色氨酰-tRNA合成酶基因WRS1调控水稻种子发育

doi:10.16819/j.1001-7216.2020.0302

摘要/Abstract

摘要：

【目的】氨酰-tRNA合成酶（aminoacyl-tRNA synthetases, aaRSs）与遗传信息传递密切相关,已发现植物中aaRSs家族蛋白在维持翻译功能之余,还参与配子发生与胚发育、质体的早期发育以及免疫信号的感知与病害防御等生物学过程。本研究利用水稻胚乳发育缺陷突变体,分析水稻色氨酰-tRNA合成酶（WRS1）在胚乳发育中的作用,证明WRS1基因编码一个影响水稻胚乳发育的关键因子。【方法】本研究通过甲烷磺酸乙酯（ethyl methane sulfonate, EMS）诱变籼稻（Oryza sativa subsp. indica）品种N22,筛选到一个稳定遗传的水稻粉质胚乳突变体（wrs1）,图位克隆获得目标基因。对wrs1成熟种子进行形态学观察以及淀粉相关理化性质测定,利用细胞学切片分析wrs1发育中胚乳的结构,利用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）和GUS活性染色分析基因表达模式,通过qRT-PCR比较野生型与突变体花后12 d胚乳中淀粉合成相关基因表达情况,免疫印迹检测野生型与突变体成熟种子中淀粉合成酶蛋白积累情况,使用全自动氨基酸分析仪测定游离氨基酸含量。【结果】 wrs1突变体幼苗表现出明显的发育滞后且逐渐蔫萎死亡,从杂合突变体（WRS1wrs1）中分离到的粉质籽粒呈现明显的腹部皱缩,粒厚、千粒重下降,同时总淀粉含量下降,糊化淀粉的峰值黏度和崩解值均低于野生型。wrs1突变体发育胚乳中复合淀粉颗粒变小,排列疏松。WRS1定位于第12染色体长臂约183 kb的区间内,测序发现编码色氨酰-tRNA合成酶（tryptophanyl-tRNA synthetase, WRS）基因的第6外显子上发生单碱基替换,导致一个保守位置上的甲硫氨酸被替换。wrs1突变体中大部分淀粉合成相关基因表达量下调,且野生型与突变体间基因表达的变化与相应蛋白积累的差异存在不一致的趋势。wrs1突变体籽粒中蛋白质积累降低,而游离氨基酸含量显著升高。【结论】 WRS1编码色氨酰-tRNA合成酶,该基因突变后通过影响氨基酸稳态和蛋白质合成,造成淀粉合成相关基因异常表达从而影响淀粉的合成与积累,导致种子发育缺陷。

关键词: 粉质胚乳, 造粉体, 氨酰-tRNA合成酶, 蛋白翻译

Abstract:

【Objective】 Aminoacyl-tRNA synthetases (aaRSs) are closely related to the transmission of genetic information. Besides translation, aaRSs in plants participate in gametogenesis and embryo development, early plastid development, immune signal perception and disease defense. In this study, we used a rice endosperm defective mutant to analyze the function of tryptophanyl-tRNA synthase (WRS1) during seed development, proving that WRS1 gene encodes a key factor affecting rice endosperm development. 【Method】 In this study, a stably-inherited rice floury endosperm mutant (wrs1) was screened from the mutant library of indica cultivar N22 (Oryza sativa subsp. indica) induced by ethyl methane sulfonate (EMS). Map-based cloning and complementation test identified the target gene. Morphological observation and starch physicochemical properties of wrs1 mature seeds were analyzed. Semi-thin sections were prepared to observe the developing endosperm structure with a scanning electron microscope. qRT-PCR and GUS staining were performed to analyze the expression of WRS1. The expression of starch synthesis related genes in the endosperm at 12 days after flowering was determined by qRT-PCR, and these protein expression levels in mature seed were detected by immunoblotting. The free amino acid contents of mature seeds were measured with a fully automatic amino acid analyzer. 【Result】 The seedlings of wrs1 were featured by obvious delay in development and finally withered and died. The floury grains isolated from the heterozygous mutant (WRS1wrs1) showed shrunken belly, decreased grain thickness and thousand-grain weight. Total starch contents, the peak viscosity and breakdown viscosity of pasting starch were lower in wrs1. The compound starch granules in developing endosperm of wrs1 were smaller and loosely arranged. WRS1 was restricted to the 183 kb region of the long arm of chromosome 12. Sequencing revealed a single base substitution in exon 6 of the tryptophanyl-tRNA synthetase gene (WRS1), resulting in a substitution of methionine. The expression of most starch synthesis-related genes in wrs1 was down-regulated, while these proteins showed different accumulation levels. The contents of proteins in wrs1 grains were decreased, while free amino acids contents were significantly increased. 【Conclusion】 WRS1 encodes tryptophanyl-tRNA synthase. Mutation of this gene affects amino acid homeostasis and protein synthesis in rice endosperm, resulting in abnormal expression of the genes in starch synthesis pathway which affects starch synthesis and accumulation, and eventually lead to seed development defects.

