Welcome to Acta Agronomica Sinica，

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)

RichHTML331

33

PDF

622

PDF

44

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)."

[1] Zhang S J, Song G Q, Li Y L, Gao J, Liu J J, Fan Q Q, Huang C Y, Sui X X, Chu X S, Guo D , Others. Cloning of 9- cis-epoxy-carote- noid dioxygenase gene( TaNCED1) from wheat and its heterologous expression in tobacco. Biol Plant, 2014,58:89-98
[2] Bie X M, Wang K, She M Y, Du L, Zhang S X, Li J R, Gao X, Lin Z S, Ye X G . Combinational transformation of three wheat genes encoding fructan biosynthesis enzymes confers increased fructan content and tolerance to abiotic stresses in tobacco. Plant Cell Rep, 2012,31:2229-2238
doi: 10.1007/s00299-012-1332-y pmid: 22911265
[3] Khoshro H H, Taleei A, Bihamta M R, Shahbazi M, Abbasi A, Ramezanpour S S . Expression analysis of the genes involved in accumulation and remobilization of assimilates in wheat stem under terminal drought stress. Plant Growth Regul, 2014,74:165-176
doi: 10.1007/s10725-014-9908-x
[4] Ritsema T, Joling J, Smeekens S . Patterns of fructan synthesized by onion fructan: fructan 6G-fructosyltransferase expressed in tobacco BY2 cells-is fructan: fructan 1-fructosyltransferase needed in onion? New Phytol, 2003,160:61-67
doi: 10.1046/j.1469-8137.2003.00858.x
[5] Van den Ende W, Clerens S, Vergauwen R, Boogaerts D, Le Roy K, Arckens L, Van Laere A . Cloning and functional analysis of a high DP fructan: fructan 1-fructosyl transferase from Echinops ritro( Asteraceae): comparison of the native and recombinant enzymes. J Exp Bot, 2006,57:775-789
doi: 10.1093/jxb/erj065 pmid: 16449376
[6] 胡翠 . 果糖基转移酶融合基因转化玉米胚乳中果聚糖的积累 . 四川农业大学硕士学位论文, 四川成都, 2009
Hu C . Fructan Accumulation in Maize Endosperm Transformed with Fused Fructosyltransferase Genes. MS Thesis of Sichuan Agricultural University, Chengdu, Sichuan, China 2009 ( in Chinese with English abstract)
[7] Edelman J, Dickerson A G . The metabolism of fructose polymers in plants: transfructosylation in tubers of Helianthus tuberosus L. Biochem J, 1966,98:787-794
[8] Chalmers J, Johnson X, Lidgett A, Spangenberg G . Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass ( Lolium perenne). J Plant Physiol, 2003,160:1385-1391
[9] Van Den Ende W, Van Wonterghem D, Verhaert P, Dewil E, Van Laere A . Purification and characterization of fructan: fructan fructosyl transferase from chicory ( Cichorium intybus L.) roots. Planta, 1996,199:493-502
doi: 10.1109/38.79453
[10] Van den Ende W, Van Laere A, Le Roy K, Vergauwen R, Boogaerts D, Figueiredo-Ribeiro R C , De Carvalho M A M. Molecular cloning and characterization of a high DP fructan: fructan 1-fructosyl transferase from Viguiera discolor( Asteraceae) and its heterologous expression in Pichia pastoris. Physiol Plant, 2005,125:419-429
doi: 10.1111/j.1399-3054.2005.00579.x
[11] Gao X, She M Y, Yin G X, Yu Y, Qiao W H, Du L P, Ye X G . Cloning and characterization of genes coding for fructan biosynthesis enzymes (FBEs) in Triticeae plants. Agric Sci China, 2010,9:313-324
doi: 10.1016/S1671-2927(09)60099-7
