Functional Investigation Of Salt Tolerance Associated MATE Transporter In Rice And Map Based Cloning Of White Stripe Leaf Gene OsMetRS1

As one of the world’s top ten food crops,the yield and quality of rice are essential to human survival.The discovery of rice salt-tolerant genes and the molecular mechanism of internal salt-response in rice will not only lay a theoretical foundation for the cultivation of salt-tolerant rice,but also have important application value for solving the problems of saline-alkali land resource utilization and grain production.A salt-tolerant MATE transporter was identified on chromosome 7 of rice by Genomewide association analysis in our previous laboratory,which was tentatively named OsMATE(t)(LOC_Os07g31884).This topic takes OsMATE(t)as the research object and explores the function of this gene by means of molecular biology.The main research results are as follows:1.The expression pattern of OsMATE(t)in different tissues of rice was analyzed by qRT-PCR,and it was found that OsMATE(t)as constitutively expressed,and the highest expression was found in leaves.At the same time,we detected the expression level of this gene after NaCl treatment at different times.The results showed that salt treatment could induce the expression of OsMATE(t)in roots and leaves of rice.The expression level of OsMATE(t)was significantly up-regulated in the aboveground part after 8 hours of induction,and in the underground part after 12 hours of induction.2.Transient expression of OsMATE(t)in tobacco indicated that the protein was located in the plasma membrane.Twelve transmembrane domains of OsMATE(t)were predicted by TMHMM Sever software.At the same time,we tested the ion transport ability of OsMATE(t)with yeast defective strain,and the results showed that the protein had the function of salt ion transport.3.In order to further explore the biological function of OsMATE(t)under salt stress,we constructed OsMATE(t)knockout vector.And transgenic positive plants were obtained.The results of hydroponic experiments at seedling stage and planting identification in salt-alkali field showed that OsMATE(t)knockout families were more sensitive than wild-type ones under salt stress.4.Previous studies have showed that there were three different haplotypes in the coding region of this gene,and protein structure prediction showed that there was no difference in protein structure among different haplotypes.Therefore,the promoter sequencing analysis of rice varieties with different haplotypes showed that the number of acting elements contained in the promoter region was different in different haplotypes.As the expression level of OsMATE(t)gene was different in different haplotypes,it was speculated that the expression level of OsMATE(t)gene might be related to the difference of elements in the promoter region.Chloroplast is the energy conversion station of plant photosynthesis,and the photosynthetic rate of leaves is directly related to the yield of rice.Chlorophyll in chloroplasts acts as a "catalyst" for photosynthesis,and chlorophyll biosynthesis is a tightly controlled enzymatic reaction process.Leaf color variation,a phenotypic trait that can be easily identified,can be used as a morphological marker in rice molecular breeding.At the same time,the study of leaf color variation is of great significance for understanding chlorophyll synthesis and chloroplast genetic differentiation.In this study,a japonica rice variety Ningjing 4 was mutated by EMS,and a white striped leaf mutant osmetrs was screened from the progeny.Phenotypic observation showed that the leaves of osmetrs mutants were white striped at seedling stage and gradually returned to green at two-leaf single-leaf stage.The gene was identified by map-based cloning to encode a methithionyl tRNA synthase,so it was named OsMetRS1(LOC_OS03G11120).The main research results of this mutant osmetrs1are as follows:1.According to the phenotypic investigation,the mutant osmetrs showed a white striped state in the seedling stage,while the leaves gradually changed from white striped state to all-green state in the field under natural growth conditions.The growth of the mutant was delayed in the seedling stage due to the lack of green leaf color,which resulted in significant differences in plant height,tiller,effective panicle number and seed setting rate in the mature mutant compared with the wild type.2.The gene was located in the 218kb region of the long arm of chromosome 3 by map-based cloning.Through gene annotation and gene sequencing in this region,the gene with mutant effect was identified as a gene encoding methionyl-tRNA synthase.Sequencing results showed that a single base substitution from C to T occurred on the 8th exon of this gene in both the wild type and the mutant,resulting in the change of amino acid.At the same time,the site predicted that the three-dimensional structure of the mutant protein had also changed significantly.3.In order to identify the target gene,we constructed a gene complementary vector,and transformed osmetrs1 mutants through Agrobacterium-mediated infection.The complementary positive lineage restored the mutant’s leaf color to green at the seedling stage.4.OsMetRS1 was constitutively expressed and the highest expression was found in leaves.OsMetRSl is a chloroplast locational protein.Homologous sequence alignment and phylogenetic tree analysis showed that OsMetRS1 is a functionally conserved protein,which is ubiquitous in monocotyledonous and dicotyledonous plants.Quantitative analysis of chlorophyll biosynthesis related genes in the wild-type mutants showed that the expression of genes involved in chlorophyll biosynthesis was changed after OsMERTS1 mutation.