Dissertation > Excellent graduate degree dissertation topics show

Cloning and Expression Analysis of Walnut (Juglans Regia L.) JrVTE1Gene

Author: WangCanCan
Tutor: YangKeQiang
School: Shandong Agricultural University
Course: Tree Genetics and Breeding
Keywords: Walnut (Juglans regia L) Tocopherol cyclase Prokaryotic expression Transgenic jujube Transgenic pear
CLC: S664.1
Type: Master's thesis
Year: 2013
Downloads: 7
Quote: 0
Read: Download Dissertation

Abstract


Vitamin E is an essential lipid soluble compound for humans which is synthesized onlyby oxygenic photosynthetic organisms, and walnut (Juglans rehia L.) is a major Vitamin Esource. In this study, the tocopherol cyclase gene was isolated from walnut cultivar‘Xiangling’, cloned to pET28a and was transformed to E.coli BL21(DE3) for expression;Agrobacterium-mediated genetic tansformation system was established, and the JrVTE1genewas transformed into jujube (Zizyphus spinosus Hu) and pear (Pyrus communis L.)respectively. It has the important practical significance to improve crop nutrition value,excavate the excellent genetic resources of walnut. There are several works in the follow.1. Molecular cloning and characterization of JrVTE1gene from Walnut. The tocopherolcyclase gene was isolated from Walnut by RACE, and designated as JrVTE1. Sequence dataof JrVTE1had been deposited at GenBank under accession numbers of KC751543. Thefull-length cDNA sequence of JrVTE1contained1,749base pairs (bp). It was predicted toencode a protein of451amino acids. Multiple alignment of amino acid sequences indicatedthat JrVTE1shared high sequence identity with the VTE1s from Eucalyptus gunnii, Heveabrasiliensis, Glycine max, Arabidopsis Thaliana, Medicago truncatula and Sesamum indicum,which was about80%. JrVTE1was belonged to the tocopherol cyclase family, which wasused to catalyze2-methyl-6-based-benzoquinone (2M6PBQ) to δ-tocopherol or catalyze2,3-two methyl5-based-benzoquinone (2,3DM5PBQ) to γ-tocopherol in the synthesis of vitaminE.2. Expression study of real-time fluorescent quantitative of JrVTE1gene in J. Regiaembryonal tissue. Real-time fluorescent quantitative RT-PCR analysis showed that JrVTE1gene was expressed in three important periods, respectively. And it was highest in90d afterflorescence.3. Prokaryotic expression of JrVTE1gene. Prokaryotic expression vectorpET28a-JrVTE1was constructed and was transformed into E. coli BL21(DE3) for expression.The culture conditions of recombinant cells was optimized in37℃and was induced by 1mmol·L-1IPTG. The SDS-PAGE analysis showed that there was a specific protein in thecells which had recombinant plasmid pET28a-JrVTE1after2hours. And the quantity of theprotein was increased with the extension of time. But there was no specific expression proteinin the control ones. The molecular weight of protein expressed by pET28a-JrVTE1was49.5kD which was equal to the expected result, so it was the target protein.4. Plant expression vectors was constructed and transformed to wild jujube and pearsuccessfully. JrVTE1gene was cloned to pRI101-AN vector and was transformed into jujubeand pear tender leaves respectively by Agribacterium-mediated transformation. The JrVTE1transgenic jujube and pear were both identified by Kan-resistant and PCR, and there were9and25transgenic JrVTE1jujube and pear positive plants respectively.

Related Dissertations

  1. Expression of D-AtCGS in E. Coli and Preparation of Polyclonal Antibody Against D-AtCGS,Q943.2
  2. Cloning and Expression Analysis of GPx, GST and SAHH Genes in Chlamydomonas Sp. ICE-L from Antarctica,Q943.2
  3. cDNA Cloning, Expression of vp5 and vp7 Genes and Subcecullar Localization of VP5 and VP7 Proteins in Grass Carp Reovirus,S941.41
  4. Cloning, Expression of vp6 and ns38 Genes and Immunogenicity of VP6 and NS38 in Grass Carp Reovirus,S941.41
  5. Functional Analysis of Proteins Encoded by RNA2 of Wheat Yellow Mosaic Virus,S435.121
  6. Comparative Study on Reproductive Biological Characteristics of Helicoverpa Armigera and Helicoverpa Assulta (Lepidoptera:Nuctuidae),S433
  7. Cloning and Expression Analysis of Scavenger Receptor Class B Type Ⅰ and Antifreeze Proteinstype Ⅱ Genes in Lutjanus Sanguineus,S917.4
  8. Identification of B.melitensis 16M Protein Immunodominant Antigens,S852.61
  9. Isolation and Identification of Infectious Bronchitis Virus and Sequence Analysis of Its S1 Gene and N Gene,S852.65
  10. Isolationand Identification of Porcine Parvovirus and Parts of Its Biological Characteristics,S852.65
  11. Prokaryotic Expression and Purification of Human Beta-Defensin-9,Q78
  12. Biochemical Characterization and Molecular Modification of a Novel Marine Mud-derived Pyrethroid Hydrolase,X172
  13. The Prokaryotic Expression of Melittin Gene and Its Targeting Transcription in Hela Cells,R346
  14. Genetic Study of IL-15 in All and Generation of Anti-IL-1α Antibody and Related Study of IL-1α,R733.71
  15. Functional Research of Soybean Bioactive Peptide -Prokaryotic Expression, Purification and Bioassay of Lunasin,Q946.1
  16. Biological Functions of Two-component Signal Transduction System CiaRH in Highly Pathogenic Streptococcus Suis Serotype 2 Strain 05ZYH33,S852.611
  17. Development of PCV2 DNA Vaccine and Establishment of Indirect ELISA Method,S858.28
  18. Isocitrate lyase gene cloning and expression characteristics of,R346
  19. Expression of the Extracellular Domain of Human Osteostat in E. Coli and Its Bioactivity Analysis,R378
  20. Construction of Expression Vectors for Human Interferonα-2b Gene in Both Nucleus and Chlorplast of Tobacco and the Transformation,S572
  21. Gene Clone, Expression and Purification of Murine Flt3L as Well as Identification of Its Biological Activity,Q78

CLC: > Agricultural Sciences > Gardening > Fruit trees gardening > Nuts (shell fruit ) > Walnut ( Juglans )
© 2012 www.DissertationTopic.Net  Mobile