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【目的】对水稻穗退化突变体spd11进行遗传分析及候选基因鉴定,以便了解水稻穗发育的调控机制。【方法】用化学诱变剂甲基磺酸乙酯(EMS)处理粳稻品种中花11的直立密穗突变体dep2,从突变体库中筛选到一份穗退化突变体spd11。观察该突变体表型,并调查其主要农艺性状。由于突变体不能结实,将可分离出spd11突变植株的株系分单株收种、种植,并对后代株系的分离情况进行调查统计,分析该突变性状的遗传行为。将spd11杂合植株与冈46B杂交的F2后代作为定位群体,对spd11突变体进行基因定位,遴选候选基因并进行DNA测序验证;同时,对不同物种中spd11候选基因的同源基因所编码蛋白进行进化树和序列比对分析。【结果】与其对照亲本相比,spd11植株剑叶长度增加23%;穗部一次枝梗明显缩短,且一次枝梗数量减少58%。小穗几乎完全退化为白色絮状物,偶尔可见个别退化不完全的颖花着生,且该颖花仅由一个完全闭合的颖壳组成,不能正常结实。除此以外,spd11的分蘖数及剑叶宽等农艺性状无显著差异。遗传分析表明,在可分离出spd11突变株的后代中,一部分株系无分离,全部植株均为正常株,而另一部分株系有突变株分离,并且正常植株与突变植株分离明显,分离比例经卡方(χ~2)测验符合3﹕1,表明spd11的突变性状由一对隐性核基因控制。利用分子标记将该突变基因定位于第1染色体长臂2个In/Del标记ch1-2295和ch1-2299之间约43.2 kb的区域内,遗传距离分别为0.23 cM和0.46 cM,该区间内共有8个预测基因。测序分析发现,spd11突变体中OsLOG编码区第116位碱基G突变为碱基A,造成编码蛋白的第39位半胱氨酸(C)突变为酪氨酸(Y)。同源蛋白比对和系统进化分析表明LOG蛋白在不同物种中都是高度保守的,并且spd11的突变发生在非常保守的氨基酸上。对已报道的多个log等位突变体的突变位点和突变表型严重程度的比对分析表明,spd11突变位点可能处于OsLOG蛋白功能的关键位点。【结论】SPD11可能是细胞分裂素激活酶基因OsLOG的等位基因,spd11在OsLOG外显子上一个关键位点发生了突变,导致OSLOG蛋白功能受损,使细胞分裂素的活化进程受阻,从而产生了穗退化的突变表型。
【Objective】 Genetic analysis and candidate gene identification of spd11, a rice panicle degenerative mutant, were conducted to understand the regulatory mechanism of panicle development in rice. 【Method】 Eighteen upright mutant mutants of japonica rice Zhonghua 11 were treated with ethyl methylsulfonate (EMS) as chemical mutagen, and a spikelet degeneration mutant spd11 was screened from the mutant pool. Observe the phenotype of the mutant and investigate its major agronomic traits. Because the mutant can not be strong, the strains of spd11 mutant can be isolated and planted, and the isolation of offspring strains is investigated and statistically analyzed. The genetic behavior of the mutant is analyzed. Spd11 hybrid plants and Okazaki 46B hybrid F2 offspring as a locus group, spd11 mutants gene mapping, selection of candidate genes and DNA sequencing validation; the same time, different species of spd11 candidate genes encoded by the homologous genes were Phylogenetic tree and sequence alignment analysis. 【Result】 The results showed that the length of flag leaf of spd11 increased by 23% compared with that of control. The number of primary branch of spd11 was significantly shortened and the number of primary branch decreased by 58%. Spikelets almost completely degenerated into white flocs, occasionally showing some incompletely degenerated spikelets, and the spikelets consisted of only one fully closed glume shell, which was not normally robust. In addition, there were no significant differences in agronomic traits such as tiller number and width of spd11. Genetic analysis showed that among the offspring of spd11 mutant isolates, some of them were not segregated, all of the plants were normal, while the others were separated by mutants, and the normal plants were separated from the mutated plants clearly. The chi square (χ ~ 2) test conformed to 3: 1, indicating that the mutant trait of spd11 was controlled by a pair of recessive nuclear genes. The molecular marker was used to locate the mutant gene in the region of 43.2 kb between the two In / Del markers ch1-2295 and ch1-2299 on the long arm of chromosome 1. The genetic distances were 0.23 cM and 0.46 cM respectively. Eight predicted genes. Sequencing analysis revealed that the base G of the codon 116 in the spd11 mutant was mutated to base A, resulting in the mutation of the cysteine at position 39 (C) to tyrosine (Y). Homologous protein alignment and phylogenetic analysis indicated that the LOG protein is highly conserved in different species and the spd11 mutation occurs on very conserved amino acids. The analysis of the reported multiple log allele mutants and the severity of the mutant phenotype showed that the spd11 mutation may be the key site of OsLOG protein function. 【Conclusion】 SPD11 may be the allele of cytokinin-activating enzyme gene OsLOG. The mutation of spd11 in a key site of OsLOG exon resulted in the impaired OSLOG protein function and blocked the activation of cytokinin A mutated phenotype of panicle developed.