scholarly journals Transformation of Amorphadiene Synthase and Antisilencing P19 Genes into Artemisia annua L. and its Effect on Antimalarial Artemisinin Production

2020 ◽  
Vol 10 (3) ◽  
pp. 464-471
Author(s):  
Elfahmi Elfahmi ◽  
Fany Mutia Cahyani ◽  
Tati Kristianti ◽  
Sony Suhandono
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Artemisia Annua ◽  
Pcr Analysis ◽  
Sequencing Analysis ◽  
Pcr Products ◽  
Transformed Hairy Root ◽  
Polymerase Chain ◽  
Infiltration Method

Purpose : The low content of artemisinin related to the biosynthetic pathway is influenced by the role of certain enzymes in the formation of artemisinin. The regulation of genes involved in artemisinin biosynthesis through genetic engineering is a choice to enhance the content. This research aims to transform ads and p19 gene as an antisilencing into Artemisia annua and to see their effects on artemisinin production. Methods: The presence of p19 and ads genes was confirmed through polymerase chain reaction (PCR) products and sequencing analysis. The plasmids, which contain ads and/or p19 genes, were transformed into Agrobacterium tumefaciens, and then inserted into leaves and hairy roots of A. annua by vacuum and syringe infiltration methods. The successful transformation was checked through the GUS histochemical test and the PCR analysis. Artemisinin levels were measured using HPLC. Results: The percentages of the blue area on leaves by using vacuum and syringe infiltration method and on hairy roots were up to 98, 92.55%, and 99.00% respectively. The ads-p19 sample contained a higher level of artemisinin (0.18%) compared to other samples. Transformed hairy root with co-transformation of ads-p19 contained 0.095% artemisinin, where no artemisinin was found in the control hairy root. The transformation of ads and p19 genes into A. annua plant has been successfully done and could enhance the artemisinin content on the transformed leaves with ads-p19 up to 2.57 folds compared to the untransformed leaves, while for p19, cotransformed and ads were up to 2.25, 1.29, and 1.14 folds respectively. Conclusion: Antisilencing p19 gene could enhance the transformation efficiency of ads and artemisinin level in A. annua.

scholarly journals Biological activity and hairy roots induction of Hibiscus sabdariffa L.

2019 ◽  
Vol 2 (6) ◽  
pp. 66-74
Author(s):  
Vu Thi Bach Phuong ◽  
Cao Minh Dai ◽  
Pham Thi Anh Hong ◽  
Quach Ngo Diem Phuong
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Reducing Power ◽  
Root Production ◽  
Pcr Analysis ◽  
Root Extract ◽  
Root Induction ◽  
Hibiscus Sabdariffa ◽  
Transformed Hairy Root ◽  
Pharmaceutical Production

Hibiscus sabdariffa L. has been used traditionally in many countries of the world as food, especially as a flavouring agent in food industry. H. Sabdariffa is used for treating heart, nerve, liver disease, high blood pressure, arteriosclerosis, sore throat, cough, hypoglycaemia, laxative, diuretic, kidney stone, scurvy... The aims of this study are evaluation of bioactivities of H. Sabdariffa and the production of transformed hairy root of H. Sabdariffa for pharmaceutical production. In this study, Yen and Duh method showed the reducing power of ethanol the root extract and leaf extract are higher than that of stem. The root extract showed the α-glucosidase inhibitory activity with IC50 at 0.2 mg/mL is higher than those of stem and leaf. These results shows that root has higher bioactivites than stem and leaf. In this study, hairy roots of H. Sabdariffa were successfully induced via Agrobacterium rhizogenes ATCC 15834 in the plant cells. The frequency of hairy root and number of hairy root induction from the wounded sites of leaves are the highest (100% and 12.89 roots). The stable introduction of rolB and rolC genes of A. rhizogenes ATCC 15834 into H. Sabdariffa plants was confirmed by PCR analysis. Besides, the absence of virG gene confirmed hairy roots as bacteria-free. Subsequently, these results demonstrated that H. Sabdariffa, particularly the roots, has great potential as pharmacological values and hairy root production can be used as pharmaceutical sources.  

