水稻叶片快速衰老控制基因RLS3的克隆及功能分析

发布时间：2018-06-08 01:48

本文选题：水稻 + 突变体　；参考：《中国农业大学》2016年博士论文

【摘要】：叶片是作物最主要的光合作用器官,其生长、发育和衰老与作物产量密切相关。叶片提前衰老使作物粒重下降,结实率降低,严重影响产量和品质。本研究从元江野生稻渗入系YIL18的EMS突变体库中,筛选出一个叶片变红快速衰老的突变体rls3,并采用图位克隆的方法获得了控制水稻快速衰老的基因RLS3,结果如下：突变体rls3与YIL18相比,株高变矮、茎秆变细、叶片变红枯萎、叶绿素含量下降、光合效率降低及结实率明显下降。透射电镜结果显示,播种后60天,YIL18的叶绿体较大,基质片层排列规则紧密,此时突变体叶绿体较小,皱缩于细胞膜的边缘,基质片层垛叠混乱且变得膨大。播种后100天,YIL18的叶绿体也开始变小皱缩,而突变体rls3细胞内的叶绿体几乎完全降解。利用粳稻品种中花17与突变体rls3进行杂交,构建F2代分离群体,遗传分析结果表明,突变性状受一对隐性基因控制,位于水稻第3染色体短臂,进一步精细定位将该基因定位在约134 kb的区间内。通过比对YIL18和rls3的序列,发现在LOC_Os03g38990基因第10个内含子与第11个外显子的剪接位点处有一个G到A的突变,该突变导致第11个外显子中第一个碱基被剪切掉,阅读框发生改变,最终引起编码的蛋白质提前终止。为了验证突变体rls3表型是否由LOC_Os03g38990基因控制,构建了过表达载体,并将其转入突变体rls3。结果表明,过表达转基因植株叶片恢复绿色,株高和穗长增加,结实率明显上升。同时,构建了RNAi载体,并将其转入中花17。结果表明,干扰转基因植株出现了与突变体类似的表型,即叶片变红,株高变矮,穗长变短及结实率降低。这表明LOC_Os03g38990就是RLS3基因,该基因的突变会引起叶片变红并快速衰老。qRT-PCR的结果表明,RLS3在被检测的部位(叶片、叶枕、叶鞘、茎基和根)中均有表达,叶片中表达量最高。RNA原位杂交结果显示,杂交信号主要呈现在叶肉细胞中。RLS3蛋白的亚细胞定位结果显示,RLS3被定位于叶绿体中,表明RLS3可能在叶绿体发挥作用。RLS3基因编码一个含有AAA 11结构域的蛋白,该结构域包含Walker A和Walker B两个基序,这是AAA蛋白家族的标志性特征。叶片衰老过程中,RLS3可能通过降解光合受损蛋白,起到光保护并维护光合装置的作用,进而维持叶绿体的正常功能。RLS3基因的克隆和功能解析,对进一步揭示水稻衰老调控的分子机理及产量性状的遗传机理具有重要的意义。
[Abstract]:Leaf is the most important organ of photosynthesis, and its growth, development and senescence are closely related to crop yield. Early senescence of leaves decreased grain weight and seed setting rate, which seriously affected yield and quality. In this study, an EMS mutant library of YIL18, a wild rice infiltration line from Yuanjiang, was studied. A mutant rls3 for rapid senescence of red leaves was screened, and the gene RLS3 was obtained by map cloning. The results are as follows: compared with YIL18, the mutant rls3 has shorter plant height, thinner stems, and red and withered leaves. Chlorophyll content decreased, photosynthetic efficiency decreased and seed setting rate decreased obviously. The results of transmission electron microscope showed that the chloroplasts of YIL18 were larger and the matrix lamellar arrangement was regular at 60 days after seeding. The chloroplast of the mutant was smaller, crumpled to the edge of the cell membrane, and the matrix lamellar stack was confused and expanded. The chloroplasts of YIL18 also began to shrink and the chloroplasts in the mutant rls3 cells almost completely degraded 100 days after seeding. Japonica rice variety Zhonghua 17 was hybridized with mutant rls3 to construct F _ 2 segregated population. Genetic analysis showed that the mutant traits were controlled by a pair of recessive genes and were located at the short arm of rice chromosome 3. Further fine mapping of the gene in the region of about 134 kb. By comparing the sequences of YIL18 and rls3, we found that there was a G to A mutation at the splicing site between the 10th intron and the 11th exon of the LOC03g38990 gene, which caused the first base of exon 11 to be cut off and the reading frame changed. Eventually, the encoded protein terminates early. In order to verify whether the rls3 phenotype of the mutant was controlled by the LOCSCI Os03g38990 gene, the overexpression vector was constructed and transformed into the mutant rls3. The results showed that the overexpressed transgenic plants returned to green leaves, plant height and ear length increased, and seed setting rate increased significantly. At the same time, the RNAi vector was constructed and transferred into Zhonghua 17.1%. The results showed that the interfered transgenic plants had similar phenotypes to the mutants, that is, the leaves became red, the plant height became shorter, the spike length became shorter and the seed setting rate decreased. This indicates that LOCOs03g38990 is the RLS3 gene. The mutation of the LLS3 gene causes red leaves and rapid senescence. The results of qRT-PCR show that RLS3 is expressed in the detected sites (leaf, pillow, sheath, stem and root). The results of in situ hybridization showed that the signal of hybridization mainly appeared in the subcellular localization of the. RLS3 protein in mesophyll cells. The results showed that RLS3 was located in chloroplasts. It is suggested that RLS3 may play a role in chloroplast. RLS3 gene encodes a protein containing AAA 11 domain, which contains two motifs, Walker A and Walker B, which are the iconic features of AAA protein family. In the process of leaf senescence, RLS3 may protect and maintain the photosynthetic apparatus by degrading photosynthetic damaged protein, and then maintain the normal function of chloroplast. RLS3 gene cloning and functional analysis. It is of great significance to reveal the molecular mechanism of rice senescence regulation and the genetic mechanism of yield traits.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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