水稻侧根突变体Oslrd2的表型分析和基因克隆

侧根是水稻胚后发育的重要器官,具有吸收养分和稳固植株的作用。本研究从甲基磺酸乙酯(EMS)诱变的水稻Kasalath突变体库中筛选得到一个水稻侧根突变体Oslrd2,对其进行了表型和遗传分析、图位克隆、候选基因测序及转基因互补验证。结果表明,与野生型相比,Oslrd2幼苗期的苗高和根系伸长都受到明显抑制,侧根短且数量少。通过对侧根原基的亚甲基蓝染色和解剖观察,发现相较于野生型,Oslrd2的侧根原基数目降低,且原基和侧根的形态发生膨胀变形;其成熟期的株高、有效穗数、每穗实粒数和千粒重也受到显著抑制。遗传分析表明,Oslrd2的突变性状受一对隐性核基因控制。利用SSR和InDel分子标记将突变基因定位在水稻第11号染色体InDel 3与InDel 4之间,物理距离约为10 kb,该定位区间内包含一个编码α-微管蛋白的基因(LOC_Os11g14220)。测序比对发现,突变体Oslrd2中该基因编码序列(CDS)1 176 bp处缺失了一个胞嘧啶(C),以及在1 179 bp处的胞嘧啶(C)突变为腺嘌呤(A)。上述突变导致其氨基酸序列中第392位的天冬氨酸(D)变为谷氨酸(E),第393位的异亮氨酸(I)变为终止密码子TAA,翻译提前终止。通过遗传互补验证的转基因植株表型和逆转录PCR(RT-PCR)分析,证实突变体Oslrd2的表型变化是由该基因突变引起的。研究结果明确了OsLRD2基因在水稻根系发育过程中的重要作用,为解析水稻侧根发育的分子机制提供了理论基础。

Phenotypic Analysis and Gene Cloning of a Lateral Root Mutant Oslrd2 in Rice(Oryza sativa L.)

Lateral roots are important organs for rice post-embryonic development,which play roles in nutrients absorptions and plant stabilization.In this study,a rice lateral root mutant Oslrd2(Oryza sativa lateral root defective 2)was identified from a mutant library derived from Kasalath by ethyl methyl sulfonate(EMS).The phenotypes of the mutant were studied and genetic analysis,map-based cloning,candidate gene sequencing and transgenic complementation were conducted.Compared with the wild type,the seedling height and root elongation of Oslrd2 was clearly inhibited in the seedling stage,and the lateral roots were also less and shorter.Through methylene blue staining and anatomical examination,the lateral root primordia in Oslrd2 were less,and the primordia and lateral roots were swelled.Moreover,the plant height,effective panicles number,the number of grains per panicle,and thousand-grain weight of Oslrd2 at the maturation stage were significantly impaired.Genetic analysis showed that the mutant phenotype of Oslrd2 was controlled by a pair of recessive nuclear genes.And the mutant gene was located between two markers InDel 3 and InDel 4 on rice chromosome 11 with a physical distance of about 10 kb.A gene encodingα-tubulin(LOC_Os11g14220)was found within this region.Sequencing analysis revealed a deletion of C¹¹⁷⁶ and a transition of C¹¹⁷⁹ to A in its CDS sequence of Oslrd2,which resulted in the replacement of residue D³⁹² by E³⁹² and I³⁹³ by a stop codon in the encoded protein sequence,respectively,leading to premature termination of translation.Complementation test confirmed that the Oslrd2 mutant phenotype was caused by this gene.Our results clarify the critical role of OsLRD2 in rice root development and provide a theoretical basis to dissect the molecular mechanism of lateral root development in rice.