| Cotton is an important natural fiber crop and economic crop in China,and its fiber quality directly determines the quality of cotton textiles.Single fiber quality is one of the main factors restricting the development of cotton industry.Cultivating new varieties with good fiber quality has become the main goal of cotton breeding.At present,many genes related to fiber development have been cloned and some transgenic cotton has been obtained,but there is still a lack of genes and varieties with real application value.Exploring the genes related to cotton fiber development and their biological functions will not only help to better study the elongation and development mechanism of cotton fiber,but also provide a theoretical basis for cultivating new cotton varieties with good fiber quality.Therefore,this study mainly focused on the screening and functional analysis of genes related to cotton fiber development and the cultivation of transgenic cotton with high-quality fiber,in order to provide new genetic resources for cotton fiber quality improvement genetic engineering and to cultivate new transgenic cotton varieties.The main results of this study are as follows:1.In this study,transcriptome databases of different tissues?root,leaf,anther,stigma?and different fiber developmental stages?7 DPA,14 DPA,26 DPA?of Gossypium hirsutum have been constructed.The size of seven transcriptome databases were 4.43 Gb5.20 Gb,about twice the genome size of Gossypium hirsutum?2.5 Gb?,and 83.32%88.22% of Clean Reads could be aligned to the reference genome of Gossypium hirsutum TM-1.Through differential gene expression analysis of transcriptome libraries of fibrous and non-fibrous tissues?root,leaf,anther and stigma?,the number of genes significantly up-regulated in 7 DPA,14 DPA and 26 DPA was 1,205,1,135 and 937,respectively.GO enrichment analysis and KEGG metabolic pathway analysis showed that these genes were mainly involved in catalytic activity,carbohydrate metabolism,cell membrane and organelles,signal transduction and other functions and metabolic pathways.36 genes were randomly selected from the significantly up-regulated genes in fibre.The results of expression patterns of 36 genes analyzed by qRT-PCR showed that these genes were fibre-specific or dominant genes.2.The endogenous bidirectional gene pairs?Ghrack1 and Ghuhrf1?were found for the first time in cotton when we studied the fiber dominant expression gene Ghrack1.The results of qRT-PCR showed that the bidirectional genes had similar expression patterns and were predominantly expressed in the early stage of fibrocyte differentiation.Sequence analysis showed that the intergenic sequence?GhRU?of the bidirectional genes Ghrack1 and Ghuhrf1 was 1,073 bp,with a high GC content?38.7%?and conformed to the sequence characteristics of bidirectional promoters.Using gus and gfp as reporter genes,a series of plant expression vectors have been constructed and transformed into Arabidopsis thaliana by dipping-flower method.In Arabidopsis,GhRU was initially proved to be a bidirectional promoter,which could simultaneously drive the expression of gus and gfp in vigorously growing tissues.It was speculated that GhRU might be a bidirectional promoter for the predominant expression of fibers.3.In this study,bidirectional gene pairs and their bidirectional promoters in Gossypium hirsutum genome have been systematically analyzed.By searching for bidirectional gene pairs in Gossypium hirsutum genome,1,383 pairs of bidirectional genes have been obtained.Combining with transcriptome data of different tissues of cotton,1,986 bidirectional genes were screened to have tissue expression specificity,among them,236 pairs of bidirectional genes were predominantly expressed in cotton fibers.The intergenic sequences of 30 bidirectional genes were randomly cloned,and gus and gfp reporter genes were linked to the end of the intergenic sequence to construct plant expression vectors.25 intergenic regions of bidirectional genes showed bidirectional promoter activity in tobacco transient expression.The existence of bidirectional gene pairs in the genomes of Gossypium barbadense,Gossypium arboretum and Gossypium raimondii have been further analyzed.1,091,940 and 1,249 pairs of bidirectional gene pairs were found respectively.The functional enrichment and homology analysis showed that the bidirectional genes in different subspecies of cotton were conservative in function and sequence structure.4.In this study,two fiber-specific or dominant genes,CotAD24232?GhXET?and CotAD22544?GhKTN?,were selected as the key research objects from above 36 cotton fiber-specific or dominant expression genes.Plant expression vectors with fiber-specific promoter E6 have been constructed and transformed into cotton by Agrobacterium-mediated transformation.16 and 10 transgenic cotton plants with GhXET and GhKTN genes were obtained respectively.The copy number of target genes in transgenic cotton plants was detected by droplet digital PCR and Southern blot.12 and 8 transgenic cotton plants?GhXET and GhKTN?with single-copy gene insertion were screened respectively.The results of qRT-PCR showed that the expression levels of these two genes in 7 DPA,14 DPA and 26 DPA fibers of transgenic cotton plants were significantly higher than those of recipient plants.The mature fiber length of the transgenic GhXET and GhKTN cotton plants increased significantly compared with the recipient plants,increasing by 4.7 mm and 2.5 mm,respectively.The flanking sequences of exogenous genes at insertion sites of cotton genome were obtained by means of junction ligation and nested-PCR in transgenic cotton with GhXET and GhKTN genes.Based on the obtained information about flanking sequence,a specific detection method and a homozygote detection method for single locus insertion transformation events were established.The sensitivity of the specific PCR detection method can reach 44 copies,which provide effective scientific data and technical support for the safety assessment of transgenic cotton. |
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