拟南芥叶绿体分裂突变体<i>x</i>17-3和<i>pd</i>50的基因鉴定与分析

李劲宇; 高原; 张琴; 刘小敏; 高宏波

doi:10.13332/j.1000-1522.20170433

拟南芥叶绿体分裂突变体x17-3和pd50的基因鉴定与分析

Genetic identification and analysis of chloroplast division mutants x17-3 and pd50 in Arabidopsis thaliana

摘要

摘要:
目的叶绿体起源于内共生的蓝细菌，它通过与细菌类似的分裂方式进行增殖以维持遗传稳定。叶绿体分裂需要起源于原核和真核细胞的蛋白高度协调。拟南芥是研究叶绿体分裂的模式植物。在过去的20年中，人们对拟南芥叶绿体分裂蛋白复合体进行了初步的研究。然而, CRL基因在叶绿体分裂中的功能还不清楚。
方法本研究通过突变体筛选和图位克隆鉴定得到2个新的crl突变体，分别为x17-3和pd50。通过显微观察和分子生物学方法分析了x17-3和pd50中叶绿体分裂表型、CRL基因剪接方式和mRNA含量以及叶绿素含量。最后通过转化互补和RNA干扰(RNAi)技术进一步确认了基因功能。
结果x17-3和pd50的叶绿体形态与野生型相比有较大差异，表现为叶绿体体积增大，细胞中叶绿体数量减少。x17-3叶绿体数量为野生型的40%，而pd50叶绿体减少到只有1~4个，植物生长也受到明显抑制。通过粗定位及测序分析发现x17-3和pd50的CRL基因存在突变，突变位点在内含子并且影响mRNA剪接，最终导致阅读框移码突变。通过实时荧光定量PCR分析发现，pd50中CRL mRNA含量比野生型和x17-3明显降低。遗传互补实验进一步验证了x17-3和pd50中叶绿体分裂和植物生长抑制表型是CRL基因突变导致的。应用RNAi技术抑制CRL基因表达也能产生明显的叶绿体分裂异常表型。此外，pd50和x17-3的叶绿素含量比野生型明显降低。
结论本项工作为进一步揭示CRL基因的功能提供新的研究材料和实验依据。

Abstract:
ObjectiveChloroplasts are originated from endosymbiontic cyanobacteria. During cell proliferation, chloroplasts divide by binary fission, a manner like that in their bacterial ancestor, to ensure the stable inheritance. Chloroplast division requires highly coordinated actions of proteins with both prokaryotic and eukaryotic origin. Arabidopsis thaliana is a model plant for the study of chloroplast division. The chloroplast division machinery has been preliminarily studied in Arabidopsis thaliana in the past two decades. However, the function of CRL gene in chloroplast division is still unclear.
MethodIn this study, we identified two new crl mutants x17-3 and pd50 by mutant screening and genetic mapping. We compared the chloroplast phenotype, gene splicing, mRNA level and chlorophyll contents of x17-3, pd50 and the wild type by microscopic observation and molecular biology techniques, respectively. Further gene function confirmations were done by transformation complementation and RNAi.
ResultCompared with the wild type, x17-3 and pd50 showed an obviously different chloroplast phenotype. They had much larger chloroplast size and a lower number of chloroplasts per cell than those in the wild type. The chloroplast number of x17-3 was 40% of the wild type. pd50 contained only 1 to 4 chloroplasts per cell and showed a severe growth inhibition. Genetic mapping and DNA sequencing analysis revealed mutations of the CRL gene in x17-3 and pd50, which were present in intron and caused altered splicing and open reading frame shift mutations. The mRNA level of CRL decreased in pd50 compared with the wild type and x17-3 by real-time quantitative RT-PCR analysis. Complementation experiment result confirmed that the chloroplast division and plant growth defects of x17-3 and pd50 were due to the mutations in CRL gene. The CRL RNAi lines with decreased levels of the CRL mRNA also showed chloroplast division defect. In addition, the content of chlorophyll decreased in x17-3 and pd50 compared with that in the wild type.
ConclusionOur study provides new material and some useful information for the study of the function of CRL.

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