Functional Analysis Of Gene AtMYBC1 In Arabidopsis Thaliana Under Abiotic Stresses

Stressed environmental conditions such as high salt,drought,and low temperature are the major factors that limit plant productivity severely,which threaten the agriculture industry in China as well as the whole world.That is why enhancing the crop's resistant ablity to osmotic stress become one of the urgent tasks in the worldwide.With the accomplishment of sequencing for the genome of Arabidopsis thaliana,amount of information about gene sequences has been accumulated in the public database,though 90%of them was still unidentified.The reverse genetic technologies such as complementation,overexpression,antisense inhibition,gene knock-out,gene trapping,and gene activation,functional incapacitation mutation has been used to acquire mutant,which can be use to study the direct relationship between the gene and the phenotype.So the reverse genetic technology is one of the most powerful and direct evidence for gene function analysis.Based on the research by Yong Li,who had predicted the stressed response candidate genes by the characters of the cis-element upstream,realtime PCR was used to analyze the expression of the candidate genes under salt,dehydration and cold stresses.Stresses response gene AtMYBC1 was choosed for further study.T-DNA mutant of AtMYBC1 were used to study the phenotype when treated with salt,drought,cold and ABA to study the function of AtMYBC1.AtMYBC1 was cloned by RT PCR,and the plant expression vector that carrys the AtMYBC1 has already been constructed, and the transgenic homozygous plant that overexpress AtMYBC1 were obtained,all of these had made great preparation to further study the function and mechanism of AtMYBC1.The main results was summarized as follows:1.Bioinformatic analysis of the gene AtMYBCAtMYBC1 protein is similar to MYB transcription factor,and belongs to the R1/2-Myb family of transcription factors,Osmotic response element such as ABRE,CGTCA motif and ERE can be found in the promotor which indicated that gene AtMYBC1 involved in the abiotic stresses as regulator.2.Expression analysis of gene AtMYBC1 under stressedRealtime PCR results indicated that AtMYBC1 response to salt,drought and low temperature rapidly and transiently.The expression of AtMYBC1 is similar to genes encode regulatory protein factors which indicated that gene AtMYBC1 may also work as a transcriptor. Gene AtMYBC1 is up regulated under salt and dehydration stresses and have a same profile which indicated that the regulation mechanism may be also same under the two stresses.But the expression profile is different in the aerial parts and the root,which indicated that gene may be also involved in other regulation system. Semi-quantitive RT-PCR results showed that gene AtMYBC1 is a ABA response gene.The expression of gene under ABA treatment is as the same as the expression under salt and dehydration treatment which also reached a peak at 3h.So we concluded that ABA works as the signal to induce the expression of gene AtMYBC1 when the plant attacked by salt and dehydration.The expression of gene AtMYBC1 of cold stress is different from that of ABA, which may be caused by the time delay or the other signal that co-regulated the expression of AtMYBC1.3.Analysis of T-DNA inserted mutantT-DNA mutant of AtMYBC1,were obtained from NASC.PCR identification indicated that 13 homozygous were obtained which was further confirmed by RT-PCR identification.The T-DNA inserted homogygous were named atmybcl,atmybcl mutants were more tolerant to cold than the wild type,and the ion-leakage of the atmybcl was lower than the wild type when kept in -12℃,which indicated that gene AtMYBC1 negatively regulate the cold tolerance, while no differences are found under salt and dehydration stresses,which may result from the gene redundancy,homologs in the same gene families retrive the the loss of gene AtMYBC1. Further study using transgenic plant that overexpress AtMYBC1 will fully understand the relationship between AtMYBC1 and salt stress,cold stress.Mutant atmybcl is hyper sensitive to ABA in seed germination assays which show that gene AtMYB1C may negatively regulate cold stress in ABA dependent way.Further study of the mechanism of gene in cold stress would contribute to regulation mechanism of cold stress.We have cloned gene AtMYBC1 by PCR.We also constructed the plant expression vector cassette pFGCN-A1.Transgenic homozygous that overexpress AtMYBC1 from 4 different transformation assays were obtained,using the genetic analysis and PCR identification.All of these had made great preparation for further studying the function and mechanism of AtMYBC. Phenotype analysis of the plant that overexpress AtMYBC1 gene is ongoing.