scholarly journals Neurocysticercosis Diagnosed by Taenia solium PCR on Brain Biopsy

2020 ◽  
Vol 2020 ◽  
pp. 1-4
Author(s):  
Sean Wei Xiang Ong ◽  
Jean-Marc Chavatte ◽  
Jonathan Wei Zhong Chia ◽  
Ramez Wadie Kirollos ◽  
Yih Yian Sitoh ◽  
...  
Keyword(s):  
Taenia Solium ◽  
Granulomatous Inflammation ◽  
Brain Biopsy ◽  
Onset Seizure ◽  
Diagnostic Role ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Temporal Lobe Lesion ◽  
New Onset

Neurocysticercosis is a common cause for brain lesions and adult-onset epilepsy in endemic countries. However, diagnosis is challenging in the absence of typical radiologic or histopathologic features. In this case report, we present a case of a 35-year-old male with a new-onset seizure and a rim-enhancing temporal lobe lesion. Radiologic features were nonspecific, and brain biopsy was performed. Histologic features showed only nonspecific granulomatous inflammation, and the diagnosis of neurocysticercosis was confirmed only with polymerase chain reaction (PCR) testing on brain biopsy tissue demonstrating PCR products consistent with Taenia solium. This case highlights the diagnostic role of PCR in such clinical situations whereby the diagnosis is unclear after initial routine evaluation.

RT-PCR analysis of Na+/H+ exchanger mRNAs in rat medullary thick ascending limb

1995 ◽  
Vol 268 (6) ◽  
pp. F1224-F1228 ◽  
Author(s):  
P. Borensztein ◽  
M. Froissart ◽  
K. Laghmani ◽  
M. Bichara ◽  
M. Paillard
Keyword(s):  
Rat Kidney ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Pcr Analysis ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Medullary Thick Ascending Limb ◽  
Pcr Products ◽  
Polymerase Chain ◽  
Nhe Isoforms

The thick ascending limb (TAL) of rat kidney absorbs bicarbonate secondary to proton secretion, but displays both basolateral and luminal Na+/H+ exchange (NHE) activity. Several NHE genes, including NHE-1, NHE-2, NHE-3, and NHE-4, are expressed in the kidney. To identify the NHE isoforms expressed in the rat medullary TAL (MTAL), we used the reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNAs for NHE in microdissected MTAL. RT-PCR amplification from total RNA was performed between two specific primers for each NHE isoform. In rat kidney homogenate, the four NHE isoform mRNAs were detected, and the identity of the PCR products was demonstrated by the sizes of the fragments, digestion with restriction enzymes, and Southern blot analysis. In microdissected rat MTAL, NHE-3 was strongly expressed and NHE-1 mRNA was also detected, whereas NHE-2 and NHE-4 mRNAs were not detected. Therefore, NHE-3 could be the apical Na+/H+ exchanger, and NHE-1 could be the basolateral isoform in the MTAL.

scholarly journals Changes of antioxidative enzymes in Impatiens walleriana L. shoots in response to genetic transformation

Genetika ◽  
2015 ◽  
Vol 47 (1) ◽  
pp. 71-84
Author(s):  
Snezana Milosevic ◽  
Milena Lojic ◽  
Dragana Antonic ◽  
Aleksandar Cingel ◽  
Angelina Subotic
Keyword(s):  
Genetic Transformation ◽  
Biomass Production ◽  
Hairy Root ◽  
Antioxidative Enzymes ◽  
Hairy Roots ◽  
Plant Viruses ◽  
Pcr Analysis ◽  
Root Induction ◽  
Dependent Manner ◽  
Impatiens Walleriana

