ผลของอุณหภูมิต่อกลไกการสังเคราะห์ mRNA ที่มีขนาดแตกต่างกันของยีน desA ใน Spirulina platenis สายพันธุ์ C1

Temperature Regulation of the Expression of the Two IsoformmRNAs of the desA Gene in Spirulina platensis Strain C1

Name: สุธาดา มุ่งภักดี

Organization : มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

keyword: Spirulina platensis

; desA gene

; temperature regulation

; transcriptiontermination

; repetitive DNA sequence

; ribonuclease enzyme

Abstract: The expression of the desA gene for A1 2 desaturase was previously shown toyield two isoforms of mRNA at sizes of 1.5 and 1.7 kb. Upon the downward shift oftemperature from 35OC to 22OC, the level of 1.5-kb mRNA was unaffected, whereasthe level of 1.7-kb mRNA increased 2.5-fold. In order to understand how two desAmRNAs are synthesized in Spirulina platensis strain C1, the molecular mechanismof desA gene expression was investigated in the present work. As examined bySouthern blot and Northern blot analysis using unconserved and non coding regionof desA gene as probes, it was found that the two mRNAs were synthesized by thesame gene. The DNA analysis of upstream region found that both of desA mRNAspossesses the same 5' end, which might be controlled by the same promoter. Fromthe prediction, this putative promoter is similar to constitutive promoter of E. coli,Therefore, this result suggests that the promoter of this gene was not the key factorthat controlled the increasing of 1.7-kb desA mRNA at low temperature. Thedownstream regions of the desA mRNAs were also determined by using S1 nucleaseassay. It was found that the two termination sites were detected at positions, 1,175and 1,455 bp, which corresponded to 1.5- and 1.7-kb mRNA, respectively. Based onthe determined 3' ends, the untranslated region of 1.5-kb mRNA contained 1 copy ofrepetitive (RP) sequence, while 1.7-kb mNA contained 3 copies. Furthermore, theeffect of temperature on the mRNA stability was investigated by using RNaseprotection assay. The results demonstrated that the secondary structure at 3' end(formed by RP sequence) of 1.5-kb mRNA was more stable than that of 1.7-kbrnRNA. The change in temperature did not affect the stability of this mRNA. Bycontrast, the stability of 1.7-kb mRNA increased at low temperature due to twoeffects, the decrease in ribonuclease activity and effect of cellular factor that mightbind to 3 copies of RP sequence and protect mRNA from exoribouclease. As aresult, the level of 1.7-kb mRNA appeared at low temperature. Determination of themRNA stability in vivo indicated that 1.5-kb mRNA was not generated from 1.7-kbmRNA. Thus, the key factors that controlled the differentiation of desA geneexpression would be the transcription termination and stability of mRNA. The 1.5-kb mRNA was a major mRNA and its expression did not depend on temperature. Itwas also observed that the higher accumulation of 1.5-kb mRNA either at 35OC or22Oc resulted from the presence of 1 copy of RP sequence. By contrast, the 1.7-kbmRNA having 3 copies of RP sequence was unstable at 35OC. However, its stabilitywas increased when the temperature was shifted to 22OC and this enhancement wasfound to be due to the protection effect of cellular factor induced at 22OC . Thisimplies that the accumulation of 1.7-kb mRNA at low temperature might involve incold adaptation.

มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

Address: กรุงเทพมหานคร (Bangkok)

Email: info.lib@mail.kmutt.ac.th

Name: Dr.Patcharaporn Wongsa

Role: Supervisor

Created: 2544

Issued: 2005-10-26

Modified: 2006-05-24

วิทยานิพนธ์/Thesis

ISBN: 9746289721

CallNumber: BIT358

tha

DegreeName: วิทยาศาสตรมหาบัณฑิต

Level: ปริญญาโท

Descipline: เทคโนโลยีชีวภาพ

Grantor: มหาวิทยาลัยเทคโนโลยีพระจอมเกล้าธนบุรี

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