[12] 张小芸, 何近刚, 孙学辉, 吴金霞 . 转果聚糖合成关键酶基因多年生黑麦草的获得及抗旱性的提高. 草业学报, 2011,20(1):111-118
doi: 10.11686/cyxb20110116
Zhang X Y, He J G, Sun X H, Wu J X . Transformation of Lolium perenne with a fructan:fructan 1-fructosyltransferase gene from Agropyron cristatum and enhancement of drought tolerance in transgenic plants. Acta Pratacult Sin, 2011,20(1):111-118 (in Chinese with English abstract)
doi: 10.11686/cyxb20110116
[13] Knipp G, Honermeier B . Effect of water stress on proline accumulation of genetically modified potatoes ( Solanum tuberosum L.) generating fructans. J Plant Physiol, 2006,163:392-397
doi: 10.1016/j.jplph.2005.03.014 pmid: 16455352
[14] Li H J, Yang A F, Zhang X C, Gao F, Zhang J R . Improving freezing tolerance of transgenic tobacco expressing sucrose: sucrose 1-fructosyltransferase gene from Lactuca sativa. Plant Cell Tissue Organ Cult, 2007,89:37-48
[15] Ruelland E, Vaultier M, Zachowski A, Hurry V. Cold signalling and cold acclimation in plants. In: Jacquot J, Delseny M, eds. Advances in Botanical Research, Salt Lake City: Academic Press, 2009. pp 35-150
[16] Szabados L, Savouré A . Proline: a multifunctional amino acid. Trends Plant Sci, 2010,15:89-97
[17] Hellwege E M, Raap M, Gritscher D, Willmitzer L, Heyer A G . Differences in chain length distribution of inulin from Cynara scolymus and Helianthus tuberosus are reflected in a transient plant expression system using the respective 1-FFT cDNAs. FEBS Lett, 1998,427:25-28
[18] 陈漱阳, 张安静, 傅杰 . 普通小麦与华山新麦草的杂交. 遗传学报, 1991,18:508-512
Chen S Y, Zhang A J, Fu J . The hybridization between Triticum aestivum and Psathyrostachys huashanica. Acta Genet Sin, 1991,18:508-512 (in Chinese with English abstract)
[19] Zhang W, Zhang R, Feng Y, Bie T, Chen P . Distribution of highly repeated DNA sequences in Haynaldia villosa and its application in the identification of alien chromatin. Chin Sci Bull, 2013,58:890-897
[20] 刘成 . 多年生簇毛麦基因组新重复序列的分离及其在小麦抗病新种质鉴定中的应用. 电子科技大学博士学位论文, 四川成都, 2010
Liu C . Isolate New Repetitive DNA Sequence from Daypyrum breviaristatum Genome and Its Application in Identifying New Resistant Wheat Germplasm. PhD Dissertation of Electronic Science and Technology of China, Chengdu, Sichuan, China 2010 ( in Chinese with English abstract)
[21] De Pace C, Snidaro D, Ciaffi M, Vittori D, Ciofo A, Cenci A, Tanzarella O A, Qualset C O, Mugnozza G S . Introgression of Dasypyrum villosum chromatin into common wheat improves grain protein quality. Euphytica, 2001,117:67-75
doi: 10.1023/A:1004095705460
[22] Mcguire P E, Dvorak J . High salt-tolerance potential in wheatgrasses. Crop Sci, 1981,21:702-705
doi: 10.2135/cropsci1981.0011183X002100050018x
[23] Pilon-Smits E A, Ebskamp M J, Paul M J, Jeuken M J, Weisbeek P J, Smeekens S C . Improved performance of transgenic fructan-accumulating tobacco under drought stress. Plant Physiol, 1995,107:125-130
doi: 10.1104/pp.107.1.33 pmid: 12228347
[24] Heyer A G, Wendenburg R . Gene cloning and functional characterization by heterologous expression of the fructosyltransferase of Aspergillus sydowi IAM 2544. Appl Environ Microbiol, 2001,67:363-370
