Cloning and characterization of rbcL gene in sweet potato

(Ipomoea batatas)

Rubylyn D. Mijan∗1 and Maria Carmina Manuel2

1Agricultural Sciences Department, College Agriculture, University of Southern Mindanao (USM),Kabacan, North Cotabato 9407, Philippines 2Institute of Biological Sciences, College of Arts andSciences, University of the Philippines Los Ban˜os (UPLB), College, Laguna 4031, Philippines

Email: Rubylyn D. Mijan∗- mijanrubylyn@yahoo.com; Maria Carmina Manuel- mcdcmanuel@yahoo.com;

∗Corresponding author

Abstract

Plants would not able to undergo photosynthesis without

rubisco, which is a powerful tool in phylogenic analysis,

species diversity estimates, varietal identification and

population analysis. Transformation is a parasexual method of

introducing new genes into an organism. pGEM-T easy vector,

containing multiple cloning region, flanked by recognition sites

for the restriction enzymes HaeIII and HinfI, are employed for

cloning PCR product and to transform bacterial strain E. coli JM-

109 which is deficient in B-galactosidase activity due to deletion

in both genomic and episomal copies of lacZ gene. Amp r and

lacZ gene are used for recombinant selection. On the other hand

cloning of the gene for sequence divergence amongst species and

genera is also a powerful tool in comparison to direct

sequencing of PCR product. The rbcL gene was isolated from

Ipomoea batatas. The sequence of rbcL gene found more or less

similar in Ipomoea species. After sequencing, rbcL probe may be

used for screenable related taxa as well as the taxa which have

the low photosynthetic rate, the insertion of rbcL gene through

recombinant DNA technology in higher amount may increase

photosynthesis rate. The information obtained in this study may

be used in crop improvement either for qualitative or

quantitative traits.

Keywords: Sweet potato, Rubisco, Cloning , Transformation, E.coil, pGEM® - T

Easy vector systems.

Introduction

Sweet potato (Ipomoea batatas L.) is the sixth most important

crop for food and industrial materials worldwide after rice,

wheat, potato, maize and barley, and it is the fifth most

important crop in many developing countries

(http://cipotato.org/sweetpotato/ and http://www.fao.org/). It

is a warm season crop which is extensively cultured in tropical,

subtropical and temperate regions. Sweet potato is a cash crop

with high yield, drought tolerance and wide adaptability to

various climates and farming systems (Van Heerden and Laurie,

2008 and Zu K et al., 2010). Sweet potato is available all year

round and is easily propagated from stem cuttings. Specifically,

the leaves have been shown to have nutritive and anti-nutritive

effects. The leaves contain cyanide, tannins, oxalate and phytic

acid as antinutrients and a couple of minerals (calcium,

magnesium, iron, zinc, potassium, manganese, phosphorus, copper

and sodium) and vitamins (vitamin A, B6, B12, C and D). A study

showed that the purple leaves of camote contain 6 g of phenolics

and 21.5 μg of β-carotene per 100 g. Thus, studies on sweet

potato are focused on increasing biomass production by improving

photosynthesis.

Leaf is the general location of photosynthesis and acts as

a source of carbohydrate for sink nutrients to support growth in

sink organs of plants. Leaf maturation is an important event in

the process of leaf development and is closely related to

photosynthesis efficiency, which is regulated by various

proteins (Chen et al., 2010). Ribulose-1,5-bisphosphate

carboxylase/oxygenase (RuBisCO) is a key enzyme of

photosynthetic carbon assimilation, and catalyzes the first step

of photosynthetic carbon assimilation and photorespiratory

pathways (Lawlor, 2002; Dejimenez et al., 1995; Hong et al., 2005) .

