Welcome to Acta Agronomica Sinica，

Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (02): 218-226.doi: 10.3724/SP.J.1006.2018.00218

• Orginal Article • Previous Articles     Next Articles

Cloning and Expression Analysis of Galactosyltransferase Gene GhGalT1 Promoter in Cotton

Li-Xia QIN, Jing LI, Huan-Yang ZHANG, Sheng LI, Meng-Jie ZHU, Gai-Li JIAO, Shen-Jie WU*   

  1. Institute of Cotton, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, Shanxi, China;
  • Received:2017-04-11 Accepted:2017-09-10 Online:2018-02-12 Published:2017-10-27
  • Contact: Shen-Jie WU
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (31601350), the Shanxi Province Fundamental Research Foundation (2015021152), and the National Major Project for Developing New GM Crops (2016ZX08005).

RichHTML319

14

PDF

1397

Abstract:

Glycosytransferases (GTs) transfer an activated sugar donor to a specific acceptor to form glucosidic bond, which are regulated by various abiotic and biotic stresses, and may play a role in plant responses to changes in living conditions. In this study, a 539 bp fragment of GhGalT1 5′-flanking sequence was isolated from upland cotton Coker 312 by PCR, designated pGhGalT1. Analysis of pGhGalT1 sequence by PlantCARE revealed it contained not only putative CAAT box, TATA box sequence, but also MBS, HSE, TC-rich repeats, MYCCONSE and CGTCA-motif cis-acting element which involved in drought, heat, dehydration, defense and stress responsiveness. Thus, we constructed it into pBI101-GUS vector and formed pGhGalT1::GUS fusion expression vector (pBI101-pGhGalT1-GUS), then transferred the vector into Arabidopsis by the Agrobacterium-mediated floral dip method, and successfully obtained positive transgenic plants by screening test of resistance to kanamycin and PCR detection. Histochemical assay of T3 generation of transgenic Arabidopsis revealed that GUS activities were mainly accumulated in root tips of primary and lateral roots in 5- to 15-day-old seedlings, and less strongly in cotyledons and rosette leaves. The histochemical staining results and the assay of quantitative GUS activity and GUS gene expression under abiotic stresses and hormone treatments revealed that the GhGalT1 promoter was salt-/osmotic-/6-BA-/MeJA-/BL-inducible. These findings contribute to the selection of a suitable promoter for crop molecular improvement.

Key words: cotton (Gossypium hirsutum L.), glycosytransferase, promoter, cis-acting element, GUS histochemical staining, quantitative GUS assay

Table 1

Primers used in this study"

引物名称
Primer name		 上游引物序列
Forward primer sequence (5′-3′)		 下游引物序列
Reverse primer sequence (5′-3′)
pGhGalT1 GGGGTCGACTAACAACGGTGAAGAAAGAA GGTGGATCCAACTGTGAATTTGAAGAGAAG
GUS ATGTTACGTCCTGTAGAAACC TCATTGTTTGCCTCCCTGCTGC
NPTII ATGATTGAACAAGATGGATTGC TCAGAAGAACTCGTCAAGAAGGC
AtActin2 GAAATCACAGCACTTGCACC AAGCCTTTGATCTTGAGAGC
GUS-RT ATGTTCTGCGACGCTCAC CCCGGCTAACGTATCTGT

Fig. 1

Map of construction of the pBI101-pGhGalT1-GUS expression vector"

Fig. 2

Amplified fragment of GhGalT1 gene promoter in cotton M: DNA marker DL2000; 1: negative control; 2: GhGalT1 promoter sequence."

Fig. 3

Sequence and predicted cis-acting elements of GhGalT1 promoter"

Table 2

The cis-acting elements and predicted functions in the GhGalT1 promoter sequence"

