Molecular Cloning, Expression And Tissue Distribution Of Insulin-like Growth Factor Ⅰ In Endangered Animals

Wild animal is an important component of natural ecosystem, it is very important to protect wild animal particularly endangered animals to conserve the biodiversity and ecological balance. Captive breeding is an essential strategy to enlarge their population. However, in captive, most of animals exhibit reproductive problem such as lower fertility rate, lower cub survival rate which restrict the development of captive breeding, particularly for giant panda, due to high specialization upon its reproductive capability and cub-raising behavior, it is very crucial to resolve the lower reproduction capability and cub survival rate.One reason for the lower survival rate of the panda cub is that the cub iscongenitally immature with underdeveloped immunity system, the other is that somepanda mother couldn't breed the newborns with enough panda milk. So far, there arejust a few reports on the molecular mechanism of the panda's reproduction ability andcub's survival rate. This study is mainly focused on the lower cub survival rate andthe lower reproduction rate of giant panda, lesser panda, Amur tiger and goldenmonkey, the project is aimed at: (1) Cloning, sequence analysis and expression ofIGF-I which play vital roles in growth, reproductive and milk secreting regulation. (2)The significance and possibility of a further study towards the production of theirrecombinant IGF-I by recombinant DNA technique, and their application in assistedreproduction and breeding of the endangered species. (3) Tissue distribution at IGF-ImRNA and peptide level and distribution abundance differences among variousimportant tissues which will reveal the possible physiological function in differenttissues.The main results were as the following:1. By RT-PCR, liver IGF-I from giant panda and lesser panda encoding 153 amino acids respectively were isolated, both with predicted molecular mass of 17kDa. The two IGF-I cDNAs showed high degree of homology with other reported sequences at nucleotide and amino acids levels. Two differences in giant panda amino acidssequences were found from sequence alignment. Whether these differences affect the molecular conformation or function for the growth factor's processing or these changes in the molecular structure may be responsible for the abnormal ability of growth and reproductive by influencing the function of IGF-I have not yet been determined. Among the species compared, the lesser panda shared the highest homology with giant panda. There was only one amino acid residue difference between them. From the phylogenetic tree constructed for IGF-I, the homology was as high as 95%, which revealed the relative relation between them was more closely than with others and give molecular biology support for the resolution of position and classification of lesser panda. However, we do think more research should be done to decide if the lesser panda belongs to the same subject with the giant panda, since the results was possible due to the high conservation of IGF-I among species.2. The DNA fragment encoding the mature peptide of giant panda and lesser panda IGF-I were subcloned to the pET-DsbA expression vector and highly expressed in E. colx. BL21 with IPTG induction. The expression fusion proteins (DsbA-GPIGF-I and DsbA-RPIGF-I) were mostly existed in soluble form and both of them were 33kDa. The induced cells culture were proved to be active IGF-I antigens. The soluble crude proteins were directly highly purified to by Metal Affinity Resins eluted at 150mM imidazole concentration. The soluble form, high yield and easily purification of DsbA-IGF-I provide foundation for further research on the biological activity of the recombinant IGF-I which could be used for breeding improvement after the cleavage with thrombin.3. The tissue distribution, cellular location and expression half- quantification were studied on the giant panda's liver, heart, kidney, muscle, lung, spleen, stomach with the peptides and mRNA of IGF-I by IMC and ISH method. The average IOD of different tissue slides fields which represent the expression abundance for the IMC and ISH were calculated by Image Pro Plus software and followed with statistics analysis. The results showed that there was not any significant differences of the IGF-I peptide expression level on the liver, lung, kidney and heart in giant panda.However, there were significant differences of mRN A expression level in these tissues. The level in liver was the highest than in any other tissues. The levels in heart and muscle were higher than those in lung, kidney, esophagus, spleen and stomach. There was no significant difference of expression level in the later five tissues. We verified that the IGF-I peptide and mRN A were widely distributed in various tissue of the cub giant panda and that the distribution sites of IGF-I peptide and the mRN A in the cub giant panda were nearly consistent although the mRNA abundance were different in the tissues. All of the above demonstrated the giant panda IGF-I should play an important role in Endocrine, paracrine/autocrine which indicated that the IGF-I was very important to the cell proliferation and differentiation, organ growth and development in giant panda and should be further study its physiological function in giant panda.4. Tissue distribution, cellular location and expression half- quantification were alsostudied on the lesser panda's liver, heart, kidney, muscle, lung, spleen, stomach withthe peptides and mRNA of IGF-I by IMC and ISH method. Calculation of the meanIOD and the statistical analysis were the same as those in giant panda. As the samedistribution level in giant panda, there was not any significant difference of the IGF-Ipeptide level on the liver, lung, kidney and heart in lesser panda. It was different forthe IGF-I mRNA abundance in various tissues of red panda. The abundances in liverand muscle were both the highest than in any other of the other six tissues. There wasnot any significant difference among the later six tissues. We verified that the IGF-Ipeptide and mRNA were widely distributed in various tissue of the newborn lesserpanda and that the distribution sites of IGF-I peptide were nearly consistent withIGF-I mRNA. We demonstrated the lesser panda IGF-I should play an important rolein Endocrine, paracrine/autocrine which indicated that the IGF-I was also veryimportant to the growth and development of lesser panda newborn and was worthy forfurther study its physiological function in lesser panda. The differences of tissuedistribution pattern between lesser panda and giant panda were worthy to be furtherstudied.