In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

马铃薯in vitro 块茎发育的研究及其遗传转化受体系统的建立

研究了BAP在SLM→TIM、MA→TIM和切段诱导系统中与in vitro块茎发育的关系。在SLM→TIM系统中BAP没有促进块茎发育的作用;在MA→TIM系统中BAP具有促进块茎发育的作用;切段培养在20 ℃、8小时光照条件下则其块茎发育对BAP有依赖。系统地研究了光周期、蔗糖浓度和外源细胞分裂素在促进切段块茎发育方面的作用及其交互影响。确定了由切段诱导块茎的最佳培养条件以及用于BAP吸收代谢研究的实验条件。讨论了切段诱导系统在理论研究上的价值和生产上应用的前景。研究了无菌苗、长切段及切段BAP吸收运转代谢特点及其与块茎发育关系。马铃薯植物系统对BAP的吸收运转是需能代谢过程。BAP在植物系统运转性差与其在组织的代谢特点有关。在块茎诱导早期有标物质在小块茎或匍匐茎末端积累，这可能促使细胞分裂从而诱发块茎发育;但在成熟块茎中放射性物质浓度很低。外源细胞分裂素在切段或匍匐茎局部积累不是块茎发育的充分条件。在有利于块茎发育的条件下代谢早期BAP活性代谢产物含量明显地高于对照，代谢一定阶段后二种处理的切段BAP代谢谱趋于接近，这一代谢特点与BAP促进块茎发育的生理效应有关。运用同位素示踪技术研究了切段蔗糖吸收特点，外源细胞分裂素促进蔗糖在切段系统积累从而诱导体细胞储藏组织生化分化。短日照（黑暗）、较高浓度蔗糖、合适浓度BAP都有促进离体切段块茎发育作用，这几个因子在提高切段诱导水平方面具有协同效应。诱导早期切段中有块茎专一性糖蛋白Patatin痕量的存在。当切段发育了较大块茎后（诱导约15天）切段中Patatin含量明显增加。诱导3天切段系统中即有Patatin mRNA高水平地转录。诱导7天阶段Patatin mRNA含量迅速下降。块茎发育很可能是通过几种激素（包括块茎诱导因子）协同地对植物系统同化物尤其是蔗糖源库关系的调节而实现的。 建立了向马铃薯植物引入外源基因的受体系统。叶园盘与农杆菌（pGV2260:: pGV941）共培养。转化植株在选择培养基上培养大约3周即可从愈伤组织或直接从叶片边缘产生。在含有高浓度卡那霉素的培养基上转化植株大多表型正常，切段能够发育块茎，叶片能够形成愈伤组织。转化再生植株均含有NPT－II活性而未转化Desiree无菌苗没有NPT－II活性。Southern分析表明NPT－II基因已整合入转化植物基因组中。这一实验系统的建立为向马铃薯植物引进具有重要经济价值的外源基因创造了条件。