SIL 3001

Genetics & Molecular Biology

Institute of Biological Sciences
Faculty of Science
University of Malaya

UNDERGRADUATE RESEARCH THESIS PROPOSAL: SEM1 2021/2022

A PROJECT IDENTIFICATION  

1 Name Mim Mohd Adzrien bin Abdul Bakar  

2 Matric number SIL190013 @ 17204184  

Genome-wide identification of AP2/EREBP gene family
3 Project title in Boesenbergia rotunda; a medicinal plant   
             

B OBJECTIVES OF THE PROJECT  

1 Specific objective(s) of the project
 
(i) To identify coding sequences of AP2/EREBP genes in B. rotunda and
the conserved motifs shared among the genes.
(ii) To predict the protein properties and putative function of the
AP2/EREBP genes.
(iii) To determine the exon and intron arrangements in AP2/EREBP genes
and the evolutionary relationship of AP2/EREBP proteins.
(iv) To identify a list of candidate genes predicted to be involved in abiotic
stress responses and biosynthetic pathways.
   

2 Hypothesis
Bioinformatic analysis can identify and characterize the AP2/EREBP gene family
in the genome of B. rotunda and allow prediction of properties and functions of
these genes.

3 Literature Review (Research background of the project)  

   
Boesenbergia rotunda (commonly known as Fingerroot ginger) is a culinary and
ethnomedicinal herb that belongs to the plant family Zingiberaceae. It is commonly
found in many Asian countries (Kanna & Krishnakumar, 2019). Its medicinal usage
has received much interest from scientists worldwide as the purified secondary
metabolites from the plant have shown promising antiviral and anticancer
properties (Eng-Chong et al, 2012; Jing et al. 2010). Fingerroot can reach a height
of 46-61 cm. The red leaf sheaths give way to ovate-oblong or elliptic-lanceolate
leaves. As one of the Zingiberaceae family members, it produces pseudostems
rather than true stems, and the plant propagation is accomplished through division
of the rhizome. Pink flowers normally appear from the peak of the pseudostem and
are hidden below the leaves.

Taxonomy
The taxonomy of B. rotunda is in the Kingdom: Plantae; Subkingdom:
Viridiplantae; Infrakingdom: Streptophyta; Superdivision: Embryophyta; Division:

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Tracheophyta; Subdivision: Spermatophytina; Class: Magnoliopsida; Superorder:

Liliane; Order: Zingiberales; Family: Zingiberaceae; Genus: Boesenbergia;
Species: B. rotunda. (Guala, 2019).

Nomenclature
Boesenbergia rotunda is native to Southeast Asia due to the little seasonal variation
near the equator as the plant could not withstand low temperature environment
during the winter. Its common name, fingerroot comes from its rhizome that looks
similar to human fingers. The genus for the plant was initially known as
Gastrochilus. It was then identified that Gastrochilus Wall. is a homonym of
Gastrochilus Don (1825; Orchidaceae) before its name wais changed to
Boesenbergia. However, the genus name Gastrochilus is still used in some of the
Zingiberaceae studies. Schlechter (1913) emphasized the importance of
implementing the nomenclatural change into Boesenbergia for the ginger species of
Gastrochilus and he thus made the necessary transfer.

Chemical compounds
Research has shown that B. rotunda plant has anti-allergic, antibacterial, anticancer,
anti-inflammatory, antioxidant, and antiulcer properties, as well as wound healing
properties. The flavour and medicinal properties of B. rotunda are is due toplant
contains chemical compounds such as flavonoids including alpinetin, boesenbergin,
cardamonin, geraniol, krachaizin, panduratin, pinostrobin, pinocembrin,
rotundaflavone, and silybin (Ching et al. 2007; Morikawa et al. 2008; Yusuf et al.
2013). The plantIt contains essential oils such as nerol, camphor, cineole, fenchene,
hemanthidine, and limonene (Baharudin et al. 2015). Besides, it also contains
alkaloids, and phenolics.

