Cloning,expression And Function Of Dwarf Gene Rht18 In Tetraploid Wheat

Wheat is the main food crop of human beings.The lodging and pests problems greatly reduce the yield of wheat.Dwarf gene is the key gene in wheat high-yield breeding.Carrying dwarfing gene Rht18,durum wheat ANW16G is an important tetraploid dwarfing gene resource.Rht18 is a dominant semi-dwarf gene,which is sensitive to gibberellin.Map cloning shows that the dwarf gene Rht18 is the gene GA2ox-A9encoding gibberellin 2 oxidase（GA2-oxidase,GA2ox）.Rht18 has been preliminarily shown to have certain agronomic potential and breeding value in hexaploid wheat.At present,the function and dwarfing mechanism of Rht18 gene are not clear,and its utilization in breeding is also very limited.In order to enhance the utilization of tetraploid dwarf wheat and dwarf gene Rht18,using durum ANW16G and its plant height near isogenic line LD222（Langdon222）as materials,the response of ANW16G to exogenous gibberellin and the content of endogenous gibberellin were conducted firstly.Then Rht18 gene was cloned and its expression level in stem internodes at different developmental stages was detected.Furthermore,the function of Rht18 gene was preliminarily studied by transgenic Arabidopsis thaliana.The main results are as follows:1.The dwarf ANW16G was sprayed with exogenous gibberellin starting at the jointing stage until the mature stage once a week.The results showed that the plant height increased by 68.5%,and the spike length and each stem internode elongated to a certain extent,but the elongation ability of each stem internode was different.The elongation of the inverted second stem internode was the largest,reaching 166.4%,followed by the inverted third and fifth stem internodes,which were 124.9%and104.7%,respectively,suggesting that the decrease of ANW16G plant height was mainly caused by the inverted second stem internode,followed by the inverted third and fifth stem internodes.2.The GA₄ contents in stem of ANW16G and LD222 at jointing stage,booting stage and heading stage were determined respectively.The GA₄ content of dwarf ANW16G was lower than that of tall LD222 in all three stages,especially at booting stage,followed by heading stage and jointing stage.These results indicated that endogenous gibberellin GA₄ lack or deficiency led to ANW16G dwarfing,and the plant height reducing mainly occurred at the middle or later stages of growth and development.3.The inverted second stem internode at heading stage was selected for paraffin section analysis.The results of cross section showed that the diameter of internodes,the diameter of epidermal cells,the area of epidermal cells and the number of vascular bundles in ANW16G were relatively larger than those of the tall control LD222.After ANW16G was sprayed with gibberellin,due to the elongation of internodes,the diameter of internodes,the diameter of epidermal cells,the area of epidermal cells and the number of vascular bundles were closer to those of the tall LD222.The results of longitudinal section showed that there was no significant change in the internode cell length in ANW16G compared with LD222,but the number of cells per unit area（100μm²）decreased significantly.After ANW16G sprayed with gibberellin,the internode cell length did not change significantly,while the number of cells per unit area（100μm²）increased significantly,which indicated that the decrease in plant height of ANW16G was due to the decrease of cell number rather than the shortening of cell length.4.The results of cloning and sequencing showed that two types of GA2ox-A9（Rht18）gene DNA sequences were obtained in ANW16G and LD222,which were 2531bp and 2546 bp,named GA2ox-A9-1 and GA2ox-A9-2,respectively.The GA2ox-A9（Rht18）genes of the two materials contained 3 exons and 2 introns,the open reading frame（ORF）was 1044 bp in length and encoded 347 amino acids.The amino acids of GA2ox-A9-1 protein were different at position 89,275,279.The amino acids of GA2ox-A9-2 protein were different at position 182,186,251,336,the proteins encoded were typical conserved domains of 2-ketoglutarate-dependent dioxygenase.Phylogenetic analysis showed that durum wheat GA2ox-A9 had high evolutionary conservatism with other species,and was closest to Triticum urartu.Real-time PCR results showed that the expression of GA2ox-A9 gene was the highest in ANW16G of dwarf wheat,especially in the stem at jointing stage,which was much higher than that of LD222,which indicated that the high level expression of GA2ox-A9 gene,that is,Rht18 gene,was related to dwarfing traits.5.By transforming Rht18 into Arabidopsis thaliana,18 transgenic positive lines were obtained.The expression of Rht18 gene was extremely low in wild-type Col-0,but very high in transgenic lines.Compared with wild-type Col-0,the most significant change for transgenic lines was the decrease of plant height which showed extreme dwarfing.And transgenic lines also had the characteristics of growth retardation and delayed maturation.To sum up,plant height reduction of Rht18 gene is the result of the significant shortening of each stem internode,especially the inverted second stem internode.The shortening of stem internodes is due to the decrease of the number of cells,not the shortening of cell length.Rht18 gene negatively regulates the synthesis of GA₄,and high expression of Rht18 leads to plant dwarfing.The effects of Rht18 genes on plant height were confirmed by the study of Rht18 transformed Arabidopsis thaliana.The results will provide a reference for further analysis of the dwarfing mechanism of Rht18gene and the utilization of Rht18 gene.