Isolation of Banana Genes

Involved in the Developmental

Control of Banana Ripening

PCASTRD- DOST
Funding Agency

IPB, Crop Science Cluster CA, UPLB

Implementing Agency
Personnel

• Eureka Teresa Ocampo – Project Leader

• Laarni Grace Corales –MS Student
• Emelita Dalisay – Support Staff
• Susana Samatra – Support Staff
Budget – PhP 471,460
released 1st week of Jan 15, 2008
Duration – October 16, 2007 -
April 15, 2009 (18 months)
 Bananas
• Number one fruit crop in the
Philippines
• Annual revenues
• Export market - US$ 350M
• Local consumption – US$1B
• Local area of production
• 10% are large farms
• 90% are small family –owned farms
 Bananas
• Production constraints
• Low yields due to pests and diseases
• Large postharvest losses
• Postharvest treatments are expensive
• No postharvest facilities
• Cultivars don’t have good postharvest
keeping qualities
• Local varieties are not exported
• Banana prices are high
Improvement of Postharvest
Quality
Conventional breeding ****
• Triple genome/parthenocarpy

Genetic engineering
• Identify genes responsible for
desired traits
• Understanding genetic control of
ripening
Banana ripening

Climacteric pattern of fruit ripening

Control of Ripening via Coordinated Signalling Pathways

MADS-Box Genes

o Transcription factors
o Roles in floral development
are well established
o Modular structure for DNA
binding
 Specific Objectives

To isolate and sequence fruit-specific

MADS-box genes from banana

To isolate the genomic DNA region

encoding for identified MADS-box
genes and to characterize promoter
region
 Outputs
Primer designs
Full MADS-box gene sequences
Several isoforms identified
Differ in the 3’ UTR region
Map of promoters region for
Cavendish, Saba, Lakatan and Señorita
 Primer Designs
MaMADS 2
MADS2F 5’-ATGGGGAGGGGGAGAGTGGAGCTG-3’
MADS2R 5’- AGCAAGCCATCCTGGCACGTA-3’.

Promoter region
MH5R (5’ GCAGAACTCGTACAGCTTCC 3’).
Gene Sequences of MaMADS2 in
Lakatan and Saba are now available
 In Lakatan, the MaMADS 2 gene was
found to have three isoforms; Types I, II
and III
 Lakatan and Saba have the same
translated region
 The isoforms have similar nucleotide
sequences with insertions and deletions on
some sites
 Very notable that the isoforms have clear
differences in the 3’ UTR regions
Three isoforms of Lakatan MaMADS2 gene vs Cavendish MaMADS2
Type_I
MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIVFSN 50
RGKLYEFCSSSSMLKTLERYQKCNYGAPETNIISREIQTSQQEYLKLKAR 100
VEALQRSQRNLLGEDLGPLSIKELEQLERQLDASLRQIRSTRTQCMLDQL 150
ADLQRR-EQMLCEANKALKIRMDEGNQANQQQLWDP-NAHAVAYCRHQPQ 198
Type_II
MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIIFSN 50
RGKLFEFCSSSSMLKTLERYQKCNYGAPETNVMSRETQNSQQEYLKLKAR 100
VEALQRSQRNLLGEDLGPLNIKELEQLERQLDASLRQIRSTRTQYMLDQL 150
TDLQRR-EQMLCEANKALKRRLEESNQANQQQLWDP-NT----YGRQQPQ 194
Type_III
MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIIFSN 50
RGKLFEFCSSSSMLKTLERYQKCNYGAPETNVISRETQSSQQEYLKLKAR 100
VEALQRSQRNLLGEDLGPLNIKELEQLERQLDASLRQIRSTRTQYMLDQL 150
TDLQRR-EQMLCENKALKRRLEESNQANQQQLWDP-NT----YGRQQPQ 194
M.acuminata_MADS2
MGRGRVELKRIENKINRQVTFAKRRNGLLKKAYELSVLCDAEVALIVFSN 50
RGKLYEFCSSSSMLKTLERYQKCNYGAPETNIISREIQTSQQEYLKLKAR 100
VEALQRSQRNLLGEDLGPLSIKELEQLERQLDASLRQIRSTRTQCMLDQL 150
ADLQRR-EQMLCEANKALKIRMDEGNQANQQQLWDP-NAHAVAYCRHQPQ 198

Putative amino acid sequences of the isoforms

Putative map of the promoter region for the gene
MaMADS2 DNA fragment B14
 Cis-acting regulatory elements
Consensus
Motif Description
Sequence
Binding site of TGA6 transcription factor expressed in flowers of
ASF1MOTIFCAMV TGACG Arabidopsis
CCWWWWWWG Motif found in the promoter of SOC1, an Arabidopsis flowering time
CARGATCONSENSUS G MADS box transcription factor
Cis element of transcription factors involved in activation of
EBOXBNNAPA CANNTG phenylpropanoid biosynthesis genes
GTGANTG10 GTGA Motif found in the promoter of tobacco late pollen gene g10
Binding site of a MYB.Ph3, a petunia petal epidermis specific
MYBCORE CNGTTR transcription factor
Cis element of transcription factors involved in activation of
MYCCONSENSUSAT CANNTG phenylpropanoid biosynthesis genes
One of two co-dependent regulatory elements responsible for pollen
POLLEN1LELAT52 AGAAA specific activation of tomato lat52 gene

Binding consensus sequence of Arabidopsis transcription factor, RAV1

RAV1AAT CAACA which has an AP2-like domain found to been conserved in Arabidopsis
APETALA2 (AP2) which is involved in flower development
Motif found in promoter of Solanum tuberosum KST1 gene expressed in
TAAAGSTKST1 TAAAG guard cells and in flowers
Thank you very much