doi: 10.1128/AEM.67.1.363-370.2001 pmid: 11133467
[25] Chen H, Nelson R S, Sherwood J L . Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques, 1994,16:664-668
[26] He X L, Chen Z Z, Wang J W, Li W X, Zhao J X, Wu J, Wang Z H, Chen X H . A sucrose:fructan-6-fructosyltransferase ( 6-SFT) gene from Psathyrostachys huashanica confers abiotic stress tolerance in tobacco. Gene, 2015,570:239-247
[27] 贺晓岚, 王建伟, 李文旭, 陈真真, 赵继新, 武军, 王中华, 陈新宏 . 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. 作物学报, 2016,42:389-398
He X L, Wang J W, Li W X, Chen Z Z, Zhao J X, Wu J, Wang Z H, Chen X H . Cloning of 6-SFT gene from Leymus racemosus and analysis of tolerance to drought and cold stresses in transgenic tobacco. Acta Agron Sin, 2016,42:389-398 (in Chinese with English abstract)
[28] Fales F W . The assimilation and degradation of carbohydrates by yeast cells. J Biol Chem, 1951,193:113-124
doi: 10.1515/bchm2.1951.286.1-6.270 pmid: 14907695
[29] Bates L S, Waldren R P, Teare I D . Rapid determination of free proline for water-stress studies. Plant & Soil, 1973,39:205-207
[30] Suárez-González E M, López M G, Délano-Frier J P, Gómez-Leyva J F . Expression of the 1-SST and 1-FFT genes and consequent fructan accumulation in Agave tequilana and A. inaequidens is differentially induced by diverse (a)biotic-stress related elicitors. J Plant Physiol, 2014,171:359-372
[31] Lasseur B, Schroeven L, Lammens W, Le Roy K, Spangenberg G, Manduzio H, Vergauwen R, Lothier J , Prud’Homme M P, Van den Ende W. Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase. Plant Physiol, 2009,149:327-339
doi: 10.1104/pp.108.125559
[32] Chazen O, Neumann P M . Hydraulic signals from the roots and rapid cell-wall hardening in growing maize ( Zea mays L.) leaves are primary responses to polyethylene glycol-induced water deficits. Plant Physiol, 1994,104:1385-1392
[33] Ravikumar G, Manimaran P, Voleti S R, Subrahmanyam D, Sundaram R M, Bansal K C, Viraktamath B C, Balachandran S M . Stress-inducible expression of AtDREB1A transcription factor greatly improves drought stress tolerance in transgenic indica rice. Transgenic Res, 2014,23:421-439
doi: 10.1007/s11248-013-9776-6 pmid: 24398893
[34] Kawakami A, Sato Y, Yoshida M . Genetic engineering of rice capable of synthesizing fructans and enhancing chilling tolerance. J Exp Bot, 2008,59:793-802
doi: 10.1093/jxb/erm367
[1] ZHOU Wen-Qi, QIANG Xiao-Xia, WANG Sen, JIANG Jing-Wen, WEI Wan-Rong. Mechanism of drought and salt tolerance of OsLPL2/PIR gene in rice [J]. Acta Agronomica Sinica, 2022, 48(6): 1401-1415.
[2] ZHANG He, JIANG Chun-Ji, YIN Dong-Mei, DONG Jia-Le, REN Jing-Yao, ZHAO Xin-Hua, ZHONG Chao, WANG Xiao-Guang, YU Hai-Qiu. Establishment of comprehensive evaluation system for cold tolerance and screening of cold-tolerance germplasm in peanut [J]. Acta Agronomica Sinica, 2021, 47(9): 1753-1767.
[3] MENG Jiang-Yu, LIANG Guang-Wei, HE Ya-Jun, QIAN Wei. QTL mapping of salt and drought tolerance related traits in Brassica napus L. [J]. Acta Agronomica Sinica, 2021, 47(3): 462-471.
