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International Journal of Comprehensive Leading Research In Science ( ISSN: 2455-4693 )

Vol. 2 Issue 8, pp: (1-13), August 2016, Available at: www.ijclris.com

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MOLECULAR CHARACTERIZATION OF AMARANTHUS

RETROFLEXUS RBCL GENE

Jayanthi.M, Thamillarasi.k, Ramya.I, Janani.R,Amutha.R*

Department of Microbiology ,Vivekanandha College of arts & Sciences for Women,

Thiruchencode, Namakkal, Tamilnadu.

* Corresponding Author

ABSTRACT:

People perception is usually influenced by the appearance of the food by colouring. Different

foods are associated with different colours by people. Artificial food colouring agent cause

human health problems, Carcinogen, Intestinal bleeding, Diarrhoea & excessive

menstruation, mainly cause [ADHD] Attention defict hyperactivity disorder in children.

Natural colouring cannot cause these problems. In the present study Amaranthus leaf

pigment (betacyanin) was used as food colouring agent. Amaranthus is defined as “never-

fading flower” in Greek. Several species of Amaranthus are often considered as weeds,

people around the world worth Amaranthus as leaf vegetables, cereals and ornamentals.

DNA was extracted from leaf tissue using a modification of the cetyl trimethyl ammonium

bromide (cTAB) method. We developed and tested a set of primer for the specific

amplification of rbcl gene segments from Amaranthus by PCR. The rbcL gene encodes

ribulose-1.5-biphosphate carboxylase/oxygenase large subunit .The PCR amplification

profile was checked on agarose gel and the product size was compared with a size ladder of

100bp. The PCR product was sequenced by Sanger sequencing method. Phylogenetic

analysis the sequences were aligned using Clustal W, and the genetic distances were

computed according to the Kimura 2-Parameter (K2P) model. The rbcL gene has 700 bp in

length and has conserve primer in PCR. DNA bar coding the genus of the sample is

Amaranthus. The primer used for the analysis showed similarity to retroflexus species. Thus

we can conclude that the given sample might be Amaranthus retroflexus.

Keywords: Amaranthus retroflexus, rbcL gene, PCR.

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INTRODUCTION:

Colour is an important characteristic to choose food by people’s. The visual aspect of

food products can be important factor in a people’s decision. Colour thus making the product

more attractive and increasing people’s acceptability. For that reason the use of dye’s and

artificial colouring. Artificial colour additives are widely used to enhance the appearance of

food and beverages. However because of concerns about the potential health risk from the

consumption of artificial food colourings. Health problems such as physical and mental

disorders mainly in children’s, Impulsiveness, inattentiveness, DNA damage, chromosomal

aberration’s and causing intestinal problems in human. To avoid this problems, the use of

natural dyes or natural colouring from the naturally occuring sources such as plants, animals,

insects, and minerals. In this way medicinal plants are used as food additives.

Medicinal plant’s does not produce any human health problems. They are rich in

minerals (calcium, iron, magnesium, phosphorus, potassium, zinc, copper and manganese)

and vitamins (vitamin A, vitamin B6, vitamin C, riboflavin and foliate) (Gerold Jerz et

al.,2007). It will produce the natural colour and act as a food additives. So medicinal plants

have much attention in recent times because of their diversity in treating disease and its

medicinal properties. Dependence on herbs as medicine for treatment of disease. The

increasing awareness towards traditional medicine in human and animal healthcare paved the

way to research on efficacy of the herbs used in the treatment of illness. The WHO supports

and accepts the traditional medicine as a valuable resource for primary health care (Manila S,

1993). Plants are a source of many potent and powerful drugs used medicinally in different

countries (Hashim et.al.,2010). A wide range of medicinal plants parts is used to extract as

raw drugs and they possess varied medicinal properties (Arora DS et al.,2007).

Amaranthus is a medicinal plant, this plant leaf have a pigment, it is used for food

colouring agent.The plant has several active constituents like alkaloids, flavonoids,

glycosides, phenolic acids, steroids, amino acids, terpenoids, lipids, saponins, betalains, B-

sitosterol, stigmasterol, linoleic acid, rutin, catechuic tannins and carotenoids. It also contains

amaranthoside, a lignin glycoside, amaricin, a coumaroyl adenosine along with stigmasterol

glycoside, betaine such as glycinebetaine and trigonelline (Azhar-ul-Haq et al., 2006;

Blunden et al., 1999). Antioxidants protect cells against damage caused by molecules known

as free radicals.Oxidative damage is implicated in most disease processes such as




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cardiovascular disease, cancer, inflammatory conditions, asthma, liver disease and macular

degeneration. (Willcox et al.,). Betalains are a class of nitrogen-containing pigments, the

betalain pigment also interacts with their environment, such as attraction of pollinators and

protection of tissue against damaging UV-radiation as well as increase pathogen resistance

and improve viral defense especially for under gorund growing part like red beet root.