Impatiens walleriana L. shoots were inoculated with Agrobacterium rhizogenes A4M70GUS and the effects of genetic transformation on the catalase (CAT), superoxide dismutase (SOD) and peroxidase (POX) activities in wounded region of stems and unwounded leaves were evaluated 10, 24, 240 and 720 hours after inoculation. Following Agrobacterum infection activities of plant antioxidative enzymes changed in a time-dependent manner indicating that dynamic processes occurred during plant-Agrobacterium interaction, plant cell transformation and formation of hairy roots. Appearance of hairy roots on wound sites of shoots was observed ten days after inoculation with A. rhizogenes and the root induction frequency was 100%. Among selected hairy root lines significant differences in growth rate and biomass production were observed and an average 3-fold increase in biomass production was observed for the best growing hairy root line compared with the untransformed roots. PCR analysis showed presence of uidA, rolB, rolC and rolD genes in all analyzed I. walleriana L. hairy root lines, while amplification fragment of rolA gene was detected in 83.3% transformed lines. Efficient transformation protocol for I. walleriana L described in this work offer possibilities to generate hairy root cultures for in vitro propagation of plant viruses.

Effects of testicular interstitial fluid on the proliferation of the mouse spermatogonial stem cells in vitro

Zygote ◽  
2013 ◽  
Vol 22 (3) ◽  
pp. 395-403 ◽  
Author(s):  
Peng Wang ◽  
Yi Zheng ◽  
Ying Li ◽  
Hua Shang ◽  
Guang-Xuan Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Interstitial Fluid ◽  
Culture Medium ◽  
Physiological Role ◽  
Spermatogonial Stem Cells ◽  
Proliferation Rate ◽  
Pcr Analysis ◽  
Polymerase Chain

SummarySpermatogenesis is a process in adult male mammals supported by spermatogonial stem cells (SSCs). The cultivation of SSCs has potential value, for example for the treatment of male infertility or spermatogonial transplantation. Testicular interstitial fluid was added to culture medium to a final concentration of 5, 10, 20, 30 or 40%, in order to investigate its effects on proliferation of mouse SSCs in vitro, Alkaline phosphatase (AKP) assay, reverse transcription polymerase chain reaction (RT-PCR) analysis and indirect immunofluorescence of cells were performed to identify SSCs, and the proliferation rate and diameters of the SSCs colonies were measured. The results showed that the optimal addition of testicular interstitial fluid to culture medium was 30%. When medium supplemented with 30% testicular interstitial fluid was used to culture mouse SSCs, the optimum proliferation rate and diameter of the cell colonies were 72.53% and 249 μm, respectively, after 8 days in culture, values that were significant higher than those found for other groups (P < 0.05). In conclusion, proliferation of mouse SSCs could be promoted significantly by supplementation of the culture medium with 30% testicular interstitial fluid. More research is needed to evaluate and understand the precise physiological role of testicular interstitial fluid during cultivation of SSCs.

scholarly journals Kultur akar rambut Cinchona ledgeriana dan C. succirubra dalam kultur in vitro Hairy root culture of Cinchona ledgeriana and C. succirubra by in vitro culture

2016 ◽  
Vol 72 (2) ◽  
Author(s):  
Nurita TORUAN-MATHIUS ◽  
. REFLINI ◽  
. NURHAIMI-HARIS ◽  
. JOKO-SANTOSO ◽  
A PRIANGANI-ROSWIEM
Keyword(s):  
Hairy Root ◽  
Hairy Roots ◽  
Basal Medium ◽  
Root Culture ◽  
Hairy Root Culture ◽  
Chain Reaction ◽  
Ri Plasmid ◽  
Polymerase Chain ◽  
Cinchona Ledgeriana

