發(fā)布時(shí)間：2018-06-08 01:48

  本文選題：水稻 + 突變體　； 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：葉片是作物最主要的光合作用器官,其生長、發(fā)育和衰老與作物產(chǎn)量密切相關(guān)。葉片提前衰老使作物粒重下降,結(jié)實(shí)率降低,嚴(yán)重影響產(chǎn)量和品質(zhì)。本研究從元江野生稻滲入系YIL18的EMS突變體庫中,篩選出一個(gè)葉片變紅快速衰老的突變體rls3,并采用圖位克隆的方法獲得了控制水稻快速衰老的基因RLS3,結(jié)果如下：突變體rls3與YIL18相比,株高變矮、莖稈變細(xì)、葉片變紅枯萎、葉綠素含量下降、光合效率降低及結(jié)實(shí)率明顯下降。透射電鏡結(jié)果顯示,播種后60天,YIL18的葉綠體較大,基質(zhì)片層排列規(guī)則緊密,此時(shí)突變體葉綠體較小,皺縮于細(xì)胞膜的邊緣,基質(zhì)片層垛疊混亂且變得膨大。播種后100天,YIL18的葉綠體也開始變小皺縮,而突變體rls3細(xì)胞內(nèi)的葉綠體幾乎完全降解。利用粳稻品種中花17與突變體rls3進(jìn)行雜交,構(gòu)建F2代分離群體,遺傳分析結(jié)果表明,突變性狀受一對隱性基因控制,位于水稻第3染色體短臂,進(jìn)一步精細(xì)定位將該基因定位在約134 kb的區(qū)間內(nèi)。通過比對YIL18和rls3的序列,發(fā)現(xiàn)在LOC_Os03g38990基因第10個(gè)內(nèi)含子與第11個(gè)外顯子的剪接位點(diǎn)處有一個(gè)G到A的突變,該突變導(dǎo)致第11個(gè)外顯子中第一個(gè)堿基被剪切掉,閱讀框發(fā)生改變,最終引起編碼的蛋白質(zhì)提前終止。為了驗(yàn)證突變體rls3表型是否由LOC_Os03g38990基因控制,構(gòu)建了過表達(dá)載體,并將其轉(zhuǎn)入突變體rls3。結(jié)果表明,過表達(dá)轉(zhuǎn)基因植株葉片恢復(fù)綠色,株高和穗長增加,結(jié)實(shí)率明顯上升。同時(shí),構(gòu)建了RNAi載體,并將其轉(zhuǎn)入中花17。結(jié)果表明,干擾轉(zhuǎn)基因植株出現(xiàn)了與突變體類似的表型,即葉片變紅,株高變矮,穗長變短及結(jié)實(shí)率降低。這表明LOC_Os03g38990就是RLS3基因,該基因的突變會(huì)引起葉片變紅并快速衰老。qRT-PCR的結(jié)果表明,RLS3在被檢測的部位(葉片、葉枕、葉鞘、莖基和根)中均有表達(dá),葉片中表達(dá)量最高。RNA原位雜交結(jié)果顯示,雜交信號(hào)主要呈現(xiàn)在葉肉細(xì)胞中。RLS3蛋白的亞細(xì)胞定位結(jié)果顯示,RLS3被定位于葉綠體中,表明RLS3可能在葉綠體發(fā)揮作用。RLS3基因編碼一個(gè)含有AAA 11結(jié)構(gòu)域的蛋白,該結(jié)構(gòu)域包含Walker A和Walker B兩個(gè)基序,這是AAA蛋白家族的標(biāo)志性特征。葉片衰老過程中,RLS3可能通過降解光合受損蛋白,起到光保護(hù)并維護(hù)光合裝置的作用,進(jìn)而維持葉綠體的正常功能。RLS3基因的克隆和功能解析,對進(jìn)一步揭示水稻衰老調(diào)控的分子機(jī)理及產(chǎn)量性狀的遺傳機(jī)理具有重要的意義。
[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.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S511

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1 林延慧;水稻葉片快速衰老控制基因RLS3的克隆及功能分析[D];中國農(nóng)業(yè)大學(xué);2016年

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本文編號(hào)：1993833