Subcellular Localization And Specific Expression Analysis Under Salt Stress Of QSTS8,a Quantitative Trait Locus Associated With Salt Tolerance At Seedling Stage In Rice

Soil salinity is a major abiotic stress that limits rice productivity.Rice(Oryza sativa L.)is an annual gramineous plant and is the staple food of more than half of the world's population.Rice is a salt-sensitive crop,particularly at the seedling and reproductive stages.In the present study,based on the initial mapping of salt-tolerant QTL qSTS8 at seedling stage in rice,the fine mapping of this locus was carried out to predict candidate genes and clone gene sequence.Through the bioinformatics analysis of candidate genes,specific analysis of stress expression and subcellular location analysis,which will lay the foundation for the locus gene function research.The main research results are as follows:1.The BC3F2population were derived from a cross between a salt-sensitive variety Nipponbare and a salt-tolerant variety Faguodao.By two-step substitution mapping,qSTS8 was finally narrowed down to a 30-kb region that contains three predicted genes in cultivated rice between the two markers MK8 and OSSR8.Through sequence blasting of these three genes between Faguodao and Nipponbare,the predict gene LOC_Os08g33154 and LOC_Os08g33160 were finally determined as candidate genes for the locus.2.Use of the conventional gene cloning method,the full-length CDS sequences of the LOC_Os08g33154 and LOC_Os08g33160 genes were obtained from the rice variety Nipponbare.Bioinformatics analysis showed that the LOC_Os08g33154 gene encodes 112 amino acids,with a relative molecular weight of 13269.19 and a theoretical PI value of 7.87,suggesting that this gene may encode a hydrophilic non-transmembrane protein.The LOC_Os08g33160 gene encodes 432 amino acids,with a relative molecular weight of 45378.88 and a theoretical PI value of 9.47.It is speculated that this gene may encode a non-secretory protein with hydrophilic instability.3.The temporal and spatial expression specificity of candidate genes LOC_Os08g33154 and LOC_Os08g33160 were analyzed by four stress treatment experiments and real-time fluorescence quantitative PCR technology.The results showed that the LOC_Os08g33154 and LOC_Os08g33160 genes were expressed in the roots,stems and leaves of the cultivated rice.Both LOC_Os08g33154 and LOC_Os08g33160 were induced by NaCl,ABA,PEG-6000 and the low temperature stress(4?),and the expression levels were significantly different between the salt-tolerant variety Faguodao and the salt-sensitive variety Nipponbare.Interestingly,the expression level of the LOC_Os08g33160 gene in different tissues of the salt-tolerant variety Faguodao and the salt-sensitive variety Nipponbare showed significant or extremely significant differences at nearly all the salt stress periods.4.Use of the subcellular localization technology,LOC_Os08g33154 gene encoding protein fused with GFP fluorescent protein C-terminus,and the fusion protein was expressed in the cells of rice protoplasts and tobacco leaves through transient transformation technology.The results showed that the LOC_Os08g33154 protein was located on the endoplasmic reticulum in rice cells which will lay a foundation for the functional study of the LOC_Os08g33154 encoded protein.5.Use of the subcellular localization technology,LOC_Os08g33160 gene encoding protein fused with GFP fluorescent protein N-terminus,and the fusion protein was expressed in the cells of rice protoplasts and tobacco leaves by transient transformation technology.The results showed that the protein encoded by the LOC_Os08g33160 gene was located in the nucleus which will provide a theoretical basis for further research on the function of the LOC_Os08g33160 encoded protein.