Screening And Gene Cloning Of Less Lateral Root Mutant Induced By ABA In Arabidopsis

As vital post-embryonic development organ, lateral root plays an important role on root architectureestablishment in arabidopsis. Although we have known that lateral root development can be affected byplant oneself nutrition level and the outside biological, biological environment conditions, molecularmechanisms of lateral root development about these factors, especially ABA (abscisic acid), are poorlyunderstood. Accordingly, key gene cloning and studies on molecular mechanism of lateral rootdevelopment regulated by ABA are of great significance. After largely and repeatedly screening relatedmutant to lateral root development in ABA stress condition, we got4related mutants whose phenotypeswere stable. This paper focuses on one T-DNA insert mutant named raa2-1whose lateral root number isless than that of wild type in ABA stress condition. Through the TAIL-PCR, we learned that theinformation about the gene RAA2, and at the same time we got another T-DNA insertion mutant materialson the gene RAA2from the salk seed bank, named raa2-2.Through phenotype analysis, genetic analysis, gene cloning and simple function analysis on mutants,we get the main research results, as following:(1) By screening from280kinds of T-DNA insertionmutants (from Jianru Zuo lab) and40EMS mutagenic mutants (from Chunpeng Song lab) in ABA stresscondition, we finally obtained4related mutants to LR development whose phenotypes were relativelystable. They are t237, t245, e28and e45. In MS medium containing5μM ABA condition, their lateral rootnumber is less than that of wild type (WT) Columbia (Col-0), while their phenotypes show no significantdifference from phenotype of wild type in MS medium condition.(2) Function depletion mutation ofRAA2results that formation of mutants lateral root primordia is reduced more largely in the exogenous5μM ABA existence condition. In different types and concentrations sugar existence condition, mutant rootsystem phenotype is not significantly different from wild type’s.(3) Germination experiment treated with0.5μM and1μM ABA shows that the germination ratio of mutant seeds is lower than that of wild typeseeds, and mutants are more sensitive to ABA. Water loss and infrared experiments show that mutantstomatal opening may be bigger than wild type’s.(4) Genetic analysis shows that mutants are single generecessive mutations. Through TAIL-PCR we have cloned RAA2gene which is located in the first chromosome. The results of bioinformatics analysis on the gene RAA2show that protein RAA2withmultiple transmembrane domains belongs to LrgB-like family, and the membrane proteins homologous tothe bacteria lrgAB operons have two conservative LrgA and LrgB domains. Function of the membraneprotein RAA2is unknown at present. The T-DNA insertion site into the gene RAA2in mutant raa2-1islocated in the second exon. The T-DNA insertion site into the gene RAA2in mutant raa2-2is located in thefirst intron.(5) Analysis results on transcription level of the gene RAA2show that the gene RAA2is bothknocked out in two mutants. We also constructed the complement vector and the super expression vectorfor the gene RAA2in order to understand further its function.By the above experimental results, we guess that the root architecture signal transduction processregulated by ABA is rather complex, and its specific mechanism may not be related to sugar signal. Wealso found that the gene RAA2may participate in other signal transduction process regulated by ABA. Inaddition, we constructed the complement vector and the super expression vector for the gene RAA2to dobasic work well.