Deteksi Gen Ketahanan Hawar Daun Bakteri Xa21 pada Padi (Oryza sativa L.) Hitam dan Merah Lokal Indonesia

Beras berpigmen mulai popular dikonsumsi oleh masyarakat sebagai bahan pangan fungsional. Tetapi, terdapat faktor pembatas produksi beras berpigmen yaitu penyakit hawar daun bakteri yang disebabkan oleh Xanthomonas oryzae pv. Oryzae (Xoo). Penggunaan varietas tahan yang memiliki gen ketahanan Xa dinilai efektif untuk menanggulangi masalah penurunan hasil padi. Gen Xa ini antara lain terdiri dari gen Xa21. Penelitian ini bertujuan untuk mengetahui keberadaan gen ketahanan hawar daun bakteri Xa21 pada padi hitam kultivar Sembada Hitam, Cempo Ireng, Melik dan Hitam Toraja serta padi merah kultivar Aek sibondang, Merah Sumbawa, Segreng, dan Pari Eja di Indonesia. Metode penelitian meliputi isolasi genom padi, pengecekan hasil isolasi DNA dengan elektroforesis gel agarosa (0,8%), pengukuran konsentrasi dan kemurnian DNA, amplifikasi DNA primer pTA248, dan analisis data. Hasil penelitian ini mendeteksi keberadaan gen Xa21 pada semua kultivar padi hitam dan merah tersebut dengan dengan sifat tahan

Optimization of qRT-PCR Annealing Temperature of WRKY45 Gene for Detection of Resistance Genes Against Xanthomonas oryzae pv. oryzae on Black Rice Cempo Ireng

Plant pathogens constrain the development of black rice farming. One of these pathogens is Xanthomonas oryzae pv. oryzae (Xoo), causing a bacterial leaf blight disease. The disease disrupts crop growth and reducing yields. Cempo ireng is a local pigmented rice cultivar from Yogyakarta, Indonesia, which is reported for its high resistance to Xoo. One of the rice resistance mechanisms to Xoo infection is a molecular defense employing plant resistance genes such as WRKY45. Comparing the expression of resistance-related genes of the resistant cultivar to the susceptible ones is needed to elucidate the resistance mechanism of the black rice to Xoo. For this purpose, the expression of WRKY45 gene at the level of mRNA can be performed using qRT-PCR. The success of qPCR analysis is greatly influenced by the accuracy of the annealing primer temperature of the corresponding gene. This study aimed to optimize the primer's annealing temperature for WRKY45 gene. The optimization was done by a temperature gradient PCR. Determination of the optimal annealing temperature was selected based on the profile of the amplification curve, melt curve, melt temperature and the Ct value obtained. The annealing temperature gradient used in this study was ranging from 52°C to 60°C. The results showed that the best annealing temperature for WRKY45 gene primers is 58.3°C based on the amplification curve, melt curve, melt peak and Ct value of 29.21

Optimization of heat shock temperature and time on the transformation of pRGEB32 into Escherichia coli DH5α

Genome editing technique is one of the methods for studying the expression of gene, eliminating unfavorable traits or phenotypes and generating the new characters of species. The pRGEB32 plasmid is one of the vectors that used in genome editing with carrying the Cas9 gene, restriction site of sgRNA (single guide RNA) and specific promoters that can be expressed in plants. The first step in the genome editing process is inserting pRGEB32 into Escherichia coli for propagation. The large size of the plasmid molecule becomes a challenge to determine the right method in the transformation process. This study aims to determine the temperature and time of heat shock transformation of plasmid pRGEB32 into E. coli. The transformation of pRGEB32 into plasmids was carried out with variations in temperature and time, 42℃ (30 seconds and 60 seconds) and 55℃ (30 seconds and 60 seconds). The results showed that a heat shock temperature of 55℃ with a time of 60 seconds was the best temperature for the transformation of pRGEB32 into E. coli. This optimization of heat shock condition will increase the transformation efficiency, which is in the range of 3322-10.989 cfu/µg.

Identifying Single Nucleotide Polymorphisms (SNPs) in OsFER1 and OsFER2 Genes Linked to Iron accumulation in Pigmented Indonesian Rice (Oryza sativa L.)

Iron (Fe) is an essential micronutrient for the well-being of plants, animals, and bacteria. In plants, iron plays a pivotal role in a myriad of metabolic processes, encompassing redox reaction, photosynthesis, respiration, chlorophyll synthesis, and nitrogen fixation. For humans, iron is indespensable for several metabolic functions, particularly in the synthesis of haemoglobin. Iron deficiency can lead to health issues on a global scale, therefore identifying key crops, such as rice for providing sufficient iron in diet intake is very important. In rice, the maintenance of iron homeostasis is orchestrated by various genes, with OsFER1 and OsFER2 acting as iron accumulator genes in leaves, stems, flowers, and grains. The primary objective of this study was to ascertain the single nucleotide polymorphisms (SNP) in the OsFER1 and OsFER2 and to assess the iron content in Indonesian local rice cultivars. To achieve this, we examined partial sequences of OsFER1 and OsFER2 to identify SNPs in the Indonesian rice cultivars used (Cempo Ireng, Pari Ireng, Hitam Kalsel, Merah Pari Eja, and Ciherang). Concurrently, the iron content in the seeds was quantified using Atomic Absorption Spectrophotometry (AAS). The analysis revealed that the OsFER1 gene sequence, specifically exon 5, exhibited a SNP in the form of a transition. In contrast, the OsFER2 gene sequences, specifically in intron 2 displayed SNPs in the form of insertions. Notably, the iron content in the seeds was highest in Cempo Ireng (black rice), while it was lowest in Merah Pari Eja (red rice) and Ciherang (non-pigmented rice). Importantly, the identified SNPs in these partial gene sequences did not exert any discernible influence on iron levels or the formation of ferritin protein.