Summary Problems encountered in hairy root culture  of  C. ledgeriana and C. succirubra are low percentage of transformation of explants by Agrobacterium rhizogenes and slow growth of hairy root. The objective of this research was to evaluate the potential of several A. rhizogenes strains for initiation  hairy roots of  C. succirubra and C. ledgeriana, and to obtain the best medium for hairy root culture of Cinchona spesies. Axenic shoot and leaves explants of eight-month-old of C. ledgeriana and  C. succirubra seedlings were inoculated with A. rhizogenes strain ATCC-15834, ATCC-8196,    R-20001, 07-20001, A4, R-MAFFA, TISTR509, TISTR510 and LBA9457. Inoculated explants were cultured in solid MS medium with the addition of 100 mg/L amphicylin. Subculture of the hairy root was performed by transferred of root pieces into fresh liquid basal medium MS, B5, White and Heller. Hairy roots from the best of basal medium were subcultured on the same medium with the addition of 50  and 100 mg/L   L-tryptophane, three or five times concentration of MS vitamins. The integration of T-DNA of   A. rhizogenes in hairy root was confirmed with specific primer for TL and TR-DNA of plasmid by Polymerase Chain Reaction analysis. The results showed that only A. rhizogenes strain  LBA 9457 were effective for  transformation of explants from both Cinchona species. The fastest hairy roots growth were found  in MS medium, while growth in others medium was poor. Hairy roots of  C. ledgeriana has vigor and growth better than hairy roots of C. succirubra. MS with the addition of 50 mg/L  L-tryptophane and  three times the concen-trations of vitamin  is the best medium for hairy root growth and vigor. Hairy roots of  C. succirubra and C. ledgeriana used in this studies were confirmed that hairy roots  contained TL and TR-DNA region of Ri plasmid with molecular weight 780 and 1600 bp.  The results showed that strain of A. rhizogenes, plant species, source of explant and composition of medium affect the initiation, growth, development  and vigor of hairy roots.Ringkasan Masalah dalam kultur akar rambut  C. ledgeriana dan C. succirubra adalah rendahnya tingkat keberhasilan transformasi eksplan dengan Agrobacterium rhizogenesdan pertumbuhannya yang lambat. Penelitian ini bertujuan untuk mengevaluasi  potensi dari beberapa galur A. Rhizogenes untuk inisiasi, mendapatkan komposisi medium terbaik untuk pertumbuhan akar rambut C. ledgeriana dan C. succirubra, serta konfirmasi terintegrasinya TR dan TL-DNA Ri plasmid ke dalam jaringan eksplan.  Eksplan batang  dan  daun  berasal  dari kecambah aksenik C. ledgeriana dan C. succirubra berumur delapan bulan diinokulasi dengan A. rhizogenes galur 15834, 8196, R-20001, 07-20001, A4, R.MAFFA,TISTR 509, TISTR 510 dan LBA 9457. Eksplan yang sudah diinokulasi dikulturkan dalam medium MS padat. Subkultur dilakukan dengan cara mentransfer potongan ujung akar rambut ke dalam medium cair MS, B5, White dan Heller. Akar rambut dari medium kultur yang terbaik kemudian disubkultur ke dalam medium yang sama dengan penambahan 50 dan 100 mg/L L-triptofan dengan konsentrasi vitamin sebanyak tiga kali dan lima kali dari konsentrasi normal MS. Integrasi T-DNA dalam akar rambut dikonfirmasi meng-gunakan Polymerase Chain Reaction  dengan primer spesifik untuk TL dan TR-DNA plasmid. Hasil yang diperoleh menunjukkan bahwa hanya A.rhizogenes galur LB9457 yang efektif menginfeksi eksplan baik batang maupun daun dari kedua spesies kina. Induksi, pertumbuhan dan vigor akar rambut yang terbaik diperoleh dari medium MS dengan penambahan 50 mg/L L-triptofan dan tiga kali konsentrasi vitamin. Hasil konfirmasi akar rambut baik dari batang maupun daun menggunakan PCR, menunjukkan bahwa TL dan TR-DNA dari Ri plasmid  A. rhizogenes mampu menghasilkan pita-pita DNA dengan BM780 dan 1600 pb. Hasil yang diperoleh menunjukkan bahwa galur  A. rhizogenes, spesies tanaman, sumber eksplan dan komposisi medium berpengaruh terhadap inisiasi, pertumbuhan,  perkembangan dan vigor akar rambut.

