Сибирский федеральный университет

Тип публикации: статья из журнала

Год издания: 2008

Ключевые слова: apoplast, Protein secretion, thaumatin II

Аннотация: In order to achieve high-level apoplastic accumulation of a biologically active thaumatin II protein in transgenic tobacco plants, a chimerical gene lrth encoding a thaumatin II recombinant precursor has been constructed. The precursor contains the N-terminal signal peptide from RS-AP radish defensin and a sequence for the thaumatin mature form. The lrth chimerical gene was transferred to the binary vector pGD121 in order to perform the agrobacterial transformation of the plant. Sixteen transgenic tobacco lines harboring the lrth gene and 12 lines with unmodified thauII gene (from an earlier constructed pBIThau vector) were obtained. All transgenic plants exhibited the normal phenotypes. Three thauII- and four lrth-containing transgenic lines were randomly selected for the further investigations. Analysis of the thauII and lrth transcripts using RT-PCR showed that in all cases, a specific transcript that was equal in size to the mature protein was detected. Pre-mRNA transcribed from the chimerical lrth gene that contained a 91-bp intron from the radish rs-ap gene was spliced correctly in the transgenic tobacco plants. Total protein and protein from apoplast and remnant tissue was isolated from leaves of the selected tobacco lines. Western-blotting of total, apoplastic and remnant tissue extracts indicated that plants that had been transformed with the wild-type construct retained most of the produced thaumatin intercellularly. On the contrary, the major portion of thaumatin in the lrth-transformed tobacco lines was detected in the intercellular washing fluid fraction. Unlike the protein in control, the secreted from the transgenic plants thaumatin was sweet, probably indicating the conservation of the conformational state in the recombinant thaumatin.

Журнал: Biotechnology in Russia

Выпуск журнала: № 6

Номера страниц: 40-54

Место издания: Москва

Издатель: Федеральное государственное бюджетное учреждение "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов"

• Pushin
• Ovchinnikova E.V.
• Shul'ga O.A.
• Firsov A.P.
• Dolgov S.V.

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