Map-based Cloning And Phenotypic Analysis Of A Set Of Salt-Sensitive Mutants In Arabidopsis

Stress is major limiting factor in agricultural production, abiotic stress such as high salt, drought, cold, heat seriously influence the growth and development of plants. In the process of plant growth, the expression of a series of genes involved in abiotic stress response could be regulated to improve the resistance of plants. The flowering plant Arabidopsis thaliana is an important model plant, the identification of its genes and their functions could provide a rapidly systematic way to identify stress response genes for research on agricultural crops.Ethylene-responsive factor 3 (ERF3) encodes a member of the ERF (ethylene response factor) subfamily B-1 of ERF/AP2 transcription factor family (ATERF-3). Generally, ERF3 promoter is suppressed by transcription inhibitors in plant, and its expression level is very low, but ERF3 can be induced by high salt, ABA and other abiotic stress, and its expression could be corresponding increased. To investigate the essential stress signaling mediating components in plants, Professor Yang's lab constructed the transgenic Arabidopsis thaliana plants sy24, which carrying the firefly luciferase reporter gene under the control of the ERF3 promoter. Then transgenic seeds were mutagenized with MES and finally got a set of mutants. A supersensitive CCD camera could be used to screen mutants that exhibited higher level of LUC density in response to stresses (such as high salt, etc). In this research, three mutants are finally screened. They are P13H09, sy29, sy30. Genetic analysis showed that these three mutants are all recessive mutation controlled by a single gene.Fluorescent phenotypic analysis showed they are all more sensitive than the wildtype when treated with NaCl, ABA, Sorbitol, cold, but insensitive to high temperature. Phenotypic analysis showed that:(1) With high salt treatment, the seed germination of sy30 was inhibited, but had no effect on P13H09 and sy29, and the root growth of the three mutants were all inhibited by NaCl; (2) with ABA treatment, the seed germination of sy30 was restrained, there were no effect on P13H09 and sy29% the root growth of sy30 was inhibited, on the contrary, root growth of sy29 was not sensitive to ABA; (3) sy30 were more sensitive to high pH; (4) the three mutants were all not affected by high temperature.In this experiment, the method of map-based cloning was used for the positioning of this set of mutant genes. The results showed that the P13H09 mutant gene was in chromosome 4, between CER450392 and CER448513, and the genetic distance was 0.23M, RNA sequencing analysis showed that in this area the gene At4gxxxxx had a point mutation from G to A, amino acid had changed frome Gly to Asp; the sy29 mutant gene was in chromosome 4, between CIW7 and CER466462, and the genetic distance was 0.15M, RNA-Seq analysis showed that in this area the gene At4gxxxxx in UTR had a point mutation from G to A; the sy30 mutant gene was in chromosome 5, between CER457578 and CER457835, and the genetic distance was 0.24M, sequencing analysis showed that in this area the gene At5gxxxxx had a point mutation from C to T, amino acid had changed frome Gln to stop codon. These results also showed that these genes in Arabidopsis play important roles in plant stress resistance.This study established a feasible method of map-based cloning in Arabidopsis. Meanwhile, the study of salt resistance genes in Arabidopsis can lay foundation for identifying of a wide range of specific plant genes and a more comprehensive understanding of gene functions.