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Cloning of Putative DHCR and CYP85 Genes in Spinach (Spinacia oleracea L.)

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Author(s)
주초이
Issued Date
2009
URI
http://dcoll.jejunu.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000004806
Abstract
Phytoecdysteroids (PEs) are a group of ecdysteroid produced in plant kingdom compared to zooecdysteroid that are synthesized by insect or nematode. In insects, ecdysteroids function as a hormone which control molting, metamorphosis, and other physiological processes related to development at nearly every stage. In contrast in insects, ecdysteroids show no apparent hormonal activities in plants instead they are postulated to be important as a chemical defense against non-adopted insect species. To elucidate the exact function of PEs, understanding of their metabolism is prerequisite. In plants, the content and distribution of ecdysteroids were illustrated, but the biosynthetic pathway is not clear until now. Most of the previous works in ecdysteroid biosynthesis have been investigated by metabolomic approach. In this work a genomic approach was used to identify genes which are essential for molecular biological analysis of ecdysteroid biosynthesis in plants. Two enzymes, 7-dehydrocholesterol reductase (DHCR) and CYP85 were focused on, which are supposed to catalyze formation of double bond at C7 ? C8 and oxidation at C6, respectively, in the ecdysteroid ring structure. Candidate genes for these two enzymes were cloned and their expressional characteristics were discussed in spinach.
The partial gene of So-DHCR was cloned by the degenerate RT-PCR of first strand cDNA synthesized from mRNA using specific degenerate primers which were designed based on the conservative amino acid sequences of known DHCR?s in other plants. The partial gene was confirmed in NCBI and ExPASy database and it showed high homology to other plant DHCR?s with a similarity of 90% on the amino acid level. RACE technique was used for amplifying the ends of cDNA for obtaining full-length gene. The obtained full length of So-DHCR gene was 1,691 bp encoding 434 amino acids. Its theoretical pI was 7.89, and the molecular weight was near 45 kDa. The deduced amino acid sequence of So-DHCR cloned from spinach showed up to 86% similarity to those in ecdysteroid-negative plants. The sequence homology of So-DHCR was expected to be higher among the ecdysteroid-positive plants because they supposed to be evolved from the same ancestor. As expected the homology of DHCR between two ecdysteroid-positive plants, spinach and Achyranthes japonica, was much higher with 94%. Ecdysteroid biosynthetic ability of spinach leaves was known to vary with their age, the data obtained from this study, however, showed that there was no significant difference among leaf ages in So-DHCR expression.
In the case of So-CYP85, the partial gene was cloned by the degenerate RT-PCR using specific primers designed based on the conservative amino acid sequences of CYP85 family known in other plants. When the partial gene was analyzed against NCBI and ExPASy databases, the amino acid sequence homology to known CYP85 family in other plants was 74%. RACE technique was used for amplifying the ends of cDNA for obtaining full-length CYP85 gene as in DHCR. The full length gene of the putative So-CYP85 was 1,696 bp encoding 464 amino acids. Its theoretical pI was 9.30, and the molecular weight was near 54 kDa. There was a heme-biding domain (FGGGTRQCPG) at the site of 407-426 bp. The protein expressed in E. coli exhibited a maximum absorption at 409nm in UV-visible spectroscopy. The deduced amino acid sequence of the cloned So-CYP85 from spinach showed highest homology of 79% to that in Vitis vinifera, an ecdysteroid-negative plant. As expected in So-DHCR, the homology of So-CYP85 between two ecdysteroid-positive plants, spinach and Achyranthes japonica, was much higher with 92%. The expression of the putative So-CYP85 in spinach was higher in older leaf and lower in younger one which was consistent with the ecdysteroid biosynthetic abilities of leaves at different age as described in the previous paper. The level of So-CYP85 expression was inversely proportional to the 20E content in the leaf and this supported the idea for the phytoecdysteroid biosynthesis to be down-regulated by the end-product 20E. The above results from this study showed several positive evidences for the cloned DHCR and CYP85 genes to be involved in PE metabolism. In order to obtain direct evidences for their role in PE biosynthesis, however, further transgenic and metabolomic studies are necessary.
Affiliation
제주대학교 대학원
Department
대학원 생명공학과
Advisor
Zung Riu
Awarded Date
2009. 8
Table Of Contents
SUMMARY 1
INTRODUCTION 3
MATERIALS AND METHODS 7
Plant Materials 7
Bacterial Strains and Plasmids 7
Isolation of RNA from Plant 7
Degenerate RT-PCR for Partial Gene Cloning 8
Cloning of Full Length cDNA 10
Protein Expression of So-CYP85 in E. coli 11
Analysis of Gene Expression in Spinach 13
Analysis of Phytoecdysteroid by HPLC 13
RESULTS 17
Isolation of RNA 17
Cloning and Expression of putative DHCR 18
Cloning of candidate gene 18
Homology of the cloned gene to other known genes 21
Expression of the endogenous gene within spinach 23
Cloning and Expression of putative CYP85 26
Cloning of candidate gene 26
Homology of the cloned gene to other known genes 29
UV absorption spectrum of the gene product 31

Expression of the endogenous gene within spinach 32
DISCUSSION 36
Characteristics of the Putative So-DHCR Gene 36
Characteristics of the Putative So-CYP85 Gene 37
Conclusion 39

REFERENCES 41
ACKNOWLEDGMENT 50
Degree
Master
Publisher
제주대학교 대학원
Citation
주초이. (2009). Cloning of Putative DHCR and CYP85 Genes in Spinach (Spinacia oleracea L.)
Type
Dissertation
Appears in Collections:
Faculty of Biotechnology > ETC
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