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Effect of long-term cultivation on resveratrol accumulation in a high-producin cell culture of Vitis amurensis

100%

Dubrovina A. S. , Kiselev K. V.

nr 3

Resveratrol is a polyphenol, present in grapes, peanuts, and other plant sources, with a wide range of valuable biological activities. We established a Vitis amurensis cell culture accumulating high levels of resveratrol by introducing the rolB gene of Agrobacterium rhizogenes in the V. amurensis genome, and studied the stability of resveratrol accumulation during 27 months of continuous subculturing. This study demonstrates a decline in the high level of resveratrol production by the rolB transgenic cell line during its long-term cultivation. Elicitation of the rolB transgenic calli with methyl jasmonate and salicylic acid, which are known to stimulate the production of plant secondary metabolites, resulted in a recovery of resveratrol accumulation in the rolB transgenic cell culture, while the empty vector-transformed culture with trace starting content of resveratrol exhibited low inducibility to the treatment.

Changes of CDPK genes expression during embryogenesis in Panax ginseng rolC-expressing cell culture 2c3

100%

Turlenko A. V. , Kiselev K. V.

nr 2

True and false alternative transcripts of calcium-dependent protein kinase CPK9 and CPK3a genes in Vitis amurensis

80%

Dubrovina A. S. , Aleynova O. A. , Kiselev K. V. , Novikova G. V.

nr 07

Calcium-dependent protein kinases (CDPKs) are major Ca²⁺ sensors in plants playing important roles in plant development and stress responses. In the present study, RT-PCR analysis revealed that VaCPK3a and VaCPK9 cDNAs lacking extensive regions in the kinase, autoinhibitory, and Ca²⁺-binding domains were numerous in probes derived from wild grapevine Vitis amurensis. Most of the VaCPK3a and VaCPK9-modified transcripts lacked canonical splice sites and possessed short direct repeated sequences (SDRs) instead. Three VaCPK9 transcript variants were generated using canonical 5'GT and 3'AG splice sites and lacked several subdomains in the kinase domain, including ATP-binding site, which is known to be indispensable for kinase activities. These observations indicate that post-transcriptional mRNA processing might lead to production of CDPKs with abolished phosphorylation activities. Recombinant proteins VaCPK3aSF2, lacking autoinhibitory and Ca²⁺-binding domain, and VaCPK3aSF3, lacking VIII–XI kinase subdomains and autoinhibitory domain, phosphorylated exogenous substrate in a Ca²⁺- independent manner. However, reverse transcription at +65°C using heat-stable reverse transcriptase (RT) markedly lowered abundance of the unusual VaCPK3a and VaCPK9 cDNAs with SDRs, while it did not eliminate VaCPK9 cDNAs generated using canonical splice sites. The results show that VaCPK9 gene undergoes unproductive alternative splicing mediated by canonical splice sites to generate three mRNA isoforms lacking important catalytic kinase subdomains. The unusual VaCPK3a and VaCPK9 transcripts with SDRs are likely to be false alternative transcripts generated by RT template switching in vitro. The data demonstrate that using non-thermostable RTs for studying alternative splicing are not appropriate.