顺式作用元件
cis-acting element		 核心序列
Core sequence		 位置
Location (bp)		 功能
Function
3-AF binding site CACTATCTAAC -472 to -462 光响应元件 Light responsive element
ATCT-motif AATCTAATCT -401 to -392 光响应元件
Part of a conserved DNA module involved in light responsiveness
Box 4 ATTAAT -505 to -499 光响应元件
Part of a conserved DNA module involved in light responsiveness
Box 1 TTTCAAA -387 to -381 光响应元件Light responsive element
GAG-motif AGAGAGT -246 to -240 光响应元件 Part of a light responsive element
MYCCONSE CACCTG -298 to -293 脱水响应 Dehydration responsiveness
CGTCA-motif CGTCA -175 to -171 茉莉酸甲酯响应元件 MeJA responsive element
MBS CAACTG -141 to -136 干旱诱导的MYB 结合位点
MYB binding site involved in drought-inducibility
TC-rich repeats ATTTTCTTCA -460 to -451 防御及胁迫响应元件
Cis-acting element involved in defense and stress responsiveness
HSE AAAAAATTTC -249 to -259 热胁迫响应元件 Heat stress responsiveness
Sp 1 CC(G/A)CCC -152 to -147 光响应元件Light responsive element
Skn-1 motif GTCAT -520 to -524, -325 to -321,
-206 to -202		 胚乳特异表达
Endosperm specific expression
TCCC-motif TCTCCCT -156 to -148 光响应元件 Light responsive element
Circadian CAANNNNATC -364 to -355 调控生物节律
Cis-acting regulatory element involved in circadian control

Fig. 4

Vector construction of GhGalT1 promoter M: DNA marker DL2000; A: electrophoregram of pBI101-pGhGalT1-GUS plasmid detected by PCR; 1 and 2: negative control; 3: positive control; 4-5: pBI101-pGhGalT1-GUS plasmid; B: electrophoregram of pBI101-pGhGalT1-GUS plasmid digested by Sal I and BamH I."

Fig. 5

Kanamycin resistance screening of T1 generation transgenic positive plants Arrowheads indicate seedlings with kanamycin resistance."

Fig. 6

Positive detection in transgenic Arabidopsis lines by PCR A-C: electrophoregram of positive detection by PCR in some T1 generation of transgenic Arabidopsis with NPTII primers (A), pGhGalT1 primers (B) and GUS primers (C), respectively; D: electrophoregram of positive detection by PCR in T3 generation of transgenic Arabidopsis with pGhGalT1 primers; 1: wild type; 2: positive control; 3-11: different lines from T1 and T3 generations of transgenic plants."

Fig. 7

Histochemical assay of GUS activity in pGhGalT1:GUS transgenic Arabidopsis A-B: five-day-old seedlings; B: magnification of root regions of five-day-old seedlings; C-D: ten-day-old seedlings; D: magnification of root tip regions of ten-day-old seedlings; E-F: 15-day-old seedlings; F: magnification of root tip regions of 15-day-old seedlings; G-H: mature leaf; H: magnification of mature leaf regions of G."

Fig. 8

Histochemical assay of GUS activity of GhGalT1 promoter in transgenic Arabidopsis under stresses A-C: ten-day-old seedlings in mock treatment; D-N: ten-day-old seedlings treated with 150 mmol L-1 NaCl (D-E), 300 mmol L-1 mannitol (F-G), 100 μmol L-1 6-BA (H-J), 50 μmol L-1 MeJA (K-L) or 500 nmol L-1 BL (M-N). B, C: magnification of root tip and cotyledon in ten-day-old seedlings in mock treatment; E: magnification of root tip in ten-day-old seedlings under 150 mmol L-1 NaCl treatment; G: magnification of root tip in ten-day-old seedlings under 300 mmol L-1 mannitol treatment; I, J: magnification of root tip and cotyledon in ten-day-old seedling with 100 μmol L-1 6-BA treatment; L: magnification of root tip in ten-day-old seedlings with 50 μmol L-1 MeJA treatment; N: magnification of root tip in ten-day-old seedlings under 500 nmol L-1 BL treatment."

Fig. 9

Quantification of GUS activity and GUS gene in transgenic Arabidopsis under different stresses NaCl: sodium chloride; MeJA: methyl jasmonate; BL: brassinolide; 6-BA: 6-Benzylaminopurine. Mean values and SE (bar) were shown from three independent experiments (n > 50). The capitals and lowercase indicate significant difference between stress treatment and the control at the 0.01 and 0.05 probability levels, respectively (t-test). WT: wild type; L2 and L6: pGhGalT1:GUS transgenic lines 2 and 6."

