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Construction Of Expression Vector Of Insulin-like Growth Factor-1 Regulated By Matrix Attachment Regions And Its Transformation In Cabbage

Posted on:2005-08-11Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y H ZhangFull Text:PDF
GTID:1103360152955718Subject:Vegetable science
Abstract/Summary:PDF Full Text Request
Insulin-like growth factor-1 (IGF-1) is a single-chain polypeptide of 70 amino acids, which is mainly secreted by the liver as a result of stimulation by growth hormone (GH). Many studies showed that IGF-1 is a multifunctional cell proliferation and regulation factor, which has very wide biological activities. It mediates most of the in vivo cell division and metabolic effects of growth hormone. Almost every cell in human body is affected by IGF-1, especially cells in muscle, cartilage, bone, liver, kidney, nerves, skin, and lungs. Recently IGF-1 has been used to treat diabetes, insulin-resistant syndrome, dwarfism, some nervous system diseases and other obstinate diseases. The clinic outcomes were rather encouraging. Since the content of IGF-1 in human and animal bodies is very low, it cannot meet the demands of basic researches and clinical uses by separation of natural products. Therefore, it is inevitable to produce large amounts of biologically active IGF-1 by recombinant DNA technology. The expression of IGF-1 has been performed in bacterium and yeast systems. In both foreign host cells, most of the recombinant protein was unable to fold properly and often accumulated to form inactive inclusion bodies. There are also some limitations, such as the risk of zoonoses and suppressing the normal growth of hosts by expressed products, in the expression systems like transgenic animals and mammalian cell cultures. Plants remained to be studies as a potential transgenic production system of choice for the expression of rIGF-1.In this research project, the DNA sequence of coding IGF-1 was modified on the basis of cabbage (Brassica oleracea var. capitata L.) codon usage bias. IGF-1 gene was then semisynthesized according to the optimized sequence. Meanwhile, two segments of matrix attachment regions (MARs) were cloned from tobacco (Nicotiana tabacum L.) and cabbage. The function of these MARs in transgene expression was tested. The results showed that the MARs increased the levels of transgene expression obviously. Therefore, the MARs were used to construct the high efficiency expression vectors of IGF-1. It was the first time to transform cabbage with IGF-1. Transgenic plants were obtained and this breaks a new path of developing cabbage as a medicinal vegetable in the treatment of some diseases. The experiments involved the following aspects:1 Isolation of matrix attachment regions (MARs)Matrix attachment regions (MARs) are DNA elements usually 300 to 1000 base-pairs long that attach the chromatin to the nuclear matrix. MARs widely exist in eukaryotic genome and subdivide the eukaryotic genome, in both structure and function, into loop domains. They not only can make chromatin form into chromatin loop, but also can act as the origin of DNA replication and regulategene expression in eukaryotic cells. They have recently attracted much attention because of their perceived capacity to prevent transgene silencing and increase the levels of transgene expression in both plants and animals. In the present research, using CTAB extraction procedure, we isolated DNAs from tobacco and cabbage. Based on the reported MAR sequence in the tobacco plant, primers P1/P2 were designed. HindIII-EcoR I sites were added to 5'-end of PI so as to construct the vectors with facility. The MAR was then isolated from tobacco genomic DNA by PCR method. To compare the functions of different MARs in transgene expression, another primer pair P3/P4 were designed according to a short interspersed nuclear element (SINE) sequence in Brassica. EcoR I -Hindlll sites were attached to the 5'-side of P3 for the aim of constructing expression vectors. Also using PCR method, we got the MAR from cabbage. Sequencing analysis showed that tobacco MAR was 688 bp long and the content of AT was 73.1%, the SINE was 527 bp long with a 74.8% AT. Both of them contained "90% AT box" and other several MAR-related motifs.2 Construction of GUS expression vectorsThe objective for constructing a series of GUS expression vectors was to test and compare the effects of different orig...
Keywords/Search Tags:Insulin-like growth factor-l(IGF-l), Cabbage Matrix attachment region(MAR), Transgenic plant
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