大赖草6-SFT基因的克隆及其转基因烟草抗旱和抗寒性分析

摘要

The fructan biosynthesis enzyme (6-SFT) plays an important role in plant in response to abiotic stresses. In this study, a full-length cDNA encoding sucrose:fructan-6-fructosyltransferase, designated as Lr-6-SFT (GenBank accession No. KT387273), was cloned from Leymus racemosus (2n=4x=28, NsNsXmXm) using reverse transcriptase PCR (RT-PCR) and rapid-amplifica-tion of cDNA ends (RACE) techniques. The full-length open reading frame comprises 1863 bp and encodes 620 amino acids. The predicted protein structure of the gene contains a conserved fructosyltransferase domain. Multiple sequence alignment and phy-logenetic analysis showed that the Lr-6-SFT protein shared high similarity with 6-SFT proteins from Psathyrostachys huashanica, Triticum aestivum, Aegilops searsii, and Hordeum vulgare subsp. vulgare. The Lr-6-SFT gene was transferred into tobacco (Nico-tiana tabacum L. cv. W38) via Agrobacterium-mediated transformation. The screened plants were tested by PCR and RT-PCR, and the transgenic tobacco plants exhibited much higher tolerance to drought and cold compared with the non-transgenic plants. Under drought and cold stresses, the Lr-6-SFT expressions were associated with the increased accumulation of stored carbohy-drate and proline and the decreased malondialdehyde storage. These results suggest that Lr-6-SFT is a typical member of the gly-coside hydrolase 32 (GH32) family and may be linked to enhanced tolerance to drought and cold stresses.%果聚糖合成酶(6-SFT)在植物抵御逆境胁迫中起重要作用。利用 RACE 结合 RT-PCR 技术从大赖草(Leymus racemosus,2n=4x=28, NsNsXmXm)中克隆到6-SFT基因,其完整开放阅读框为1863 bp。该基因被命名为Lr-6-SFT (GenBank 登录号 KT387273),编码620个氨基酸,其推导氨基酸序列含有保守的果糖基转移酶结构域。氨基酸序列比对及进化树分析表明,大赖草6-SFT与华山新麦草、普通小麦、西尔斯山羊草和大麦6-SFT具有高度相似性。采用基因重组技术构建 p1300-35SN-Lr-6-SFT表达载体,利用农杆菌介导法将该载体转入烟草品种 W38中。对经过抗性筛选、PCR和RT-PCR验证的转基因植株进行抗旱和抗寒性鉴定,发现转基因植株与对照相比,抗旱和抗寒性明显增强；在逆境胁迫条件下,转基因植株的果聚糖、可溶性糖、脯氨酸含量都显著高于对照,而丙二醛的含量显著低于对照。本研究表明, Lr-6-SFT基因是典型的GH32家族成员,其表达能够提高烟草对干旱和寒冷胁迫的抗性。