Author: N. S. Chavan*, Sachin Bhor., S. T. Waghmare
Article Summary: Climatic aberrations such as drought and flood are causing threat to food production. Development of stress tolerant varieties is essential for maximum food production. Stress tolerance is complex and multigenic phenomenon. Transcription factors (TFs) play an vital role in regulating stress response transcription factors induce the expression of stress response genes (SOS genes) which causes physiological changes in plants hence, engineering transcription factors in crops is an effective and efficient way to induce drought tolerance. Under abiotic stress conditions most of the miRNA gets differentially expressed miRNAs that positively induce the expression of transcription factors can be used as candidate genes for production of stress tolerant plants by transgenic technology.
In the present scenario yield loss due to abiotic stress conditions is major problem in agricultural crop production. Climatic aberrations such as drought and flood are causing threat to food production. Development of stress tolerant varieties is essential for maximum food production. Stress tolerance is complex and multigenic phenomenon. miRNA engineering is novel approaches to acquire tolerance against abiotic factor. Transcription factors (TFs) play an vital role in regulating stress response transcription factors induce the expression of stress response genes which causes physiological changes in plants hence, engineering transcription factors in crops is an effective and efficient way to induce drought tolerance. Along with transcription factors MicroRNAs are included in the family of gene regulators as they are also involved in regulation of gene expression at post transcriptional level. MiRNAs are small non coding RNAs containing 18-22 nucleotides. Scientific studies revealed that under abiotic stress conditions most of the miRNA gets differentially expressed and targets genes which codes for transcription factor for e.g MiR 169 which gets upregulated under stress condition and codes for CBF/DREB transcription factor). So the miRNAs that positively induce the expression of transcription factors can be used as candidate genes for production of stress tolerant plants by transgenic technology.
2. Strategies for identification miRNA in Crop Plant
2.1 . Identification of novel stress-regulated microRNAs from rice
To identify novel stress regulated miRNAs from rice, small RNA libraries with size range of 18-28 nucleotides from respective stress treatments (salt, drought, temperature, cold, etc.) along with control will be created. Based on the cDNA sequence and various Bioinformatics tools, rice stress regulated miRNAs will be predicted. Differentially expressed miRNAs will be further confirmed by northern blot hybridization.
2.2 . Gene expression analysis and characterization of stress responsive Transcription factors in Rice
Global gene transcript level analysis can be done by microarray in stress sensitive and tolerant cultivars. Data from this experiment will be used to identify transcription factors differentially expressed under stress condition. Coding sequences will be obtained from transcription factor database. In silico analysis of candidate transcription factors will be done based on the data from microarray and qRT-PCR. The transcription factors with consistent expression pattern across microarray and qRT-PCR platform will be selected for study of miRNA.
2.3 . Identification of miRNAs that targets sequences which codes for Transcription factors
Identification of entire sets of miRNAs and their targets will lay the foundation that is needed to unravel the complex miRNA mediated regulatory networks controlling development and other physiological processes such as salt stress. The targets of plant miRNAs often belong to families of transcription factors involved in the control of genes associated with a particular trait. Coding sequences from miRNAs that positively regulate the expression of TFs will be used to generate over expression vector for Agrobacterium mediated transformation in rice. Whereas, coding sequences of miRNAs that mimic the target TFs expression during stress conditions will be obtained. artificial miRNAs (amiRNA) will be designed by using Web MicroRNA Designer ( www.md3.weigelworld.org) online tool. This amiRNA will be used to generate a rtificial miRNAs (amiRNA) knockdown construct to enhance the expression of TFs.
3. Strategies for transfer and screening of miRNA in crop plant
- Development of transgenic rice Transgenic rice plants with altered TFs expression levels will be generated by genetic transformation of rice with Agrobacterium harboring miRNA over expression vector and amiRNA knockdown constructs.
- Genotypic and phenotypic analysis of transgenic rice Transgenic lines will be selected for tolerance against abiotic stress conditions and abiotic stress assays. Expression of transgene during stress condition is analysed.
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About Author / Additional Info:
I am currently working as Asssistant professor at KK Wagh college of agri-biotech. Sarswatinagar. Nashik