Effect Of DAD Gene Promoter Sequence Variation On Upland Cotton Domestication

Apoptosis(APO)is the physiological process of cell death that is regulated by the internal mechanism of organisms.It is common during the growth and development of multicellular organisms.Anti-apoptotic gene(Defender against Apoptotic Death,DAD)is a highly conserved gene that inhibits apoptosis in animals and plants.At present,accumulated studies of the DAD genes were mainly focused on animals and limited model plants.However,studies on the whole evolutionary pattern of the genes in plants and detailed functions in specific plants are still less poor.Upland cotton(Gossypium hirsutum L.),a tetraploid species with AADD genome,is the most common commercial cotton and is an important economic crop in China.In this study,using this species as the experimental material,we compared expressions of the DAD genes between cultivated and wild cottons,specifically explored the influences of sequence divergences in promoters of the DAD genes on cotton domestication.The main research contents and results are as follows:1.To fully understand the evolution of DAD genes in plants,the nucleotide and amino acid sequences of the anti-apoptotic genes(DAD1,DAD2)from the Arabidopsis thaliana in GenBank were used as the target sequences to blast,respectively,in NCBI and PLAZA.Finally,58 DAD homologous sequences were obtained.Through bioinformatics methods,the copy number,gene structure,chromosome location of DAD genes in different plant species and their evolutionary trends in seed plants were analyzed.Results indicated that DAD gene was a low copy gene with only 1~3 copies in different seed plants,and the length of different DAD gene varied from 108 to 201 aa.Phylogenetic and synteny analyses further showed that the evolution of the DAD gene in these seed species had an obvious lineage-specific characteristic.2.In order to explore the expression changes of DAD gene during domestication,based on our previous transcriptome data,we used the RPKM method to comprehensively compare the DAD gene expressions in different organs(leaves,petals,seeds,and fiber)between cultivated and wild cottons.The results showed that the DAD gene in upland cotton had two loci: DAD1 and DAD2.Each locus also contained homoeologs on the A and D genomes.In contrast to DAD1,the expression levels of DAD2 in leaves,petals,seeds,and fibers of cultivated cottons were all higher than that of wild ones.In addition,the expression levels of DAD2 in A genome were mostly higher than that in the D genome.3.The expression of plant genes is meticulously controlled at all aspects,of which the roleof promoters is particularly important.Above studies have shown that the expression divergence of A genome homoeolog of DAD2 between the cultivated and wild cottons is relatively larger,but the identity of coding sequences of two cottons is conserved.Therefore,it is firstly investigated whether the difference of DAD2 expression is related to the sequence divergence in promoters between the cultivated and wild cottons.About 1500 bp upstream of the two DAD2 genes were cloned and sequenced.Except that a 30-bp short fragment was inserted in the promoter of cultivated cotton(like a TATA-box-element),17 SNPs were found in the upstream regions between the cultivated and wild cottons.In addition,from the point of view of gene function,the cultivated cotton obtained 2 new cis-acting elements through 2 SNP variations,which were a gibberellin reaction element and an anaerobic induction related element,respectively.4.To explore whether the expression change of DAD is related to the sequence mutations in promoter,we replaced the CaMV 35 S promoter of PBI121 with that of DAD2 genes of the two cotton accessions,and then fused them with a GUS gene,respectively.Plant expression vectors(PDAD2-M and PDAD2-T)were constructed while using the PBI121 as the positive control.Tobacco was then transformed by three vectors via the Agrobacterium-mediated method,respectively.The results of GUS histochemical staining were consistent with those of GUS enzyme activity assay: DAD2 gene promoter activity in cultivated cotton was significantly higher than that in wild cotton(P<0.05).5.To investigate whether a cis-acting element,which related to gibberellin response,in promoter of the cultivated cotton is functional,the expression levels of DAD2 in both cultivated and wild cottons were compared after treating with 50 ug/mL Gibberellin at different time points(30 min,1 h,2 h,6 h,12 h,and 24 h).Results of Quantitative Real-time PCR showed that gibberellin treatment promoted expressions of the DAD2 genes in both cultivated and wild cottons,but relative expression of DAD2 gene in cultivated cotton was significantly higher than that of the wild cotton(P<0.05).