小麦近缘属植物1-FFT基因的克隆及功能分析

Molecular Cloning and Functional Analysis of 1-FFT in Wheat Relatives

果聚糖与植物碳素分配和抗逆性有关,在逆境胁迫中具有保护植物细胞免受伤害的作用。为明确不同来源的果聚糖1-果糖基转移酶基因在植物抗逆中的作用,本研究分别以小麦近缘属植物华山新麦草(Psathyrostachys huashanica,2n=2x=14,Ns Ns)、簇毛麦(Dasypyrum villosum,2n=2x=14,VV)、大赖草(Leymus racemosus,2n=4x=28,Ns Ns Xm Xm)为材料,利用RACE技术克隆获得3个果聚糖1-果糖基转移酶1-FFT基因,分别命名为Ph-1-FFT、Dv-1-FFT和Lr-1-FFT。3个基因的完整开放阅读框长度分别为1989、1950和1989 bp,编码662、649和662个氨基酸,其编码氨基酸序列均含有果糖基转移酶保守结构域。氨基酸序列比对及进化树分析表明,Ph-1-FFT和Lr-1-FFT高度同源,与Dv-1-FFT位于不同的分支,而Dv-1-FFT与普通小麦、圆锥小麦、西尔斯山羊草和乌拉尔图小麦1-FFT具有高度同源性。采用基因重组技术构建p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT表达载体,利用农杆菌介导法将3个载体分别转入烟草品种W38中。对经过抗性筛选、PCR和RT-PCR验证的转基因植株鉴定发现,其抗旱和抗寒性明显高于对照,不同来源的1-FFT转基因植株的抗逆性差异不明显;在逆境胁迫条件下,转基因株系的果聚糖、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,不同来源的1-FFT转基因植株的果聚糖、可溶性糖、脯氨酸和丙二醛含量差异不显著。本研究表明,Ph-1-FFT、Dv-1-FFT和Lr-1-FFT基因均是典型的GH32基因家族成员,其表达可能对提高烟草抗旱和抗寒性起作用。

Fructan is closely related to carbon distribution and the response to adverse stresses in plant, protecting plant cell membrane stabilization in stress conditions. To determine the effects of fructan：fructan-1-fructosyltransferase genes from different species, we cloned three genes of full-length c DNA from Psathyrostachys huashanica（2 n = 2 x = 14, Ns Ns）, Dasypyrum villosum（2 n = 2 x = 14, VV） and Leymus racemosus（2 n = 4 x = 28, Ns Ns Xm Xm）, designated Ph-1-FFT, Dv-1-FFT, and Lr-1-FFT, respectively, using reverse transcriptase PCR（RT-PCR） and rapid-amplification of c DNA ends（RACE） techniques. The full-length open reading frames（ORF） are 1989, 1950, and 1989 bp in length, encoding 662, 649, and 662 amino acids, respectively. The predicted protein contains a conserved fructosyltransferase domain. Multiple sequences alignment and phylogenetic analysis indicated that the Dv-1-FFT shared a high similarity with 1-FFT from Triticum aestivum, T. turgidum subsp. durum, Aegilops searsii and T. urartu, Ph-1-FFT and Lr-1-FFT were clustered into the same evolutionary branch, which was different from those of Dv-1-FFT. Furthermore, the plant expression vector p CAMBIA1300-35 SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT was constructed and transferred into tobacco（Nicotiana tabacum） cv. W38 via Agrobacterium-mediated transformation. The validation with PCR and RT-PCR assay showed that drought and cold tolerance was obviously improved in all types of transgenic tobacco compared with the wild type, and no significant difference between transgenic genotypes. Under drought and cold stresses, the contents of carbohydrate and proline were significantly higher in the transgenic tobacco lines than in the wild type, whereas, malondialdehyde content was significantly lower in the transgenic lines, which was no significant difference between the transgenic plants with 1-FFT from different species. These results suggest that Ph-1-FFT, Dv-1-FFT, and Lr-1-FFT are typical members of the gene family coding glycoside hydrolase 32（GH32） and play a role in drought and cold resistance in tobacco.