[4] JIANG Shu-Kun,WANG Li-Zhi,YANG Xian-Li,LI Bo,MU Wei-Jie,DONG Shi-Chen,CHE Wei-Cai,LI Zhong-Jie,CHI Li-Yong,LI Ming-Xian,ZHANG Xi-Juan,JIANG Hui,LI Rui,ZHAO Qian,LI Wen-Hua. Detection of QTLs controlling cold tolerance at bud bursting stage by using a high-density SNP linkage map in japonica rice [J]. Acta Agronomica Sinica, 2020, 46(8): 1174-1184.
[5] HENG You-Qiang,YOU Xi-Long,WANG Yan. Pathogenesis-related protein gene SfPR1a from Salsola ferganica enhances the resistances to drought, salt and leaf spot disease in transgenic tobacco [J]. Acta Agronomica Sinica, 2020, 46(4): 503-512.
[6] Shan-Bin CHEN, Si-Fan SUN, Nan NIE, Bing DU, Shao-Zhen HE, Qing-Chang LIU, Hong ZHAI. Cloning of IbCAF1 and identification on tolerance to salt and drought stress in sweetpotato [J]. Acta Agronomica Sinica, 2020, 46(12): 1862-1869.
[7] ZHANG Huan, YANG Nai-Ke, SHANG Li-Li, GAO Xiao-Ru, LIU Qing-Chang, ZHAI Hong, GAO Shao-Pei, HE Shao-Zhen. Cloning and functional analysis of a drought tolerance-related gene IbNAC72 in sweet potato [J]. Acta Agronomica Sinica, 2020, 46(11): 1649-1658.
[8] Long LI,Xin-Guo MAO,Jing-Yi WANG,Xiao-Ping CHANG,Yu-Ping LIU,Rui-Lian JING. Drought Tolerance Evaluation of Wheat Germplasm Resources [J]. Acta Agronomica Sinica, 2018, 44(7): 988-999.
[9] Bin YU,Hong-Yu YANG,Li WANG,Yu-Hui LIU,Jiang-Ping BAI,Feng ZHANG,Di WANG,Jun-Lian ZHANG. Relationship between Potato Canopy-air Temperature Difference and Drought Tolerance [J]. Acta Agronomica Sinica, 2018, 44(7): 1086-1094.
[10] Li-Li WAN, Zhuan-Rong WANG, Qiang XIN, Fa-Ming DONG, Deng-Feng HONG, Guang-Sheng YANG. Enhanced Accumulation of BnA7HSP70 Molecular Chaperone Binding Protein Improves Tolerance to Drought Stress in Transgenic Brassica napus [J]. Acta Agronomica Sinica, 2018, 44(04): 483-492.
[11] Pin LYU, Hai-Feng YU, Jian-Hua HOU. QTL Mapping of Yield Traits Using Drought Tolerance Selected Backcrossing Introgression Lines in Sunflower [J]. Acta Agronomica Sinica, 2018, 44(03): 385-396.
[12] FANG Yan,SUN Wan-Cang,WU Jun-Yan，LIU Zi-Gang,DONG Yun,MI Chao,MA Li,CHEN Qi,HE Hui-Li. Response of Membrane Fatty Acid Composition and ATPase Activity in Brassica rapa L. to Temperature in North China [J]. Acta Agron Sin, 2018, 44(01): 95-104.
[13] LYU Pin,YU Hai-Feng,YU Zhi-Xian,ZHANG Yong-Hu,ZHANG Yan-Fang,WANG Ting-Ting,HOU Jian-Hua. Construction of High-density Genetic Map and QTL Mapping for Seed Germination Traits in Sunflower under Two Water Conditions [J]. Acta Agron Sin, 2017, 43(01): 19-30.
[14] HE Xiao-Lan,WANG Jian-Wei,LI Wen-Xu,CHEN Zhen-Zhen,ZHAO Ji-Xin,WU Jun,WANG Zhong-Hua,CHEN Xin-Hong. Cloning of 6-SFT Gene from Leymus racemosus and Analysis of Tolerance to Drought and Cold Stresses in Transgenic Tobacco [J]. Acta Agron Sin, 2016, 42(03): 389-398.
[15] DU Hao,DING Lin-Yun,HE Man-Lin,CAI Cai-Ping,GUO Wang-Zhen*. Cloning and Functional Identification of Promoter Region of GhWRKY64Induced by Multi-stresses in Cotton (Gossypium hirsutum) [J]. Acta Agron Sin, 2015, 41(04): 593-600.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No. 80 Xueyuan South Rd, Beijing 100081, P. R. China E-mail: zwxb301@caas.cn
Supported by Beijing Magtech Co.Ltd