Kloning parsial gen penyandi P5CS dari tebu (Saccharum officinarum L.) Cloning of P5CS-encoding gene fragment from sugarcane (Saccharum officinarum L.)

Abstract

Abiotic stress such as drought stress is one of the important factors that affect plant growth. Plants have an adaptation mechanism to overcome the stress condition by accumulating osmoprotectant compounds. Proline is a well known compatible solute and can be accumulated to a high concentration in plant cells under drought or osmotic stress. One of the important enzymes in proline biosynthesis is ∆1 - pyrroline-5-carboxylate synthetase (P5CS) encoded by P5CS gene. This research is aimed to clone partial length of P5CS gene from S. officinarum, variety PSJT 941. The amplification of P5CS gene fragment was done by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), using specific primers. DNA fragment of 984 bp, 975 bp and 1725 bp were cloned into Escherichia coli XL1-Blue using pGEMT Easy plasmid vector. Results from BLAST analysis showed that the P5CS sequences have high homology (99%) with the P5CS gene of S. officinarum in the GenBank database. AbstrakCekaman abiotik seperti kekeringan merupakan salah satu faktor penting yang mempengaruhi pertumbuhan tanaman. Tanaman mempunyai strategi adaptasi dalam mengatasi cekaman tersebut dengan mengakumulasi senyawa osmoprotektan yang terakumulasi dalam konsentrasi tinggi. Prolin merupakan salah satu senyawa osmoprotektan yang dapat melindungi tanaman dari cekaman kekeringan maupun osmotik. Salah satu enzim yang berperan penting dalam biosintesis prolin adalah ∆1 -pyrroline-5- carboxylate synthetase (P5CS) yang disandi oleh gen P5CS. Penelitian ini bertujuan untuk mengklon fragmen gen P5CS dari S. officinarum varietas PSJT 941. Amplifikasi fragmen gen P5CS dilakukan dengan teknik Reverse TranscriptionPolymerase Chain Reaction (RT-PCR) menggunakan primer spesifik gen P5CS. Fragmen DNA hasil RT-PCR berukuran 984 bp, 975 bp, dan 1725 bp diklon ke dalam Escherichia coli XL 1-Blue menggunakan vektor plasmid pGEM-T Easy. Hasil analisis BLAST menunjukkan bahwa sekuen fragmen gen produk RT-PCR yang berasal dari S. officinarum PSJT 941 memiliki homologi yang sangat tinggi (99%) dengan gen P5CS pada S. officinarum yang ada dalam pusat data Genbank.

Registration of ‘L 14‐267’ sugarcane

“…The stalks were taken from the middle row to minimize the effect of environmental factors, such as direct sunlight, on stalk color. Quantitative measurements were based on an average of 10 stalks, and morphological characteristics were according to Artschwager and Brandes (1958). Color was described based on the Munsell color chart for plant tissues (Munsell Color Co., 2012).…”

“…The botanical description of L 14‐267 as per Artschwager and Brandes (1958) is summarized in Table 9. A key distinguishing characteristic of L 14‐267 is its canopy appearance.…”

Transformation of DHN1 gene and DHN promoter constructs into sugarcane calli, regeneration of the calli, and acclimatization of the plantlets

“…Sugarcane shoots of the Kidang Kencana (KK) variety were grown on solid MS1 (Murashige & Skoog) media and incubated in a dark culture room for 4-6 weeks up to callus formation. After 2-4 subculture cycles, embryogenic calli derived from the shoots were then used as an explant for Agrobacteriummediated genetic transformation of the DHN gene construct and its promoter following previously developed methods (Minarsih et al, 2012).…”