色氨酰-tRNA合成酶基因WRS1调控水稻种子发育

Tryptophanyl-tRNA Synthetase Gene WRS1 Regulates Rice Seed Development

【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。

【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.