CDNA Cloning And Tissue Expression Of FoxO1 In Pig

Transcriptional factor, a type of protein molecule, plays an important role in organism development and metabolism. It controls gene expression by regulating the expression of target genes. Forkhead (Fox) transcription factor is a new protein family, which is issued and named in 2000. FoxO play an important role in the growth and development of animals, cell differentiation, metabolism, apoptosis and immunization through transcriptional regulation and signal transduction pathway. FoxO1 is the earliest members be found, and has closely related to myoblast differentiation and adipocytes metabolism. It has been as a potential factor involving the differentiation of skeletal muscle and the expression of muscle fiber type 1 related gene. Thus, FoxO1 is a key candidate to study development and metabolism of muscle and adipose. In this study, pig was used as experimental animal. FoxO1 was cloned. Meanwhile, the tissue expression of FoxO1 in different pig breeds and different types of skeletal muscle was investigated. The main results were as follows:1. The partial cDNA of FoxO1 was cloned from pig's liver by RT-PCR, which primer was designed according to the homolog blast among human, chimpanzee and rat. Pig FoxO1 cDNA sequence had 87%, 87%, 85%, 87% and 87% homology with human, chimpanzees, rats, cattle and dogs, respectively. GenBank accession number was DQ673620.2. Tissue-specific expression analysis by SQ RT-PCR showed that FoxO1 was widely expressed in liver, lung, kidney, spleen, heart, stomach, subcutaneous fat, visceral fat, the longest back muscle, cutaneus and quadriceps muscle and other tissues from 1-day-old piglet and 9-month-old pig. However, the expressional level of FoxO1 was different in various tissues and different developmental stages. The expression of FoxO1 was highest in visceral adipose and lower in heart and skeletal muscle from 1-day-old piglets. In addition, the expression of FoxO1 not only showed significantly difference between smooth muscle and skeletal muscle, but also had significance among the different types of muscle from 9-month-old pigs. This shows that the expression of FoxO1 may be related to the type of skeletal muscle and exercise intensity.3. The distribution of FoxO1 was further investigated using real-time quantitative PCR. FoxO1 showed the differential expression in various tissues and pigs of different days, which was consistent to the results from RT-PCR detection. FoxO1 was abundant in heart, liver, skeletal muscle, subcutaneous fat and visceral fat tissue from 1-day-old piglets, hinting that FoxO1 has intimately relationship with lipid metabolism. Furthermore, the expression of FoxO1 in 180-day-old pigs was higher than 1-day–old pigs (P<0.05). FoxO1 expression in muscle from 180-day-old and 1-day-old pigs showed no significant difference.4. Total RNA was extracted from soleus muscle, gastrocnemius muscle and extensor digitorum longus in hind leg of the 6-month-old male Bamei pig, Landrace, and crossbred pigs (Landrace×Bamei). The results showed that the expression level of FoxO1 mRNA was different in various economic pig breeds and skeletal muscle types. The expression of FoxO1 in skeletal muscle of Bamei and the crossbred pigs was significantly higher than Landrace(P<0.01). Moreover, FoxO1 was lowest expressed in soleus muscle, which mainly containingⅠtype muscle firber; And most abundant in extensor digitorum longus, which mainly containingⅡtype muscle fiber. This suggests that FoxO1 expression has the negative correlation withⅠtype muscle firber. Pig breeds of the different economic types may be closely related to the regulation of FoxO1 gene.5. The correlation of FoxO1 and MyoD mRNA expression in muscle tissue from pigs of different economic type was tested. The results showed that the expression of MyoD was decreased accompanying the increase of FoxO1. The expression of FoxO1 and MyoD mRNA existed negative correlation in muscle development (r =0.728 , P<0.05). This suggests that the up-regulation of FoxO1 may cause to the expression reduce of MyoD mRNA in muscle tissue and negative modulate the development of skeletal muscle and expression of I type muscle fiber related genes.