水稻粉质胚乳突变体M37的基因定位及变异分析

Gene mapping and mutation analysis of floury endosperm mutant M37 in rice

在粳稻品种Kitaake的60Co辐射突变体库中筛选到1个稳定遗传的粉质胚乳突变体M37,其粒厚、千粒重、总淀粉和直链淀粉含量均显著降低。对成熟及发育中胚乳淀粉颗粒结构进行观察,发现突变体的胚乳中淀粉颗粒排列松散,产生大量小单粒淀粉颗粒。利用M37/Dular的F2群体中分离出的682个粉质极端个体将目标基因定位到水稻第1染色体长臂的51.6 kb,该区间含有7个开放阅读框(ORFs)。其中ORF6编码腺苷二磷酸葡萄糖焦磷酸化酶大亚基2 (AGPL2)。对该基因测序分析发现突变体M37在AGPL2第4外显子上发生1个单碱基G到A的替换,导致该位点氨基酸由丙氨酸(Ala)变成苏氨酸(Thr)。氨基酸序列分析发现AGPL2的同源基因主要存在于单子叶中,突变位点在不同物种中均非常保守。该基因与已报道的水稻w24、gif2、eb6和eb7为等位基因。荧光定量PCR分析发现突变体中多个淀粉合成相关基因的表达水平发生显著变化。

A floury endosperm mutant, M37, was isolated from a japonica rice cultivar Kitaake through 60Cogamma irradiation.The seed thickness, 1000-seed weight, starch content and amylose content in M37 significantly decreased compared with wild type. By observing the structure of mature and developing endosperm, we found that the starch granules in M37 were smaller and loosely packed. The target gene was mapped to 51.6 kb on the long arm of rice chromosome 1 using 682 recessive individuals isolated from the F2 population of M37/Dular. This region contains 7 open reading frame(ORFs).Within this region, ORF6 encoded the large subunit of ADP-glucose pyrophosphorylase(AGPL2). Sequence analysis revealed that there was only one nucleotide substitution of Guanine(G) to Adenine(A) in the 4 rd exon of M37, which resulted in an amino acid replacement of Alanine(Ala) to Threonine(Thr) in AGPL2. Amino acid sequence analysis showed that the homologous gene of AGPL2 mainly existed in monocotyledons, and the mutation site was very conservative in different species. The M37 was allelic to w24, gif2, eb6 and eb7. In addition, the expression of starch synthesis-related genes was also obviously altered in M37 developing endosperm. This study analyzed the effect of mutant sites in AGPL2 gene on rice endosperm phenotype, which deepens understanding of AGPL2 regulating starch synthesis and amyloplast development.