scholarly journals Efficient genetic transformation of Impatiens hawkerii Bull. (Balsamiaceae) using agrobacterium rhizogenes

2009 ◽  
Vol 61 (3) ◽  
pp. 467-474 ◽  
Author(s):  
Snezana Milosevic ◽  
Angelina Subotic ◽  
A. Cingel ◽  
Sladjana Jevremovic ◽  
Slavica Ninkovic
Keyword(s):  
Agrobacterium Rhizogenes ◽  
Hairy Root ◽  
Hairy Roots ◽  
Lateral Roots ◽  
Basal Medium ◽  
Growth Patterns ◽  
Pcr Analysis ◽  
Gus Assay ◽  
Hairy Root Clones ◽  
First Time

Transformation of Impatiens hawkerii Bull. mediated by Agrobacterium rhizogenes strain A4M70GUS was studied. Hairy roots developed 10 days after inoculation were excised from the shoot explants and transferred onto Murashige and Skoog's (MS) basal medium lacking plant growth regulators. More than 20 hairy root clones were established and eight of them were further analyzed. Each clone differed significantly from the others in growth capacity and lateral branching. Clone C2 showed the highest biomass (20.6 g L-1) as well as the highest number of lateral roots (37 ? 2.2). The transgenic nature of the established hairy root clones was confirmed by GUS assay and PCR analysis. In conclusion, hairy roots were developed for the first time in I. hawkerii Bull., and transgenic hairy root clones showed a distinct morphological nature and growth patterns.

scholarly journals Detection and characterization of clustered regularly interspaced short palindromic repeat-associated endoribonuclease gene variants in Vibrio parahaemolyticus isolated from seafoods and environment

2019 ◽  
Vol 12 (5) ◽  
pp. 689-695
Author(s):  
Pallavi Baliga ◽  
Malathi Shekar ◽  
Moleyur Nagarajappa Venugopal
Keyword(s):  
Polymerase Chain Reaction ◽  
Vibrio Parahaemolyticus ◽  
Genotypic Variation ◽  
Gene Variants ◽  
Chain Reaction ◽  
Pcr Products ◽  
Short Palindromic Repeat ◽  
Polymerase Chain

Aim: In Vibrio parahaemolyticus, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated cas6 endoribonuclease gene has been shown to exhibit sequence diversity and has been subtyped into four major types based on its length and composition. In this study, we aimed to detect and characterize the cas6 gene variants prevalent among V. parahaemolyticus strains isolated from seafoods and environment. Materials and Methods: Novel primers were designed for each of the cas6 subtypes to validate their identification in V. parahaemolyticus by polymerase chain reaction (PCR). In total, 38 V. parahaemolyticus strains isolated from seafoods and environment were screened for the presence of cas6 gene. Few representative PCR products were sequenced, and their phylogenetic relationship was established to available cas6 gene sequences in GenBank database. Results: Of the 38 V. parahaemolyticus isolates screened, only about 40% of strains harbored the cas6 endoribonuclease gene, among which 31.6% and 7.9% of the isolates were positive for the presence of the cas6-a and cas6-d subtypes of the gene, respectively. The subtypes cas6-b and cas6-c were absent in strains studied. Sequence and phylogenetic analysis also established the cas6 sequences in this study to match GenBank sequences for cas6-a and cas6-d subtypes. Conclusion: In V. parahaemolyticus, the Cas6 endoribonuclease is an associated protein of the CRISPR-cas system. CRISPR-positive strains exhibited genotypic variation for this gene. Primers designed in this study would aid in identifying the cas6 genotype and understanding the role of these genotypes in the CRISPR-cas immune system of the pathogen.

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