Cloning, Expression Analysis And Development Of A Functional Maker Of CO-like Gene TaCO9in Wheat(Triticum Aestivum L.)

Photoperiod is one of important factors that trigger the change from vegetative growth toreproductive growth. CONSTANS (CO) is a key role in of the Photoperiod Pathway in themodel plant Arabidopsis. The former researchers payed more attention to the venalizationgene, but the CO family especially the forth member needs further research. To learn moreabout the CO gene in wheat, the homologous cloning method was used to isolate the relatedgene. The results are followed:1. Here a novel gene TaCO9(CO homolog) that encoding CO-like protein was clonedfrom hexaploid wheat (Triticum aestivum L.) based on the sequence of HvCO9in barely.There were three homologous genes of TaCO9in Xinong889(Winter) and all of them were870bp in length, encoding289amino acids composed of CCT domain, but not the B-boxdomain. The construction of TaCO9which contains two exons of480bp,396bp and a singleintron is similar to HvCO9.2. The tertiary tructure was predicted based on the amino acids sequence of TaCO9. TheNF-YA2of CCT domain was conserved among TaCO9, HvCO9and Ghd7. A phylogeneticanalysis showed that the TaCO9protein was located on the same branch of the CO-like treesuch as rice Ghd7and the barely HvCO9proteins, which are all negative regulators offlowering.3. Prokaryotic expression vector for TaCO9gene in common wheat (pEASYE1-TaCO9)was constructed and expressed in E. coli BL21(DE3) after deduced by IPTG, the targetprotein was detected by SDS-PAGE. The result indicated that a36.0kDa protein consistentwith the predicted molecular weight based on the amino acids edcoded dy cDNA wasobtained by IPTG inducting for10h.4. The expressions of TaCO9were detected in root, stem, leaf and young spike of wheatat heading stage by RT-PCR, the order of the expression level was leaf> stem> young spike.5.6bp insertion near the CCT domain in the second exon was detected in a spring habitvariety, Ningchun4, and a marker based on the divergence in the downstream sequence ofintron specialized in inserted sequence was developed. Using the maker and Chinese Springnulli–tetrasomic families, the mutant TaCO9gene in Ningchun4was located on wheat chromosome1A, named TaCO9-1A.6. One codominant maker was developed based on the6bp insertion in TaCO9-1A andconfirmed by25different varieties. The codominant maker was highly correlated with notonly spring and winter habit but also the heading, flowering time of wheat cultivated atYangling for two years. This maker was expected to apply for identifying the growth habit ofwheat.