Expression, isolation and identification of submergence induced Genes from FR13A through RT-PCR

doi:10.15406/mojbm.2017.01.00036

MOJ

eISSN: 2574-9722

Biology and Medicine

Short Communication Volume 1 Issue 6

Expression, isolation and identification of submergence induced Genes from FR13A through RT-PCR

Ray BP,¹

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Uddin MI,² Razia S,³ Hosne Ara Begum⁴

¹Department of Biotechnology and Senior Scientific Officer, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh
²Department of Biotechnology and Chief Scientific Officer, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh
³Department of Biotechnology and Scientific Officer, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh
⁴Chief Scientific Officer and Director (Research), Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh

Correspondence: Ray BP, Department of Biotechnology and Senior Scientific Officer, Bangladesh Institute of Nuclear Agriculture (BINA), Bangladesh

Received: May 16, 2017 | Published: September 25, 2017

Citation: Ray BP , Uddin MI, Razia S, et al. Expression, isolation and identification of submergence induced Genes from FR13A through RT-PCR. MOJ Biol Med. 2017;1(6):164–165. DOI: 10.15406/mojbm.2017.01.00036

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Abstract

The present study was undertaken to identify submergence-induced genes,OsARP (Antiporter regulating protein) from the submergence tolerant rice (Oryza sativa L.) cultivar, Indica (FR13A). We analyzed submergence-tolerant genetic material FR13A to improve flooding tolerance in rice and, specifically, complete submergence tolerance. Healthy leaf samples collected from plant material, FR13A through complete submergence of 0, 1, 3, 5 & 7days, the gene was expressed in the shoot. We isolated mRNA through Trizol kit and mRNA converted to cDNA through Reverse Transcriptase (RT). The OsARPgene was first expressed into E. coli and antibody was produced by using purified recombinant protein. The expression of OsARP protein was detected under submergence stresses. We examined the expression and subcellular localization of OsARP in a submergence tolerant rice cultivar FR13A. Gradient PCR of cDNA with these three genes (OsARP, OsGGT, OsMGD ) were amplified from 3, 7days submerged by annealing temperature (0C) of 62, 64 and 66. This PCR containing cDNA of OsARPgene was present clear amplification by Tm of 640C than otherOsGGT, OsMGD genes. A time-course study showed that OsARP-gene expression increased in FR13A during submergence of 7 days. This indicates that OsARPis a membrane bound protein of rice, which is expressed under submergence stresses.

Keywords: cDNA, submergenc, OsARP, mRNA, indica, environments

Introduction

Rice (Oryza sativa L.) is the only cereal that can be cultivated in the frequently flooded river deltas of South-East and South Asia. Flash-flooding adversely affect at least 16% of the rice lands of the world (~22mha) and 1 billion USD annual loss in South & South-east Asia. Submergence can result in yield losses of up to 100% depending on different climatic & agronomic factors.¹ More than 2.0 million-hectare areas are affected by different grades of flash floods and reduces 5% average yield in Bangladesh.² In this circumstance, improvement of germplasm is likely the best option to withstand submergence and stabilize productivity in these environments. High frequency regeneration of plants from in vitro cultured tissues and cells are a pre-requisite for successful application of tissue culture and genetic engineering technologies for crop improvement.³ The present study was undertaken to identify submergence-induced genes from the submergence-tolerant rice cultivar, indica (FR13A) for crop improvement through genetic engineering technologies. Rice has adapted to flood-prone environments, i.e. where excessive flooding limits growth and yields, by either submergence tolerance or elongation ability.⁴ The functional roles of this OsChaC (OsARP) protein lie in the tolerance of submergence by regulating membrane bound transporter/antiporter in higher plants. In addition, it was confirmed that three isoforms like this OsARP gene are present also in Arabidopsis genome. Therefore, expression analysis of ChaC/OsARP gene will be conducted in different rice cultivars and subsequently transgenic crops will be developed through Agrobacterium mediated transformation.

Materials and methods

Healthy leaf samples collected from plant material, FR13A through complete submergence of 0, 1, 3, 5 & 7 days, the gene was expressed in the leaves through stress. RNA was extracted from the leaves of FR13A using the Trizol Reagent at Biotechnology Lab., Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Primer mix incubated for 5min at 65ºC and then placed on ice for 1min. cDNA synthesis mix was added to primer mix. The mixture was spinned down at 3000 rpm for 1minute by centrifuge, then gently mixed and incubated for 50 min at 50ºC. The reaction was terminated for 5min at 85ºC and placed on ice for 1 min. The mixture was spinned down at 3000 rpm for 1 minute by centrifuge and then 1µl RNase free H₂O was added. The tube was incubated for 20min at 37ºC. The cDNA samples were stored at-20°C. Finally, mRNA converted to cDNA through Reverse Transcriptase (RT) of 3 & 7days submerged samples. cDNA samples were evaluated both quantitatively and qualitatively using spectrophotometer and agarose gel electrophoresis, respectively.

Results and discussion

We analyzed submergence-tolerant genetic material FR13A to improve flooding tolerance in rice and, specifically, tolerance of complete submergence. The rice (O. sativa L. subsp. indica) cation transporter gene OsCTP (accession No. AB112061) was reported earlier; it encodes a protein of 137 amino acids having an atomic mass of 15kDa⁵ under salt, drought and submergence stresses. Healthy leaf samples collected from plant material, FR13A through complete submergence of 0,1,2,3,4,5,6 & 7 days, the gene was expressed in the shoot. We isolated mRNA through Trizol kit and cDNA was then synthesized from this mRNA through Reverse Transcriptase (RT). We examined the expression and sub cellular localization of OsARP in a submergence tolerant rice cultivar FR13A. The target genes, OsARP, OsGGT, OsMGD we examined the expression using cDNA through RT-PCR during submergence of 3, & 7 days. Gradient PCR of cDNA with these three genes were cloned from 3, & 7days submerged by annealing temperature (°C) of 58, 60, 64, 66, 68, and 70. This PCR containing cDNA of OsARP gene was present clear amplification by Tm of 64°C than another two OsGGT, OsMGD genes. A time-course study showed that OsARP-gene expression increased in FR13A during submergence of 7 days. This indicates that OsARP is a membrane bound protein of rice, which is expressed under submergence stresses. The study of abiotic stress response has advanced considerably in recent years. Analyzing of a single stress in plants can be very different from the conditions encountered by plants in the field where several stresses may be occurred simultaneously.⁷ This can alter plant metabolism in a novel manner that may be different from that caused by each of the different stresses applied individually, and may require a new type of response that would not have been induced by each of the individual stresses.⁸ Therefore, some genes that can be expressed by specific stress might be expressed in other stress as well. The expression of putative vacuolar antiporter regulator OsARP was wide spread in all the plant parts under salt stress. The salt stress increased the transcripts levels of OsNHX1 in roots and shoots indicating that ionic stress played major role for the expression of this gene in different plant parts.⁸ Therefore, expression analysis of OsARP gene will be conducted in different rice cultivars and subsequently transgenic crops will be developed through Agrobacterium mediated transformation.