水稻短穗小粒突变体sps1的鉴定与基因精细定位

Identification and Gene Mapping of a Shorten Panicle and Seed Mutant sps1 in Rice(Oryza sativa L.)

【目的】通过对一个水稻短穗小粒突变体的鉴定与基因精细定位,为水稻等禾本科作物的籽粒发育及分子改良奠定基础。【方法】在水稻EMS诱变体库中鉴定到一个短穗小粒突变体,暂命名为sps1(shorten panicle and seed 1)。成熟期观察野生型和sps1的形态变化,考察株高、节间长、穗实粒数、结实率和千粒重等农艺性状;对野生型和sps1籽粒外稃内外表皮中部进行扫描电镜观察,并利用石蜡切片进一步分析野生型和sps1籽粒的形态变化;配制缙恢10号/sps1杂交组合进行遗传分析,并利用其F2群体进行基因精细定位;对野生型和sps1两叶一心期的叶鞘进行油菜素内酯(brassinolide,BR)敏感性试验;抽穗期分析SPS1在水稻根、茎、叶、鞘和穗中的表达,并对籽粒发育相关基因和BR相关基因进行q PCR分析。【结果】sps1穗和倒1、2、3的节间长度均极显著短于野生型,导致株高半矮化;此外,sps1穗枝梗数、结实率和千粒重也显著降低;扫描电镜观察发现sps1外稃中部内外表皮细胞长度极显著小于野生型,宽度则极显著变大,石蜡切片观察进一步证实了sps1籽粒宽短是由细胞变短、变宽造成的;籽粒发育相关基因q PCR分析发现,部分通过调控细胞分裂和扩展进而影响水稻籽粒发育的基因表达量发生了显著变化,在sps1中,AFD1、SLG、HGW和GS3的表达量显著上调,GW7和GID1显著下调;选取符合3﹕1分离比例的F2代分离群体中的突变单株进行基因定位,最终将调控基因精细定位在第7染色体上标记sps1-3和sps1-2之间134 kb的物理范围内,包含19个注释基因;经测序,与野生型相比,发现sps1中的Os07g0616000在编码区有一个A-T的碱基替换,致使编码的赖氨酸变成了终止密码子,导致蛋白翻译提前终止,初步确定为候选基因。q PCR分析发现SPS1在水稻的根、茎、叶、鞘和穗中均有表达,且在茎秆中的表达量最高;生物信息学分析发现,SPS1是DEP2的一个新等位基因。sps1对外源BR的敏感性降低,BR钝感基因D1的表达极显著下调;推测SPS1/DEP2可能通过BR信号传导途径调控水稻籽粒和株型的发育。【结论】sps1是一个水稻短穗小粒突变体,SPS1编码一个表达蛋白,是DEP2的新等位基因,通过BR信号传导途径调控水稻籽粒和株型的发育。

【Objective】Identification and gene mapping of a shorten panicle and seed mutant is significant for rice functional genomics research and molecular breeding. 【Method】A shorten panicle and seed mutant（sps1） was identified from the EMS-induced library. At the maturity stage, agronomic traits such as plant height, panicle length, seed number per panicle, filled grain number per panicle, seedsetting rate and 1000-seeds weight were measured. Scanning electron microscopy was carried out to analyze the inner and outer epidermis between the wild type and sps1 and the grain morphology of wild type and sps1 was observed by paraffin section. The sps1 was crossed with Jinhui 10, and the F1 and F2 generations were used for genetic analysis. Sensitivity test of brassinolide（BR） was carried out on sheath of wild-type and sps1. The root, stem, leaf, sheath and spike were collected, and the genes associated with grain development and the genes associated with BR were analyzed in q PCR.【Result】The sps1 was dwarf in all phases of plant development, and the panicle length, the first, second and third internodes were significantly shorter than those of the wild type. In addition, there were significant decrease in the branch number, seed-setting rate and the 1000-seeds weight in the sps1 mutant. The results of scanning electron microscopy showed that when compared with the wild type, the inside and outside the central lemma of epidermal cells of sps1 was shorter and broader, and paraffin section showed that the smaller seed size of sps1 was caused by reduced cell length, increased cell width and cell number. There were significant changes in the expression of genes regulating rice grain size by controlling cell extension and division, such as the AFD1, SLG, HGW and GS3 significantly increased, the GW7 and GID1 significant reduced. Choose the single mutant strains in F2 generation of Jinhui10/sps1 accord with the 3：1 separation ratio to conduct gene localization. And SPS1 was finally mapped on chromosome 7 with a 134 kb physical distance between markers sps1-3 and sps1-2. There are 19 annotated genes in the fine mapping region. Sequencing indicated that a single nucleotide substitution from A to T occurred in Os07 g0616000, and a lysine was changed into termination codon, and then leaded to protein coding early termination in sps1. The root, stem, leaf, sheath and spike of the wild type and sps1 were analyzed by q PCR, and the results showed that the target gene was expressed in each organ of the plant, and the expression was highest in the plant stem. Bioinformatics analysis showed that SPS1 was a new allele of DEP2. The sensitivity of sps1 to the exogenous BR was reduced, and the expression of D1 was significantly reduced. It is speculated that SPS1/DEP2 may regulate the development of rice grain and plants by BR signaling pathway.【Conclusion】sps1 was a shorten panicle and seed mutant. SPS1 encoded an expression protein and is a new allele of DEP2, which regulated the development of rice grain and plant type by BR signaling pathway.