The Studies On Cloning And Expression Of Amphisox9in Amphioxus Branchiostoma Belcheri Tsingtauense

Sox9is the first member of Sox gene family which has intron fragments after the discovery of SRY gene. About671Sox9gene sequences are recorded in the Genebank,which widely distribute in many species from mammals and bird to coelenterate. Sox9gene is a multifunctional gene which play key role in sex determination, chondrogenesis, formation of nervous system and pancreas. Furthermore, this gene can also take part in oncogenesis.This paper reviewed the research achievements about structure, function and evolution of Sox9gene, and was expected to provide reference materials for further study of Sox9gene.The function of Sox9gene and his mechanisms are mainly concentrated in higher animal such as human and mice. In vertebraes Sox9gene takes part in sex determination, while in the invertebrates this gene is rarely reported about sex determination.Amphioxus belongs to cephalochordate, which is the existent closest relative groups between invertebrate and vertebrate, since it has part of the morphological pattern of the vertebrates and the copy numbers of the same gene in the family members are much less than that of vertebrates. So amphioxus is regarded as the model animal for insights into the gene origin and evolution, and early organogenesis of the vertebrates. It is a well-known model organism widely used for interspecies comparative genome studies and developmental homology analysis.Since the Amphioxus has been firstly reported in1774, none of the report on Sox9in amphioxushas been seen.Therefore, to study Sox9gene of amphioxus is of great theoretical significance, which provides the theory referrence on the elucidation of the origins of Sox9gene in vertebrates and the research of comparative biology.According to the sequences of different species which are closely related to amphioxus such as Brachydanio rerio, Styela clava herdman, Andrias davidianus, Epinephelus, Megalobatrachus dividianus and so on from GeneBank, we obtain the conservative sequence of Sox9gene by using software of MEG A3.1. Designing a pair of specificity primer with help of Primer primer5.0software and using PCR method, we get the segment of Sox9gene in amphioxus. Splicing gene segments which come from3end and5end unknown flanking sequence amplified by genome walking method, a full-length of3162bp Amphisox9in amphioxus is obtained. Using PCR method and labeling the Digoxin on the probe, the tissue expression analysis of this gene in amphioxus is examined by methods of tissue slice and in situ hybridization. Based on the results of tissue in situ hybridization, total RNA from tissues which express Amphisox9is extracted. Furthermore, the full length cDNA sequences of Amphisox9is get, which exists an intron in coding sequence.