Recently, natural plant pigments have been widely used as natural colorants to replace

synthetic dyes. They are largely used as food colorants in food products like yogurts, ice

cream and other products (Zhong et al., 2005 ) and (Stintzing et al., 2002)Nature has been a

source of medicinal agents for thousands of years. Although advances have been made in

pharmacology and synthetic organic chemistry, this reliance on natural products, particularly

on plants, remains largely unchanged (Trevor et al., 2001).

DNA was extracted from leaf tissue using a modification of the cetyl trimethyl

ammonium bromide (cTAB) method of Doyle and Doyle (1990). Polymerase chain reaction

(PCR)-based techniques have been developed to evaluate DNA polymorphisms in a

wide variety of organisms (Welsh & McClelland, 1990; Williams et. al., 1990).The rbcL

gene encodes ribulose-1.5-biphosphate carboxylase/oxygenase large subunit (Ellis, 1979).

The rbcL gene has 700 bp in length and has conserve primer. The rbcL sequence can be used

for cogeneric analysis(Kres et.al.,2005). The rbcL gene is an adaptive gene to environment

heterogeneity and widely used for plant evolutionary and plant adaptation mechanism

(Galmez et.al, 2005; Sem, 2011).




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MATERIALS METHODS

PLANT COLLECTION

The plant Amaranthus retroflexus leaves was bought from the own land in anthiyur

Erode (Dt). Good conditioned leaf sample was collected, due to morphological condition.

DNA EXTRACTION

The leaf sample was collected and it was extracted with the help of liquid nitrogen,

the extract was made in to paste. And then add warm cTAB extraction buffer and mixed well.

It was incubated at 10 to 60 minutes at 65ºC. Add equal volume of isoamyl alcohol was

added, mixed well by the inversion position, and centrifuged 8000rpm for 5minutes at 4ºc.

The supernatant was collected, and it was added 1/10 volume of cTAB - NaCl solution and

mixed well, then extract with equal volume of chloroform, isoamylalcohol. Centrifuged and

collect the upper phase, 1volume of cTAB precipitation solution was added. Mixed well,

centrifuged 1000rpm in 5 minutes at 4ºC, and then precipitate was collected adding 0.6

volume of isopropanol mixed well and centrifuged for 8000rpm in 15minutes at 4ºc. The

pellet was washed with 70% ethanol, dried, and resuspended in minimal volume of TE

buffer.

PCR AMPLIFICATION

Polymerase chain reaction (PCR) of the isolated sample DNA barcode gene was

carried out in a 20μl reaction mix containing 1X Taq buffer, Taq DNA polymerase, and

genomic DNA. The PCR was performed using Agilent sure cycler 8800.

Agarose gel electrophoresis

Prepare agarose solution in the buffer provided (0.5xTEB) boil the agarose, until it is

completely dissolved. When the gel temperature is around 40ºC, add 2µl ethidium bromide

and mix properly. Pour the agarose mixture in to tray and solidify. Then keep the gel tray

containing 0.5x TEB buffer with the wells in the cathode (Negative) side. The buffer level in

the tank should be maintained above the gel tray. Connect the cords between the

electrophoresis tank and the power pack before loading the samples. To prepare samples for

electrophoresis, add 5µl of gel loading dye in to the sample and mix well. Load 20µl of the

sample and 3µl of DNA marker in to the well. After loading switch on the power pack and




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adjust the voltage to 50V or 100V. Continue the electrophoresis until the dye reach the gel

and observe the bands under UV trans illuminator.

Sequence Information:

The PCR products were purified and sequenced by Bangalour laboratory in QTlomics

traints to genes.

NCBI: Nucleotide Blast (BLASTn) output:

The sequences obtained were subjected to BLAST using the BLAST N tool of NCBI

to obtain the closest hit of the query sequence by Bangalor laboratory in QTlomics traints to

genes.

Phylogenetic Analysis: (Altschul et al., 1997)

The rbcL gene sequences obtained from the Amaranthus strains were initially

sequences available in the National Center for Biotechnology Information database using

BLAST network services to determine their approximate phylogenetic affiliations. The

sequences were aligned using Clustal W and the genetic distances were computed according

to the Kimura 2-Parameter (K2P) model. The similarity values between the sequences were

calculated from distance matrices by reversing the Jukes-Cantor distance formula.

