水稻长护颖突变体基因的克隆与表达分析

doi:10.3864/j.issn.0578-1752.2015.11.001

摘要/Abstract

摘要： 【目的】利用EMS对水稻（Oryza sativa L.）保持系品种宜香1B进行诱变，筛选出3份长护颖突变体。通过基因定位和克隆，探明控制该性状的遗传基础以及分子机理，并在不同器官进行表达分析，了解该基因表达特点。【方法】以3份水稻长护颖突变体Oslg-1、Oslg-2和Oslg-3为材料，进行表型分析、等位性鉴定、基因定位、生物信息学分析，以及qRT-PCR定量表达分析。【结果】Oslg-1突变体小穗在幼穗发育早期与野生型无明显差异，但在成熟期其护颖的远轴表皮细胞凸起且粗糙，形成的结节轴向对齐排列，且毛状物较多，形成垂直相间的横纵沟，与外稃表皮细胞结构相似。遗传分析表明，该类突变表型受1对隐性基因控制，OsLG定位于第7染色体短臂SSR标记RM5344和RM20934之间，遗传距离分别为1.11和0.82 cM，物理距离为246.3 kb。对该区域候选基因分析和测序，发现LOC_Os07g04670基因在编码区第182位碱基（T→A）改变，导致其编码氨基酸第61位（Leu→His）的改变。等位性分析表明，Oslg-2、Oslg-3与Oslg-1属等位变异，进而对突变体Oslg-2和Oslg-3的OsLG测序，突变分别发生在第316位（T→A）和119位（T→C）碱基，导致其编码的氨基酸第106位（Trp→Arg）和第40位（Leu→Pro）突变。对该基因进行同源进化分析和序列比对，表明该基因可能调控水稻护颖伸长。对本突变材料的候选基因和另一控制护颖性状的PAP2进行实时荧光定量PCR（qRT-PCR）分析，结果表明，OsLG在水稻的叶片、穗、叶鞘和根中均有表达，且在穗部表达最高，而PAP2在除穗部以外的其他部位几乎不表达，表明2个控制护颖性状的基因均具有组织特异性，且PAP2的特异性更强；在长护颖突变体中，2个基因表达量均下调，表明其具有协同表达特点。【结论】3份水稻长护颖突变体OsLG与已报道的G1为同一基因，其功能结构域内氨基酸的突变导致长护颖发育；OsLG和PAP2在穗部具有协同表达的特点。

关键词: 水稻, 长护颖突变体, 等位变异, 基因克隆, 基因表达

Abstract: 【Objective】 Three long empty glumes mutants were screened from a maintainer line Yixiang 1B(Oryza sativa L. ssp. indica) by ethyl methanesulfonate (EMS) mutagensis. This study is to explore the genetic basis and molecular mechanism for controlling the development of long empty glumes by gene mapping and cloning. And the temporal and spatial expression of related genes were also concerned. 【Method】 Three long empty glumes mutants, Oslg-1, Oslg-2 and Oslg-3, were analyzed by phenotypic observation, allelism identification, gene mapping, bioinformatics analysis, and expression analysis by quantitative PCR. 【Result】The glumes showed no obvious difference at the stage of early floret differentiation between wild type and mutant. However, the epidermal cells of glume presented significant difference during floret maturation. At the heading date, epidermal cells of abaxial glume presented bulge and rough nodules, and formed axially alignment. The epidermal hairs became more, and bulges arranged at a regular intervals, which were similar to the structure of epidermal cells of lemma. Genetic analysis suggested that the mutant Oslg-1 was controlled by one recessive nuclear gene. The Oslg-1gene was mapped between SSR markers RM5344 and RM20934, on the short arm of rice chromosome 7, with genetic distances of 1.11 cM and 0.82 cM, respectively, and with a physical interval of 246.3 kb. By analyzing and sequencing for the candidate genes in this genomic region, it was found that there was a single nucleotide change at 182 base (T→A) in exon of LOC_Os07g04670 gene, which caused a missense mutation at 61 amino acid (Leu to His) in the encoded product. Allelism analysis by inter-crosses showed that Oslg-1, Oslg-2 and Oslg-3 were allelic variation. Detection of the gene OsLG in Oslg-2 and Oslg-3 mutants was made and found that there was a single nucleotide change (316, T→A and 119, T→C) in exon of OsLG gene, which caused a missense mutation (106, Trp to Arg and 40, Leu to Pro) in the encoded product, respectively. These results suggested that the candidate gene may regulate the elongation of rice glume by the homologous gene sequence alignment and phylogenetic analysis. Besides, the relative expression content involved in OsLG gene and another PAP2 genecontrolling glume trait was also analyzed by real-time PCR (qRT-PCR). The results showed that OsLG gene was expressed in roots, leaf sheath and panicle, and presented rather higher expression in panicle than that in root and leaf sheath. By contrast, PAP2 gene was only detected in panicle. Therefore, both of OsLG and PAP2 were tissue-specificaly expressed, and PAP2 was much more higher expressed in panicle than OsLG. It was deduced that these two genes functioned in a synergistic pattern to control the development of long empty glumes as the expression level of OsLG and PAP2 were down in OsLG mutants. 【Conclusion】The Oslgmutants are allelic with G1 reported previously, and the mutation of a single amino acid in functional domain leads to the development of long sterile glume. OsLG and PAP2 are synergistically expressed in panicle.

Key words: rice, long empty glumes, allelic variation, gene cloning, gene expression

朱玲，陈晓琼，杜康兮，韩保林，冉秀华，张红宇，徐培洲，吴先军. 水稻长护颖突变体基因的克隆与表达分析[J]. 中国农业科学, 2015, 48(11): 2085-2095.

ZHU Ling, CHEN Xiao-qiong, DU Kang-xi, HAN Bao-lin, RAN Xiu-hua, ZHANG Hong-yu, XU Pei-zhou, WU Xian-jun. Gene Cloning and Expression Analysis of Long Empty Glumes Mutants in Rice[J]. Scientia Agricultura Sinica, 2015, 48(11): 2085-2095.

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