水稻穗退化突变体spd11的遗传分析及候选基因鉴定

doi:10.3864/j.issn.0578-1752.2016.10.001

摘要/Abstract

摘要： 【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（χ²）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。

关键词: 水稻, 穗退化突变体, 遗传分析, 候选基因鉴定, OsLOG

Abstract: 【Objective】 The objective of this study was to gain a better understanding of the regulation mechanism of early rice panicle development by genetic analysis and candidate gene identification of panicle degradation mutant spd11. 【Method】 A panicle degradation mutant, spd11, was isolated by ethyl methanesulfonate mutagenesis from the mutant library of japonica rice dense and erect mutant dep2 of Zhonghua 11. Phenotypes and the main agronomic traits of spd11 were analyzed under field conditions in Chengdu, Sichuan. Because spd11 can not seed, we individually harvested each plant of the isolate spd11 mutant strains lines and planted each of the plants as an independent line for genetic analysis. We investigated the segregation of each strain and analyzed the genetic pattern of spd11. The mutant gene of spd11 was mapped using F₂ populations by crossing spd11+/spd11-plants with Gang 46B. Then, we conducted DNA sequencing, phylogenetic and alignment analysis of LOG proteins in different species. 【Result】 Compared with the control, spd11 exhibited long flag leaf blade lengths. The lengths of primary branches were shortened along with a 58% decrease in branch numbers. The florets of spd11 almost degenerated to white floc, though incompletely degenerated florets consisting only of a completely closed husk could be found occasionally. Other agronomic traits such as tiller number and flag leaf blade width showed little difference across lines. Genetic analysis showed that some lines were wild-type plants, but other lines isolated mutant plants. The segregation ratios between wild-type and mutant plants were all fitted to 3:1, indicating that spd11 was controlled by a single recessive nuclear gene. Then, the mutant gene was mapped to a region of 43.2 kb between two In/Del markers ch1-2295 and ch1-2299 on the long arm of chromosome 1, and the genetic distance were 0.23 cM and 0.46 cM, respectively, containing 8 predicted genes. Sequencing analysis revealed that a single base was changed (G116A) in the coding region of the OsLOG gene which encodes a cytokinin-activating enzyme, causing a missense mutation (C39Y). Alignment and phylogenetic analysis indicated that the LOG proteins were highly conserved in different species, and the mutation in spd11 was located at a very important amino acid site. Comprehensive analysis of the mutation sites and the phenotypes of reported log allelic mutants hinted that this site (C39Y) may lie within OsLOG. 【Conclusion】 SPD11 may be allelic to the OsLOG gene, and the mutation on a key site of OsLOG could affect cytokinin activation in spd11 mutant, eventually leading to the panicle degradation phenotype.

Key words: rice, panicle degradation mutant, genetic analysis, candidate gene identification, OsLOG

钟萍，陈璞睿，王迁，肖富良，张宽，马芙蓉，黄美菱，王平荣，邓晓建，孙昌辉. 水稻穗退化突变体spd11的遗传分析及候选基因鉴定[J]. 中国农业科学, 2016, 49(10): 1835-1843.

ZHONG Ping, CHEN Pu-rui, WANG Qian, XIAO Fu-liang, ZHANG Kuan, MA Fu-rong, HUANG Mei-ling, WANG Ping-rong, DENG Xiao-jian, SUN Chang-hui. Genetic Analysis and Candidate Gene Identification of the Panicle Degradation Mutant spd11 in Rice[J]. Scientia Agricultura Sinica, 2016, 49(10): 1835-1843.

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