Key words: floury endosperm, amyloplast, aminoacyl-tRNA synthetase, protein translation

中图分类号:

唐小涵, 刘世家, 刘喜, 田云录, 王云龙, 滕烜, 段二超, 张元燕, 江玲, 张文伟, 王益华, 万建民. 色氨酰-tRNA合成酶基因WRS1调控水稻种子发育[J]. 中国水稻科学, 2020, 34(5): 383-396.

Xiaohan TANG, Shijia LIU, Xi LIU, Yunlu TIAN, Yunlong WANG, Xuan TENG, Erchao DUAN, Yuanyan ZHANG, Ling JIANG, Wenwei ZHANG, Yihua WANG, Jianmin WAN. Tryptophanyl-tRNA Synthetase Gene WRS1 Regulates Rice Seed Development[J]. Chinese Journal OF Rice Science, 2020, 34(5): 383-396.

图/表 10

表1 wrs1转基因家系鉴定所用引物

Table 1 Markers used for identification in wrs1 transgenic lines.

引物名称 Primer name	正向引物序列 Forward (5′-3′)	反向引物序列 Reserve (5′-3′)	产物大小 Product size / bp
N4	TCCCTGATTCCATTTCTC	TTATGCCTCCATTGTTGA	404
N10	GGCCATCTCATCCCCTTCAT	AATTGAGTCCCGGCCACC	633
Hyg	CTATTTCTTTGCCCTCGGAC	ATGCCTGAACTCACCGCGAC	1083

图1 野生型与wrs1突变体表型分析 A, B—野生型（A）和wrs1突变体（B）成熟种子外观比较,标尺为2 mm;C~H—野生型（C, D, E）和wrs1突变体（F, G, H）成熟种子横断面的扫描电镜观察（局部）,由上到下依次对应野生型和wrs1突变体中的黑框、红框、白框,标尺为5 μm;I, J—野生型（I）和wrs1（J）突变体种子吸胀后的胚（30℃下吸胀10 h）,标尺为1 mm;K~M—野生型和突变体不同时期的发育情况,分别为30℃催芽24 h（K）、发芽后3 d（L）,发芽后20 d（M）的种子/幼苗发育状况,K, L标尺为5 mm,M标尺为5 cm;N—千粒重比较,n = 3;O—籽粒大小比较,n = 20。所有数值为平均值±标准差,t检验,*表示在P<0.05水平上差异显著。

Fig. 1. Phenotypic analysis of wild-type and the wrs1 mutant. A, B, Comparison of mature seeds of wild-type (A) and the wrs1 mutant (B). Bars = 2 mm. C~H, Scanning electron microscopic (SEM) analysis of cross-sections of wild-type (C-E) and wrs1 mutant (F-H) mature seeds. C-E and F-H correspond to the black frame, red frame, and white frames in wild-type and wrs1, respectively. Bars = 5 μm. I, J, Embryos of wild-type (I) and wrs1 mutant (J) seeds after imbibition (30℃, 10 h). Bars = 1 mm. K~M, Developmental status of wild-type and the wrs1 mutant. 24 hours after seed germination (K), 3 days after germination (L), and 20 days after germination (M). Bars = 5 mm in K and L, 5 cm in M. N, 1000-grain weight, n = 3. O, Grain length, width and thickness, n = 20. All values are means ± SD. *indicates significant difference at P<0.05 level(Student’s t-test).

图2 野生型和wrs1突变体种子淀粉含量和理化特性分析 A, B—N22和wrs1突变体胚乳中总淀粉（A）与直链淀粉（B）含量,n = 3;C—野生型和wrs1突变体黏度特性比较;D—不同尿素浓度下N22和wrs1突变体米粉的膨胀情况;E—野生型和wrs1突变体米粉膨胀体积比较,n = 3。所有数值取平均值±标准差,t检验,*和**分别表示在0.05和0.01水平上差异显著。

Fig. 2. Content and physicochemical characteristics of starches in mature seeds of wild-type and the wrs1 mutant. A, B, Contents of total starch (A) and amylose (B) in wild-type and the wrs1 mutant. n = 3. C, Analysis of RVA characteristic values of starch in wild-type and the wrs1 mutant. D, Swollen volume of the wild-type and wrs1 starch in urea solution. E, Comparation of the swollen volume in the wild-type and wrs1. n = 3. Values are means ± SD. * and **indicate significant difference at 0.05 and 0.01 level, respectively(Student’s t-test).

