水稻雌性败育基因FA的图位克隆

doi:10.16819/j.1001-7216.2018.8023

摘要/Abstract

摘要：

目的对水稻胚囊突变体的表型观察、遗传定位和基因克隆,将为研究植物生殖发育奠定理论基础。方法从粳稻品种宁粳4号突变体库中筛选出一个雌性败育突变体female abortion(fa),对野生型和突变体不同发育时期的胚囊进行细胞学观察并统计胚囊类型。以突变体杂合型为母本,N22为父本构建定位群体,对该表型进行遗传分析,采用图位克隆方法精确定位目的基因。结果表型分析显示,突变体雌蕊在外观上没有表现出差异,但成熟时植株完全不育。与野生型相比, 该突变体胚囊发育异常,不能形成七细胞八核胚囊结构,而且形成多种类型的异常胚囊。遗传分析表明,该突变性状受一对隐性核基因控制。我们将该基因初定位在第1染色体的分子标记L1和L3之间,通过扩大定位群体,最终将基因定位在分子标记L10和L11之间,物理距离为117 kb。测序分析发现该区间内LOC_{_}Os01g68870外显子上有一个单碱基替换,由胸腺嘧啶(T)替换成胞嘧啶(C),氨基酸由亮氨酸变为脯氨酸,从而导致该表型的出现。qRT-PCR结果显示,相对于OsMADS13、OsAPC6、OsTDL1A基因在突变体fa胚囊中表达量而言,OsDEES1基因在突变体fa胚囊中表达量变化最为显著,FA可能是OsDEES1的上游基因。亚细胞定位结果显示,该蛋白定位于质膜。结论 FA是多孢囊基因MULTIPLE SPOROCYTE 1(MSP1)的新等位基因。本研究进一步明确了该基因对于水稻胚囊发育的重要性,可为探明它所在的调控网络体系提供新的线索。

关键词: 雌性败育突变体, 遗传分析, 图位克隆, 水稻

Abstract:

【Objective】Phenotypic observation, genetic mapping and gene cloning of rice embryo sac mutants will lay a theoretical basis for recovering plant reproductive development.【Method】A female abortion (fa) mutant was obtained from ethyl methylsulfonate (EMS)-treated Ningjing 4. The embryo sacs of the wild-type and mutant were observed at different developmental stages, and the types of mutant embryo sacs were also calculated. The heterozygous mutant was used as the female parent and N22 as the male parent to construct the mapping population. The phenotype was analyzed by genetic analysis, and the target gene was precisely mapped by map-based cloning method. 【Result】Phenotypic analysis showed that the mutant’s pistil showed no difference in appearance, but the plants showed complete sterility at maturity. Compared with the wild type, the development of the mutant embryo sacs was abnormal, which could not form seven-cell eight-nuclear structure. Genetic analysis showed that the fa mutant traits were controlled by a single recessive gene. The gene was initially mapped between the L1 and L3 markers on chromosome 1 and finally between markers L10 and L11 by expanding the mapping population, with a physical distance of 117 kb. Sequencing analysis showed that LOC_Os01g68870 had a single base substitution in exon, from thymine(T) to cytosine(C), leading to amino acid change from leucine to proline and this phenotype. The results of qRT-PCR showed that compared with OsMADS13, OsAPC6 and OsTDL1A, the expression level of OsDEES1 in the mutant fa embryo sacs was the most significant, and FA might act at OsDEES1 upstream. Subcellular localization showed that the FA protein is localized to the plasma membrane. 【Conclusion】 According to the present study, FA gene was a new allele of MSP1. Our work further elucidates the importance of this gene in the development of embryo sacs and provides new clues in regulation the network system.

Key words: female abortion mutant, genetic analysis, map-based cloning, rice

中图分类号:

吕凤, 唐倩莹, 王启明, 郑海, 尤世民, 柏文婷, 肖晏嘉, 赵志刚, 万建民. 水稻雌性败育基因FA的图位克隆[J]. 中国水稻科学, 2018, 32(6): 519-528.

Feng LÜ, Qianying TANG, Qiming WANG, Hai ZHENG, Shimin YOU, Wenting BAI, Yanjia XIAO, Zhigang ZHAO, Jianmin WAN. Map-based Cloning of Female Abortion (FA) Gene in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(6): 519-528.

图/表 8

表1 本研究定位和定量PCR所用引物序列

Table 1 Primer for gene mapping and qRT-PCR.