Plants would not be able to undergo photosynthesis without

rubisco. Rubisco is composed of eight large and eight small

catalytic subunits separately. The large subunits are encoded by

the chloroplast genome and the small subunits are encoded by the

nuclear genome. The variation in the nucleotide sequence of the

genes coding for the large subunit of rubisco ( rbcL) which

exist in the chloroplast genome has warranted its use as a

powerful tool in phylogenetic analysis, species diversity

estimates, varietals identification and population analysis.

Cloning of the rbcL gene for sequence divergence amongst Ipomoea

species is a powerful tool in comparison to direct sequencing of

the PCR products. The advantages of cloning are that the gene is

there for longer periods of times and fixes the ambiguities of

PCR reaction by miss incorporation of bases by Taq polymerase as

well as resolves the problem of repeated amplification using

genomic DNA which is critical in case of fossilized specimens as

well as for materials which are exotic.

It is clear that, sweet potato exhibits the characteristics

of efficient and longstanding photosynthesis even they are grown

under stress conditions. The insertion of rbcL gene through

recombinant DNA technology or other recent similar technologies,

in higher amount may increase photosynthesis rate. In order to

better understand the mechanism of efficient photosynthesis in

sweet potato, we cloned and characterized the rbcL genes in

sweet potato (Ipomoea batatas).

Methods

Plant materials

Three local strains of sweet potato (Ipomoea batata L.) were

used in this study. Plants grown under natural conditions at

University of the Philippines, Los Baños were selected. Expanded

young leaves of sweet potato were harvested (Fig. 1) early in

the morning and frozen immediately in liquid nitrogen.

Figure 1. Young leaves of Ipomoea batatas (samples 1, 2 and 3, respectively).

Genomic DNA isolation

Plant genomic DNA isolation methodology of Milligan (1992)

was used. Young leaves of sweet potato were washed and dried

with paper towels to eliminate excess dirt. One hundred

milligram of young leaf tissues were ground to a fine powder in

liquid nitrogen and was then placed in 1.5-mL microtubes

containing 500 µL 2% CTAB (cetyltrimethyl ammonium bromide)

extraction buffer [20 mM EDTA, 100 mM Tris-HCl, pH 8.0, 1.4 M

NaCl, 2% CTAB]. The solution was incubated at 60ºC for 45 min

with periodic gentle swirling; 500 µL of chloroform-

isoamylalcohol (24:1) was added to the tubes and gently mixed by

inversion 10x for 1 min. Samples were centrifuged for 10 min. at

5,000 rcf; 200-300 µL of the supernatant was then transferred to

a fresh tube following the addition of 500 µL chloroform-

isoamylalcohol (24:1); this procedure was repeated twice. 200-

300 µL of the supernatant was then transferred to a fresh tube

with 700 µL of cold isopropanol (-20ºC). Samples were gently

mixed by inversion and centrifuged at 5,000 rcf for 10 min, and

so it was possible to visualize the DNA adhered to the bottom of

the tube. The liquid solution was then released and the DNA

pellet washed with 700 µL of 70% ethanol to eliminate salt

residues adhered to the DNA, and set to dry for 2-4 minutes,

with the tubes inverted over a paper towel. The pellet was then

re-suspended in 30 µL 1x TE buffer (10 mM Tris-HCl pH 8.0, 1 mM

EDTA pH 8.0) plus 2 µL ribonuclease (RNAse A 10 mg mL–1) in each

tube. The integrity of the genomic DNA isolated was checked by

running 2 µL on a 1% agarose gel using gel electrophoresis.

PCR Ampli cationfi

The rbcL gene was ampli ed in a 20 µl reaction volume,fi

consisting of: 1X PCR buffer, 2.5 mM MgCl2, 0.8 mM each of dNTP,

0.3 µM each of forward and reverse primers, 1 U/µl Taq

polymerase and 100 ng of template DNA. The primers used had the

following sequences:

rbcL_f: ATGTCACCACAAACAGAGACTAAAGCrbcL_r: GTAAAATCAAGTCCACCRCG

The program set-up was run and the cycling profile was as follows:

954 9430s 551 721 9430s 551 721 7210

5X 30X

After the PCR run, the PCR products were analyzed using gel

electrophoresis at 1% agarose at 100V for 30 minutes. The

amplified rbcL PCR product was digested with restriction enzymes

HaeIII and HinfI and was rapidly and efficiently puri ed usingfi

QIAquick PCR Purification Kit. The puri ed DNA obtained afterfi

elution was used for cloning.

