碱蓬PEPCase基因的克隆与分析

采用cDNA末端快速扩增(RACE)技术首次获得碱蓬(Suaeda glauca)中含PEPCase基因完整编码区的cDNA序列,长度为3 038 bp。获得的序列采用生物信息学方法和系统进化方法进行分析。结果表明,获得的cDNA序列包含2 898 bp的完整开放阅读框,编码的966个氨基酸序列含有两个PEPCase活性位点以及6种其他的活性位点;预测蛋白质的相对分子质量为109 785.3,等电点为5.51,属于不稳定的亲水性蛋白;含量相对较多的氨基酸是Leu、Glu、Arg、Asp、Ser,不含Pyl和Sec;不包含跨膜结构和信号肽序列,推断为非分泌性蛋白;二级结构以α螺旋为主,三级结构为紧密球状结构;分析结果还表明获得的碱蓬PEPCase基因应该属于C3型。通过对碱蓬PEPCase基因的克隆及序列分析从而为后期碱蓬PEPCase蛋白表达的研究及获得高油量的转基因碱蓬植株奠定了基础。

Study on the Cloning and Isolation of sus scrofa GPX2 Gene by RACE Method

[Objective] Using molecular biotechnology to clone the sus scrofa GPX2 gene. [Method] Using total RNA of sus scrofa duodenum as template, degenerated primer pairs were designed according to the homology alignment analysis of GPX2 gene of human, rat, mouse, dog and cattle. A sus scrofa GPX2 gene sequence of 330 bp was obtained by RT-PCR application method. Primes were designed respectively according to the known sequence, sus scrofa GPX2 gene was isolated and cloned by 3-RACE and 5-RACE method and analyzed the gene sequence. [Result] A mRNA sequence of 924 bp was successfully cloned and isolated in this research. This sequence contained complete 3＇end and had higher sequence homology with human,mouse,cattle and dog GPX2 gene, and there was codon called TGA which encoding Sec on the position of No. 114-116 gene. [Conclusion] Sequence alignment analysis showed that the cloned gene was sus scrofa GPX2 gene （ NCBI GenBank database, the sequence number was D098982）.

毛尖紫萼藓泛素延伸蛋白基因克隆及序列分析

目的:该文首次从毛尖紫萼藓中克隆到泛素延伸蛋白基因的全长,为深入研究其功能打下基础。方法:提取植物的总RNA,经反转录得到cDNA,根据实验室得到的毛尖紫萼藓泛素延伸蛋白基因的EST序列设计引物,基于3’RACE(Rapid Ampli-fication of cDNA End)技术克隆得到全长序列,使用分子生物学软件进行序列分析。结果:获得了cDNA全序列,GenBank登录号为JQ659260,序列全长为673bp,开放阅读框为471 bp,编码156个氨基酸残基。结论:通过Blast P对其编码的氨基酸序列进行比对结果显示:毛尖紫萼藓泛素延伸蛋白与小立碗藓、北美云杉、葡萄、蓖麻、毛果杨、紫茎泽兰的泛素延伸蛋白的同源性均在96%以上。

Cloning and Expression of a Profilin Gene from Rapeseed

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA clone (designated Repro) encoding profilin gene was isolated from rapeseed ( Brassica napus L. cv. canadian Tween) using RT-PCR technique. Sequence analysis showed 82% similarity to Zea mays L. ZmPro3, 85% to Arabidopsis AthPRF1, 82% to Nicotiana tabacum L. NTPRO, 81% to Oryza sativa L. profilin A. A new full-length cDNA was obtained by 5'-RACE and 3'-RACE techniques. Sequence analysis showed that the size of full-length cDNA is 672 bp which contains a major open reading frame of 134 amino, acids, 5' and 3' untranslated regions and a long Poly (A) tail. Northern blot analysis showed that the profilin gene is a pollen and anther specific gene.

Cloning of Syntaxin Gene in Limonium sinense Kuntzet

[Objective] The aim was to clone Syntaxin genes in Limonium sinense Kuntze. [Method] Limonium sinense Kuntze leaves were used as materials and total RNA was extracted and transcribed reversely. Nested primers were designed based on EST sequences at 5’ region of Syntaxin, and cDNA obtained through reverse reaction was taken as the template. Sequences of Syntaxin gene at 3’ region were obtained through two rounds of PCR amplifications. [Result] DNA fragments （1 096 bp） were obtained. For LsSyntaxin, open reading frame （ORF） was 816 bp and the encoded amino acids were 271. The relative molecular weight of Syntaxin was 30 254.3 Da and isoelectric point in theory was 5.55. [Conclusion] Syntaxin genes from Limonium sinense Kuntze were cloned. The research laid foundation for the study on Syntaxin gene function in Limonium sinense Kuntze and salt-secreted process.