水稻质体发育调控基因（OsFLN2）的克隆及初步功能分析

发布时间：2019-04-16 15:16

【摘要】：叶绿体是绿色植物进行光合作用的重要场所,同时也是光合色素的载体,广泛分布在叶片绿色组织细胞中。高等植物叶绿体的发育是一个非常复杂的调控过程,需要核基因编码蛋白和叶绿体基因编码蛋白协调完成,叶色突变是研究质体、叶绿体发育的重要材料,相关调控机制的研究结果在基础研究和生产实践中都将发挥非常重要的作用。水稻白穗突变体qp(t)1是由粳稻品种“日本晴”经EMS诱变而来,该突变体在3叶期之前表现为完全的白化,随着植株的生长,3叶期之后新抽出的叶片开始转绿,而老叶片的叶绿体发育也缓慢恢复,呈现白色条纹状,抽穗期穗部白化。图位克隆发现位于第3号染色上的AP(t)1基因(LOC_Os03g405500)第五个外显子出现一个单碱基的替换突变,由G突变为A,引起第537个氨基酸由色氨酸(Trp)突变为终止密码子,导致蛋白编码提前终止。将互补载体转化突变体后,突变表型得以恢复正常,进一步证实AP(t)1基因突变确实是造成水稻白化的内在原因。生物信息学分析结果表明AP(t)1与拟南芥AtFLN2基因同源。Real-time PCR实验表明,OsFLN2基因在水稻中各个部位都有表达,但主要在叶片中表达。ap(t)1突变体中OsFLN2基因的表达量显著下降,而互补转基因株中的表达量与野生型基本一致。OsFLN2基因突变后对质体编码的RNA聚合酶(PEP)参与编码的基因影响较大,突变体中的PEP类基因的表达量较野生型显著下降,而核编码的RNA聚合酶(NEP)类基因变化不大。亚细胞定位实验发现OsFLN2定位于水稻原生质体的叶绿体上。运用CRISPR-Cas9技术根据目的基因的序列对野生型进行定点敲除,产生的纯合型敲除阳性转基因株表型与突变体类似,而且定点突变后的白化转基因株后期并不能转绿,说明定点突变株中OsFLN2的功能可能遭到了更为严重的破坏。对FLN2功能的解读,将为进一步揭示质体及叶绿体发育机制,阐明植物光合复合体的分子作用机理奠定基础。
[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.
【学位授予单位】：浙江师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

【参考文献】