Cloning and expression analysis of TaTDR-Like genes in wheat

【Objective】 Wheat tapetum degeneration retardation-like(TaTDR-Like)genes were cloned and their expression patterns were analyzed between different tissues and different developmental stages of anthers in order to gain insight into the molecular mechanisms underlying abnormal wheat pollen development.【Method】 In this study,orthologues of rice(Oryza sativa L.)gene TDR(Os02t0120500)were cloned from the common wheat cultivar,MF-XN1376. Bioinformatics was used to analyze the characteristics of the proteins and cis-acting elements of the gene promoters. Moreover, real-time fluorescence quantitative PCR(qRT-PCR)was used to assess the tissue-specific expression of TaTDR-Like genes in different tissues(anther,leaf,ovary,stem and spike)and expression pattern of TaTDRL-D genes in anthers at different developmental stages. The subcellular localization of TaTDRL-D protein was observed,and yeast two-hybrid technology was used to detect its self-activation. The plant fusion expression vector 35S-TaTDRLD-EGFP was constructed and transferred into tobacco leaf epidermal cells through agrobacterium-mediated transfer.【Result】 It was found that there were three homologous copies,namely TaTDRL-A,TaTDRL-B and TaTDRL-D,encoding 550,553 and 557 amino acids(aa),corresponding to molecular weights of 58.50,58.90 and 59.21 kD and isoelectric points(pI)of 4.77,4.66 and 4.63,respectively. TaTDR-Like proteins all have a bHLH conserved domain at the C-terminal. TaTDRL-A and TaTDRL-B had 100% and 96% similarity with TaTDRL-D,respectively,while TaTDRL-D showed 98% and 88% similarity with OsTDR and AtAMS,respectively. A phylogenetic tree analysis showed that TaTDR-Like proteins were closely related to rice TDR(Os02g0120500),Brachypodium distachyum TDR(XP014756384), barley(KAE8787147.1)and Arabidopsis AMS(AT2G16910.1). Promoter analysis results showed that TaTDRL-A,TaTDRL-B and TaTDRL-D promoters contain many cis-acting elements,such as those involved in the response to light conditions,abiotic stress and phytohormones,which may participate in plant growth,development and the response to abiotic stress. Tissue-specific expression analysis showed that TaTDRL-D had the highest expression level in anthers,followed by young panicles and stems,while TaTDRL-A and TaTDRL-B showed higher expression levels in young panicles. Further studies revealed the expression level of TaTDRL-D to be the highest at the early mononuclear stage of anther development after which it showed a gradual decrease. The expression level in cytosolic male sterility tissues(CMS-XN1376)was higher than that in male fertile tissues(MF-XN1376)from the late mononuclear stage to the tri-nuclear stage. The results showed that TaTDRL-D had transcriptional activation activity. Subcellular localization showed that the TaTDRL-D protein was localized in the nucleus.【Conclusion】 TaTDRL-D gene may be involved in anther development and negatively regulate anther fertility in wheat.