Cloning And Functional Analysis Of Chloroplast Development Essential Gene TSV2 Under Low Temperature Conditions In Rice

Chloroplast is semi-autonomous organelles in plants that carry out photosynthesis,which is controlled by nuclear and chloroplast genes.When mutations occur in related with these genes,the abnormal chloroplasts development and the malfunctioning synthesis of photosynthetic pigments may show,contributing to leaf color abnormality and ultimately leading to death.Low temperature stress is the main abiotic stress which affects rice growth,leading to a range of physiological and metabolic abnormalities.Especially in the stage of seedling development,low temperature will reduce the germination rate of seeds,delay the period of leaf formation and turn into green.In this experiment,the low-temperature sensitive albino mutant tsv2 obtained by ⁶⁰Co?-rays irradiation of japonica variety Jiahua No.1.Through the analysis of physiological characteristics,map-based cloning,complementation,knockout,subcellular localization,bioinformatics prediction and relevant functional analysis,the main conclusions are as follows:1.Under the condition of 20?,the mutant tsv2 exhibits the albino phenotype,malformed chloroplast and eventually died after the 4-leaf stage.As the temperature increased,the leaf color gradually recovered,and the change of the leaf color in the mutant was consistent with the chlorophyll?Chl?content and chloroplast development.At 32?,the leaf color of the mutant tsv2 returned to normal green.2.Genetic analysis of F₂ generation from cross of Pei'ai 64S and tsv2 revealed that the mutant was controlled by a recessive nuclear gene.The mutant gene was located in the 60 kb region between marker ID19911 and ID13097 in rice chromosome 2 by map-based cloning with two populations?Pei'ai 64S/tsv2,93-11/tsv2?.The candidate gene in this region was sequenced and sequence alignments revealed that a base substitut on the eighth exon of the gene LOC_Os02g33500?TSV2?.The molecular complementation and CRISPR/Cas9 genome editing techniques showed that TSV2 is responsible for the mutant phenotype.3.The gene TSV2 encodes a threonyl-tRNA synthetase belonging to class IIa of the aminoacyl-tRNA synthetase family.Conservative analysis on amino acid sequence of TSV2 protein revealed that it was high sequence similarity in different higher plants.Further analysis of the evolution relationship between more homologous proteins,showing that TSV2 protein is more closely related to monocotyledonous plants,rather than dicotyledons.4.TSV2 was abundantly expressed in leaves and mainly localized to the chloroplasts.Transcriptional level analysis showed that the mutation of TSV2evidently reduced the expression levels of certain chloroplasts-associated genes and prevented the assembly of ribosome in chloroplasts at low temperature?20??,whereas they recovered to nearly normal levels at high temperature?32??.These results indicated the vital role of TSV2 for chloroplast development under low temperature environments,and its disorder would lead to an aberrant chloroplast and abnormal expressions of chloroplasts-associated genes.It is important to study the physiological metabolism and molecular regulatory mechanism of rice under low temperature stress,and it can also help improve the low temperature tolerance of rice.