PLAN RESEARCH

TRAN NGUYEN NGOC ANH

Email: trannguyenanh.184@gmail.com

Designing an brief experimental proposal for

"Identification and molecular characterization of transcription factors

regulating vascular cambium development in pine tree”

INTRODUCE
Secondary growth is one of the most important biological processes on Earth.
Its product, wood, is of primary importance to humans as timber for construction,
fuelwoods, and wood-pulp for paper manufacturing. However, despite its
economic and environmental significance, secondary growth has received little
research interest, mainly because most agricultural products are derived from seeds
or roots. Furthermore, the biology of wood formation is surprisingly understudied
because of the inherent problems of tree species: long generation time, large size,
and lack of genetically pure lines. Study of wood formation at the molecular level
using real trees has begun in recent years. A genomics approach has been
successfully used to examine global gene expression patterns in developing xylem
tissues of black locust (Yang et al., 2003), pine (Allona et al., 1998; Lorenz and
Dean, 2002), and poplar (Sterky et al., 1998; Hertzberg et al., 2001). In addition,
plant vasculature is required for the transport of water and solutes throughout the
plant body. The primary constituents of vascular tissues, xylem, and phloem, are
derived from the meristematic vascular procambium and cambium. Almost all of
the developmental fate decisions in this process are regulated by transcription
factors. However, current understanding of the molecular mechanisms of wood
formation in trees is still limited. This plan together the literature describing the
roles that these genes play in vascular cambium development
METHODOLOGY
+ Indentification of gene families of transciption gene in pine tree genome
We used representative genes from appropriate model plant or poplar gene
families as the basis to search for orthologs in pine tree. Sequences were retrieved
from NCBI Genbank: The sequences were retrieved and evaluated for the presence
of transcription factors domains by searching against the conserved domain database
(CDD), using BLAST tool at NCBI genbank and BioEdit sorfware. Genome was
addressed by the identification of only a single gene copy with the highest similarity
to the corresponding homologs in poplar or Arabidopsis from multiple gene copies.

+ Cluster and protein sequence analysis

Extracting protein from leaves of Pine tree, and Mega 7 software to import the
amino acid sequences of genome familises the MEGA7 program and multiple
sequence alignment analysis was conducted using MUSCLE with default
parameters. Construction of cluster trees was performed with the neighbour-joining
algorithm method by the MEGA7 program using the bootstrap values resulted from
1000, Poisson correction and pairwise deletion, Conserved motifs in TFs were
identified with the online tool, MEME using the following parameters: optimum
width, 6–200 amino acids; with any number of repetitions and maximum number of
motifs set at 25
+ Analysis of gene structure and gene ontology annotation
Extracting DNA from leaves of Pine tree by CTAB. From DNA sequences, to
design primers. After that using them to PCR and analyse genome (if length of
genome higher than 1000 kb, cloning by E.coli before analyse)
The genomic and coding DNA sequences of the identified transcription factors
were retrieved from the Phytozome. To evaluate the gene ontology (GO) annotation
of the identified TFs, their amino acid sequences were imported into the Blast2GO
suite. Blastp search was performed against pine plants, protein sequences at NCBI.
The resulting hits were mapped to obtain the GO terms, which were annotated to
assign functional terms to the query sequences. Plant GOslim was used to filter the
annotation to plant-related terms

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+ Making gene expression atlas
Using Mega 7. The phylogenetic tree was constructed with the neighbour-
joining algorithm and the bootstrap values resulted from 1000 replicates and are
shown as percentages at the nodes. The 0.1 scale denotes 10% change.
+ Vector Construction and Plant Transformation
Leaves were excised from in vitro plantlets, cut into disks and dipped in the
diluted Agrobacterium culture. Transgenic plants were selected with hygromycin.
Rooted plantlets were acclimatized in pots placed inside a humid chamber and
finally transferred to the greenhouse.
+ Molecular analysis of transgenic plants
Extracting DNA from leaves of Pine tree by CTAB. From DNA sequences, to
design primers. After that using them to PCR and analyse genome ( if length of
genome higher than 1000 kb, cloning by E.coli before analyse).
Polymerase chain reaction products were resolved on a 1% (w/v) agarose gel and
visualized after ethidium bromide staining.
+ RNA extraction and RT-PCR analysis
RNA was extracted from shoot tip samples of transgenic and non-transgenic
control lines using Tri-Reagent, and 3 µg of the RNA was treated with DNase-I.
HighCapacity cDNA Reverse Transcription kit was used for the synthesis of first-
strand cDNA.
Power SYBR Green PCR master mix (Applied Biosystems) was utilized to
conduct quantitative reverse transcription polymerase chain reaction (qRT-PCR)
analysis according to the manufacturer’s protocol
+ Histology
Poplar petioles and stems were sectioned using a vibrating microtome to
generate 100-µm serial sections. For histochemical analysis of lignification, sections
were stained with 1% (w/v) phloroglucinol in 6 N HCl, viewed under the microscope

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REFERENCES
Allona I, Quinn M, Shoop E, et al. 1998. Analysis of xylem formation in pine by
cDNA sequencing. Proceedings of the National Academy of Sciences, USA 95, 9693
- 9698.
Jung Hyun Yang and Huanzhong Wang, 2016. Molecular Mechanisms for Vascular
Development and Secondary cell wall formation. Front. Plant Science 7
Liam Campbell and Simon Turner, 2017. Regulation of vascular cell division.
Journal of Experimental Botany 68, 27–43
Melis Kucukoglu, 2015. Molecular Regulation of Vascular Cambium Identity and
Activity. Swedish University of Agricultural Sciences
Sookyung Oh, Sunchung Park & Kyung-Hwan Han, 2003. Transcriptional
regulation of secondary growth in Arabidopsis thaliana. Journal of Experimental
Botany 393, 2709 - 2722
Yang J, Park S, Kamdem DP, Keathley DE, Retzel E, Paule C, Kapur V, Han K-H.
2003. Novel insight into trunk-wood gene expression profiles in a hardwood tree
species, Robinia pseudoacacia. Plant Molecular Biology(in press).