[1] Campbell J A, Davies G J, Bulone V V, Henrissat B.A classification of nucleotide-diphospho-sugar glycosyltransferases based on amino acid sequence similarities.Biochem J, 1997, 326: 929-939
[2] Lao J, Oikawa A, Bromley J R, McInerney P, Suttangkakul A, Smith-Moritz A M, Plahar H, Chiu T Y, Gonzalez Fernandez-Nino S M, Ebert B, Yang F, Christiansen K M, Hansen S F, Stonebloom S, Adams P D, Ronald P C, Hillson N J, Hadi M Z, Vega-Sanchez M E, Loque D, Scheller H V, Heazlewood J L. The plant glycosyltransferase clone collection for functional genomics.Plant J, 2014, 79: 517-529
[3] Coutinho P M, Deleury E, Davies G J, Henrissat B.An evolving hierarchical family classification for glycosyltransferases.J Mol Biol, 2003, 328: 307-317
[4] Jackson R G, Kowalczyk M, Li Y, Higgins G, Ross J, Saiidberg G, Bowles D J.Overexpression of anArabidopsis gene encoding a glucosyltransferase of indole-3-acetic acid: phenotypic characterisation of transgenic lines. Plant J, 2002, 32: 573-583
[5] Jackson R Q, Lim E K, Li Y, Kowalczyk M, Saiidberg G, Hoggett J, Ashford D A, Bowles D J.Identification and biochemical characterization of anArabidopsis indole-3-acetic acid glucosyltransferase. J Biol Chem, 2001, 276: 4350-4356
[6] Tognetti V B, Van Aken O, Morreel K, Vandenbroucke K, van de Cotte B, De Clercq I, Chiwocha S, Fenske R, Prinsen E, Boerjan W, Genty B, Stubbs K A, Inze D, Van-Breusegem F. Perturbation of indole-3-butyric acid homeostasis by the UDP-glucosyltransferase UGT74E2 modulatesArabidopsis architecture and water stress tolerance. Plant Cell, 2010, 22: 2660-2679
[7] Martin R C, Mok D W, Sniels R, Van Oiickelen H A, Mok M C. Development of transgenic tobacco harboring a zeatin O-glucosyltransferase gene fromPhaseolus. In Vitro Cell Dev Biol Plant, 2001, 37: 354-360
[8] Wang J, Ma X M, Kojima M, Sakakibara H, Hou B K.N-glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response inArabidopsis thaliana. Plant Cell Physiol, 2011, 52: 2200-2213
[9] Wang J, Ma X M, Kojima M, Sakakibara H, Hon B K.Glucosyltransferase UGT76C1 finely modulates cytokinin responses via cytokinin N-glucosylation inArabidopsis thahana. Plant Physiol Biochem, 2013, 65: 9-16
[10] Poppenberger B, Fujioka S, Soeno K, George G L, Vaistij F E, Hiranuma S, Seto H, Takatsuto S, Adam G, Yoshida S, Bowles D J.The UGT73C5 ofArabidopsis thaliana glucosylates brassinosteroids. Proc Natl Acad Sci USA, 2005, 102: 15253-15258
[11] Suzuki H, Fujioka S, Takatsuto S, Yokota T, Murofushi N, Sakurai A.Biosynthesis of brassinolide from teasterone via typhasterol and castaserone in cultured cells ofCatharanthus roseus. J Plant Growth Regul, 1993, 13: 21-26
[12] Husar S, Berthiller F, Fujioka S, Rozhon W, Khan M, Kalaivanan F, Elias L, Higgins G S, Li Y, Schuhmacher R, Krska R, Seto H, Vaistij F E, Bowles D, Poppenberger B.Overexpression of the UGT73C6 alters brassinosteroid glucoside formation inArabidopsis thaliana. BMC Plant Biol, 2011, 11: 51
[13] Glauser G, Boccard J, Rudaz S, Wolfender J L.Mass spectrometry-based metabolomics oriented by correlation analysis for wound-induced molecule discovery: identification of a novel jasmonate glucoside.Phytochem Anal, 2010, 21: 95-101
[14] Lim C E, Ahn J H, Lim J.Molecular genetic analysis of tandemly located glycosyltransferase genes, UGT73BI, UGT73B2, and UG17383, inArabidopsis thaliana. J Plant Biol, 2006, 49: 309-314