Health benefits
Fingerroot provides protection against Helicobacter pylori infection. H. pylori is a
bacterial pathogen that causes dyspepsia, gastritis, peptic ulcers and is responsible
for the formation of colon and stomach cancers (Dore & Graham, 2008). The
essential oil from B. rotunda aids in inhibiting Helicobacter activity. The ginger
also contains flavonoid components that act as a drug to prevent H. pylori infection
(Chong et al. 2012). Besides, B. rotunda could be helpful for HIV patients as the
chloroform extracts of the plant could inhibit propagation of amobas and prevent a
disease called Amoebiasis that leads to chronic diarrhea in the HIV patients. The
methanolic extracts of B. rotunda also prevents teeth cavity formation. B.rotunda
has bactericidal activity against the oral bacteria Streptococcus mutans and
Lactobacillus that are the cause for tooth decay and dental caries (Chong et al.
2012). B. rotunda also has antiulcer properties which could help in ulcers
treatment. The methanolic extract of B. rotunda contains pinostrobin, which has
cytoprotective properties that helps to fight ulcers. Pinostrobin also has antioxidant
propery and may reduce the amount of thiobarbituric acid reactive compounds
(Chong et al. 2012). The ethanolic extracts from the rhizomes also aid in the
healing of wounds. According to the findings from previous research, wounds
treated with the rhizome extract and intrasite gel healed swiftly, resulted in a scar
that was less visible, and it could prevent and cure a variety of disorders
(Ruttanapattanakul et al. 2021). In addition, B. rotunda is also known for its
aphrodisiac properties where it increases sexual desire, attraction, pleasure, or
behavior due to the compounds boesenbergin, krachaizin, panduratin, and

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pinostrobin contained in the plant (Yotarlai et al. 2011). Various crude extracts of
B. rotunda plant components, such as the leaf, stem, and rhizomes, have been
studied and shown to be pharmacologically active in promoting aphrodisiac action.
Research was conducted on the effects of B. rotunda on male rats. They discovered
that ethanolic extract dosages of 0.06, 0.12, and 0.24 g/kg increased the diameter of
seminiferous tubules as well as the weights of the testicular and seminal vesicles
(Sudwan et al. 2007).

Despite its high bioactivity, the genomic information of B. rotunda is still scarce,
and this limits the access to the regulatory mechanisms and biosynthetic pathways
for its bioactive metabolites.

AP2/EREBP gene family

The APETALA2/ethylene-responsive element binding protein (AP2/EREBP)
superfamily is one of the largest groups of plant-specific transcription factors (Liu
& Zhang, 2017). The proteins play important roles in plant growth and
development, as well as responses to various environmental stresses, including high
and low temperature shocks, drought, high salinity and pathogen infection. It is
believed that the proteins in this superfamily are essential for B. rotunda to achieve
optimal growth and development, as well as stable synthesis of various secondary
metabolites and bioactive compounds. Nonetheless, little is known about this
superfamily in B. rotunda.

Genes in AP2/EREBP family

There was a total of 149 AP2/EREBP genes found and these genes can be
categorized into four subfamilies which are known as ethylene responsive factor
(ERF), dehydration responsive element binding gene (DREB), RAV (related to
ABI3/VP), and AP2 (Chen et al. 2006).

ERF subfamily
The ERF subfamily in Arabidopsis has been reported to help in the induction or
repression of gene expression in response to ethylene, cytokinin, and abiotic
stresses such as injury, cold, excessive salt, and drought (Rashotte et al. 2010). This
is because the ERF is able to recognize the GCC-box (Allen & Mark, 1998). ERF
gene also plays an important role in fruit ripening. Climacteric fruits exhibit a surge
in respiration and manufacture of the gaseous hormone ethylene at the start of
ripening, but non-climacteric fruits show no substantial change in respiration and
ethylene production during the shift from unripe to ripe. The plant hormone
ethylene is recognised to have an important role in influencing most aspects of fruit
ripening in climacteric fruits such as apples, kiwifruits, and tomatoes (Aghdam et
al., 2018).

DREB subfamily
In Arabidopsis, the DREB genes activate many stress-sensitive genes by interacting
with the C-repeat/dehydration responsive element (CRT/DRE) in the promoters of
target genes, which contains a core motif of A/GCCGAC (Huang et al. 2012;
Yamaguchi-Shinozaki & Shinozaki, 1994). They control stress-responsive genes in
response to a variety of abiotic conditions such as low temperature (Jaglo-Ottosen
et al. 1998), heat (Novillo et al. 2004), drought (Sakuma et al. 2006), and excessive
salt (Qin et al, 2007). As a result, they are ideal candidates for genetic modification

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to improve crop stress tolerance.