Cloning and Sequence Analyses

PCR product was ligated to pGEM-T Easy vector ( Promega

Inc., USA) and transformed to E. coli JM109 competent cells

( Promega Inc., USA ) through heatshock treatment at 42oC for 50

s. Screening of recombinant plasmids was performed using

blue/white screening method. The putative clones were grown in

Luria-Bertani Broth at 37oC with shaking. Plasmid DNA was

isolated using alkaline lysis miniprep (Maniatis et al., 1982).

In order to verify the presence of insert in the isolated

plasmid, HaeIII and HinfI digestion was performed. PCR products

were sent to MACROGEN, Inc. (Korea) for DNA sequencing. The

nucleotide sequence of rbcL gene was edited to discard the vector

sequences at either ends and compared with published sequences

in the National Center for Biotechnology Information (NCBI)

database using BLASTN programmes (Altschul et al., 1990). BLAST

(Basic Local Alignment Search Tool) of NCBI (Altschul et al.,

1997) was used to identify significant homologues, similar

nucleotides and protein sequences from the databases. The

nucleotide sequences were then translated to amino acid

sequences by using the ExPASy Translate tool (www.expasy.org).

For further protein structure and function prediction, protein

sequences were submitted to protein homology/analogy recognition

engine V2.0 (Phyre2) to interpret protein structure and disorder

prediction and alignment views (Kelley and Sternberg, 2009).

ClustalX were then used for alignment and cladogram construction

of amino acid sequences (Tohmpson et al., 1997).

Results and discussion

Genomic DNA Isolation

DNA extraction is a routine step in many biological studies

including molecular identification, phylogenetic inference,

genetics, and genomics. DNA yield or quantity is one of the most

important criteria in efficiency evaluation of DNA extraction. A

variety of methods have been established to isolate DNA

molecules from biological materials and many DNA extraction kits

are commercially available (Milligan BG, 1998). Different

methods have various effects on DNA extraction (Chen M et al.,

2008). An ideal extraction technique should optimize DNA yield,

minimize DNA degradation, and be efficient in terms of cost,

time, labor, and supplies. It must also be suitable for

extracting multiple samples and generate minimal hazardous

waste.

Cetyltrimethyl ammonium bromide (CTAB) method is commonly

used for DNA extraction from diverse organisms (Milligan BG,

1998). This method is relatively time-consuming and require a

fume hood to operate because of the phenol and chloroform

involved. It is expected that CTAB method isolated considerable

amounts of genomic DNA with acceptable quality. In this study,

intact bands of genomic DNA was observed from the all samples of

sweet potato leaves but still all samples examined were smear

positive indicating higher DNA degradation (Figure 2). According

to Dean, M. and Ballard, J.W.O., 2001, there are many factors

that affect DNA degradation which include tissue preservation

methods, exposure to UV radiation, temperature, pH, and salt

concentration of the environment. Shahjahan et al., 1995, found

that higher temperature for lysis could also cause DNA

degradation.