[15] Lim C E, Choi N J, Kim A, Lee S A, Huang Y S, Lee C H, Lim J.Improved resistance to oxidative stress by a loss-of-function mutation in theArabidopsis UGT71C1 gene. Mol Cells, 2008, 25: 368-375
[16] Kim A, Heo J O, Chang K S, Lee S A, Lee M H, Lim C E, Lim J.Overexpression and inactivation of UGT73B2 modulate tolerance to oxidative stress inArabidopsis. J Plant Biol, 2010, 53: 233-239
[17] Song J T, Koo Y J, Seo H S, Kim M C, Choi Y D, Kim J H.Overexpression ofAtSGTl, an Arabidopsis salicylic acid glucosyltransferase, leads to increased susceptibility to Pseudomonas syringae. Phytochemistry, 2008, 69: 1128-1134
[18] 李田, 孙景宽, 刘京涛. 植物启动子研究进展. 生物技术通报, 2015, 31(02): 18-25
Li T, Sun J K, Liu J T.Research advances on plant promoter.Biotechnol Bull, 2015, 31(02): 18-25 (in Chinese with English abstract)
[19] Li F, Han Y Y, Feng Y N, Xing S C, Zhao M R, Chen Y H, Wang W.Expression of wheat expansin driven by the RD29 promoter in tobacco confers water-stress tolerance without impacting growth and development.J Biotechnol, 2013, 163: 281-291
[20] Pino M T, Skinner J S, Park E J, Jeknic Z, Hayes P M, Thomashow M F, Chen T H.Use of a stress inducible promoter to drive ectopic AtCBF expression improves potato freezing tolerance while minimizing negative effects on tuber yield.Plant Biotechnol J, 2007, 5: 591-604
[21] Qin L X, Rao Y, Li L, Huang J F, Xu W L, Li X B.CottonGalT1 encoding a putative glycosyltransferase is involved in regulation of cell wall pectin biosynthesis during plant development. PLoS One, 2013, 8: e59115
[22] Li X B, Lin C, Cheng N H, Liu J W.Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fibers.Plant Physiol, 2002, 130: 666-674
[23] Clough S J, Bent A F.Floral dip: a simplified method forAgrobacterium mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16: 735-743
[24] Qin L X, Li Y, Li D D, Xu W L, Zheng Y, Li X B.Arabidopsis drought-induced protein Di19-3 participates in plant response to drought and high salinity stresses.Plant Mol Biol, 2014, 86: 609-625
[25] Jefferson R A.Assaying chimeric genes in plants: theGus gene fusion system. Plant Mol Biol Rep, 1987, 5: 387-405
[26] Li X B, Fan X P, Wang X L, Cai L, Yang W C.The cottonActin1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell, 2005, 17: 859-875
[27] Wang X, Tan Y P, Zhou J, Wang C T, Liu X Q.Expression of a tobacco glycosyltransferase gene driving promoter in transgenic tobacco.Agric Sci Technol, 2010, 11: 83-85
[28] Luo K, Zhang G, Deng W, Luo F, Qiu K, Pei Y.Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco.Plant Cell Rep, 2008, 27: 707-717
[29] Singh H, Sen R, Baltimore D, Sharp P A.A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes.Nature, 1986, 319: 154-158
[30] Li J J, Herskowitz I.Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system.Science, 1993, 262: 1870-1874
[31] 贾笑英, 向云, 张金文, 王蒂. 马铃薯损伤诱导型启动子Wun1基因的克隆及其GFP表达活性. 分子植物育种, 2006, 4: 333-338
Jia X Y, Xiang Y, Zhang J W, Wang D.Cloning of potato wound-inducible promoterWun1 and its GFP express activity. Mol Plant Breed, 2006, 4: 333-338 (in Chinese with English abstract)
[32] 周潇, 姜航, 屈汉金, 邓子牛, Gentile A, 龙桂友. 柑橘冷诱导基因及其启动子表达载体构建与瞬时表达分析. 果树学报, 2015, 32: 353-358
Zhou X, Jiang H, Qu H J, Deng Z N, Gentile A, Long G Y.Construction of plant vectors with promoter and cold-induced genes in citrus and transient expression verification. J Fruit Sci, 2015, 32: 353-358 (in Chinese with English abstract)
[33] 魏桂民, 张金文, 王蒂, 张俊莲, 陆艳梅, 高宜峰. 马铃薯Sgt1基因启动子的结构及功能分析. 中国生物化学与分子生物学报, 2013, 29: 969-977
