水稻窄卷叶突变体nrl4的表型分析与基因定位

发布时间：2018-02-12 13:34

本文关键词： 水稻(Oryza sativa L.) 窄卷叶突变体(nrl) 表型分析遗传分析基因定位　出处：《中国农业科学》2016年20期 　论文类型：期刊论文

【摘要】：【目的】研究水稻卷叶突变体叶形变化的分子机理,鉴定出新的水稻卷叶基因。【方法】利用EMS诱变籼稻品种浙农34,获得一个窄卷叶突变体,命名为nrl4(narrow and rolling leaf 4)。在抽穗期随机选取野生型浙农34和nrl4各10株,对其进行表型观察和主要农艺性状调查等,并测定叶绿素含量;同时用Zeiss荧光显微镜观察剑叶中部叶片横切面维管束的数目并统计泡状细胞数量。以多代自交稳定的突变体nrl4为母本与野生型浙农34杂交,观察植物F_1和F_2叶片表型,统计F_2中性状分离比并作卡方测验,分析突变表型的遗传行为。利用nrl4与粳稻品种浙农大104杂交,采用F_2分离群体进行基因精细定位。利用定量表达对定位区间内5个预测的基因进行相对表达量的分析。【结果】与野生型相比,窄卷叶突变体nrl4抽穗期时全部叶片卷曲且变窄、叶绿素含量升高;株高稍有增加,结实率增大,籽粒明显变长、变窄。窄卷叶突变体nrl4功能叶夹角不同程度减小,叶形更为直立。突变体叶近轴表面特有的泡状细胞数目降低、体积变小,导致叶片向内卷曲。突变体nrl4的叶脉数减少,叶片变窄,中脉一侧2个大维管束之间的小维管束平均为4.5个,而野生型为6.0个。遗传分析表明,突变体nrl4和浙农34杂交的F_1表型正常,F_2群体中正常植株与窄卷叶突变植株的分离比符合3㑳1,表明突变体nrl4的突变性状受1对隐性核基因控制;利用SSR和In Del分子标记将nrl4定位在水稻第3染色体长臂3M11103和3M1115之间、物理距离约为53 kb的区间。在这一区间内,有5个预测注释基因,序列比对和表达分析表明在野生型浙农34和突变体nrl4之间,这些基因序列及启动子序列均未发生变化,但是LOC_Os03g19770在突变体叶片中的表达量显著增加。【结论】nrl4叶片变窄与维管束数目减少有关,叶片发生内卷与泡状细胞数目减少、体积变小有关。水稻窄卷叶突变体nrl4的性状由1对隐性核基因控制,该基因位于第3染色体In Del标记3M11103和3M1115物理距离约为53 kb的区间内,预测区间内基因序列及5′UTR区未发现碱基变异,但LOC_Os03g19770在突变体叶片中的表达量达到了野生型植株叶片的17.5倍,推测LOC_Os03g19770为候选基因。
[Abstract]:[objective] to study the molecular mechanism of leaf shape change in rice leaf curl mutant, and to identify a new rice leaf roll gene. [methods] A narrow coiled leaf mutant was obtained by EMS mutagenesis in indica rice variety Zhejiang Nong 34. It was named nrl4(narrow and rolling leaf 4. At heading stage, 10 wild type Zhejiang nong 34 and 10 nrl4 were randomly selected, phenotypic observation and main agronomic characters investigation were carried out, and chlorophyll content was measured. At the same time, the number of vascular bundles and the number of bubbly cells were observed by Zeiss fluorescence microscope. The phenotypes of plants FStu1 and F2 were observed by crossing the stable mutants nrl4 with wild type Zhejiang Nong 34. The segregation ratio of characters in FStu2 was counted and chi-square test was performed to analyze the genetic behavior of mutant phenotypic. Nrl4 was used to cross with japonica rice variety Zhejiang Nongda 104. F-2 isolated population was used for gene fine mapping, and quantitative expression was used to analyze the relative expression of 5 predicted genes in the locational interval. [results] compared with wild type, all leaves of narrow coiled mutant nrl4 were curled and narrowed at heading stage. Chlorophyll content increased, plant height slightly increased, seed setting rate increased, grain became longer and narrower. The angle between functional leaves of narrow curly leaf mutants decreased to varying extent, leaf shape became more upright, and the number of vesicular cells peculiar to the near axial surface of leaves of the mutant decreased. The number of leaf veins in the mutant nrl4 decreased and the leaf became narrower. The average number of small vascular bundles between the two large vascular bundles on the midvein side was 4.5, while the number of wild type was 6.0. Genetic analysis showed that. The segregation ratio of normal plants and narrow curly leaf mutant plants in F1 normal phenotypic population of nrl4 and Zhejiang Province 34 hybrids was 3? 1. It shows that the mutant nrl4 is controlled by a pair of recessive nuclear genes, and that the nrl4 is located between the long arm 3M11103 and 3M1115 of chromosome 3 of rice by SSR and in Del molecular markers, and the physical distance is about 53 kb. The sequence alignment and expression analysis of five predictive annotated genes showed that there was no change in the sequence of these genes and promoter sequences between the wild type Zhejiang nong 34 and the mutant nrl4. But the expression of LOC_Os03g19770 in mutant leaves increased significantly. [conclusion] the narrowing of nrl4 leaves is related to the decrease of vascular bundles, and the number of involute and vesicular cells in leaves is decreased. The character of nrl4 is controlled by a pair of recessive nuclear genes, which are located in the region of 3M11103 and 3M1115 physical distance of 3M11103 and 3M1115 on chromosome 3. No base variation was found in the predicted region of gene sequence and 5UTR region, but the expression of LOC_Os03g19770 in mutant leaves was 17.5 times higher than that in wild-type plants. It was suggested that LOC_Os03g19770 was a candidate gene.
【作者单位】：浙江大学农业与生物技术学院;浙江大学农生环测试中心;
【基金】：浙江省重大科技攻关专项(2012C12901-2) 浙江省科技厅水稻产业科技创新服务平台高等学校学科创新引智计划(Grant B14027) 教育部创新团队资助项目(IRT1185)
【分类号】：S511

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