Figure 2: Genomic DNA extracted from the leaves of sweet potato(Ipomoea batatas) using CTAB method. M: DNA ladder(1 Kb plus); Lane 1-2:

Ipomoea batatas Sample 1; Lane 3-4: Ipomoea batatas Sample 2; Lane 5-6:Ipomoea batatas Sample 3; Lane 7: Negative control

PCR Amplification and cloning of rbcL gene

The rbcL gene, which encodes the large subunit of ribulose-

1,5-bisphosphate carboxylase/oxygenase (RUBISCO), has been

widely sequenced from numerous plant taxa, and the resulting

data base has greatly aided studies of plant phylogeny (Palmer

et al., 1998; Clegg and Zurawski 1991; Chase et al., 1993). Such

phylogenies, based on rbcL sequences, were successfully obtained

at the family level (e.g., see Zurawski et al., 1984; Soltis et

al., 1990; Wilson et al., 1990; Jansen et al., 1991; Bousquet et al.,

1992b; Michaels et al., 1993; Morgan and Soltis 1993) and also at

higher levels (Bousquet et al., 1992a; Gaut et al., 1992; Chase et

al., 1993). The rbcL gene in higher plants was rst cloned andfi

sequenced in maize (McIntosh et al., 1996). Several studies

regarding phylogenetic relationships using rbcL sequences have

also been inferred at lower taxonomic levels (inter- and

intrageneric) in the Cornaceae (Xiang et al., 1993), the

Cupressaceae (Gadek and Quinn 1993), the Ericaceae (Kron and Chase

1993), the Geraniaceae (Price and Palmer 1993), the Onagraceae

(Conti et al., 1993), and the Saxifragaceae (Soltis et al., 1993),

indicating that rbcL can be used at the generic level. In this

study, rbcL gene in sweet potato (Ipomoea batatas) was

successfully cloned and characterized in order to contribute in

the better understanding of the efficient photosynthesis in the

plant. Prior to cloning, rbcL gene was ampli ed using thefi

designed primers. Ampli cation produced band sizes offi

approximately ~554 bp for samples 1 and 2 (lane 1, 2-3) of

Ipomoea batatas however, no band was amplified in sample 3 (lane

4) (Figure 3). This may require optimization of the PCR protocol

because there were instances that the protocol may not work in

another organism of the same species. Ampli ed products forfi

rbcL gene in sample 1 and sample 2.1 and 2.2 of I. batatas were

used for cloning.

Figure 3: PCR ampli cation of rbcL gene using the gDNA. M: DNAfiladder (1 Kb plus); Lane 1: Ipomoea batatas Sample 1; Lane 2-3:

Ipomoea batatas Sample 2; Lane 4: Ipomoea batatas Sample 3; Lane 5:Negative control

The cloning vector used was pGEM-T Easy vector ( Promega

Inc., USA) with a size of 3,015 bp. The T-overhang feature of

this plasmid made it an efficient vector for cloning of PCR

products since it was compatible to the A-overhang generated in

a non-template fashion by a thermostable polymerase (Promega,

2010). Furthermore, because of the multiple cloning sites (MCS)

located in the α-peptide coding region of β-galactosidase gene,

recombinant clones could be screen using blue/white screening

method. Restriction sites in this region would allow restriction

digestion to verify the presence of an insert. In this study,

restriction digestion using HaeIII and HinfI was performed to

con rm the presence of insert in the plasmid. Based on thefi

result, rbcL gene insert was identi ed in all samples of fi Ipomoea

batatas using HaeIII only (Figure 3). No insert was found using HinfI

enzyme (Figure 4). The reason may be due to incubation time was

short, too few units of enzyme used or HinfI didn’t cleave

completely.

Figure 4: Restriction enzyme digestion using HaeIII to determine presence ofrbcL gene insert in the plasmid. M: DNA ladder (1 Kb plus); Lane 1: Ipomoeabatatas Uncut Sample 1 PCR; Lane 2: Ipomoea batatas Sample 1; Lane 3: Ipomoeabatatas Sample 2 (B1.1); Lane 4: Ipomoea batatas Sample 3; Lane 5: R1

Figure 5: Restriction enzyme digestion using HinfI to determine presence ofrbcL gene insert in the plasmid. M: DNA ladder (1 Kb plus); Lane 1: Ipomoeabatatas Uncut Sample 1 PCR; Lane 2: Ipomoea batatas Sample 1; Lane 3: Ipomoeabatatas Sample 2 (B1.2); Lane 4: Ipomoea batatas Sample 3; Lane 5: R2

