水稻甜质胚乳突变体m5788的鉴定及基因定位

doi:10.16035/j.issn.1001-7283.2022.04.003

摘要/Abstract

摘要：

胚乳发育是种子形成的关键,其决定水稻的外观品质和食味品质。m5788是从粳稻品种中花11的组织培养后代中发现的甜质胚乳突变体,其籽粒皱缩,千粒重与穗粒数均显著降低,淀粉合成受阻,可溶性糖含量显著增加。通过对m5788与IRAT129杂交产生的F₂代群体分析表明,甜质胚乳性状受1对隐性核基因控制。对569个F₂隐性极端单株进行连锁分析和定位,将目的基因定位在8号染色体长臂端Z8-25.8和Z8-25.9之间110kb的区域内。该区间内存在1个与玉米甜质基因Sugary 1氨基酸序列相似性高达82.2%的基因LOC_Os08g40930,编码一个属于淀粉去分支酶（DBE）途径的异淀粉酶ISA1。测序结果表明,该基因序列和启动子在野生型和m5788中不存在碱基差异。qRT-PCR分析结果表明,与野生型相比,突变体中LOC_Os08g40930的表达量明显降低。同时,DBE途径中支链淀粉酶的编码基因表达量也显著降低。因此,m5788携带的isa1基因是一个新发现的等位变异。

关键词: 水稻, 胚乳发育, 精细定位, 淀粉合成

Abstract:

Starch synthesis in endosperm development determines the appearance and flavor quality of rice grains. m5788 is a shriveled grain sweet embryo mutant derived from the tissue culture progeny of Japonica var. Zhonghua 11. The 1000-grain weight and the number of grains per panicle decreased significantly. Due to the blocking of starch synthesis, the level of soluble sugar increased significantly. Genetic analysis revealed that m5788 was determined by a pair of recessive nuclear genes. Linkage analysis localized the candidate gene to the 110kb region flanked by indel markers of Z8-25.8 and Z8-25.9 on chromosome 8 in 569 F₂ mutant individuals. In this region there was a putative isoamylase encoding the gene LOC_Os08g40930, which was 82.2% homologous to maize Sugary 1 and was involved in the DBE pathway of the starch debranching enzyme ISA1. The sequencing results showed that no genomic sequence change occurs in LOC_Os08g40930 between wild type and m5788. However, the qRT-PCR analysis showed that the decrease in the expression level of the gene LOC_Os08g40930 in the mutant m5788 was presented together with the expression level of the coding-gene of pullulanase-type in same DBE pathway. All results suggest that isa1 carried by m5788 is a novel allele.

Key words: Rice, Endosperm development, Fine mapping, Starch synthesis

郑思怡, 杨晔, 宋远辉, 花芹, 林泉祥, 张海涛, 程治军. 水稻甜质胚乳突变体m5788的鉴定及基因定位[J]. 作物杂志, 2022 (4): 14–21

Zheng Siyi, Yang Ye, Song Yuanhui, Hua Qin, Lin Quanxiang, Zhang Haitao, Cheng Zhijun. Identification and Fine Mapping of Sugary Endosperm Mutant m5788 in Rice (Oryza sativa L.)[J]. Crops, 2022(4): 14–21

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标记Marker	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
Z8-25.6	CTCGTGCGCGCGCACCGTTGG	GCAGATGGATGGGACGGAGT
Z8-25.7	TTTGGGGCTGGAGCACCTTG	CATCATAGAATGTGCTGA
Z8-28.8	TTTTTCTTCTTCTCATGCCTT	TTTAGCTTCTCTTCCATTCCA
Z8-25.9	GAAGAGAGCTGATTCAGGAGAGAGC	ATAGTTAGGCAGCAACAGCAACG
Z8-26.2	ATGATGGTCCATAAGAGTCG	CAGTGTTCAAGATGGTCATTG

引物Primer	正向引物Forward primer (5′-3′)	反向引物Reverse primer (5′-3′)
ISA1	GTACTTCGATGTCTCCAATGTCG	ATACTCTCCTCGGCTTATCACTG
PUL	CCTAAAGCTTACTACCCATGCAT	ACATGTCCTTGTCTCCACTTCTT

农艺性状 Agronomic trait	野生型 Wild type	突变体 Mutant
株高Plant height (cm)	117.0±2.8	100.2±2.7^**
分蘖数Tiller number	9.5±2.1	12.6±1.9^*
有效穗数Effective tiller number	9.3±1.7	13.1±2.7^**
穗长Panicle length (cm)	24.2±1.3	20.6±1.0^**
一次枝梗数Number of primary branches	14.0±1.8	12.9±1.1
二次枝梗数Number of secondary branches	45.8±4.0	43.0±6.6
穗粒数Grain number per panicle	278.0±45.8	184.0±12.3^*
结实率Seed setting (%)	97.0±0.0	95.0±2.0
千粒重1000-grain weight (g)	29.6±1.95	15.6±0.87^**

品质指标Quality index	野生型Wild type	突变体Mutant
水分Moisture	9.99±0.0011	9.89±0.0004
蛋白质Protein	5.67±0.0005	7.58±0.0012^**
直链淀粉Amylose	19.26±0.0035	19.62±0.0008^*
脂肪酸Fatty acid	14.67±0.0113	16.19±0.0092^**
胶稠度Gel consistency	50.63±0.0073	35.97±0.0227^**

基因Gene	基因注释Gene annotation
LOC_Os08g40830	含有pumilio结构域的蛋白PPD1
LOC_Os08g40890	与精胺合酶有关
LOC_Os08g40940	表达蛋白
LOC_Os08g40840	胞外复合亚基EXO70
LOC_Os08g40910	表达蛋白
LOC_Os08g40950	表达蛋白
LOC_Os08g40850	线粒体载体蛋白
LOC_Os08g40919	表达蛋白
LOC_Os08g40860	RNA假尿苷合酶
LOC_Os08g40930	α淀粉酶,含有蛋白质的催化结构域
LOC_Os08g40990	受体蛋白激酶1
LOC_Os08g40870	五肽重复蛋白
LOC_Os08g40880	含有蛋白质的RNA识别基序
LOC_Os08g40900	生长素响应因子