正向引物 Forward sequence(5'-3')	反向引物序列 Reverse sequence(5'-3')	实验目的 Purpose
ACGCTGAAAAGCAAGGATGT	TTCTAGCCCTCCTCTTTGACA	初定位 Preliminary mapping
AATTCGGTCACTCGCTGTCACG	CTGCGGACGAAATTGCTTAGCC	初定位 Preliminary mapping
GGGATTATTTGAAATCTTTGC	ATATAGCATTGCCAGTTTGC	初定位 Preliminary mapping
GTCGACGGCTTCCTCAAGATTGG	TGAGACCTCTGTGAAGGCACTCG	初定位 Preliminary mapping
CGAGTTCGTCCAAGTGAGC	CATCCACCATTCCACCAATC	精细定位Fine maping
GAAAGGTGATGGGAGAGCAG	TGACACCCTCTCTCCACCTC	精细定位Fine maping
AAGCCAGCAATGTTATGAGC	CATTACCAGCAGCGGAGTA	精细定位Fine maping
CATGGGCCAGAATTAAGAGG	CATCCACTTTCCTCTCCTGC	精细定位Fine maping
GCATGCTACCACCTTGACTGC	GTGAGTAGCGAGACCGAGAGTGC	精细定位Fine maping
AGCTGTGCGAGGAATCCAAACG	CTCCGATCCCAGCAGCTACTCC	精细定位Fine maping
GTTATATGGCTCTGGCACGC	TTGTTTAGGACACCGCTTGC	精细定位Fine maping
TGTTGTGCAAGTGATTGGCA	CAAGACACATCGTACCGCTG	精细定位Fine maping
TTGCTCCTCCCTACAACAGT	AGCTATGGCAAGAAGAGGTGA	精细定位Fine maping
TGGTTACCTGCCTTGATCGA	CTTTCTGCTCCGTTTGTTGC	精细定位Fine maping
TGTGGCTGAGAAACCGAGCA	CGACTTCATGTCCCGGTGGA	qRT-PCR
ACGACGACTGCCTCCTCAAC	GAGGCGTAGCAAATGGTGAG	qRT-PCR
TGCCAGGAATAAGTCTGCTG	TAATACTTCGGCAAGCAACG	qRT-PCR
CCCGGTAAGTGAGGGTACAA	TGATCCAAAACCACTCCAGA	qRT-PCR

图1 野生型和fa 突变体表型^ A–野生型和突变体fa的株型和抽穗形态,标尺为20 cm。B–野生型和突变体fa颖花表型,标尺为2 mm。le–外稃;pa–内稃;gl–护颖。C–野生型和突变体fa的雌蕊表型,标尺为1 mm。D–野生型和突变体fa的成熟穗,标尺为2.5 cm。

Fig. 1. Phenotypes of the wild type and the fa mutant.^ A, Plant morphology of the wild type(WT) and the fa mutant during heading, bar=20 cm. B, Spikelet of WT and the fa mutant, bar=2 mm. le, Lemma; pa, Palea; gl, Glume. C, Pistil of WT and the fa mutant, bar=1 mm. D, Mature spikelet of WT and the fa mutant, bar=2.5 cm.

图2 野生型和fa 突变体的胚囊发育过程观察及其不同类型的胚囊统计^ A~D,I~L为野生型胚囊发育过程;E~H,M~T代表突变体fa胚囊发育过程。A和E–孢原细胞期;B和F–大孢子母细胞期;C和G–四分体期;D和H–功能大孢子期;I和M–二核胚囊期;J和N–四核胚囊期;K和O–八核胚囊期;L和P~T–成熟胚囊期。箭头指示核。U–统计野生型(n=156)和突变体fa(n=216)成熟胚囊中不同类型的胚囊。An–反足细胞;Ar–胞原细胞;Ec–卵细胞;MMC–大孢子母细胞;Pn–极核。A~P标尺为50 µm,Q~T标尺为100 µm。

Fig. 2. Embryo sac development of the wild type(WT) and fa mutant and statistics of different types of embryo sacs.^ A to D and I to L, Wild type; E to H and M to T, fa mutant. A and E, Archesporial cell stage; B and F, Megaspore mother cell stage; C and G, Tetrad stage; D and H, Functional megaspore stage; I and M, Bi-nuclear embryo sac; J and M, Tetra-nuclear embryo sac; K and O, Eight-nuclear embryo sac; L and P to T, Mature embryo sac. Arrows indicate nuclei. U, Statistics of different types of embryo sacs in mature embryo sac of the wild type (n=156) and the mutant (n=216). An, Antipode cell; Ar, Archesporial cell; Ec, Egg cell; MMC, Megaspores mother cell; Pn, Polar nuclei; NES, Normal embryo sac; MNES, Multi-nucleated embryo sac; DES, Degenerated embryo sac; EES, Empty sac with no embryo formation; Others, Other abnormal embryo sacs. Bar= 50 µm in A to P and 100 µm in Q to T.