Sequence Analyses of rbcL gene

Based on the result of BLAST nucleotide analysis, rbcL gene

shares 99% nucleotide sequence similarity with Ipomoea

cordatotriloba (KF242497.1), Ipomoea batatas cultivar Xushu (KP212149.1)

and Ipomoea trifida (KF242496.1) and 98% nucleotide sequence in

Ipomoea splendor-sylvae (KF242493.1) and Ipomoea tricolor (KF242495.1)

with an E-value = 0 ( Table 1) suggesting a homologous and

highly similar relationship among them.

The three database protein sequences in FASTA format were

aligned using ClustalW2.1 multiple alignment (Figure 6).

Alignment result of the three protein sequences exhibited high

level of conservation in the entire column (represented by *).

Analyses in phylogenetic tree found out that all three

samples share a common ancestor. However, sample 3 (M3_rbcL_R)

formed another cluster with sample 1 and 2 (M1_rbcL_R and

M2_rbcL_R, respectively). This result correlated with the

morphology of all samples where sample 3 looks differently from

sample 1 and 2 (Figure 1).

VIRT7510 ------------------------------------------------------------VIRT10204 MSPTTETKASVGFKAGVKDYKLTYYTPEYQTKDTDILAAFRVTPQPGVPPEEAGAAVAAEVIRT18765 MSTTTETKASVGFKAGVKDYKLTYYTPEYQTKDTDILAAFRVTPQPGVPPEEAGAAVAAE

VIRT7510 -------------------------------------------------------MFTSIVIRT10204 SSTGTWTTVWTDGLTSLDRYKGRCYRIERVIGEKDQYIAYVAYPLDLFEEGSVTNMFTSIVIRT18765 SSTGTWTTVWTDGLTSLDRYKGRCYRIERVIGEKDQYIAYVAYPLDLFEEGSVTNMFTSI *****

VIRT7510 VGNVFGFKALRALRLEDLRIPTAYIKTFQGPPHGIQVERDKLNKYGRPLLGCTIKPKLGLVIRT10204 VGNVFGFKALRALRLEDLRIPTAYIKTFQGPPHGIQVERDKLNKYGRPLLGCTIKPKLGLVIRT18765 VGNVFGFKALRALRLEDLRIPTAYIKTFQGPPHGIQG----------------------- ************************************

VIRT7510 SAKNYGRAVYECLLKKRGPVIRT10204 SAKNYGRAVYECLRGG---VIRT18765 -------------------

Figure 6. Multiple sequence alignment of putative PCR-amplified rbcLgene

Figure 7. Cladogram tree by ClustalX

All three protein sequences resulted to a 100% confidence

by the single highest scoring template. The three top sequences

in the database also showed a 100% confidence of the 96% of the

sequence as shown in Figure 3. However sample 3 showed lower

percent identity which ranges from 41-96% compared to 60-98% and

64-96% of sample 2 and 3, respectively. This highly related

match suggests that the protein of rbcL gene strongly belongs to

the Ipomoea species.

Discussion

Figure 8. Three predicted protein structure- template model (Phyre2). Sample1, 2 and 3 respectively

Conclusion

Ribulose-bisphosphate carboxylase (rbcL) gene in sweet

potato (Ipomoea batatas) was successfully cloned and characterized

using the p-GEM T vector. Chloroplast carries genetic

information for the larger subunit of ribulosel, 5- biphosphate

carboxylase that play a central role in photosynthesis and also

30-40% of the total leaf protein in many plants. The ribulosel,

5-biphosphate carboxylase gene comparatively most abundantly

found protein because rubisco is a very insufficient catalyst

with allowed specific activity (1 mol/min-1 protein) therefore

large amount of rubisco are required to support high

photosynthetic rate. Sequencing data of rbcL gene revealed out

that the gene comprises approximately 1400bp and A, C, T, G

count is more or less similar in Ipomoea species. On the other

hand rbcL sequence may be utilized for the screening of

population, species diversity estimates & vertical

identification of related taxas. If the related taxas possess

low productivity due to low photosynthetic rate, the insertion

of rbcL gene in higher amount may increase the photosynthetic

rate, therefore, may be utilized in crop improvement.