The relationship between morpho‐physiological changes and expression of transcription factors in NTT local rice cultivars as a response to drought stress

Response by plants to drought occurs through a series of mechanisms that involve transcription regulation. This research was conducted to study transcription factors (TF) and physiological changes in the drought response of local rice cultivars from East Nusa Tenggara (Nusa Tenggara Timur, NTT) during drought stress. Using three NTT local rice cultivars (Boawae Seratus Malam (BSM), Gogo Jak (GJ), and Kisol Manggarai (KM)) and the fraction of transpirable soil water (FTSW) method with two treatment levels, FTSW 1 (control) and FTSW 0.2 (severe stress), we analyzed the TF expression of OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6. Based on the result, the highest level of TF expression occurred in the BSM, followed by the GJ and KM cultivars. Analysis of physiological characteristics showed an association between TF expression levels and physiological response, with the BSM cultivar showing high pigment levels, high proline content, and lower H2O2 levels. A linkage was also found in relation to water conservation, as indicated by the higher relative water content and cell membrane stability index in the BSM cultivar in contrast to lower electronic leakage and malondialdehyde percentage when exposed to drought. Based on the results, it can be concluded that the BSM cultivar can be considered as a drought‐tolerant local cultivar according to morpho‐physiological analysis. In this study, all NTT local rice cultivars showed a subtle upregulation of stress‐responsive transcription factors OsDREB1A, OsDREB2A, OsWRKY45, and OsNAC6 as responses to drought stress

An NLR paralog Pit2 generated from tandem duplication of Pit1 fine-tunes Pit1 localization and function

NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication

Evaluation of Cells and Medium Optimization for Invitro Model of Diabetic and Electrolyte Imbalance

Metabolism syndrome has many negative impacts on human health. Various efforts and methods are attempted in the treatment of this disease. One of the methods used is CRISPR/Cas9 gene therapy. Re-testing of knock out cells using the CRISPR/Cas9 method is needed to evaluate its success. In conducting the test, the right medium is needed so that the results are optimal and can be evaluated properly. In this study, we optimized the medium for three types of cells (fibroblasts, myoblasts and macrophages) in high and low glucose medium to evaluate gene knockout results. The medium was modified by adding high concentrations of glucose and sodium. The results, in macrophage culture, giving variations in glucose concentration in low glucose medium gave a significantly different percentage of live cells between treatments, while the treatment with variations in glucose concentration in macrophages in high glucose medium and fibroblasts and myoblasts in high and low glucose medium did not show any difference in the percentage of living cells. In the treatment of various concentrations of natrium, macrophages, fibroblasts and myoblasts on high and low glucose medium all showed significantly different percentages of living cells. Therefore, DMEM low glucose medium is suitable as a medium for the treatment of high glucose and natrium induction in macrophage cells, but is not suitable for fibroblast and myoblast cells

Seed Halopriming: A Promising Strategy to Induce Salt Tolerance in Indonesian Pigmented Rice

Unfavorable environmental conditions and climate change impose stress on plants, causing yield losses worldwide. The Indonesian pigmented rice (Oryza sativa L.) cultivars Cempo Ireng Pendek (black rice) and Merah Kalimantan Selatan (red rice) are becoming popular functional foods due to their high anthocyanin contents and have great potential for widespread cultivation. However, their ability to grow on marginal, high-salinity lands is limited. In this study, we investigated whether seed halopriming enhances salt tolerance in the two pigmented rice cultivars. The non-pigmented cultivars IR64, a salt-stress-sensitive cultivar, and INPARI 35, a salt tolerant, were used as control. We pre-treated seeds with a halopriming solution before germination and then exposed the plants to a salt stress of 150 mM NaCl at 21 days after germination using a hydroponic system in a greenhouse. Halopriming was able to mitigate the negative effects of salinity on plant growth, including suppressing reactive oxygen species accumulation, increasing the membrane stability index (up to two-fold), and maintaining photosynthetic pigment contents. Halopriming had different effects on the accumulation of proline, in different rice varieties: the proline content increased in IR64 and Cempo Ireng Pendek but decreased in INPARI 35 and Merah Kalimantan Selatan. Halopriming also had disparate effects in the expression of stress-related genes: OsMYB91 expression was positively correlated with salt treatment, whereas OsWRKY42 and OsWRKY70 expression was negatively correlated with this treatment. These findings highlighted the potential benefits of halopriming in salt-affected agro-ecosystems. © 2023 by the authors