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作物学报 ›› 2018, Vol. 44 ›› Issue (6): 814-823.doi: 10.3724/SP.J.1006.2018.00814

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

小麦近缘属植物1-FFT基因的克隆及功能分析

王建伟1,贺晓岚1,*(),李文旭3,陈新宏2   

  1. 1 凯里学院大健康学院, 贵州凯里 556011
    2 西北农林科技大学农学院 / 陕西省植物遗传工程育种重点实验室, 陕西杨凌 712100
    3 河南省农业科学院小麦研究所, 河南郑州 450002
  • 收稿日期:2017-08-26 接受日期:2018-03-26 出版日期:2018-06-12 网络出版日期:2018-04-08
  • 通讯作者: 贺晓岚
  • 基金资助:
    本研究由凯里学院博士专项基金(BS201606);贵州省教育厅科技拔尖人才支持计划项目资助(黔教合KY字[2017]094)

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

Jian-Wei WANG1,Xiao-Lan HE1,*(),Wen-Xu LI3,Xin-Hong CHEN2   

  1. 1 School of Life and Health Science, Kaili University, Kaili 556011, Guizhou, China
    2 Shaanxi Provincial Key Laboratory of Plant Genetic Engineering Breeding / College of Agronomy, Northwest A&F University, Yangling 712100, Shaanxi, China;
    3 Wheat Research Institute, Henan Academy of Agricultural Sciences, Zhengzhou 450002, Henan, China
  • Received:2017-08-26 Accepted:2018-03-26 Published:2018-06-12 Published online:2018-04-08
  • Contact: Xiao-Lan HE
  • Supported by:
    This study was supported by the Special Fund for the Doctoral Program of Kaili University(BS201606);the Science and Technology Top Talents Support Program of Guizhou Provincial Education Department(黔教合KY字[2017]094)

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摘要：

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, NsNs)、簇毛麦(Dasypyrum villosum, 2n = 2x = 14, VV)、大赖草(Leymus racemosus, 2n = 4x = 28, NsNsXmXm)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。

关键词: 1-FFT基因, 抗旱性, 抗寒性, 小麦近缘属植物, 转基因烟草

Abstract:

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 cDNA from Psathyrostachys huashanica (2n = 2x = 14, NsNs), Dasypyrum villosum (2n = 2x = 14, VV) and Leymus racemosus (2n = 4x = 28, NsNsXmXm), designated Ph-1-FFT, Dv-1-FFT, and Lr-1-FFT, respectively, using reverse transcriptase PCR (RT-PCR) and rapid-amplification of cDNA 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 pCAMBIA1300-35SN-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.

Key words: 1-FFT gene, drought tolerance, cold tolerance, wheat relatives, transgenic tobacco

图1

植物表达载体p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT示意图 LB和RB: T-DNA的左、右边界; 35S°: CaMV 35S poly A; 35S2: 含双增强子的CaMV 35S启动子; p35S1: CaMV 35S启动子; Tnos: 终止子。"

图2

华山新麦草、簇毛麦和大赖草1-FFT基因PCR扩增产物电泳分析 M1: DL2000; M2: marker Ш. a: 基因保守区段扩增产物; b: 3￠ RACE扩增产物; c: 5￠ RACE 扩增产物; d: Ph-1-FFT (1~3泳道)、Dv-1-FFT (4~6泳道)和Lr-1-FFT (7~9泳道)的cDNA。"

图4

华山新麦草、大赖草和簇毛麦与其他物种1-FFT序列系统进化树分析 各节点处数值表示bootstrap值(迭代1000次)。比对物种为华山新麦草(Ph)、大赖草(Lr)、簇毛麦(Dv)、圆锥小麦(Tt)、西尔斯山羊草(As)、乌拉尔图小麦(Tu)、普通小麦(Ta)、粗山羊草(AE)和大麦(BA)。"

表1

不同来源1-FFT基因的cDNA及其编码产物的长度、分子量和等电点比较"

基因
Gene		 cDNA长度
cDNA length (bp)		 编码产物长度
Length of coding product (aa)		 分子量
Mw (kD)		 等电点
pI
Ph-1-FFT 1989 662 72.2 4.91
Dv-1-FFT 1950 649 71.3 5.00
Lr-1-FFT 1989 662 72.0 4.87

图3

华山新麦草、簇毛麦、大赖草及其他物种1-FFT序列分析 方框显示序列保守区; 星号(*)表示相同残基; 冒号(:)表示保守替换; 点号(.)表示半保守替换。比对物种有普通小麦(Ta)、圆锥小麦(Tt)、西尔斯山羊草(As)、乌拉尔图小麦(Tu)、簇毛麦(Dv)、华山新麦草(Ph)、粗山羊草(AE)、大麦(BA)和大赖草(Lr)。"

图5

T0代转基因植株的鉴定 A: 转基因植株的PCR鉴定; B: 转基因植株1-FFT的表达鉴定。M: D15000 marker; P: 阳性对照(质粒); WT: 阴性对照; NT: 转空载体的阴性对照; 1~12: 转基因植株。"

图6

转华山新麦草(Ph)、簇毛麦(Dv)和大赖草(Lr) 1-FFT基因烟草干旱和冷胁迫后的表型 a: 干旱胁迫处理35 d后; b: 冷胁迫(10°C 10 d, -20°C 35 min)处理后转移至温室培养7 d。NT为转空质粒对照。"

图7

转华山新麦草(Ph)、簇毛麦(Dv)和大赖草(Lr) 1-FFT基因烟草干旱和冷胁迫后的生理指标变化 NT为转空质粒对照。在相同条件下, 误差线上不同字母表示4类转基因植株间差异显著(P < 0.05)。"

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