水稻粉质胚乳突变体ws的表型分析及基因克隆
收稿日期: 2016-03-19
修回日期: 2016-04-28
网络出版日期: 2016-09-10
基金资助
国家转基因生物新品种培育重大专项(2014ZX08001006);国家自然科学基金资助项目(31371598);江苏省科技支撑计划资助项目(BE2014394);农业部长江中下游粳稻生物学与遗传育种重点实验室、长江流域杂交水稻协同创新中心、江苏省现代作物生产中心资助项目
Phenotyping and Gene Cloning of a Floury Endosperm Mutant ws in Rice
Received date: 2016-03-19
Revised date: 2016-04-28
Online published: 2016-09-10
从甲基亚硝基脲(1-Methyl-1-Nitrosourea, MNU)处理的粳稻品种滇粳优1号突变体库中,筛选到一个稳定遗传的胚乳粉质突变体ws,其籽粒的千粒重、籽粒大小、总淀粉含量、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。对成熟及发育中的胚乳淀粉结构进行观察,发现ws突变体的胚乳中产生大量小而不规则排布的单淀粉颗粒。利用F2群体中分离出的92个隐性极端个体将突变基因连锁在第8染色体近着丝粒位置,随后共用2025个极端个体将目标基因定位于95 kb的区间。测序发现ws突变体中编码腺苷二磷酸葡萄糖焦磷酸化酶(Adenosine diphosphate glucose pyrophosphorylase, AGPase)小亚基S2的基因发生点突变,导致编码氨基酸的替换。基因表达分析发现,突变体胚乳中编码AGPase各亚基的相关基因表达量没有发生显著改变,而Western杂交分析显示突变体中AGPS2b的蛋白含量下降。同时,ws突变体的胚乳中AGPase活性下降为野生型的一半。研究结果表明,OsAGPS2的突变导致水稻胚乳中AGPase活性降低,从而影响了淀粉合成。
潘鹏屹, 朱建平, 王云龙, 郝媛媛, 蔡跃, 张文伟, 江玲, 王益华, 万建民 . 水稻粉质胚乳突变体ws的表型分析及基因克隆[J]. 中国水稻科学, 2016 , 30(5) : 447 -457 . DOI: 10.16819/j.1001-7216.2016.6048
In this study, we obtained a stably inherited floury endosperm mutant ws from japonica variety cv. Dianjingyou 1 (DJY), with its 1000-grain weight, grain size, total starch content, and amylose content in mature seeds all decreased compared with wild-type, as well as the swelling power of starch. By observing the structure of mature and developing endosperm, we found that the starch granules in ws mutant were smaller and loosely packed. The WS locus was first mapped to a region near the centromere in chromosome 8 with 92 recessive individuals, and then was narrowed down to a 95 kb region by using 2025 recessive individuals. Sequence analysis revealed that the coding region of the small subunit of adenosine diphosphate glucose pyrophosphorylase (AGPS2) had a single nucleotide substitution, resulting in a change in the amino acid sequence. RT-PCR analysis showed no significant difference in the expression levels of genes encoding the AGPase subunits in the mutant endosperm, while immunoblot analysis revealed a reduced protein content of the AGPS2b. Meanwhile, the enzyme activity of AGPase in the mutant was decreased to half of the wild-type. These results showed that the mutation of OsAGPS2 caused the decreased activity of AGPase, thus affecting the starch biosynthesis in rice endosperm.
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