表2 野生型wrs1突变体淀粉RVA谱特征分析

Table 2 Analysis of RVA characteristic values of starch in wild-type and the wrs1 mutant.

材料 Material	峰值黏度 Peak viscosity / cP	热浆黏度 Hot paste viscosity / cP	崩解值 Breakdown viscosity / cP	冷胶黏度 Cool paste viscosity / cP	消减值 Setback viscosity / cP	糊化温度 Pasting temperature /℃	峰值时间 Peak time / min
N22	2494	1536	958	3727	1233	81.40	5.80
wrs1	2041	1506	535	3262	1221	80.65	6.13

图3 野生型和wrs1突变体发育中的胚乳半薄切片观察 A, D—半薄切片分析野生型（A）与wrs1突变体（D）开花后第8天背部胚乳中的复合淀粉颗粒结构;B, E—半薄切片分析野生型（B）和wrs1突变体（E）开花后第8天中间部位胚乳中的复合淀粉颗粒结构;C, F—半薄切片分析野生型（C）和wrs1突变体（F）开花后第8天腹部胚乳中的复合淀粉颗粒结构;标尺为50 μm。

Fig. 3. Semi-thin section analysis of developing endosperm of wild-type and the wrs1 mutant. A, D—Semi-thin section analysis of the compound starch granules of back area of wild-type (A) and wrs1 (D) mutant endosperm at 8 DAF. B, E—Semi-thin section analysis of compound starch granules of central part of wild-type (B) and wrs1 mutant (E) endosperm at 8 DAF. C, F—Semi-thin section analysis of compound starch granules of the ventral side of wild-type (C) and wrs1 mutant (F) endosperm at 8 DAF. Bars = 50 μm.

图4 WRS1的图位克隆 A—WRS1位点的精细定位,WRS1定位在第12染色体长臂标记N56-39与N56-32之间约183 kb区域内,包含16个预测基因;B—Os12g0540900基因结构和突变位点,序列中存在1处单碱基替换（红色）,导致蛋白质中第256位氨基酸由Met变成Ile;C—转基因家系CP1、CP2和CP3的PCR鉴定结果,上下图分别为转基因阳性鉴定引物N10与Hyg的扩增情况;D—N22、wrs1突变体以及突变体背景的转基因阳性家系（CP1、CP2和CP3）的籽粒表型,上侧为各转基因阳性家系成熟种子外观,下侧为对应家系的种子横断面。

Fig. 4. Map-based cloning of WRS1. A, Fine mapping of the WRS1 locus. The WRS1 locus was mapped to a 183 kb region with markers N56-39 and N56-32 on the long arm of chromosome 12, which contains 16 predicted open reading frames (ORFs). B, The structure of Os12g0540900 and the mutation site. One nucleotide substitution (red) in the sequence is indicated, which results in a substitution of Met-256 to Ile. C, PCR analyses of wrs1 complemented lines (CP1, CP2 and CP3). The upper and lower panels in turn show the amplification of the transgenic positive identification primers N10 and Hyg. D, Mature seed appearance of wild-type, the wrs1 mutant and three positive transgenic lines (CP1, CP2 and CP3). The upside shows the mature seed phenotype of each positive transgenic line, and the downside shows cross-section of the seeds in corresponding transgenic line.

图5 WRS1与其同源蛋白结构及进化树分析 A—WRS1及其同源蛋白的进化树分析。进化树用MEGA7软件构建,采用邻接法,步长检验1000次重复。蛋白名字采用相应的NCBI编号。B—WRS1及其同源蛋白结构。灰色方框代表在动物中保守的WHEP结构域,黑色方框代表罗斯曼折叠催化核心结构域,白色方框代表反密码子结合结构域,aa代表氨基酸。C—WRS1及其同源蛋白序列比对,两个灰色方框区域代表Ⅰ类氨酰-tRNA合成酶特有的保守基序,箭头位置代表突变位点;二级结构从PHYRE2网站获得,蛋白预测基于同源建模法（normal mode）,以已知的酵母色氨酰-tRNA合成酶晶体模型c3kt3D为模板;氨基酸序列采用ESPript3.0网站进行比对（默认参数）[37]。

