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Protein Disulfide Isomerase PDIL1-1 Has a Regulatory Role in Endosperm Development in Rice

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dc.contributor.advisor서학수-
dc.contributor.author김연정-
dc.date.accessioned2017-07-13T17:40:40Z-
dc.date.available2017-07-13T17:40:40Z-
dc.date.issued2013-02-
dc.identifier.other000000008560-
dc.identifier.urihttps://hdl.handle.net/10371/121034-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 식물생산과학부 작물생명과학전공, 2013. 2. 서학수.-
dc.description.abstractProtein disulfide isomerase (PDI) is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER). A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. Possible functions of rice PDI-like protein 1-1 (PDIL1-1) during seed development were identified in this study. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1∆ , identified by genomic DNA PCR and western blot analysis, displayed a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1∆ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1∆ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins related to glucose/starch metabolism and ROS (reactive oxygen species) scavenging. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1∆ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.-
dc.description.tableofcontentsCONTENTS
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I
CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . V
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI
LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . VII
GENERAL INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . 5
PDIL1-1 facilitates deposition of seed storage proteins in rice . . . . . 5
PDIL1-1 involves in ER stress and unfolded protein response . . . . . 6
The alteration of activity of seed proteins results in opaque endosperm
in cereal crops . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . 11
Genotypic analysis of T-DNA insertion knock-out mutants of PDIL1-1 11
Two-dimensional gel electrophoresis and imaging analysis . . . . . . 11
Enzymatic digestion of proteins in gels and MALDI-TOF analysis . . 14
Measurement of seed protein amount in dry seeds . . . . . . . . . . . 16
Methylene blue and iodine staining of seeds . . . . . . . . . . . . . . 16
Measurement of aleurone layer thickness . . . . . . . . . . . . . . . . 17
II
Expression and purification of C-termini of PDIL1-1 and BIP proteins
for antibody production . . . . . . . . . . . . . . . . . . . . . . 17
Evaluation of PDIL1-1 and BiP levels by western blot . . . . . . . . . 18
Quantitative real-time RT-PCR . . . . . . . . . . . . . . . . . . . . . 18
Northern blot analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Observation of starch granules and protein body . . . . . . . . . . . . 20
Determination of starch, amylose, free sugar, and lipid contents . . . . 21
X-ray diffraction analysis . . . . . . . . . . . . . . . . . . . . . . . . 22
Isolation of OsCP1 by yeast two-hybrid . . . . . . . . . . . . . . . . 22
In vitro binding assay . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Phylogenetic analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 24
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Isolation of PDIL1-1∆ mutant rice and phenotypic characterization of
its mutant seeds . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Various seed proteins accumulated in the PDIL1-1∆ mutant . . . . . 29
PDIL1-1∆ has a thick aleurone layer . . . . . . . . . . . . . . . . . . 36
Content of free sugars was altered in the PDIL1-1∆ mutant . . . . . . 37
Chaperone BiP level increased in the PDIL1-1∆ mutant . . . . . . . . 44
The PDIL1-1 gene is expressed in various tissues including immature
and mature seeds . . . . . . . . . . . . . . . . . . . . . . . . . 46
PDIL1-1 interacts with the cysteine protease OsCP1 . . . . . . . . . . 47
DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
III
ABSTRACT IN KOREAN . . . . . . . . . . . . . . . . . . . . . . . . 73
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dc.formatapplication/pdf-
dc.format.extent8228232 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectProtein disulfide isomerase-like protein-
dc.subjectPDIL1-1-
dc.subjectendosperm-
dc.subjectchalky phenotype-
dc.subjectprotein body-
dc.subjectaleurone layer-
dc.subjectfree sugar-
dc.subjectcysteine protease-
dc.subjectseed protein-
dc.subject.ddc633-
dc.titleProtein Disulfide Isomerase PDIL1-1 Has a Regulatory Role in Endosperm Development in Rice-
dc.typeThesis-
dc.contributor.AlternativeAuthorYeon Jeong Kim-
dc.description.degreeDoctor-
dc.citation.pages74-
dc.contributor.affiliation농업생명과학대학 식물생산과학부(작물생명과학전공)-
dc.date.awarded2013-02-
Appears in Collections:
College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Plant Science (식물생산과학부)Theses (Ph.D. / Sc.D._식물생산과학부)
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