Leucojum aestivum L.’den tirozin dekarboksilaz (tydc) gengngn gzalasyonu ve klonlanması
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Dosyalar
Tarih
2021
Yazarlar
Dergi Başlığı
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Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, Amaryllidaceae ailesine ait olan Leucojum aestivum L. (göl soğanı) bitkisinden, tirozin dekarboksilaz (TYDC) geninin izole edilmesi amaçlanmıştır. L. aestivum Trakya bölgesinde yayılış gösteren ve farmakolojik olarak önemli bazı alkaloidlerin, özellikle de likorin ve galantaminin doğal üreticisi olan oldukça önemli bir farmasötik bitkidir. Galantamin, Alzheimer hastalığı başta olmak üzere demans benzeri nörolojik problemlerin tedavisinde kullanılan ilaçların etkin maddesi olarak önemli bir sekonder metabolittir. TYDC enzimi; çeşitli bitki türlerinde önemli bazı sekonder metabolitlerin sentez yolağında, ilk basamaklarda etkin olan kritik bir enzimdir. Galantamin biyosentezi de, fenilalanin ve tirozin amino asitleri ile başlar ve TYDC sentez yolağındaki kritik iki enzimden biridir. Dolayısıyla TYDC geninin aydınlatılması, galantamin üretiminin artırılması yönündeki manipülasyonlara olanak tanıyacaktır. Daha önce bir çok bitkide TYDC geni incelenmiş ancak L. aestivum?da herhangi bir çalışma yapılmamıştır. Sunulan tez çalışması kapsamında galantamin üreticisi olan yakın akraba türlerin TYDC gen bilgileri incelenmiş ve genin izolasyonu için primerler tasarlanmıştır. L. aestivum?un çiçeklerinden total RNA izole edilerek tasarlanan primerler ile genin tam ORF?si elde edilmeye çalışılmıştır. v Elde edilen TYDC1 genine ait DNA fragmenti, öncelikle pJET.Blunt/1.2 klonlama vektörüne klonlanmış ve dizi analizi yaplılarak LuaTYDC1 genine ait kodlayıcı dizi elde edilmiştir. Genin kodlayıcı dizisi, galantamin üreticisi olan yakın türler Narcissus pseudonarcissus, Narcissus papyraceus, Lycoris aureus ve Lycoris longituba’un TYDC1 geni ile %87-91,6 oranında yüksek bir homoloji göstermiş, amino asit dizisi karşılaştırmalarında ise homoloji oranı %89-93,53 olarak gözlenmiştir. Daha sonra restriksiyon enzimleri ile kesilerek pJET.Blunt/1.2 vektöründen geri kazanılan LuaTYDC1 geni pQE.70 ekspresyon vektörüne bağlanarak ekspresyon ve fonksiyon analizleri için E. coli?ye transfer edilmiştir.
In this study, it was aimed to isolate the tyrosine decarboxylase (TYDC) gene from Leucojum aestivum L. (summer snowflake) plant belonging to the family Amaryllidaceae. L. aestivum is a very important pharmaceutical plant that is a natural producer of some pharmacologically important alkaloids, especially lycorine and galantamine, spread Thrace region. Galantamine is an important secondary metabolide as the active ingredient of drugs used in the treatment of dementia-like neurological problems, especially Alzheimer's disease. TYDC enzyme; It is a critical enzyme that is effective in the first steps in the synthesis pathway of some important secondary metabolites in various plant species. Galantamine biosynthesis also starts with the phenylalanine and tyrosine amino acids and TYDC is one of the two critical enzymes in the synthesis pathway. Thus, the illumination of the TYDC gene will allow manipulations to increase galantamine production. TYDC gene has been studied in many plants before, but no study has been done in L. aestivum. Within the scope of the presented thesis, TYDC genes of closely related species that are galantamine producers were examined and primers were designed for the isolation of the gene. vii The full ORF of the gene was obtained with designed primers by using isolated total RNA from the flowers of L. aestivum. The obtained DNA fragment was firstly cloned into pJET.Blunt / 1.2 cloning vector and the coding sequence of the LuaTYDC1 gene was obtained by sequence analysis. The coding sequence of the gene showed a high homology of 87-91.6% with the TYDC1 gene of Narcissus pseudonarcissus, Narcissus papyraceus, Lycoris aureus and Lycoris longituba which are galantamin producer close relative species, and the homology rate was 89-93,53% in their amino acid sequence comparisons. Later, the LuaTYDC1 gene, which was cut with restriction enzymes and recovered from the pJET.Blunt / 1.2 vector, was ligated to the pQE.70 expression vector and transferred to E. coli for expression and functional analysis.
In this study, it was aimed to isolate the tyrosine decarboxylase (TYDC) gene from Leucojum aestivum L. (summer snowflake) plant belonging to the family Amaryllidaceae. L. aestivum is a very important pharmaceutical plant that is a natural producer of some pharmacologically important alkaloids, especially lycorine and galantamine, spread Thrace region. Galantamine is an important secondary metabolide as the active ingredient of drugs used in the treatment of dementia-like neurological problems, especially Alzheimer's disease. TYDC enzyme; It is a critical enzyme that is effective in the first steps in the synthesis pathway of some important secondary metabolites in various plant species. Galantamine biosynthesis also starts with the phenylalanine and tyrosine amino acids and TYDC is one of the two critical enzymes in the synthesis pathway. Thus, the illumination of the TYDC gene will allow manipulations to increase galantamine production. TYDC gene has been studied in many plants before, but no study has been done in L. aestivum. Within the scope of the presented thesis, TYDC genes of closely related species that are galantamine producers were examined and primers were designed for the isolation of the gene. vii The full ORF of the gene was obtained with designed primers by using isolated total RNA from the flowers of L. aestivum. The obtained DNA fragment was firstly cloned into pJET.Blunt / 1.2 cloning vector and the coding sequence of the LuaTYDC1 gene was obtained by sequence analysis. The coding sequence of the gene showed a high homology of 87-91.6% with the TYDC1 gene of Narcissus pseudonarcissus, Narcissus papyraceus, Lycoris aureus and Lycoris longituba which are galantamin producer close relative species, and the homology rate was 89-93,53% in their amino acid sequence comparisons. Later, the LuaTYDC1 gene, which was cut with restriction enzymes and recovered from the pJET.Blunt / 1.2 vector, was ligated to the pQE.70 expression vector and transferred to E. coli for expression and functional analysis.
Açıklama
Yüksek lisans tezi.
Anahtar Kelimeler
gen klonlama, leucojum aestivum, gene cloning, tirozin dekarboksilaz (TYDC) geni, luaTYDC1 Leucojum aestivum, tyrosine decarboxylase (TYDC) gene