水稻脆秆突变体bc16的遗传分析和基因精细定位

发布时间：2018-04-13 05:17

本文选题：水稻 + 脆秆　；参考：《浙江大学》2016年硕士论文

【摘要】：茎秆强度作为一种重要的农艺性状,与植株的抗倒性密切相关。已有的研究表明,引起水稻倒伏的主要原因是茎秆机械强度不足。作为植株骨架的细胞壁,其纤维素、半纤维素和木质素含量与植株的机械强度密切相关。脆秆突变体属于一类纤维素、半纤维素、木质素含量改变,茎秆机械强度降低的突变体,可作为研究水稻茎秆强度和抗倒伏机理的重要材料。同时,由于脆秆突变体细胞壁的独特构成,其有可能成为一种新型牧业饲料和合成燃料乙醇的原料。目前,在水稻中已发现了多个脆秆突变体,相关脆秆基因如BC1-8、 BC10-12、BC14-15等参与了纤维素的代谢,现多数已被精细定位和克隆。因此,深入开展水稻脆秆突变体的分子机理研究,有助于进一步揭示植株机械强度变化机理,在水稻育种中具有重要的理论和实际意义。本研究利用日本晴经EMS(甲基磺酸乙酯)诱变处理后获得的一个脆秆突变体,命名为bc16(brittle culm16),进行了脆秆性状的的遗传分析和基因的精细定位。主要结果如下：1.突变体bc16茎秆和叶片的脆性在苗期就能表现,且具有株高变矮、主根和穗长变短、穗粒数减少、抽穗和成熟期推迟等特点。2.在茎秆细胞壁成分方面,突变体bc16的茎秆纤维素含量比野生型降低36.9%,而半纤维素含量则增加了23.6%,木质素含量在两者之间差异不明显。3.倒置荧光显微镜观察发现,突变体bc16的薄壁细胞形状无规则、排列紊乱,表皮层下厚壁组织的细胞壁比野生型要薄。透射电镜下观察到突变体bc16厚壁细胞次生壁和薄壁细胞壁的厚度也要比野生型薄。4.突变体bc16与野生型日本晴杂交的遗传分析表明,在F2中正常株与突变株分别为120株和38株,χ2=0.08χ；05=3.84,符合3：1的比例,说明突变体bc16的脆秆性状受一对隐性基因控制。5.利用图位克隆的方法,bc16基因初定位于Indel标记2-B和2-E之间,精细定位于水稻第2号染色体长臂端Indel标记2-F和2-H间66.6Kb的物理区间内。该区间共有7个候选基因,其中Os0冶0738900参与次生壁纤维素合成,认为是bc16最佳候选基因。测序结果表明,bc16突变体中的Os02g0738900基因从ATG开始第5113位,在第13内含子近末端发生了T→A的置换,导致转录过程中该内含子末端6个核苷酸被剪切到：mRNA中,最终导致翻译提前终止。实时荧光定量PCR结果表明Os02g0738900基因在bc16脆秆突变体根、茎、叶中的表达量降低。bc16基因可能是通过调控厚壁组织次生细胞壁和薄壁细胞初生壁的合成来影响水稻的茎秆脆性。
[Abstract]:As an important agronomic trait, stem strength is closely related to plant toppling resistance.Previous studies have shown that the main cause of rice lodging is the lack of mechanical strength of the stem.The contents of cellulose, hemicellulose and lignin in the cell wall of the plant skeleton were closely related to the mechanical strength of the plant.Brittle culm mutants belong to a class of cellulose, hemicellulose and lignin mutants, which can be used as important materials for studying the mechanism of rice stem strength and lodging resistance.At the same time, because of the unique cell wall composition of brittle culm mutants, it may become a new type of animal husbandry feed and fuel ethanol raw materials.At present, several brittle culm mutants have been found in rice. The related brittle culm genes, such as BC1-8, BC10-12, BC14-15, are involved in the metabolism of cellulose, and most of them have been mapped and cloned.Therefore, the further study of molecular mechanism of rice brittleness mutant is helpful to reveal the mechanism of plant mechanical strength change, and has important theoretical and practical significance in rice breeding.In this study, a brittle culm mutant named bc16(brittle culm 16 was obtained by EMS-induced mutagenesis. Genetic analysis and gene fine mapping of brittle culm traits were carried out.The main results are as follows: 1.The brittleness of the stem and leaf of the mutant bc16 showed that the plant height became shorter, the main root and ear length became shorter, the number of grains per ear decreased, and the heading and ripening stages were delayed.In terms of cell wall composition of stem, the content of cellulose in stem of mutant bc16 was 36.9% lower than that of wild type, while the content of hemicellulose increased 23.6.The lignin content was not significantly different between the two.Inverted fluorescence microscopy showed that the parenchyma of mutant bc16 was irregular in shape and disordered in arrangement. The cell wall of thick wall tissue in the epidermis was thinner than that in the wild type.The thickness of secondary wall and parenchyma cell wall of bc16 mutant was also thinner than that of wild type.The genetic analysis of bc16 and wild-type Japanese fine hybridization showed that there were 120 and 38 normal plants and 38 mutant plants in F2, 蠂 2 0.08 蠂 ~ (5) 5 ~ (5), which was in accordance with the 3:1 ratio, indicating that the brittleness of mutant bc16 was controlled by a pair of recessive genes.Using the method of map cloning, the gene of bc16 was initially located between Indel markers 2-B and 2-E, and fine located in the physical interval of 66.6Kb between 2-F and 2-H labeled by Indel at the long arm of chromosome 2 in rice (Oryza sativa L.).There are 7 candidate genes in this region, in which Os0 Metallurgy 0738900 is involved in the synthesis of secondary wall cellulose, which is considered to be the best candidate gene for bc16.The sequencing results showed that the Os02g0738900 gene in the mutant was located at 5113 from ATG, and the replacement of TIA at the proximal end of intron 13 resulted in 6 nucleotides at the end of the intron being cut into ATG during transcription.Finally, it leads to the early termination of translation.The results of real-time fluorescence quantitative PCR showed that the decrease of Os02g0738900 gene expression in root, stem and leaf of bc16 brittle-stem mutant. Bc16 gene may affect the brittleness of rice stem by regulating the synthesis of secondary cell wall and primary wall of parenchyma in thick wall tissue.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

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