO, Shi-hua CHENG. Characterization and Gene Mapping of an Open Hull Male Sterile Mutant ohms1 Caused by Alternative Splicing in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(5): 457-466.

图/表 8

参考文献 38

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引物名称 Primer name	上游引物 Forward primer(5'-3')	下游引物 Reverse primer(5'-3')	实验目的 Purpose
InD44	GGAATCCCTCCCTTCTTGTC	GGTCGGTAAAGACGGTGAAA	Fine mapping
KY2	GTGGGAAGAAGAACATCAACTG	GCACACAAGATAAACCCAATCAGC	Fine mapping
KY12	ACCACGAGGGTGACCGTAGA	GCGAGGGTTGATGAGATAGCA	Fine mapping
KY17	CGAGAGGCGAAGGAAATAGAACG	CTCCTCCTCCTCCTGGTTCTCC	Fine mapping
KY25	CCATGGTCGCCATTGACACG	CCTGCTATAACACTCGCACAGATGC	Fine mapping
KY26	GGTGGTGAGCCAAGAACTGACC	CCTCAAGGAATCCTCGTAAGTCG	Fine mapping
KY29	CCAAGTGTGTCCGAGCTTAGTGC	TGAGTCAAAGCGAAAGTCAACAGG	Fine mapping
RM7576	CTGCCCTGCCTTTTGTACAC	GCGAGCATTCTTTCTTCCAC	Fine mapping
InD45	CCAGGGATCTTCTCATCCAA	CCTGGCTAGCATACCACACA	Fine mapping
InD46	GCCATTGATCTTCTGCAGGT	TTTGTTGTCAATGCCCTGTT	Fine mapping
RD0304	GGCGTCACTGCTCGTA	GCCTGAAGCGTCCACA	Fine mapping
CAPS1	GCCATCGATCACCCTGAAAGTC	CTGATCAGCAAGAACAGTGC	ohms1 site detection
CKY	AGCCAAACCACACCACCATAAAG	AGGACACTGTTTGCATTGGCT	cDNA sequencing

引物名称 Primer name	上游引物 Forward primer(5'-3')	下游引物 Reverse primer(5'-3')	实验目的 Purpose
InD44	GGAATCCCTCCCTTCTTGTC	GGTCGGTAAAGACGGTGAAA	Fine mapping
KY2	GTGGGAAGAAGAACATCAACTG	GCACACAAGATAAACCCAATCAGC	Fine mapping
KY12	ACCACGAGGGTGACCGTAGA	GCGAGGGTTGATGAGATAGCA	Fine mapping
KY17	CGAGAGGCGAAGGAAATAGAACG	CTCCTCCTCCTCCTGGTTCTCC	Fine mapping
KY25	CCATGGTCGCCATTGACACG	CCTGCTATAACACTCGCACAGATGC	Fine mapping
KY26	GGTGGTGAGCCAAGAACTGACC	CCTCAAGGAATCCTCGTAAGTCG	Fine mapping
KY29	CCAAGTGTGTCCGAGCTTAGTGC	TGAGTCAAAGCGAAAGTCAACAGG	Fine mapping
RM7576	CTGCCCTGCCTTTTGTACAC	GCGAGCATTCTTTCTTCCAC	Fine mapping
InD45	CCAGGGATCTTCTCATCCAA	CCTGGCTAGCATACCACACA	Fine mapping
InD46	GCCATTGATCTTCTGCAGGT	TTTGTTGTCAATGCCCTGTT	Fine mapping
RD0304	GGCGTCACTGCTCGTA	GCCTGAAGCGTCCACA	Fine mapping
CAPS1	GCCATCGATCACCCTGAAAGTC	CTGATCAGCAAGAACAGTGC	ohms1 site detection
CKY	AGCCAAACCACACCACCATAAAG	AGGACACTGTTTGCATTGGCT	cDNA sequencing

组合 Combination	F₁结实率 Seed-setting rate of F₁/%	F₂
组合 Combination	F₁结实率 Seed-setting rate of F₁/%	野生型植株数 No. of wild-type plants			突变表型植株数 No. of mutant-type plants
ohms1/中花11 ohms1/Zhonghua 11	85.22	374	113	0.84	3.84
ohms1/02428	88.76	4658	1496	1.57

组合 Combination	F₁结实率 Seed-setting rate of F₁/%	F₂
组合 Combination	F₁结实率 Seed-setting rate of F₁/%	野生型植株数 No. of wild-type plants			突变表型植株数 No. of mutant-type plants
ohms1/中花11 ohms1/Zhonghua 11	85.22	374	113	0.84	3.84
ohms1/02428	88.76	4658	1496	1.57

一个由可变剪接造成的水稻开颖不育突变体ohms1 的鉴定及基因定位

Characterization and Gene Mapping of an Open Hull Male Sterile Mutant ohms1 Caused by Alternative Splicing in Rice

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