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Mrna Extraction, Cdna Synthesis and Tillering Specific Gene Isolation from Blb Resistant BINAshail Rice

Papia Jahan
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Ahmed Hossain
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

K. M. Nasiruddin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Sabina Yasmin
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Fahmida Khatun
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Md. Shafiullah Parvej
Department of Microbiology and Hygiene, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Bacterial Leaf Blight (BLB) caused by Xanthomonas oryzaepv. oryzae (Xoo) bacteria, is a major biotic stress in the irrigated rice belts. Genetic modification is the most effective and economical control for bacterial blight disease. For high yielding, branching capacity is most important. Molecular survey was conducted to identify the presence of tillering specific gene in BLB resistant Binashail rice. Reproducible means of Binashail rice at 21 days was designed based on the timing of full expression of the leaf. PCR with primers specific for tillering specific gene was used in the study. The cDNA of Binashail rice synthesized by an improved RT-PCR technique, derived from the total mRNAs extracted from the plant leaf. Gel documentation showed that the size of the synthesized cDNA was 250bp and tillering specific gene was 201bp in length long. The identification of tillering specific gene in Binashal rice germplasm will help in accelerating the elite breeding program in future.

Keywords: BLB; Tillering; PCR; Biotic stress

Location: Bangladesh Institute of Nuclear Agriculture and Bangladesh Agricultural University

Start Date: 00-00-0000

End Date: 00-00-0000

Program Area: Variety and Species

Commodity: Rice

Objectives:

To identify the presence of tillering specific gene in BLB resistant Binashail rice.

Methodology:

Binashail plant seeds were collected from Bangladesh Institute of Nuclear Agriculture. RM5493 primer was used to detect tillering specific genome in the sample. Molecular works was done in Microbiology lab of Bangladesh Agricultural University. Binashail seeds were germinated and seedlings in Biotechnology tissue culture lab of Bangladesh Agricultural University. Seeds of varieties Binashail was obtained from BINA. These samples were kept for germination for 21 days. RNA was extracted from fresh seedling leaves of Binashail. Two hundred grams of rice seed was rinsed with tap water and soaked for 24 hours to stimulate germination. Seeds were planted in separate seedbeds. After 21 days in the nursery, leafs were formed. RNA extracted from 100mg of plant sample using RNA was extracted using one-step RNeasy kit of QIAGEN (Germany) according to manufacturer’s instructions. First Strand cDNA Synthesis Kit 1st strand cDNA was synthesized by using Thermo Scientific DyNAmo cDNA Synthesis Kit. After thawing, mixed and centrifuged the components of the kit and stored on ice. Template RNA (2µg), Oligo (dt) primer (1 µg), Water, nuclease-free (12 µg), reagents were added into a sterilenuclease-free tube on ice in the indicated order and total volume (25µg) were used. The RNA template contained secondary structure, was mixed gently and centrifuged briefly and then it was incubated at 65°C for 5 min. It was chilled on ice, then was spin down and was placed the vial back on ice.5X Reaction Buffer (4 µl), RiboLockRNase Inhibitor (20µl) (1µl), 10 mMdNTP Mix (2µl), RevertAid H Minus M-MuLV Reverse Transcriptase (200 u/µl) (1 µl) was mixed gently and centrifuged. Oligo (dT)18 for cDNA synthesis was added and then incubated for 60 min at 42°C.The reaction was terminated by heating at 70°C for 5 min.The reverse transcription reaction product was directly used in PCR applications or stored at -20°C for less than one week. The PCR cocktail including cDNA had total volume of 25 µl. For the preparation of PCR cocktail (for each sample), at first 12.5 µl master mix were taken in a PCR tube. Then 1 µl of primer forward and 1 µl of primer reverse were added. Nuclease free water 12.5 µl was added. 2-µl template cDNA was added with PCR cocktail. The mixture was then vortexed. 2 µl of cDNA template samples were pipette into the wells of the PCR tubes for PCR amplification. Reaction was placed in the PCR tubes and run in the cDNA thermal cycler. RM5493 is a SSR marker (Forward primer 5'-CACGACGTTGTAAAACGACGCGGTAACAAACCAACCAACC-3', Reverse primer 5'-AAAGCAGGACACAGTCACACAGG-3') used for RT-PCR of tillering specific gene and annealing temperature is 58 and it shows bands at 201 bp. Loading dye (0.25% xylene cyanol, 0.25% bromophenol blue, 30% glycerol and 1 mM EDTA) and DNA marker (20bp) were used.6 µl 1xTBE buffer was placed on a piece of aluminum foil paper and 2 µl loading dye was added to it using 0.5-10 µl adjustable micropipette. Finally, 2 µl extracted DNA was added to it and mixed well using same micropipette. The samples were then added slowly to allow them to sink to the bottom of the wells. The gel was placed in the gel chamber containing 1xTBE buffer. The final level of buffer was5mm above the gel. The power supply was then connected and turned on to move the DNA from negative to positive electrode (black to red). Electrophoresis was carried out at 120v for about 11/2 hour. After the bromophenol blue dye had reached three-fourths of the gel length, the electrophoresis was stopped and the power supply was disconnected. After electrophoresis, the gel was taken out carefully from the gel chamber and the gel gently washed in running water and placed on the UV transilluminator in the 254 nm range in the dark chamber of the Image Documentation System. The image was viewed on the monitor, focused, acquired and saved in a diskette, as well as printed on paper.

Source of Information: Asian J. Med. Biol. Res. 2015, 1 (2), 265-270; ISSN 2411-4472 (Print) 2412-5571 (Online) ; www.ebupress.com/journal/ajmbr

Article Link (Online Journal):

Funding Source:

1. Government

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From the study, it is concluded Rneasy plant mini ki and RevertAid H Minis First cDNA synthesis kit that can be sensitive, simple, efficient, powerful, highly informative, mostly mono locus, co-dominant, easily analyzed and cost effective tool for screening BLB resistant gene and develop cDNA library.

Knowledge Management: Journal, Online Circulation