Determination of functionally important sequence variants in promoter sequence of homogentisate geranylgeranyl transferase from Elaeis guineensis Jacq. germplasm materials
Vitamin E consists of tocopherols and tocotrienols which are lipid-soluble compounds produced by plants with various beneficial medicinal properties. Tocotrienols have cholesterol-lowering and anticancer properties and alpha-tocotrienols can prevent inducible neurodegeneration. There is high v...
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| Format: | Thesis |
| Language: | English |
| Published: |
2020
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/98816/1/IPTSM%202021%2016%20UPMIR.pdf http://psasir.upm.edu.my/id/eprint/98816/ |
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| Institution: | Universiti Putra Malaysia |
| Language: | English |
| Summary: | Vitamin E consists of tocopherols and tocotrienols which are lipid-soluble compounds
produced by plants with various beneficial medicinal properties. Tocotrienols have
cholesterol-lowering and anticancer properties and alpha-tocotrienols can prevent
inducible neurodegeneration. There is high variability in the level of vitamin E in the E.
guineensis germplasm materials from Angola and Tanzania ranging from 300-1600 ppm,
while the level is 500-1000 ppm in the commercial variety. However, the development
of markers for tocotrienols rich oil palm is still very scarce and the understanding of the
regulation of vitamin E biosynthesis is important for genetic improvement specifically
nutritionally vitamin E rich oil palm. Therefore, this study aimed to identify the SNPs in
the promoter of HGGT that associate with the high vitamin E in E. guineensis germplasm
materials. Homogentisate geranylgeranyl transferase (HGGT) is an important vitamin E
biosynthetic enzyme that catalyses the first committed step for tocotrienols biosynthesis.
Sequence alignment of 14 accessions from Angola and Tanzania with varying levels of
vitamin E content showed the presence of an SNP (-454) that associate with high vitamin
E Angolan (AH) and Tanzanian (TH) palms and two SNPs, -781 and -113 unique to AH.
Functional characterisation to determine the roles of SNPs in influencing HGGT
promoter activity was carried out by reporter gene assay in mesocarp tissues bombarded
with four different HGGT promoter constructs. The pBGWFS7 vector used for cloning
the HGGT promoter fragments contains two reporter genes, a green fluorescent protein
(GFP) and GUS. The promoters were commercial DXP (COM), AH1, TH96 and a
mutated derivative of COM (CM, g.-454A>G) produced by introducing the variant found
in common in AH1 and TH96. The quantitative fluorometric GUS assay on GFP positive
bombarded tissues shows that the lowest expression level was obtained from COM with
GUS expression of 0.27 pmol MU min-1 mg protein-1 per copy numbers. The expression
level obtained for COM is about the same as the constitutive CaMV 35S promoter that
was used for comparison. There was no significant difference in the GUS expression
level between the COM with the CM (g.-454A>G). The results showed that AH1 gave
the highest GUS expression of 0.82 pmol MU min-1 mg protein-1 per copy numbers. It suggested that the CAAT-box unique to AH at the SNP (-113), a well-known proximal
promoter element may enhance HGGT promoter activity and the tocotrienols content in
AH1. Taken together, these studies have provided valuable information on the vitamin
E biosynthetic regulatory mechanism in oil palm germplasm as these results suggest that
the HGGT promoter is potentially useful for engineering of high vitamin E markers
through SNPs association and it is essential for the future genetic improvement effort to
produce high vitamin E oil palm. |
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