GUIDE : Dr. K. M. GothandamVaibhav Kumar Maurya

11MSROO1O

AIM :: Inhibition of ζ–carotene desaturase

gene expression in capsicum annuum.

OBJECTIVES ::A.To study the functional role of ζ-carotene

desaturase gene in chili.B.To study the effect of ζ-carotene

desaturase gene inhibition on other gene in carotenoid biosynthesis pathway.

C.To study the effect of ζ-carotene desaturase gene inhibition on other carotenoid of carotenoid biosynthetic pathway

Introduction Functional genomics attempts to answer questions about

the function of DNA at the levels of genes, RNA transcripts, and protein products.

 To understand the relationship between an organism's genome and its phenotype and study of the function of DNA as well as the nucleic acid and protein products encoded by DNA.

Genetic interaction mapping, Mutagenesis, Antisense technology are some functional genomic techniques.

Antisense refers to short DNA or RNA sequences, termed oligonucleotide, which are designed to be complementary to a specific gene sequence.

The goal is to alter specific gene expression resulting from the binding of the antisense oligonucleotide to a unique gene sequence.

RNA INTEFERENCE

RNA interference (RNAi) is a naturally occurring mechanism that leads to the “silencing” of genes resulting the respective protein is no longer synthesized.

RNAi is used to block the expression of genes and create phenotypes that can potentially yield clues about the function of these genes.

The dsRNA is introduced into the cell. This activates the DICER gene and RISC complex which eventually leads to loss of gene expression. 

The target plant chili

India ranks first in the world with an area of 8,84,183 ha and produces 10,60,345 tones per year

The natural colour extracts of chili has great value in the food items, especially in the developed nations.

Pleasant aromatic flavour , pungency and high colouring material.

The chili fruit is a rich source of vitamins C, A and E.Capsorubin and capsanthin have diverse prophylactic

and therapeutic uses in ayurvedha and allelopathic medicines.

Carotenoid Biosynthetic PathwayIsopentyl Diphosphate

Geranyl Geranyl Diphosphate

Phytoene

Lycopene

α-Carotene β-Carotene

Phytoene Synthase

cryptoxanthinzeaxanthin

LCYB- lycopene β- cyclaseLCYE- lycopene ε- cyclaseZEP - Xanthin epoxidaseCCS – Capsanthin / capsorubin synthaseZDS – ζ-Carotene desaturase

phytofluene

neurosporene

lutein antheraxanthin

violaxanthin

capsanthin

casporubin

Phytoene Desaturase

Phytoene Desaturase

ZDS

ζ-carotene

ZDS

LCYBLCYB

LCYBLCYE

ZEP

ZEP

CCS

CCS

WORK DONE ON CHILI S.N.

GENE ARTICLE AUTHOR YEAR

1 GGPP SYNTHASE

Identification of cdna for plastid located ggpp synthase from capsicum annuum

M.kuntz,S.Romer,C.Suire,P.Hunueney,B.Camara

1992

2 PHYTOENE SYNTHASE

Light and drak regulation of carotenoid biosynthesis in pepper

Andrew J.Simkin, Changfu Jhu,Marcel Kuntz,Gerhard Santman

1998

3 CAPSANTHIN/CAPSORUBIN SYNTHASE

Orange fruit color in capsicum annuum due to capsanthin/capsorubin synthasegene

Ya qin Lang,Satishi Yanaguwa,Tsuneo SasanumanTetsuo Sasakuma

2004

ζ-carotene

neurosporene

Lycopene

ZDS

ZDS

THE TARGET GENE : ζ-CAROTENE DESATURASE

•CAROTENE 7,8 DESATURASE•CONVERT ζ-CAROTENE INTO LYCOPENE

1. Phase I :TRANSFORMATION

• Insertion of desired gene (zds) to chili plant.

2. Phase II: EXPESSION ANALYSIS• To check level of gene

expression using real time PCR .

3. Phase III: METABOLIC PROFILING• Comparison between

transgenic plant and wild plant for a range of compounds by GC- MS.

PHASE 1: TRANSFORMATION1: TRANSFORMATIONSTEPS INVOLVED1.Pre-culture2.Co-culture3.Regeneration4.Hardening in green house

A. Pre-culturePre-culture• Pre-culture is the initial stage for

plant tissue culture.• This step is needed to provide

strength to the explants .B.Co-culture• This is required for the transfer of

desired gene in the explant with help of Agrobacterium tumefaciens .

Parameters studied under co-culture

1. Time period for agrobacterium infection2. Time period for co-culture3. Hormone concentration 4. Kanamycin sensitivity test 5. Cefotaxim sensitivity test

REGENERATION In this stage development of whole plant

from explant and following parameters are considered .

1.Effect of explant To select which explant is giving the best

efficiency for transformation such as cotyledon, hypocotyl or shoot tip.

2. Effect of media To optimize best media for regeneration of

explant into whole plant.

PHASE 2: EXPRESSION ANALYSIS

To check the effect of inhibition of desired gene(zds) on the enzymes of carotenoid pathway :

1.Phytoene synthase2.Phytoene desaturase

3.Lycopene β cyclase

Methodology1.Real Time PCR2.HPLC

PLASTID TRANSITIONConversion of chloroplast to chromoplast .Comparison between different stages of

fruit of transgenic plant and fruit of wild plant.

Stages of fruit development 1.Immature green2.Mature green3.Breaker (important stage for plastid transition)4.Ripe 5.Over-ripe

Methodology Light and fluorescence microscopy.

PHASE 3: METABOLIC PROFILING

To check the level of desired metabolites in both wild and transgenic plant.

Flavonoid , Phenylpropanol ,Carotenoids and Isoprenoid Analysis.

Methodology Gas Chromatography- Mass Spectroscopy.

METHODOLOGYTransformation of chili

Hardening and green house

Confirmations Of transgene using PCR

Level of gene expression in 5 different stages of fruit Real time PCR

Study of plastid types by light and fluorescence microscopy

Metabolic profiling using GC-MS

Flavonoid ,phenylpropanol, phytosterol analysis

Fruit development parameters

Abscisic acid analysis

PHASE 1

PHASE 2

PHASE 3

HPLCXanthophyllβ-CaroteneLycopeneChlorophyll

Level of expression in early stage leaves by Real time PCR

Course work1. Genetic engineering2. plant biotechnology3. Instrumentation and biostatistics4. Research methodology

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