水稻矮化小穗突变d18新等位基因的发现及生理功能分析

Identification and physiological function analysis of a new dwarf spikelet Dwarf18-allele mutant in rice

株高和穗型是决定水稻产量密切相关的农艺性状.本研究从粳稻品种台北309经甲基磺酸乙酯诱变的群体中分离出一个能稳定遗传的矮化小穗突变体,主要表现矮化、粒长增加而宽度变小和穗型变小等特点.遗传分析表明,该突变体受单隐性核基因控制.定位于水稻第1号染色体的Indel标记P4和P5之间,物理距离约为20 kb.由于该区间只有一个已经克隆的矮化基因Dwarf18(d18),其功能编码赤霉素3β-羟化酶(GA3ox2).所以,表明突变基因与d18可能等位.测序比对发现,该突变基因的第2个外显子发生了1个碱基(G)的缺失,造成无义突变,实时荧光定量PCR结果显示,OsGA3ox2基因在突变体中表达量显著下降.因此,将该突变基因暂命名为d18-1.组织切片观察结果显示,与野生型相比,突变体的茎部倒三节间纵向细胞长度显著变短,但细胞数目增多.生理功能结果分析表明,突变体对外源激素GA更敏感,但对外源激素BR不如野生型敏感.检测与GA和BR生物合成与信号传导途径相关基因的表达结果发现,与野生型相比,在突变体中参与GA失活基因OsGA2ox3出现了下调表达,GA生物合成相关基因OsGA20ox2和OsGA3ox2也出现了下调表达,GA信号传导途径中的关键基因都没有明显的差异表达变化.另外,参与BR的合成或者信号传导途径的大部分相关基因在突变体中的表达都出现了下调.由此证明,突变体矮化是由于GA激素的不能正常合成所导致,并且对外源活性BR的响应通路受损而造成对BR激素的低敏感反应.另外,对孕穗期的野生型和突变体的幼穗进行转录组测序分析表明,与野生型相比,突变体检测到1497个差异表达基因,这些差异基因涉及苯丙素的生物合成、糖代谢和碳代谢等.因此,推测突变体中的OsGA3ox2基因发生了无义突变影响了GA和BR生物合成与信号传导途径以及穗型发育相关调控途径,这为更深入研究调控水稻矮化以及穗型的遗传网络提供了新的信息.

Plant height and panicle architecture are closely related agronomic traits that determine rice yield.In this study,a genetically stable dwarf spikelet mutant was isolated from the japonica cultivar＂Taipei 309＂through ethyl methanesulfonate mutagenesis.The mutant is mainly characterized by dwarfing,long grains,small width,and small panicle architecture.Genetic analysis revealed that the mutant phenotype was controlled by a single recessive nuclear gene,which was mapped to a 20-kb region between indel markers P4and P5 on chromosome 1.Only one dwarf gene,Dwarf18（D18）,has been cloned in this region,which encodes gibberellin 3β-hydroxylase（GA3ox2）,suggesting that the mutant gene and D18 may be allelic.Sequence analysis of the mutant gene revealed the deletion of one base（G）in the second exon,causing a nonsense mutation.Quantitative real-time PCR results indicated that Os GA3ox2 expression in the mutant was significantly decreased.Therefore,we tentatively named the mutant gene＂d18-1.＂Tissue section observation revealed the mutant showed a markedly decreased length of longitudinal cells but an increased number of cells in the stem compared with the wild-type.A physiological function analysis revealed that the mutant was more sensitive to GA treatment but less sensitive to BR treatment than the wild-type.Expression analysis of genes related to GA and BR biosyntheses and signal transduction pathways revealed that GA-inactivating gene Os GA2ox3 and GA biosynthesis-related genes Os GA20ox2 and Os GA3ox2were downregulated in the mutant;however,there were no significant differences in the expression of the key genes involved in the GA signaling pathway.In addition,downregulation was observed in most of the genes involved in the biosynthesis and signal transduction of BR in the mutant.These results indicate that the dwarf phenotype observed in the mutant was caused by abnormal GA synthesis and further impairment of the response pathway to exogenously active BR,resulting in a low-sensitivity response to BR.In addition,the microarray analysis of the wild-type and mutant panicles at the booting stage detected 1497 differentially expressed genes involved in phenylpropanoid biosynthesis and carbohydrate and carbon metabolisms.Therefore,the nonsense mutation of the Os GA3ox2 gene in the mutant was considered to affect the regulation of GA and BR biosyntheses and signal transduction pathways as well as panicle architecture development.This study provides new information for further research into the regulation of rice dwarfing and the genetic network of panicle architecture.