Cloning And Expression Of TaD27and TaCCD8 In Wheat

As a novel type of plant hormone,strigolactones(SLs)inhibit tillering and shoot branching.In recent years,numerous studies about SLs biosynthesis and signaling transduction pathway emerged which makes it become a hot topic in the field of plant biology.Wheat is an important food crop,and tillering is one of the most important agronomic traits that determine wheat grain yields.Therefore clarifying the mechanism of tillering regulated by SLs in wheat is not only valuable for scientific research but also for agricultural production.In this research,wheat ?-carotene isomerase encoding gene D27(TaD27)and carotenoid cleavage dioxygenase 8 encoding gene CCD8(TaCCD8)which are key genes in biosynthesis of SLs were cloned,then their expression patterns and biological functions were analysed.The main results are as follows.1.Tow highly homologous cDNA fragments of TaD27 were cloned.One is located on the long arm of chromosome 7A(TaD27-7AL)and the other located on the long arm of chromosome 7D(TaD27-7DL).In addition,we found another locus located on chromosome 7BL encoding the third homolog(TaD27-7BL).Each of three TaD27 s contains 7 exons composing an ORF of 828bp(7AL),843bp(7DL)and 840bp(7BL)respectively and encodes a protein containing a chloroplast transit peptide at N-terminal.Analysis of phylogeny indicates that D27 s in plants clustered into three independent clades.TaD27 s share a high level of identity with orthologs in other monocots such as zea mays and Oryza sativa;as a result,they are clustered in the same clade.2.The expression level of TaD27 is relatively high in leaves,sheaths and stems;lower in young panicles;and lowest in roots.Under low phosphorus stress,the expression of TaD27 in roots is down regulated to the bottom in 6h-12 h firstly,and then it rises to the normal level after 96 h.On the contrary,the expression of TaD27 in leaves is up regulated in early stage and reaches the peak at 6h,then it continuously drops to a relatively low point at 96 h.3.Two wheat TaCCD8 s were cloned.One is located on the short arm of chromosome 6B(TaCCD8-A)and the other located on the chromosome 3B(TaCCD8-B).TaCCD8-A has two types of alternative splicing forms,which were named TaCCD8-A1 and TaCCD8-A2.Wheat TaCCD8-A and TaCCD8-B proteins contain a chloroplast transit peptide at the N-terminal which indicates that they both execute function in plastid.Analysis of phylogeny indicates that,in plant,there are two kinds of CCD8 s,Ta CCD8-A and TaCCD8-B.TaCCD8-B exists both in monocots and dicots.In contrast,TaCCD8-A exists only in monocots.4.The expression of TaCCD8-B is higher in leaves,stems and panicles,especially in leaves;lower in roots and sheaths,and the expression in leaves is about 150 times higher than that in roots.The expression of TaCCD8-A1 is higher than that of TaCCD8-B in all selected tissues except leaves.The expression of TaCCD8-A1 is highest in sheathes and is about 4 times of that in roots.Under low phosphorus stress,the expressions of TaCCD8-A1,TaCCD8-A2 and TaCCD8-B in roots are all down regulated at first,and reach the lowest point at 12-24 h around.After that,the expressions are all up regulated,and after 96 h,expressions of TaCCD8-A1 and TaCCD8-A2 are both higher than their initial state,but the expression of TaCCD8-B remains lower.5.TaCCD8-B and TaCCD8-A1 proteins with MBP tags were obtained by prokaryotic expression and affinity purification.Both MBP fusion proteins are partially soluble,and their molecular wights are around 98 kDa and 72 kDa respectively.Recombinant plant expression vectors containing three TaCCD8 s were constructed and an approach to abtain callus from immature embryos of Brachypodium distachyon was established.