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

[1]	Coen E S, Meyerowitz E M.The war of the whorls:Genetic interactions controlling flower development.Nature, 1991, 353(6339):31-37.
[2]	Colombo L, Franken J, Koetje E, et al.The petunia MADS box gene FBP11 determines ovule identity.Plant Cell, 1995, 7(11):1859-1868.
[3]	Ferrario S, Immink R G H, Shchennikova A, et al. The MADS box gene FBP2 is required for SEPALLATA function in petunia.Plant Cell, 2003, 15(4):914-925.
[4]	Ditta G, Pinyopich A, Robles P, et al.The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity.Curr Biol, 2004, 14(21):1935-1940.
[5]	Mandel M A, Yanofsky M F.The Arabidopsis AGL8 MADS box gene is expressed in inflorescence meristemsand is negatively regulated byAPETALA1. Plant Cell, 1995, 7(11):1763-1771.
[6]	Lohmann J U, Weigel D.Building beauty: The genetic control of floral patterning.Dev Cell, 2002, 2(2):135-142.
[7]	Lamb R S, Irish V F.Functional divergence with in the APETAL3/PISTILLATA floral homeotic gene lineages.Proc Natl Acad Sci USA, 2003, 100(11):6558-6563.
[8]	Jeon J, Lee S, Jung K H, et al.Production of transgenic rice plants show in greduced heading date and plant height by ectopic expression of rice MADS-box genes.Mol Breeding, 2000, 6(6):581-592.
[9]	Wang K J, Tang D, Hong L L, et al.DEP and AFO regulate reproductive habit in rice.PLoS Genet, 2010, 6(1):1-9.
[10]	Lu S J, Wei H, Wang Y, et al.Overexpression of a transcription factor OsMADS15 modifies plant architecture and flowering time in rice (Oryza sativa L.).Plant Mol Biol Rep, 2012, 30(6):1461-1469.
[11]	Fornara F, Parenicova L, Falasca G, et al.Functional characterization of OsMADS18, a member of the AP1/SQUA subfamily of MADS box genes.Plant Physiol, 2004, 135(4):2207-2219.
[12]	Kobayashi K, Yasuno N, Sato Y, et al.Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-Like MADS box genes and PAP2, a SEPALLATA MADS Box gene.Plant Cell, 2012, 24(5):1848-1859.
[13]	Sentoku N, Kato H, Kitano H, et al.OsMADS22, an STMADS11-like MADS-box gene of rice, is expressed in non-vegetative tissues and its ectopic expression induces spikelet meristem indeterminacy.Mol Gene Genomics, 2005, 273(1):1-9.
[14]	Xiao H, Wang Y, Liu D F, et al.Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference.Plant Mol Biol, 2003, 52(5):957-966.
[15]	Nagasawa N, MiyOshi M, Sano Y, et al. SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice.Development, 2003, 130(4):705-718.
[16]	Arora R, Agarwal P, Ray S, et al.MADS-box gene family in rice: Genome-wide identification, organization and expression profiling during reproductive development and stress.BMC Genom, 2007, 8:242-262.
[17]	Yadav S R, Prasad P, Vijayraghavan U.Divergent regulatory OsMADS2 functions control size, shape and differentiation of the highly derived rice floret second-whorl organ.Genetics, 2007, 176(1):283-294.
[18]	Yamaguchi T, Lee D Y, Miyao A, et al.Functional diversification of the two C-class MADS box genes OsMADS3 and OsMADS58 inOryza sativa. Plant Cell, 2006, 18(1):15-28.
[19]	Dreni L, Pilatone A, Yun D P, et al.Functional analysis of all AGAMOUS subfamily members in rice reveals their roles in reproductive organ identity determination and meristem determinacy.Plant Cell, 2011, 23(8):2850-2863.
[20]	Yamaguchi T, Nagasawa N, Kawasaki D, et al.The YABBY gene DROOPING LEAF regulates carpel specification and midrib development inOryza sativa. Plant Cell, 2004, 16(2):500-509.
[21]	Dreni L, Jacchia S, Fornara F, et al.The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice.Plant J, 2007, 52(4):690-699.
[22]	Yin L L, Xue H W.The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development.Plant Cell, 2012, 24(3):1049-1065.
[23]	Jeon J S, Jang S, Lee S, et al.An leafy hull sterile1 is a homeotic mutation in a rice MADS box gene affecting rice flower development.Plant Cell, 2000, 12(6):871-884.
[24]	Prasad K, Parameswaran S, Vijayraghavan U.OsMADS1, a rice MADS-box factor, controls differentiation of specific cell types in the lemma and palea and is an early-acting regulator of inner floral organs.Plant J, 2005, 43(6):915-928.
[25]	Khanday I, Yadav R S, Vijayraghavan U.Rice LHS1/OsMADS1 controls floret meristem specification by coordinated regulation of transcription factors and hormone signaling pathways.Plant Physiol, 2013, 161(4):1970-1983.
[26]	Cui R F, Han J K, Zhao S Z, et al.Functional conservation and diversification of class E floral homeotic genes in rice (Oryza sativa).Plant J, 2010, 61(5):767-781.
[27]	Moon S, Jung K H, Lee D E, et al.The rice FON1 gene controls vegetative and reproductive development by regulating shoot apical meristem size.Mol Cells, 2006, 21(1):147-152.
[28]	Suzaki T, Ohneda M, Toriba T, et al.FON2 SPARE1 redundantly regulates floral meristem maintenance with FLORAL ORGAN NUMBER2 in rice.PLoS Genet, 2009, 5(10):1-9.
[29]	Chu H W, Qian Q, Liang W Q, et al.The FLORAL ORGAN NUMBER4 gene encoding a putative ortholog of Arabidopsis CLAVATA3 regulates apical meristem size in rice.Plant Physiol, 2006, 142(3):1039-1052.
[30]	Xiao H, Tang J F, Li Y F, et al.STAMENLESS 1, encoding a single C2H2 zinc finger protein, regulates floral organ identity in rice.Plant J, 2009, 59(5):789-801.
[31]	Jang S, Lee B, Kim C, et al.The OsFOR1 gene encodes a polygalacturonase-inhibiting protein (PGIP) that regulates floral organ number in rice.Plant Mol Biol, 2003, 53(3):357-372.
[32]	Ren D Y, Li Y F, Zhao F M, et al.MULTI-FLORET SPIKELET1, which encodes an AP2/ERF protein, determines spikelet meristem fate and sterile lemma identity in rice.Plant Physiol, 2013, 162(2):872-884.
[33]	Yoshida A, Suzaki T, Tanaka W, et al.The homeotic gene long sterile lemma (G1) specifies sterile lemma identity in the rice spikelet.Proc Natl Acad Sci USA, 2009, 106(47):20103-20108.
[34]	卢扬江, 郑康乐. 提取水稻DNA 的一种简易方法. 中国水稻科学, 1992, 6(1):47-48.
[35]	李云峰. 水稻小穗不确定性基因LHS1-3和雄蕊雌蕊化基因PS的图位克隆与功能分析. 重庆:西南大学. 2008.
[36]	Agrawal G K, Abe K, Yamazaki M, et al.Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene.Plant Mol Biol, 2005, 59(1):125-135.
[37]	Chen J J, Ding J H, Ouyang Y D, et al.A triallelic system of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice.Proc Natl Acad Sci USA, 2008, 105(32):11436-11441.
[38]	Yang J Y, Zhao X B, Cheng K, et al.A killer-protector system regulates both hybrid sterility and segregation distortion in rice.Science, 2012, 337(6100):1336-1340.

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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