Studio della divergenza dovuta a selezione di popolazioni animali allevate attraverso analisi funzionale con microarray
Author(s)
Prosperini, Gianluca
Date Issued
February 10, 2011
Type
Doctoral Thesis
Abstract
In the recent years, the study of the gene expression profile has become a crucial element in the analysis of the genes function and cellular behaviour research. Gene expression profiles can be measured in terms of both protein and mRNA levels. In the latter case, it is known that a mRNA specific quantity is correlated with the protein production level. So, every change in gene expression level will modify both cellular behaviour and functionality. Although a relationship between expression profile and phenotype certainly exists, it is important to stress its elevated complexity. Amongst the technologies for the genetic profile quantity determination presently available, microarray technology represents the most direct and complete approach.
This technique has been used in order to analyse genetic divergence of livestock due to selection. Two swine breeds, the “Casertana” and the Large White, and two ovine breeds, the “Sarda” and the “Gentile di Puglia”, were studied. In each species, the selected breeds underwent selection for different productive attitudes and different phenotypic characteristics.
Concerning the two swine breeds, it was possible to identify genes responsible for fat deposition (up-regulated in the Casertana) and for meat tenderness (up-regulated in the Large White).
Moreover, pathway analysis showed that most of the differentially expressed genes took part to the carbohydrate metabolism. This result could be addressed to the particular lifestyle of the Casertana, which lives in a semi-wild environment, with higher energy needs than the sedentary Large White.
A good number of differentially expressed genes participate in another interesting pathway, the endocrine system regulation. This result is probably affected by the different growth rate of the two swine breeds.
In order to analyse the two sheep breeds, it was necessary to design and produce a specific chip for the ovine species, since no valid products are currently available in the market. Therefore we decided to design a high-density in situ synthesized microarray, named Aristaeus, specific for Ovis aries. It was developed starting from ESTs deposited at the NCBI, then selecting the oligonucleotides to be synthesized on the chip according to the Kane’s criteria. The microarray was produced with CustomArray 90K Combimatrix technology and contains 21743 unambiguous oligonucleotides in four copies. The oligonucleotides correspond to 10190 genes, covering about 50% of whole sheep genome.
At this point, the two ovine breeds with different attitudes (Gentile di Puglia: wool-meat; Sarda: milk) were compared, in order to: 1) evaluate chip efficiency; 2) analyse expression differences of genes potentially involved in milk production.
To evaluate chip efficiency, experiments were carried out using liver samples from individuals of the two breeds farmed in the same conditions and of same sex and age, and lactation stage (44 days after lambing). All quality control tests for the Aristaeus chip demonstrated high reproducibility of the data.
For these experiments, a cluster of differentially expressed genes resulted involved in the fatty acids oxidation, such as the peroxisomal targeting signal 1 receptor (PEX5), ATP5C1, and NDUFS1.
Fatty acids are the major source of energy for body and are stored principally in the adipose tissue as triacylglycerols (TAG). However, during periods of negative energy balance, as may occur during lactation, the levels of unesterified fatty acid (NEFA) circulating in the blood are elevated and TAG can accumulate in the liver with possible pathological consequences.
Thus the overexpression of the ATP5C1 gene in the liver of lactating Sarda individuals, may well represent a defence adaptation against the potentially harmful accumulation of TAG.
To validate microarray data we tested with Quantitative Real Time PCR six genes. The differences in gene expression between the two breeds observed by QRT-PCR confirmed microarrays data.
Afterwards, experiments aimed at the analysis of genes involved in the lactation process were performed on mammary gland tissue at two different lactation stages (stage 1: 6 days after lambing; stage 2: 44 days after lambing). The same quality checks were repeated for the second set of experiments, and also in this case all quality control tests demonstrated high reliability.
Several genes resulted differentially expressed at each lactation stage. In particular, caseins αS2, β and K were among the upregulated genes in Sarda vs Gentile at lactation stage 1. Moreover, a significant difference between the two breeds was observed in the expression of genes involved in extracellular matrix formation and cell adhesion (TJP1 upregulated in Gentile, CDH5 and TNXB upregulated in Sarda). These two processes, according with the current literature, support cells in proliferation, accurate morphological organization, as well as in milk secretion. At stage 2, some interesting genes as those encoding casein K, and involved in oxidoreductase activity (like TGOLN2 and FTH1) and in ECM-interaction (like COL1A2), resulted overexpressed in Sarda.
Finally, in Sarda, an overexpression of genes implicated in lipolysis, like lipase (DAGLB) was observed. Several studies have demonstrated that the lipolysis is particularly important in sheep cheeses making.
Through the experiments described in this thesis, we demonstrated the usefulness of microarrays for the identification of differences in gene expression associated with different production attitudes in livestock.
Additional information
Dottorato di ricerca in Ecologia e gestione delle risorse biologiche
Subjects
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