Abstract

Hop plant (Humulus lupulus L.) is an important industrial crop, grown for harvesting hop cones however, it is a host to four different viroids as well. The nature of viroid infections is not entirely clarified. In our work, we focused on analyzing viroid accumulation and their strands polarity through RNA sequencing and reverse transcription polymerase chain reaction in real time. RNA-seq data indicate that viroids amplify until saturation further demonstrating plant's biological capacity. Negative fold changes in accumulation of individual viroids between hop samples with single and multiple infections are suggesting an antagonistic relationship amongst viroids, where citrus bark cracking viroid seems to be the least and hop stunt viroid the most sensitive to the other two. RNA-seq data also show that on average (−) viroid strand is dominating over (+), especially for the citrus bark cracking viroid. Reverse transcription polymerase chain reaction in real time results from strand polarity analysis seem to be less consistent between different combinations of infection but are showing level of conformity with RNA-seq in the case of citrus bark cracking viroid.

Highlights

  • Hop plant (Humulus lupulus L.) is an important industrial crop, grown for harvesting hop cones it is a host to four different viroids as well

  • Preglednica 3: Povzetek rezultatov testa EDGE za raven akumulacije posameznih viroidov Table 3: Summary of EDGE test results for individual viroid accumulation

  • Glede na nekatere raziskave o medsebojnem vplivu patogenov v rastlini in podatke iz literature menimo, da igra rastlinska RNK interference (RNKi) in ostali mehanizmi, ki vključujejo male RNK dinamično vlogo pri interakcijah med različnimi viroidi in hmeljem

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Summary

BIOINFORMATSKA ANALIZA

V raziskavi smo izvedli bioinformatsko analizo podatkov RNK sekvenciranja, ki smo jih pridobili v sklopu projekta »Analiza odziva rastlin ob hkratnih okužbah viroidov in identifikacija odpornosti – L4-6809«, ki sta ga izvedli sodelujoči organizaciji Inštitut za hmeljarstvo in pivovarstvo Slovenije in Oddelek za agronomijo, Biotehniška fakulteta, Univerza v Ljubljani. V prvem koraku smo izvedli obratno transkripcijo molekul viroidov tako, da smo specifično prepisali ali (+) ali (−) verige viroidov. Z namenom relativne kvantifikacije okužbe smo pri vseh bioloških ponovitvah izmerili še nivo izražanja gena s konstantnim izražanjem, DRH1 (Štajner et al, 2013). V drugem koraku pa smo izvedli qPCR z uporabo Fast SYBR® Green Master Mix-a (Applied Biosystems) na napravi Applied Biosystems 7500 Fast Real-Time PCR System, pri čemer smo uporabili par začetnih oligonukleotidov specifičnih za prepisani (+) ali (−) verigi posameznega viroida oz. Na podlagi matematičnega modela (Pfaffl, 2012) smo izračunali relativne nivoje izražanja tarčnih genov z namenom, da smo lahko med seboj primerjali ravni akumulacije viroidov med različnimi biološkimi obravnavanji. Zanj smo uporabili programski paket R (paket Rcmdr, različica 2.4-x)

NIVO POMNOŽEVANJA VIROIDOV V VZORCIH HMELJA
POLARNOST MOLEKUL VIROIDOV
ZAHVALA
SKLEPI
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