Preliminary Analysis Of The Function Of The TaAgl6 In Bread Wheat(Triticum Aestivum)

Common wheat,maize and rice are all belong to major grain crops. But the biological studies of wheat are under-developed, as a result of its genome is complexed. And the progress of studies on wheat gene function is backward.The seed development of wheat is closely related to the flower development, and affects the grain yield and quality. Compared to Brachypodium distachyon and rice, the flower morphological structure and develpoment of wheat has its own characteristics, for example, a spikelet primordium can forms many florets primordium, with uncertainty. And a floret has 1 lemma, 1 palea, 3 stamens, and 1 carpel. It has theoretical significance and application value to analyse the genes’ biological functions and reveal the molecular mechanisms of flower development in wheat.Flowering is an important transition from vegetative to reproductive growth, many genes involved in this process, AGL6(AGAMOUS LIKE 6) is one of the most important subfamily. AGL6(AGAMOUS LIKE 6) gene family is an ancient MADS-box gene family in plants, involved in the regulation of flower development. In recent years, the research about AGL6 gene function has been made a lot of progress, especially in Rice(Oryza sativa), maize(Zea mays), Petunia(Petunia hybrida) and other species. There are 3 AGL6 homologous genes in common wheat genome, located in the 6A, 6B, 6D genome. However, their biological functions are not clear. Due to the differentiation of the function of AGL6 genes, the specific biological function of TaAGL6 gene in wheat flower development cannot be predicted. In this study, we analyzed the biological function of TaAGL6, layed the foundation for revealing the molecular mechanism of the flower development in wheat.The main contents and results of this thesis are as follows:Firstly, morphological characteristics of wheat flower are analyzed. In order to analyze the wheat genes’ function related to flower development, it is necessary to understand the characteristics of wheat flower morphology and development. We used the technology of parafin section and scanning electron microscope and other methods to observe the morphological characteristics of inflorescence in different growth period of Chinese Spring.Secondly, the TaAGL6 genes’ cDNA sequences were cloned and analyzed the expression pattern.We use Chinese Spring cDNA as template, cloned 3 cDNA sequences of the TaAGL6, according to their location on chromosome 6A, 6B and 6D, we named this 3 genes as TaAGL6-A, TaAGL6-B and TaAGL6-D. Sequence alignment showed that the 3 genes are conserved, in addition to SNP, TaAGL6-A, TaAGL6-D coding region in 624 have 3 bases more than TaAGL6-B.ned primers. The results of temporal and spatial expression pattern analysis of TaAGL6 genes showed that TaAGL6 expressed mainly in flowers. They also expressed in inflorescence of different lengths(represent different development stages of flowers). We found that TaAGL6 exert biological functions mainly in the flowers. In the floret, TaAGL6 genes mainly expressed in the carpel, palea and lodicules. We also designed the specific primers, respectively, analysis the expression patterns of the 3 TaAGL6 genes in floral organ, the results showed that the express pattern of the 3 TaAGL6 genes was consistent with the results of the universal primers in flower organ.Next, We constructed the over-expression vector of TaAGL6-A, TaAGL6-B and TaAGL6-D, and transformed to the Arabidopsis thaliana, respectively. The over-expression transgenic Arabidopsis showed 2 phenotypes: low plant height and No. of main stem and branches increased, blossom time earlier. Taking TaAGL6-D as an example, we analyzed TaAGL6-D and FT in the flowers of over-expression transgenic Arabidopsis plants, we see that the expression level of FT was up-regulated in transgenic Arabidopsis thaliana. Cytokinin and auxin increased in proportion in transgenic Arabidopsis stem and branches.Finally, TaAGL6 RNAi expression vector was constructed and transformed into wheat. Sequence analysis, expression pattern analysis and transgenic Arabidopsis results suggest that the 3 TaAGL6 genes are functionally redundant. This means that if only interfered with the expression of 1 or 2 genes, transgenic wheat may have no abnormal phenotype. Therefore, according to the analysis of TaAGL6 genes’ cDNA sequence in the 3’end, we designed primers and constructed the RNAi vector, silenced the expression of the 3 TaAGL6 genes at the same time.We have obtained a number of transgenic wheat. Using the sequence on the carrier as the forward primer and the sequence on the target gene as the downstream primer. Through the identification of PCR, 22 strains were positive transgenic wheat. qRT-PCR results showed that some transgenic plant target gene expression level down-regulated. Based on the phenotype of transgenic wheat, some strains were analyzed. The preliminary results showed that transgenic TaAGL6 RNAi wheat plants, appears weak, fertility decrease, blossom time delayed, the branching stems, number of carpel-like structures or structure of ovary increased and the number of stamens decreased or absent.Above all, sequence analysis and expression pattern analysis of transgenic Arabidopsis results suggest that the three copies of TaAGL6 may be redundancy on gene function, and they are involved in the regulation of flowering time and the floral organ identity of the inside two whorls. The decreased of the TaAGL6 expression level resulted in the number of carpels in florets increased, the number of stamens decreased or the stamens were abnormal. Meanwhile, the abundance of other floral organ characteristic genes in the transgenic wheats changed, the abundance of AP3 and SEP gone down, and the levels of Ta-D, TaAG, TaDL gone up. All the results suggested that TaAGL6 genes regulated flowering time and floral organ development. These genes can also influenced the expression of floral organ identity genes. The results of this study can provide resources for further understand the biological functions of MADS-box genes. Meanwhile, these results laid a solid foundation for elucidate the molecular mechanism of wheat flower development.