Map-based Cloning And Preliminary Functional Analysis Of Rice PPR Gene TCD13

Rice is the most important food crop in our country and is also a model crop for scientific research.Chloroplast is a semi-autonomous organelle in plant,and it is also an important organelle for plant growth to survive and obtain nutrients from the air.PPR proteins are a relatively large family that exists widely in various plants.Some studies have shown that the functions of most PPR proteins are highly related to the development of chloroplasts and the synthesis of chlorophyll,and their main functions are responsible for intracellular RNA.Processing modifications such as splicing and editing^[1].In this study,"Jiahua 1"was induced by EMS to a low temperature-sensitive leaf color albino mutant tcd13,and then a series of experiments were carried out to explore the following conclusions:1.The tcd13 mutant showed a completely albino phenotype at low temperature growth environment(20?),and died due to the inability to perform photosynthesis after growing to the five-leaf stage.At high temperature growth environment(32°C),the mutants can basically restore the green phenotype,which is not significantly different from the wild type.2.Under the condition of low temperature(20?),the content of photosynthetic pigments in the tcd13 mutant was extremely lower,and the chloroplast development was incomplete;while at high temperature environment(32?),the content of chlorophyll was obviously restored,and the development of chloroplast was also close to normal.3.The F₂ generation genetic population was obtained by crossing Pei'ai 64S with tcd13,and the genetic analysis and map-based cloning were carried out.It was found that the mutant gene(tcd13)was located within 68 kb between the molecular markers ID01M 04741 and ID01M 04758 of rice chromosome 1,after sequencing only found that a PLS-DYW-type PPR protein(LOC?Os01g10800)gene has a mutation site in which a base C changes to T on its exon;transgenic complementation experiments also confirmed that the albino trait of the mutant was caused by TCD13 caused by mutation.4.The subcellular localization experiment found that TCD13 protein was localized in the chloroplast,and the tissue localization and expression analysis showed that the TCD13 gene was highly expressed in its leaves and stems.5.The loss of function of the TCD13 gene led to a significant down-regulation of the expression of some PEP-dependent genes in the mutant(tcd13)under the low temperature condition of 20°C,resulting in leaf whitening.It is speculated that this is associated with abnormal chloroplast development and eventual death in mutant tcd13at low temperature.6.The results of chloroplast-related gene transcript sequencing found that TCD13 is involved in the splicing of the b6 subunit gene pet B in the cytochrome b6/f complex in the thylakoid structure responsible for synthesizing chloroplasts.It cannot assist in the post-transcriptional RNA splicing of the pet B gene,and TCD13 is not required for this splicing process at high temperature.