【摘要】：葉綠體是綠色植物進(jìn)行光合作用的重要場所,同時也是光合色素的載體,廣泛分布在葉片綠色組織細(xì)胞中。高等植物葉綠體的發(fā)育是一個非常復(fù)雜的調(diào)控過程,需要核基因編碼蛋白和葉綠體基因編碼蛋白協(xié)調(diào)完成,葉色突變是研究質(zhì)體、葉綠體發(fā)育的重要材料,相關(guān)調(diào)控機(jī)制的研究結(jié)果在基礎(chǔ)研究和生產(chǎn)實踐中都將發(fā)揮非常重要的作用。水稻白穗突變體qp(t)1是由粳稻品種“日本晴”經(jīng)EMS誘變而來,該突變體在3葉期之前表現(xiàn)為完全的白化,隨著植株的生長,3葉期之后新抽出的葉片開始轉(zhuǎn)綠,而老葉片的葉綠體發(fā)育也緩慢恢復(fù),呈現(xiàn)白色條紋狀,抽穗期穗部白化。圖位克隆發(fā)現(xiàn)位于第3號染色上的AP(t)1基因(LOC_Os03g405500)第五個外顯子出現(xiàn)一個單堿基的替換突變,由G突變?yōu)锳,引起第537個氨基酸由色氨酸(Trp)突變?yōu)榻K止密碼子,導(dǎo)致蛋白編碼提前終止。將互補(bǔ)載體轉(zhuǎn)化突變體后,突變表型得以恢復(fù)正常,進(jìn)一步證實AP(t)1基因突變確實是造成水稻白化的內(nèi)在原因。生物信息學(xué)分析結(jié)果表明AP(t)1與擬南芥AtFLN2基因同源。Real-time PCR實驗表明,OsFLN2基因在水稻中各個部位都有表達(dá),但主要在葉片中表達(dá)。ap(t)1突變體中OsFLN2基因的表達(dá)量顯著下降,而互補(bǔ)轉(zhuǎn)基因株中的表達(dá)量與野生型基本一致。OsFLN2基因突變后對質(zhì)體編碼的RNA聚合酶(PEP)參與編碼的基因影響較大,突變體中的PEP類基因的表達(dá)量較野生型顯著下降,而核編碼的RNA聚合酶(NEP)類基因變化不大。亞細(xì)胞定位實驗發(fā)現(xiàn)OsFLN2定位于水稻原生質(zhì)體的葉綠體上。運(yùn)用CRISPR-Cas9技術(shù)根據(jù)目的基因的序列對野生型進(jìn)行定點敲除,產(chǎn)生的純合型敲除陽性轉(zhuǎn)基因株表型與突變體類似,而且定點突變后的白化轉(zhuǎn)基因株后期并不能轉(zhuǎn)綠,說明定點突變株中OsFLN2的功能可能遭到了更為嚴(yán)重的破壞。對FLN2功能的解讀,將為進(jìn)一步揭示質(zhì)體及葉綠體發(fā)育機(jī)制,闡明植物光合復(fù)合體的分子作用機(jī)理奠定基礎(chǔ)。
[Abstract]:Chloroplast is an important place for photosynthesis in green plants and a carrier of photosynthetic pigments. Chloroplasts are widely distributed in green tissue cells of leaves. The development of chloroplasts in higher plants is a very complex regulatory process, which requires the coordinated completion of nuclear gene coding protein and chloroplast gene coding protein. Leaf color mutation is an important material to study the development of plastids and chloroplasts. The research results of related regulation mechanism will play a very important role in basic research and production practice. The white panicle mutant qp (t) 1 of rice was mutated by EMS of Japonica rice variety "Japan Qing". The mutant appeared to be completely albino before the 3-leaf stage. With the growth of the plant, the newly extracted leaves turned green after the 3-leaf stage. The chloroplast development of the old leaves recovered slowly, showing white stripes and albinism at heading stage. A single base substitution mutation was found in the fifth exon of the AP (t)-1 gene (LOC_Os03g405500) located on the third staining site, from G to A, causing the mutation of 537 amino acids from tryptophan (Trp) to terminating codon. Leading to early termination of protein coding. After the mutants were transformed into complementary vectors, the mutant phenotype returned to normal, which further confirmed that the mutation of AP (t)-1 gene was the internal cause of rice albinism. Bioinformatics analysis showed that AP (t)-1 was homologous to Arabidopsis thaliana AtFLN2 gene. Real-time PCR analysis showed that OsFLN2 gene was expressed in all parts of rice. The expression of OsFLN2 gene in AP (t) 1 mutant decreased significantly. OsFLN2 gene mutation had a great effect on the genes encoded by plasmid-encoded RNA polymerase (PEP), and the expression of PEP-like genes in the mutants was significantly lower than that in wild-type plants, but the expression of OsFLN2 gene in the mutants was similar to that in wild-type plants. However, the nuclear-encoded RNA polymerase (NEP) genes did not change much. Subcellular localization experiments showed that OsFLN2 was located on chloroplasts of rice protoplasts. The results showed that the homozygous knockout positive transgenic plants had the same phenotype as the mutants, and the albino transgenic plants could not turn green after site-directed mutagenesis, according to the sequence of the target gene, CRISPR-Cas9 technique was used to perform site-specific knockout of wild-type transgenic plants. The results suggest that the function of OsFLN2 in site-directed mutants may be more seriously damaged. The interpretation of the function of FLN2 will lay a foundation for further revealing the development mechanism of plastid and chloroplast and the molecular mechanism of plant photosynthetic complex.
【學(xué)位授予單位】：浙江師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

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