水稻粉质胚乳fse3突变体的表型分析及基因定位

doi:10.3864/j.issn.0578-1752.2018.11.001

摘要/Abstract

摘要： 【目的】水稻各种类型的胚及胚乳变异突变体是解析胚发育及淀粉合成和调控路径的优良材料。研究旨在通过大规模的粉质胚乳致死突变体的基因克隆和功能鉴定，获得一系列调控胚发育及淀粉合成的关键基因，为稻米品质改良提供理论指导。【方法】从化学诱变剂N-甲基-N-亚硝基脲（MNU）处理的粳稻品种宁粳3号突变体库中，筛选到一个稳定遗传的胚乳粉质皱缩致死突变体，命名为fse3（floury and shrunken endosperm 3）。将其与9311配制杂交组合，利用F2群体中隐性极端个体进行精细定位；将种子在30℃条件下清水浸种24 h，然后在35℃黑暗条件下用TTC染色2 h检测种子活力；用体视镜观察30℃吸胀9 h后的种子胚结构；从突变体杂合植株上分离粉质成熟种子，去壳后磨成糙米粉进行理化性质分析；用扫描电镜观察成熟淀粉颗粒的结构；制备半薄切片观察发育胚乳中的淀粉颗粒结构；利用qRT-PCR测定籽粒灌浆时期的淀粉合成相关基因表达量；并通过Western-blot检测相关淀粉合成酶的蛋白积累。【结果】与野生型相比，突变体籽粒的千粒重、籽粒大小、总淀粉含量、表观直链淀粉含量等指标均降低，淀粉在尿素溶液中的膨胀能力减弱，淀粉的黏度曲线与野生型也存在显著差异，fse3突变体的最高黏度、热浆黏度、冷浆黏度以及崩解值都显著高于野生型。TTC染色表明其胚活力下降，对发育中的胚纵切片观察发现突变体没有心型胚的分化。对发育中胚乳淀粉结构进行观察，发现fse3突变体的胚乳中产生大量小而不规则排布的单淀粉颗粒，复合淀粉颗粒发育滞后。成熟种子横截面观察发现fse3突变体淀粉颗粒排列疏松，大小不均匀，颗粒间存在较大间隙。利用F2群体中分离出的22个隐性极端个体将FSE3连锁在第9染色体长臂上，随后共用1 400个极端个体将目标基因定位于228 kb的区间内，包含28个开放阅读框。qRT-PCR发现在突变体中多个淀粉合成相关基因表现出不同程度的上调和下调，Western-blot结果表明部分淀粉合成酶的蛋白积累减少。【结论】FSE3是一个新的粉质胚致死基因，基因精细定位在第9染色体228 kb的物理区间，FSE3在水稻种子胚及胚乳的发育调控中起重要作用。

关键词: 水稻, 胚乳突变体, 淀粉合成, 造粉体发育, 精细定位

Abstract: 【Objective】Various types of embryo mutants and endosperm mutants in rice are excellent materials for dissecting embryo development and starch synthesis and regulation pathways. Through large scale screening and functional characterization of floury endosperm mutant with a embryo lethality phenotype, serials of genes involved in embryo development and starch biosynthesis and regulation will be obtained, which will provide the theoretical guidance for the improvement of rice quality.【Method】In this study, we obtained a stably inherited floury and shrunken endosperm mutant fse3 from japonica variety cv Ningjing 3 (NJ3), with a embryo-lethal phenotype. A hybrid F2 population of fse3 and 9311 was developed and the recessive individuals were selected to map the locus. The seeds were soaked in clear water at 30℃ for 24 h and then stained with TTC for 2 h at 35℃ in darkness to detect seed vigor. Observing the seed embryo structure which were swelled at 30℃ for 9 h with a stereo microscope. Separation of mature floury seeds from the heterozygous mutant plants, The physicochemical properties of mature grains which were grinded to brown rice flour after being peeled were analyzed. The structure of mature starch grains was observed by scanning electron microscope. Semi-thin sections were made to observe the starch grain structure of developing endosperm. The expression of starch synthesis related genes during grain filling was determined by qRT-PCR. Western-blot was used to detect the accumulation of proteins related to starch synthesis.【Result】The 1 000-grain weight, grain size, total starch content, and apparent amylose content in mature fse3 seeds were all decreased compared with wildtype, as well as the swelling power of starch. The starch viscosity curve also has significant difference with the wild type. The peak paste viscosity, the hot paste viscosity, the cool paste viscosity and the break down viscosity of the fse3 mutant are significantly higher than those of the wild type. TTC staining showed that the embryo vitality decreased and the mutant embryo can not differentiate. By observing the structure of developing endosperm, we found that a large number of small and irregular single starch granules were produced in the endosperm of fse3 mutant, and the development of compound starch granules was delayed. The cross section observation of mature seeds showed that the starch granules in the fse3 mutant were loosely packed, the size was not uniform, and there was a large gap between the particles. The FSE3 locus was first mapped to a region on the long arm of chromosome 9 with 22 recessive individuals, and then was narrowed down to a 228kb region which includes 28 open reading frames (ORFs) by using 1 400 recessive individuals. In the process of grain filling, several genes related to starch synthesis showed up-regulated and down-regulated in different degrees in the mutant. Immunoblotting showed that the protein accumulation of some amyloid synthase was reduced.【Conclusion】FSE3 is a novel floury endosperm and embryo lethality related gene, it was mapped to a 228 kb region on chromosome 9, which plays an important role in the regulation of embryo and endosperm development of rice seed.

Key words: rice (Oryza sativa L.), endosperm mutants, starch synthesis, amyloplast development, fine mapping

于艳芳，刘喜，田云录，刘世家，陈亮明，朱建平，王云龙，江玲，张文伟，王益华，万建民. 水稻粉质胚乳fse3突变体的表型分析及基因定位 [J]. 中国农业科学, 2018, 51(11): 2023-2037.

YU YanFang, LIU Xi, TIAN YunLu, LIU ShiJia, CHEN LiangMing, ZHU JianPing, WANG YunLong, JIANG Ling, ZHANG WenWei, WANG YiHua, WAN JianMin. Phenotypic Analysis and Gene Mapping of a Floury and Shrunken Endosperm Mutant fse3 in Rice[J]. Scientia Agricultura Sinica, 2018, 51(11): 2023-2037.

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