Fig. 5. Structure and phylogenetic analysis of WRS1 protein and its homologs. A, A neighbor-joining tree of WRS1 and its homologs. The tree was constructed using MEGA7 and bootstrapped with 1000 replicates. The proteins are named after their NCBI accession numbers. B, Domain analysis of WRS1 and its homologous proteins. The gray boxes represent the WHEP domain that is conserved in animals, the black boxes represent the Rossmann fold catalytic domain, the white boxes represent the anti-codon binding domain, aa represents the amino acid. C, Protein sequence alignment of WRS1 and its homologs. Two gray boxes indicate the catalytic site of Class I aaRSs; The arrowhead indicates the mutation site. The secondary structure is obtained from the PHYRE2 website by homology modeling (normal mode) with a known yeast tryptophanyl-tRNA synthetase crystal model c3kt3D as the pattern; the amino acid sequences are compared by the ESPript 3.0 website (default).

图6 WRS1的表达模式和亚细胞定位 A—WRS1在野生型的根、茎、叶、鞘、穗和不同发育天数的胚乳中的相对表达量;B—WRS1启动子驱动GUS基因表达的植株中根、茎、叶、鞘、幼穗和发育种子的GUS染色;C—35S启动子驱动下GFP-WRS1和WRS1-GFP融合表达载体在水稻原生质体中GFP荧光表达,对照为pAN580-GFP空载体,标尺为5 μm。

Fig. 6. Expression pattern and subcellular localization of WRS1. A, Relative expression levels of WRS1 in root, stem, leaf, sheath, panicle and developing seeds of wild-type. B, GUS staining patterns in root, stem, leaf, sheath, panicle and seed in transgenic plants. C, Fluorescence micrographs of the WRS1-GFP and GFP-WRS1 fusion constructs in rice protoplasts, with the empty pAN580-GFP served as a control. Bars, 5 μm.

图7 淀粉合成相关基因的表达及淀粉合成酶蛋白的积累 A—淀粉合成相关基因表达分析,结果为wrs1突变体中的基因以N22中对应基因为基准的相对表达水平,提取花后12 d种子的总RNA,用Ubiquitin作为内参基因。所有数值为平均值±标准差(n = 3),t检验,*P<0.05,**P<0.01。B—Western blotting检测野生型和突变体成熟种子中的淀粉合成相关酶蛋白含量。GBSSⅠ—颗粒结合淀粉合酶Ⅰ;AGPS2b―腺苷葡萄糖焦磷酸化酶小亚基2b;AGPL2―腺苷葡萄糖焦磷酸化酶大亚基2;BEⅠ―淀粉分支酶Ⅰ;BEⅡb―淀粉分支酶Ⅱb;PHOⅠ―质体磷酸化酶Ⅰ;SSⅡa―淀粉合酶Ⅱa;FLO4—胞质丙酮酸磷酸双激酶B,用Actin作为上样内参。

Fig. 7. Expression and immunoblotting analyses of starch synthesis-related genes. A, Expression analysis of starch synthesis-related genes. The relative expression levels of the genes in the wrs1 mutant were based on the corresponding genes in N22. Total RNA was extracted from seeds at 12 days after flowering. Ubiquitin was used as control. n = 3. All values are means ± SD. Student’s t-test, *P<0.05, **P<0.01. B, Western blotting analysis of starch synthesis related proteins in wild-type and mutant mature seeds. GBSSⅠ, Granule bound starch synthaseⅠ; AGPS2b, ADP-glucose pyrophosphorylase small subunit 2b; AGPL2, ADP-glucose pyrophosphorylase large subunit 2; BEⅠ, Starch-branching enzyme; BEⅡb, Starch-branching enzymeⅡb; PHOⅠ, Plastid phosphorylaseⅠ; SSⅡa, Starch synthase Ⅱa; FLO4(FLOURY ENDOSPERM4), cytosolic pyruvate orthophosphate dikinase B; Actin was used as control.

图8 wrs1突变体的蛋白含量与游离氨基酸含量 A—野生型N22和wrs1突变体籽粒的蛋白含量;B—野生型N22与wrs1突变体籽粒中游离氨基酸含量;C—野生型与wrs1籽粒中游离氨基酸总量;D—野生型与wrs1突变体米粉中游离氨基酸的相对含量。所有数值为平均值±标准差(n = 3),t检验,*P<0.05,**P<0.01。

Fig. 8. Contents of protein and free amino acids in wrs1. A, Protein contents in wild-type and wrs1 grains. B, Free amino acid contents in the seeds of wild-type and the wrs1 mutant. C, Total free amino acids contents in the seeds of wild-type and the wrs1 mutant; D, Relative contents of free amino acids in mature seeds of wild-type and the wrs1 mutant. n = 3. All values are means ± SD. Student’s t-test, *P<0.05, **P<0.01.

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