Studies On Phytosulfokine Chemical Synthesis, Physiological Effects And Gene Expression

Phytosulfokine,a sulfated polypeptide hormone,widely exists in plant kingdom and has shown multiple biological activities.There are two members of PSK,one is sulfated pentapeptide[H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-Gln-OH] (PSK-α)and another is sulfated tetrapeptide[H-Tyr(SO₃H)-Ile-Tyr(SO₃H)-Thr-OH] (PSK-β).In 1996,Matsubayashi et al originally found PSK in asparagus mesophyll cell cultures.PSK-αvery strongly stimulated the proliferation of plant cells.But fewer studies on PSK-αhad been reported up to now.The deeper and broader works are needed to verify its functions and existence in plant kingdom.This paper studied the PSK-αchemical synthesis,physiological effects,PSK precursor gene expression and so on.The main results were as follows:1.Synthetical pentapeptide(Tyr-Ile-Tyr-Thr-Gln)was sulfated by co-incubating it with DMF/SO₃.Sephadex G-15 column could efficiently purify the sulfated pentapeptide.The result of MS identification showed that PSK-αwas synthesized successfully.2.The relative growth rate of plant cells was considerably affected by initial cell density.This troublesome effect had interfered with the establishment of efficient plant cell culture systems,especially when only a small number of cells were expected to survive,such as in the genetic transformation of BY-2 tobacco suspension cell lines under bialaphos selection.To improve the recovery of bialaphos-resistant cells,we examined the use of the peptide plant hormone phytosulfokine(PSK),which had been shown to induce the proliferation of suspension cells.The addition of PSK to selective media increased the recovery of transformed BY-2 tobacco suspension cells.After screened by Bialaphos and identification by PCR,PCR-Southern blotting,transgenic suspension cell lines were produced.3.The transcriptional level of PSK precursor gene in wheat mature embryo and immature embryo callus tissue at different stages were detected by real-time quantitative PCR with SYBR Green I as fluorochrome.A pair of amplify primers were designed to amplify PSK precursor gene.The melting curves showed that the amplified product was very specific.The transcriptional copy number of PSK precursor gene in the immature embryo callus tissue was more than that in mature embryo callus tissue.These results indicated that the higher rate of plant regeneration of immature embryo culture possibly related to the PSK precursor gene expression.