References

Altschul, SF, Gish, W, Miller, W, Myers, EW, Lipman, DJ. Basiclocal alignment search tool. J. Mol. Biol. (1990), 215(3),403-10.

Altschul SF1, Madden TL, Schäffer AA, Zhang J, Zhang Z, MillerW, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generationof protein database search programs. Nucleic Acids Res.1997 Sep 1;25(17):3389-402.

Chen M, Zhu Y, Tao J, Luo Y (2008) Methodological comparison ofDNA extraction from Holcocerrus hippophaecolus(Lepidoptera: Cossidae) for AFLP analysis. For Study China10: 189–192.

Chen HJ, Su CT, Lin CH, Huang GJ, Lin YH. Expression of sweetpotato cysteine protease SPCP2 altered developmentalcharacteristics and stress responses in transgenicArabidopsis plants. J Plant Physiol. 2010;167(10):838–847.doi: 10.1016/j.jplph.2010.01.005.

Clegg M. T. B. S. Gaut G. H. J. Learn B. R. Morton 1994. Ratesand patterns of chloroplast DNA evolution. Proceedings ofthe National Academy of Sciences, USA 91: 6795-6801.

Dejimenez ES, Medrano L, Martinezbarajas E. Rubisco activase, apossible new member of the molecular chaperone family.Biochem-Us. 1995;34(9):2826–2831. doi: 10.1021/bi00009a012.

Hong F, Zhou J, Liu C, Yang F, Wu C, Zheng L, Yang P. Effect ofnano-TiO2 on photochemical reaction of chloroplasts ofspinach. Biol Trace Elem Res. 2005;105(1–3):269–279. doi:10.1385/BTER:105:1-3:269.

http://cipotato.org/sweetpotato/facts and http://www.fao.org/.

JWO Ballard, MD Dean. 2001. The mitochondrial genome: mutation,selection and recombination. Current opinion in genetics &development 11 (6), 667-672

Lawlor DW. Limitation to photosynthesis in water-stressedleaves: stomata versus metabolism and the role of ATP. AnnBot. 2002;89:871–885. doi: 10.1093/aob/mcf110. [PMC freearticle]

Milligan, B.G. 1998. Total DNA isolation. p. 29-64, In A.R.Hoelzel (ed.) Molecular genetic analysis of populations: Apractical approach. 2nd ed. Oxford Univ. Press, Oxford, UK.

Palmer J. D. C. F. Delwiche 1998. The origin and evolution ofplastids and their genomes. In D. E. Soltis, P. S. Soltis,and J. J. Doyle [eds.], Molecular systematics of plants II:DNA sequencing, 375–409. Kluwer Academic, Boston,Massachusetts, USA.

Van Heerden PD, Laurie R. Effects of prolonged restriction inwater supply on photosynthesis, shoot development andstorage root yield in sweet potato. Physiol Plant.2008;134(1):99–109. doi: 10.1111/j.1399-3054.2008.01111.x.

Shahjahan, A. K. M. and Rush, M. C. 1995. Identification ofphylloplane microorganisms in Louisiana rice and potentialfor biological control of rice diseases, Proceedings of the26th Rice Technical Working Group meeting, San Antonio, TX,p. 31.

Xu K, He BW, Zhou S, Li Y, Zhang YZ. Cloning andcharacterization of the Rubisco activase gene from Ipomoeabatatas (L.) Lam. Mol Biol Rep. 2010;37(2):661–668. doi:10.1007/s11033-009-9510-x.