Phylogenetic trees were then inferred by neighbour joining (NJ) using the Kimura two-

parameter model. The resulting NJ tree was evaluated by bootstrap analyses based. Finally,

an overview of the phylogenetic position of Amaranthus was created by the rbcL gene

sequences to corresponding sequences available in databases and the sequences obtained in

this study for Amaranthus retroflexus evaluated with 1,000 bootstrap replicates.




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RESULTS AND DISCUSSION

Amaranthus is a genus in the family of Amaranthaceae. The Amaranthus is

multicomponent containing several different groups of subunits and polypeptides, thus it is

characterized by essential molecular heterogeneity (Reema srivastava et.al.,2012). Studies

on the evolution relationships of the genus Amaranthus species cultivated and theirs wild

relatives have been made especially in the last 20 years, by applying various

techniques(Gabriela et.al.,).

Morphological differences among Amaranthus variant can be analyzed using

molecular marker, especially using DNA sequences. Phenotypic variation caused by

plant morphological, functional, and developmental changing because of environmental

heterogeneity to one or more genotype in one population (Radford, 1986). Medicinal

plants contain biologically useful secondary compounds, including tannins, alkaloids, and

polysaccharides, all of which can inhibit DNA extraction and amplification by co-

precipitating with or binding to DNA(M. Schori and A.M. Showalter)

PCR amplification

Polymerase chain reaction (PCR) of the given sample with the candidate DNA

barcode gene was carried out in a 20μl reaction mix containing 1X Taq buffer, Taq DNA

polymerase, and genomic DNA. The PCR was performed using Agilent sure cycler 8800.

Most of the current methods to determine genetic diversity are based on polymerase

chain reaction (PCR). Molecular methods have proved useful in the characterization of

biological material.

The low level of polymorphism in these species may reflect the high level of

inbreeding in these Amaranthus species or the fact that the primers which we have used they

were not successful in amplifying bands from members of the genus Amaranthus ( Bardini,

M.et.al., 2004).




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Amplification check on gel

The PCR amplification profile was checked on agarose gel and the product size was

compared with a size ladder of 100bp

Figure1: The PCR product wass resolved on 2% Agarose gel at 120V for approximately 60

min or till the samples reached 3/4th of the gel. The gel was visualized under UV light and

the image was captured.Lane 2: 100bp ladder, Lane 1: Amplification product of leaf sample

with rbcL

Sequence Information:

The PCR products were purified and sent for Sanger sequencing. Below is the

sequence information as obtained by Sanger sequencing.

If clean genomic DNA is obtained, appropriate primers are needed to amplify the

targeted gene region. Certain gene regions, including rbcL have universal primers that work

for most plants. There were used in this research, rbcL genes. The rbcL gene has 700 bp in

length (Radford, 1986).




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NCBI: Nucleotide Blast (BLASTn) output:

The sequences obtained were subjected to BLAST using the BLAST N tool of NCBI to

obtain the closest hit of the query sequence.

(A) rbcl




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Figure2: Showing NCBI-blast hit search result for the given leaf sample with rbcL gene

specific primer. The blast search showed similarity to genus Amaranthus. The first 10 blast

hit sequences were taken to construct a phylogenetic tree.

Phylogenetic Analysis:

The sequences were aligned using Clustal W, and the genetic distances were computed

according to the Kimura 2-Parameter (K2P) model. Phylogenetic tree was constructed using

UPGMA method on MEGA 6 software and the reliability of the tree was evaluated with

1,000 bootstrap replicates.




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These studies have allowed the development of some hypotheses about the geographical

origin of species and establish phylogenetic links between them. Also, there has beena series

of tests on the molecular characterization of germ plasm in order to obtain useful results for

breeders(Gabriela et.al.,).

(A) rbcL




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CONCLUSION

In conclusion, this study showed that in spite of antigenic and allergenic

differences among from Amaranthaceous family. This report will help us to find strategies

for marker-assisted identification of amaranth genotypes in order to conservation of amaranth

genetic resources or for different practical approaches. The results also suggest that

species usually maintained high level of polymorphism over the cultivated counterpart.

The high genetic variability caused by local adaptation and gene flow among species.

ACKNOWLEDGEMENT

The authors are thankful to Prof. Dr. M. KARUNANITHI, Chairman and secretary,

Vivekanandha Educational Institutions, and Dr.B.T.SURESHKUMAR, Principal,

Vivekanandha College of arts and Sciences for Women, Elayampalayam, Tiruchengode,

Namakkal District, Tamil nadu for providing all the facilities for our research work.

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How to cite this article:

Jayanthi.M, Thamillarasi.K, Ramya.I, Janani.R and Amutha.R, Molecular Characterization of

Amaranthus Retroflexus RBCL GENE Vol 2 Issue 8, pp : 1-13



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