Arabidopsis Resistance Against The Mutant Screening And Identification

Salt stress is one of the abiotic stress factors that seriously affect plant growth, development and production. With the increasing soil salinization and the increasing world population, the food supply becomes more and more insufficient, so the important issues about the agricultural productivity were how to develop and utilize saline land and how to increase crop yield. Arabidopsis has been used as a model plant to study gene functions because of its special biological and genetic characteristics; it has also played important roles in plant resistance research. There are two genetic methods to enhance the tolerance of stress environment and ultimately to cultivate salt-tolerant crops, namely, transferring new stress-related genes into the plants or changing the expression of the salt tolerant genes. In the world, many Arabidopsis mutant libraries have been established through various methods and have provided a good platform to study the gene function.In our reasearch, we screened Arabidopsis salt tolerance mutants from two activation tagging mutant pools which were generated with pSKI015 containing four tandem CaMV 35S enhancer close to the T-DNA right border or with the XVE activation-tagging vector pER16,an estrogen-inducible LexA-VP16-Estragon Receptor expression XVE system in the Columbia ecotype. The aim of this reasearch is to obtain the genes that involved in salt tolerance.The main physiological function of Rnase III is to control the cell type and the amount of RNA distribution; involved in the post-transcriptional RNA processing, modification and degradation; it is also involved in self-incompatibility, organogenesis, the host defense mechanisms to control tumor angiogenesis, killing tumor cells, inhibiting virus replication and so on.We had obtained T-DNA insertion lines of AT1G69340, AT2G40600, and AT5G01310 mutants from the ABRC to carry out phenotype analysis and gene function studies. The studies suggested that YmdB of E. coli interacting with a site in the RNase III catalytic region and down-regulated RNase III activity. The expression of YmdB is transcriptionally activated by both cold-shock stress .In this experiment we study the homologous genes of YmdB in Arabidopsis: AT1G69340, AT2G40600, AT5G01310, the aims are to explore these three genes'functions in stress and the relationship between the genes and RNaseⅢactivity.The study of AT1G69340 gene in the embryonic development of mutants found that many seeds of Arabidopsis embryo development were related with genes, such as SIN1,FIS1,MEA, etc. For example, studies have shown that MEA is not expressed before the formation of the embryo ( flower development period), expressed in the presence of mature gametes unfertilized fruit pod, and stop expressed in the mature seeds after fertilization. MEA expression is regulated by genomic imprinting after fertilization; the paternal gene does not reduce the embryonic lethality. In addition, the paternal gene is silence only in the endosperm, not in the vegetative organs. Moreover, maternal effects on embryonic development is independent, regardless of the dosege of the paternal genes.The main purposes of this study are to obtain salt-tolerant mutants, analysis AT1G69340, AT2G40600, AT5G01310 mutant phenotype and gene function. The main results were as follows:1. Screened the mutants from the 4200 strains of the T-DNA insertion mutant library and 4100 strains of the induction of activation tagging mutant library. In the initial screening, 56 mutants were obtained, but by the descendants of further verification, we hadn't obtained stable genetic salt-tolerant mutants.2. By processing anlysis of the mutants CS808255, CS839860 (T-DNA insertion site is AT1G69340), CS830160, CS823606 (T-DNA insertion site is AT2G40600), Salk₀24760c, Salk₀80565c (T-DNA insertion site is AT5G01310), we obtained the homozygotes except the mutant CS808255, CS839860.3. Constructied the GUS, GFP expression vector of the gene AT1G69340, AT2G40600 and searched infections of the inflorescence, we have been harvested seeds.4. Observed embryo development of the seed and tested the viability of the pollen by pollen staining of the mutants of CS808255, CS839860, and the results showed that the seeds embryo of mutant 8-9d after flowering were slightly lags behind the wild-type Seed embryo and the pollen of the mutants were similar with the WT.5. We observed the expressions of genes AT1G69340, AT2G40600, AT5G01310 in different stress treatments by real-time quantitative PCR. The gene expression was similarly except that the gene AT5G01310 increaed expression under the stress of the 300mM minotol for 3h and translate the plant from38℃(for 3h) to 25℃( for 4h).