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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (6): 814-823.doi: 10.3724/SP.J.1006.2018.00814

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2018-06-12 Published:2018-04-08
  • Contact: Xiao-Lan HE E-mail:helingzhi123@126.com
  • 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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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

Fig. 1

Structure of p1300-35SN-Ph-1-FFT/Dv-1-FFT/Lr-1-FFT transformation vectors used in the study LB and RB: left and right border of T-DNA; 35S°: CaMV 35S poly A; p35S2: CaMV 35S promoter with double enhancer sequences; p35S1: CaMV 35S promoter; Tnos: terminator."

Fig. 2

Electrophoresi s analysis of PCR products of 1-FFT gene cloned from P. huashanica, D. villosum, and L. racemosus M1: DL2000; M2: marker Ш. a: amplification of gene conserved fragment; b: PCR product of 3￠ RACE; c: PCR product of 5￠ RACE; d: cDNA of Ph-1-FFT (lanes 1-3), Dv-1-FFT (lanes 4-6), and Lr-1-FFT (lanes 7-9)."

Fig. 4

Phylogenic tree of 1-FFTs from Psathyrostachys huashanica, Leymus racemosus, Dasypyrum villosum and other plant species The bootstrap percentages (>50%) are shown next to the branches (based on 1000 replicates). The 1-FFT sequences were obtained from Psathyrostachys huashanica (Ph), Leymus racemosus (Lr), Dasypyrum villosum (Dv), Triticum turgidum subsp. durum (Tt) , Aegilops searsii (As), T. urartu (Tu), T. aestivum (Ta), Ae. tauschii (AE), and Hordeum vulgare subsp. vulgare (BA)."

Table 1

Comparison of different 1-FFT genes in cDNA length and the length, Mw, and pI of coding products"

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

Fig. 3

Sequence alignment of 1-FFTs from Psathyrostachys huashanica, Dasypyrum villosum, Leymus racemosus, and other plant species Conserved regions are boxed. Asterisks (*), colons (:), and periods (.) show the identical residues, conserved substitutions, and semiconserved substitutions, respectively. The 1-FFT sequences were obtained from Triticum aestivum (Ta), T. turgidum subsp. durum (Tt), Aegilops searsii (As), T. urartu (Tu), Dasypyrum villosum (Dv), Psathyrostachys huashanica (Ph), Ae. tauschii (AE), Hordeum vulgare subsp. vulgare (BA), and Leymus racemosus (Lr)."

Fig. 5

Validation of transgenic T0 plants A: PCR analysis of transgenic plants; B: expression patterns of 1-FFT gene; M: D15000 marker; P: positive control (plasmid); WT: negative control plants; NT: negative control (non-transgenic plants); 1-12: T0-tobacco transgenic plants."

Fig. 6

Phenotypes of tobacco plants transformed with 1-FFT from Psathyrostachys huashanica (Ph), Dasypyrum villosum (Dv), and Leymus racemosus (Lr) a: after thirty-five days of drought stress; b: recovery growth at room temperature for seven days after cold stress (10°C for 10 days and -20°C for 35 min). NT indicates the non-transgenic tobacco plant."

Fig. 7

Changes of physiological indices after drought and freezing stressed in tobacco plants transformed with 1-FFT from Psathyrostachys huashanica (Ph), Dasypyrum villosum (Dv), and Leymus racemosus (Lr) NT indicates the non-transgenic tobacco plant. Different letters above the error bars indicate significant difference among the four types of transgenic plants (P < 0.05)."

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