| Homeobox genes, also named Hox genes, are a group of genes which play an important role in determining the identity of embryonic region during the early stage of embryogenesis. Till now, Hox genes are known to be highly conserved in many species. They control the development of segment and tissue along embryo anterior-posterior axis(AP axis) and variation of its sequences as well as copy numbers will also cause the morphology change of body structure. Hox genes are typically found in an organized cluster, which structure and numbers vary in different organisms along with genome evolution. Therefore, the studies of the Hox genes is considered to be an important indicator of morphological development and evolution mechanism of some species.Pacific white shrimp(Litopenaeus vannamei) is the top economic aquaculture production species of crustacean, which morphological structure differs greatly from embryo to larval during the anamorphosis development. Thus, the study of Hox gene expression pattern at different developmental stage of Pacific white shrimp will be beneficial the understanding of its morphological development mechanism and phylogenetic position.In this study, sequences of Hox gene were obtained from the transcriptome data of the Pacific white shrimp. Firstly, we identified the members of the Hox gene family, analyzed their structure characteristics and established a phylogenetic tree by means of the bioinformatics methods. Then, to understand the expression pattern of Hox gene at embryo and larvae stage, their expression levels were analyzed according to the early developmental gene expression profiles. Finally, tissue-specificity distribution of Hox genes was further detected in adult shrimp by RT-q PCR technology.The main achievements in this thesis are as follows:1. Firstly, 27 Unigenes was obtained from the pacific white shrimp transcriptome profile. 13 Hox genes were further identified from Unigenes, and were named as Lvlab, Lvpb, Lv Hox3, Lv Dfd, Lv Scr, Lvftz, Lv Antp, Lv Ubx, Lvabd A, Lv Abd B, Lv Msx, Lv Mnx and Lv Dll, respectively. In addition, 13 Hox genes were highly conserved between orthologous genes and most of them contain the YPWM motif and homeodomain(~60 amino acids chain), which formed “helix turn helix” in the second structure. Moreover, the two parallel helix and one vertical helix are mainly composed the tertiary structure of Hox proteins. The homeodomain, as the function domain, is rich in positively charged hydrophilic amino acid, which will facilitate Hox to combine with target DNA carried negative charge, and the vertical helix will further stabilize the combination. The YPWM were formed a twisted ring in the tertiary structure of Hox proteins, as the cofactor binging site, improve the binding effienciency between the Hox and its target gene.2. All 13 Hox genes c DNA sequences were aligned with genomic sequences by BLASTn, the results indicated that most Hox genes transcriptional direction was 5’ to 3’, in contrast, the transcriptional direction of Lvftz, Lv Abd B and Lv Dll was from 3’ to 5’. In this study, 6 genes were identified that belongs to the LG41 linkage group, including Lvlab, Lv Dfd, Lv Scr, Lv Antp, Lv Ubx and Lvabd A. Clustering analysis demonstrated that, there were similarity between Hox genes homeodomain regions: neighboring Hox gene tended to cluster firstly, then clustered with other Hox genes, this observation supported the hypothesis that Hox gene originated from the same ancestor. In addition, Hox genes of the pacific white shrimp showed higher similarity with that of Hexapoda in the phylogenetic tree which in line with historical findings.3. To analysis the expression pattern of Hox gene in early developmental stage, Hox genes expression profiles were established according to the transcriptome data of the pacific white shrimp. The results indicated that abundance as well as expression pattern showed dynamic change in different early developmental stages, and the expression level also varied significantly between stages, which closely related with morphology change. In summary, Lvlab, Lvpb, Lv Hox3, Lv Dfd, Lvftz, Lv Msx, Lv Mnx and Lv Dll were mainly expressed till the nauplius, which probably involved in the regulation of head appendages and tissue development; the expression of Lv Scr, Lv Antp, Lv Ubx, Lvabd A and Lv Abd B become active from Zoea, and maybe mainly responsible for the development regulation of appendages and organ of the chest and abdomen. We also investigated the interplay between Hox genes, such as Lv Ubx and Lvabd A were co-expressed to inhibit the expression of Lv Dll and finally determined the development and function of appendages in chest and abdomen. The complex regulation mechanism of Hox genes finally determined the morphology of the pacific white shrimp.4. Expression pattern of Hox gene in adult shrimp tissue were also investigated by RT-q PCR method. The results suggested that Hox gene express significant difference in tissues. Lvlab and Lv Mnx were mainly expressed in the hepatopancreas and stomach, Lvpb was mainly expressed in stomach, Lv Hox3 was mainly expressed in eye-stalk, Lv Dfd was mainly expressed in gill, Lvftz was mainly expressed in ovary and epidermis, Lv Scr were mainly expressed in epidermis, gill and muscle, Lv Antp were expressed in the heart, Lv Ubx was detected lower expressed level in testis and gill, Lvabd A and Lv Abd B were mainly expressed in nerve, Lv Dll were expressed in the testis, blood cells and specifically in stomach, Lv Msx were only detected lower level in ovary and testis. These results indicated the functional diversity of Hox genes. Moreover, Lvlab, Lvpb, Lv Hox3, Lv Dfd, Lv Scr, Lv Antp as well as Lv Dll and Lv Mnx were expressed mainly in these tissues located in cephalothorax, while the Lvabd A and Lv Abd B were expressed in gut, nerve, ovary, testis and muscle which located in abdomen. This discovery showed that the certain colinearity between the position of Hox gene located in Hox cluster and the position of tissues, in which Hox genes were expressed, planed in shrimp body. |
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