MSC

Bitter gourd (Momordica charantia L.; 2n=2x=22) is an important vegetable crop. India produces roughly 1.2 million metric tonnes of bitter gourd from an area of 99,000 hectares. The bitter gourd's immature fruits are often eaten as vegetables, which is known for its anti-diabetic and other therapeutic properties. It is rich in Vitamin A, C, iron and phosphorous. Its enormous therapeutic benefits are attributed to the presence of phytochemicals, which are known to have immune-boosting, antiviral, anti-diabetic, antibiotic, antimutagenic, anthelminthic and cancer-preventive characteristics. Gynoecious lines have revolutionized the hybridization programmes in cucurbits. Though many gynoecious lines are reported in major cucurbits such as cucumber, gynoecious bitter gourd lines are reported from KAU, IIVR, IIHR and IARI only.
The AGAMOUS (AG) gene, leading C class gene in the ABC model of flowering, acts by activating the transcription of SPOROCYTELESS (SPL/NZZ) gene and hence SPL is considered very important for the megaspore development in flowers. For the silencing/ over expression of this gene, aimed at altering the sex form of bitter gourd, the gene must be sequence characterized. Its expression pattern in the male and female flowers can further reveal the roles of this gene in sex expression in this crop. In this background, the present study “Characterization and expression analysis of SPOROCYTELESS (SPL) gene in monoecious and gynoecious bitter gourd lines” was under taken during the period from 2020-2023 at the Department of Plant Biotechnology, College of Agriculture, Thrissur, with the objective of sequencing and analysis of SPL gene amplified from the cDNA of monoecious and gynoecious bitter gourd lines and expression analysis of the gene in the developing buds of the male and female flowers of monoecious plants and female flowers of gynoecious plants.
Monoecious bitter gourd cultivar Priyanka and gynoecious line KAU-Mc-Gy-102 were raised in pots. Initially, male and female flower buds, at differentiating phase, from the monoecious cultivar and female flowers from the gynoecious line were collected. The collected flower buds were quick frozen in liquid nitrogen, brought to laboratory, stored in -20°C for a short while and used for RNA isolation. First strand of cDNA was synthesised from the RNA isolated from each flower bud group. However, the real time PCR primers designed using the mRNA sequences of the gene has failed to amplify the gene from the cDNA. Since intronic region in the gene was found lesser, it was decided to sequence characterize the gene using the genomic DNA. DNA isolated from the leaves using CTAB method was used for PCR amplification of the SPL gene from monoecious cultivar and gynoecious line, using three primers pairs. The PCR amplicons were sequenced and contigs covering the complete length of the gene in both lines were generated. The final contigs in monoecious and gynoecious lines were 2381 2391 bp long, respectively. The sequence alignment had shown no variation between the monoecious and gynoecious lines. The BLASTn showed 100% identity with 79% query coverage, with the predicted sequence of M. charantia transcript variant (LOC111010534) (Acc. no. XM02283995.1). The BLASTx showed 99.09% identity with the sequence of M. charantia transcript variant (LOC111010534) (Acc. no. XP022139687.1). Amino acid sequence of the gene was deduced from the nucleotide sequence using ExPASy translate tool. BLASTp showed 100% identity with protein sequence of M. charantia (Acc. no. XP022139688.1) in both lines. Analysis using ORF Finder yielded 19 ORFs, with ORF4 being the longest with 768 bp coding for 255 amino acids.
For the expression analysis, RNA was isolated from the developing buds of male and female flowers of monoecious line and female buds of gynoecious line, using TRI reagent. The RNA was converted to cDNA using Oligo(dT)18 primer and reverse transcriptase enzyme. Primer pairs for SPL and Actin gene (endogenous control) were designed and real time PCR reaction based on SYBR Green chemistry was carried out. Expression of SPL gene in the developing buds of male and female flowers of monoecious cultivar and female buds of gynoecious line was analyzed. The relative expression levels of the gene was normalised with the expression of endogenous control Actin gene, following the 2-∆∆Ct method. The relative expression of the SPL gene was analysed by taking the male flower as control. Compared to male flowers, gynoecious flowers had higher fold change (276.5) followed by monoecious female flowers (21.69). The present study has sequence characterized the SPL/NZZ gene in M. charantia. The amino acid sequences were deduced and annotated. Expression analysis had shown that SPL gene is upregulated in the gynoecious flowers. Similarly, expression of this gene was higher in the female flowers in the monoecious cultivar compared to that in male flowers, proving its role in the development of megaspores in the bitter gourd flowers. Based the results, it is predicted that the monoecious lines overexpressed with SPL gene may turn gynoecious or produce more female flowers, leading to higher fruit yield.