RAV subfamily
This subfamily contains genes that consist of an AP2 domain as well as a B3 DNA
binding domain at C, terminus that acts as negative regulators in plant growth
which is critical for regulating abiotic and biotic stress responses. (Feng et al. 2014;
Fu et al. 2014; Matías-Hernández et al. 2014; Li et al. 2015). Based on a
phylogenetic analysis, the amount of RAV subfamily genes in Brachypodium
distachyon is been that almost the same as that in Setaria italica, rice, Arabidopsis,
Populus trichocarpa, Vitis vinifera, and Salix arbutifolia. These show great
conservation of this subfamily among the plants and implies that these RAV genes
in all of these plants may have shared a common ancestor prior to Brachypodium's
separation from the other plants (Chen et al. 2016).

AP2 subfamily
Apetala 2 (AP2)-family proteins are transcription factors with 60 amino acid DNA-
binding domains known as AP2 domains. The AP2 family, which includes two
AP2 domains is required for plant growth (Okamuro et al. 1997; Shigyo & Ito,
2004). In seed plants, it is further subdivided into the AP2 and ANT
(AINTEGUMENTA) groups. A study shows that the AP2-family transcription
factor AP2-L is also important in the parasite's liver-stage development. (Iwanaga
et al. 2012). Besides, AP2 is involved in the ABC model of floral development in
Arabidopsis thaliana (Riechmann & Meyerowitz, 1998).

Ongoing research at CEBAR UM has obtained and analyzed genomic and

transcriptomic data from different samples of B. rotunda. With the advent of
transcriptomics, new insights on the biosynthetic pathways of B. rotunda
metabolites can be clarified, enabling researchers to study the mechanism behind
the synthesis of bioactive compounds responsible for the medicinal properties of
the plant.

C RESEARCH APPROACH  
1 Research methodology
   
Identification of coding and protein sequences of AP2/EREBP superfamily
The project will start with the draft genome sequence and transcriptome data
obtained from an ongoing project in CEBAR on B. rotunda. All the AP2/EREBP
genes in the draft genome will be identified and the conserved motifs will be
examined. The coding and protein sequences of genes in the AP2/EREBP will be
identifed using BLASTP and TBLASTN (e-value: 1e-005) and they will be
validated using SMART and pfam database. The number of the AP2/EREBP
protein genes in the genome of B. rotunda will be determined, and the putative
genes will beare checked for the presence of the conserved AP2 domain. The gene
sequences will be classified into the RAV subfamily; and the remaining genes that
carry a single AP2/ERF domain will be assigned to the DREB/ERF subfamilies.
All or a selected group of subfamilies might be used for further analysis.

Prediction of exons and introns

The work will proceed with determining exon and intron arrangements using Gene
Structure Display Server and then phylogenetic analysis using MUSCLE and

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MEGA 7.0, towards theas well as identification of a list of candidate genes that
might be involved in abiotic stress responses and biosynthetic pathways.

Analysis of protein properties

ExPASy proteomics server is used for protein analysis.

Identifications and structural analysis of conserved motifs

MEME tool is used for conserved motifs analysis.

Functional annotation of AP2/EREBPs

BLAST2GO software is used for the annotation.

Alignment and phylogenetic analysis of AP2/EREBPs

MUSCLE and MEGA 7.0 will be used for the analysis.

D PROJECT ACTIVITY SCHEDULE

2021 2022
  Month S O N D J F M A M
  ACTIVITY                    
1. Literature review    X X               
2. Identification of coding sequences of
AP2/EREBP gene superfamily and the
conserved motifs shared among the genes
in the genome of B. rotunda                
3. Prediction of the protein properties, and
putative function of AP2/EREBP genes                
4. Determination of the exon and intron
arrangements in AP2/EREBP genes and
the evolutionary relationship of
AP2/EREBP genes              
5. Identification of candidate AP2/EREBP
genes involved in abiotic stress responses
and biosynthetic pathways                    
6. Thesis writing                    
Draft of literature review to supervisor                X    
  Draft of Materials and Methods to supervisor                  X  
Draft of results and discussion to supervisor X
Presentation slides prepared X
  Submit final copy of thesis                    X

KEY: Ongoing activity X

     

E ACTIVITY FLOWCHART

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