Cloning and Characterization of Full Length Candidate genes for Specific Physiological Traits

Presented by Shruthi H.B.

ID. No-TAD/15-17

What is a Candidate gene???• Candidate genes are sequenced genes of known

biological action involved in the development or physiology of a trait.

• Physiologists consider CG’s as “All genes involved in the expression of the physiological trait.

• Geneticist consider CG’s as “Polymorphic genes putatively involved in trait variation”.

• For Plant Geneticist CG’s is “any genes putatively involved in trait variation based on its biological function or map localization”.

How to develop a candidate gene?????

Application of Candidate gene

What is cloning of genes????

• gene cloning (DNA cloning) The production of exact copies (clones) of a particular gene or DNA sequence using genetic engineering techniques.

How is the gene cloned??• The DNA containing the target gene(s) is split into fragments using 

restriction enzymes. • These fragments are then inserted into cloning vectors, such as bacterial

plasmids or bacteriophages, which transfer the recombinant DNA to suitable host cells, such as the bacterium E. coli.

• Alternatively, complementary DNA is inserted into the vectors, or ‘naked’ DNA fragments can be taken up directly by a host bacterium from its medium (this is less efficient than vector transfer). 

• Inside the host cell the recombinant DNA undergoes replication; thus, a bacterial host will give rise to a colony of cells containing the cloned target gene. Various screening methods may be used to identify such colonies, enabling them to be selected and cultured

What is Characterization of genes?

• Characterization is the description of plant germplasm. It determines the expression of highly heritable characters ranging from morphological or agronomical features to seed proteins or molecular markers.

How is Characterization done???• New methods have made molecular analysis and genotyping

useful techniques for studying diversity. • Samples of leaf are usually collected from replicated trials. • A variety of molecular techniques are used, including cytological

markers, biochemical markers and molecular genetic markers such as SSR, EST-SSR, AFLP, RAPD.

• Their choice depends on the stage of research into molecular methodologies for the crop, facilities and expertise available in each gene bank.

• Descriptors are available to describe a genetic marker technology and collect information about genetic markers in crops that are standardized and replicable.

Case study 1- Cassava

Objective• Cassava is an important root crop to resource-poor farmers in

marginal areas, where its production faces drought stress constraints.

• This study was carried out to identify candidate drought-tolerance genes and expression-based markers of drought stress in cassava.

• One drought-tolerant (improved variety) and one drought-susceptible (farmer-preferred) cassava landrace were grown in the glasshouse under well-watered and water-stressed conditions.

Contd………….• Their morphological, physiological and molecular

responses to drought were characterized.• Ten genes that have previously been biologically validated

as conferring or being associated with drought tolerance in other plant species were confirmed as being drought responsive in cassava.

• Four genes (MeALDH, MeZFP, MeMSD and MeRD28) were identified as candidate cassava drought-tolerance genes, as they were exclusively up-regulated in the drought-tolerant genotype to comparable levels known to confer drought tolerance in other species.

Conclusion • Genes underlying MH96/0686 tolerance, which is no doubt multi-

genic, can be divided into two categories:• those whose baseline expression levels are different in DT compared

with DS (i.e. DT is primed to be less susceptible or responds more quickly to drought stress) and

• those whose expression levels under drought are significantly more changed/expressed at higher levels in DT compared with DS (i.e. they may play a role in the longer-term tolerance of MH96/0686 observed in the field).

• The more responsive genes (the second category) represent candidates for the adaptive response of MH96/ 0686 to drought and therefore important targets for further validation and for subsequent use in developing cassava varieties with enhanced drought tolerance.

Case study 2- Bajra

Objective• In the present study, they have isolated a dehydrin gene

from P. glaucum, referred to as PgDHN. The expression of PgDHN is upregulated in response to drought, salinity, heat stress, and cold stress.

• Its role in rendering tolerance to salinity and heat stress is further indicated by its expression analysis in Escherichi coli

• These results indicate that PgDHN plays a protective role under various abiotic stress conditions and could be used as a tool to improve the abiotic stress tolerance of crop plants along with other stress responsive genes.

Conclusion

• The sequence analysis of the full length protein revealed the presence of all the conserved motifs characteristic of dehydrins.

• Transcript profiles in leaves under different abiotic stress conditions showed that PgDHN is upregulated particularly in salinity stress.

• E. coli cells expressing recombinant PgDHN exhibited higher growth rates as compared to the control cells (without PgDHN) when exposed to heat and salinity stress.

• These findings suggest that PgDHN plays a crucial role in stress adaptation and provides resistance against different abiotic stresses in P. glaucum.

• Therefore, PgDHN can be used to develop transgenic crop plants to combat multiple abiotic stress conditions.

Case study 3- Turmeric

Objective• Phytocystatin, a type of protease inhibitor (PI), plays major roles

in plant defense mechanisms and has been reported to show antipathogenic properties and plant stress tolerance.

• Recombinant plant PIs are gaining popularity as potential candidates in engineering of crop protection and in synthesizing medicine.

• It is therefore crucial to identify PI from novel sources like Curcuma longa as it is more effective in combating against pathogens due to its novelty.

• In this study, a novel cDNA fragment encoding phytocystatin was isolated using degenerate PCR primers, designed from consensus regions of phytocystatin from other plant species.

Isolation of Candidate gene responsible for pest and disease resistance

Conclusion

• Cloning and characterization of candidate gene is one of the powerful tool to combat both abiotic as well as biotic stress in a specified manner.

• It also employs identification of novel CG for having stable food and nutrition security in the upcoming years if encouraged in proper way.