表2 突变体杂交组合授粉情况与成熟种子数目统计

Table 2 Combinations of the fa mutant and statistics of mature seeds.

杂交组合 Combination	授粉的花数目 No. of flowers pollinated	成熟种子数目 No. of mature seeds
fa×宁粳4 fa×Ningjing 4 fa×fa	128 143	15 0

图3 FA基因在第1染色体上的分子定位及结构分析^ A–FA基因的精细定位,P0413C03和P0678F11是基因组BAC登录号,FA基因定位在两个分子标记L10和L11间的117 kb区间内;B–FA基因的结构,黑色框代表由起始密码子ATG(+1)开始到终止密码子TAA(+1295)结束的外显子,编码1294个氨基酸,第655个氨基酸到第883个氨基酸代表LRR-RLK (Leu-rich repeat receptor-like protein kinase)结构域,第1001到第1278个氨基酸代表激酶结构域。第1074个氨基酸由亮氨酸替换成脯氨酸。蓝色框代表5′非翻译区,白色框代表3′非翻译区。

Fig. 3. FA gene mapping on chromosome 1 and structural analysis.^ A, Fine mapping of the FA gene, P0413C03 and P0678F11 are genomic BAC accession numbers, and the FA gene is mapped to a 117-kb region between two molecular markers L10 and L11; B, Genomic structure of the FA gene, black box represent exon beginning with the start codon ATG (+1) and ending with the stop codon TAA (+1295), encoding 1294 amino acids, and amino acids 655 to 883 representing LRR-RLKs(Leu-rich repeat receptor-like protein kinase) domain, and the 1001th to 1278th amino acids represent a kinase domain. The 1074th amino acid leucine is replaced by proline. The blue box represents the 5'- untranslated region and the white box represents the 3'- untranslated region.

图4 FA基因在水稻各组织中的相对表达量^以OsUbiquitin作为对照,误差线表示n=3时的标准差。S1―孢原细胞形成期,S2―大孢子母细胞形成期,S3a―二分体时期,S3b-四分体时期,S4―功能大孢子形成期。

Fig. 4. Relative expression levels of FA in rice tissues. ^ PA, Panicle; R, Root; ST, Stem; YL, Young Leaf; LS, Leaf sheath; A, Anther; PI, Pistil. OsUbiquitin was used as an internal control, Error bars show SD (n=3). S1, Archesporial cell stage; S2, Megaspore mother cell stage; S3a, Dyad stage; S3b, Tetrad stage; S4, Functional megaspore stage.

图5 FA蛋白在烟草中的亚细胞定位^ A–FA-GFP在烟草表皮细胞中的亚细胞定位;B–膜标签在烟草表细胞中的定位;C–明场视野下烟草表细胞状态;D–GFP、膜标签和明场的合并。GFP代表绿色荧光蛋白。标尺=100 µm。

Fig. 5. Subcellular localization of FA protein in tobacco. ^ A, Subcellular localization of FA-GFP in tobacco epidermal cells; B, Localization of membrane marker in tobacco epidermal cells; C, Bright field of tobacco epidermal cell status; D, The merging of GFP, membrane marker and bright field. GFP indicates the green fluorescence of proteins. Bar=100 µm.

图6 野生型和fa突变体不同时期中胚囊相关基因的表达^野生型(WT)和突变体fa胚囊发育相关基因在不同时期的表达量。以OsUbiquitin作为对照,误差线表示n=3时的标准差。*和**分别代表野生型与突变体fa差异达0.05和0.01极显著水平(t测验)。S1―孢原细胞形成期,S2―大孢子母细胞形成期,S3a―二分体时期,S3b-四分体时期,S4―功能大孢子形成期。

Fig. 6. Expression of embryo sac related genes of the wild type and fa mutant at different stages.^ Expression levels of gene related to embryo sac development in the wild type (WT) and mutant fa at different stages. OsUbiquitin was used as an internal control; Error bars show SD (n=3). * and ** indicate significant difference between the wild type and fa mutant by t-test, respectively(P<0.05, P<0.01). S1, Archesporial cell stage; S2, Megaspore mother cell stage; S3a, Dyad stage; S3b, Tetrad stage; S4, Functional megaspore stage.

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