Wei G M, Zhang J W, Wang D, Zhang J L, Lu Y M, Gao Y F.Promoter analysis of potatoSgt1 gene. J Biochem Mol Biol, 2013, 29: 969-977 (in Chinese with English abstract)
[34] 郭新勇, 程晨, 张选, 祝建波. 拟南芥冷诱导型启动子CBF3驱动IPT基因在烟草中的表达. 西北农业学报, 2012, 21: 123-131
Guo X Y, Cheng C, Zhang X, Zhu J B.Expression ofIPT gene linked with cold-induced promoter CBF3 from Arabidopsis thaliana in tobacco. Acta Agric Boreali-Occident Sin, 2012, 21: 123-131 (in Chinese with English abstract)
[35] 杨春霞, 陈英, 黄敏仁, 李火根. 拟南芥逆境诱导型启动子rd29A的克隆及活性检测. 南京林业大学学报(自然科学版), 2008, 32: 6-10
Yang C X, Chen Y, Huang M R, Li H G.Cloning of stress-inducible promoter rd29A from Arabidopsis thaliana and its activity detection in transgenic tobacco.J Nanjing For Univ (Nat Sci Edn), 2008, 32: 6-10 (in Chinese with English abstract)
[36] 杜皓, 丁林云, 何曼林, 蔡彩平, 郭旺珍. 受多逆境诱导表达的GhWRKY64基因启动子克隆与功能分析. 作物学报, 2015, 41: 593-600
Du H, Ding L Y, He M L, Cai C P, Guo W Z.Cloning and functional identification of promoter region ofGhWRKY64 induced by multi-stresses in cotton(Gossypium hirsutum). Acta Agron Sin, 2015, 41: 593-600 (in Chinese with English abstract)
[37] 扆珩, 李昂, 刘惠民, 景蕊莲. 小麦蛋白磷酸酶2A基因TaPP2AbB″-α启动子的克隆及表达分析. 作物学报, 2016, 42: 1282-1290
Yi H, Li A, Liu H M, Jing R L.Cloning and expression analysis of protein phosphatase 2A geneTaPP2AbB″-α promoter in wheat. Acta Agron Sin, 2016, 42: 1282-1290 (in Chinese with English abstract)
[1] Wang Yi-Han, Li Fu-Chang, Liu Yi, Zhu Guo-Peng. Cloning of the IbOPR2 gene promoter and identification of regulatory factors in sweetpotato [J]. Acta Agronomica Sinica, 2026, 52(4): 1268-1276.
[2] SHEN Ao, LIU Min, NI Di-An, LIU Wei. Promoter characterization and expression pattern analysis of the m6A methyltransferase gene SiMTA1 in foxtail millet [J]. Acta Agronomica Sinica, 2025, 51(7): 1969-1978.
[3] HUANG Meng-Xin, ZHUANG Ling-Ling, CHENG Pei-Pei, LI Qin, XU Jian-Tang, TAO Ai-Fen, FANG Ping-Ping, QI Jian-Min, ZHANG Li-Wu. Cloning and transcriptional activity analysis of U6 promoter in jute (Corchorus capsularis) [J]. Acta Agronomica Sinica, 2025, 51(5): 1156-1165.
[4] LIU Yong-Hui, SHEN Yi, SHEN Yue, LIANG Man, SHA Qin, ZHANG Xu-Yao, CHEN Zhi-De. Cloning and functional analysis of drought-inducible promoter AhMYB44-11- Pro in peanut (Arachis hypogaea L.) [J]. Acta Agronomica Sinica, 2024, 50(9): 2157-2166.
[5] MAO Yan, ZHENG Ming-Min, MOU Cheng-Xiang, XIE Wu-Bing, TANG Qi. Function analysis of the promoter of natural antisense transcript cis- NATZmNAC48 in maize under osmotic stress [J]. Acta Agronomica Sinica, 2024, 50(2): 354-362.
[6] ZHOU Zhi-Man, ZHANG Xiao-Yu, GAO Feng, DAI Zhi-Gang, XU Ying, CHENG Chao-Hua, YANG Ze-Mao, SU Jian-Guang, TANG Qing. Cloning and functional analysis of promoter of CsMIXTA associated with development of glandular trichome in industrial hemp [J]. Acta Agronomica Sinica, 2024, 50(11): 2754-2763.
[7] ZUO Chun-Yang, LI Ya-Wei, LI Yan-Long, JIN Shuang-Xia, ZHU Long-Fu, ZHANG Xian-Long, MIN Ling. Relative expression patterns of laccase gene family members in upland Gossypium hirsutum L. [J]. Acta Agronomica Sinica, 2023, 49(9): 2344-2361.
[8] XU Nai-Yin, WANG Yang, WANG Dan-Tao, NING He-Jia, YANG Xiao-Ni, QIAO Yin-Tao. Construction of cotton fiber quality index and weighted genotype by trait (WGT) biplot analysis [J]. Acta Agronomica Sinica, 2023, 49(5): 1262-1271.
[9] NIU Zhi-Yuan, QIN Chao, LIU Jun, WANG Hai-Ze, LI Hong-Yu. Function analysis of different Cas9 promoters on the efficiency of CRISPR/ Cas9 system in soybean [J]. Acta Agronomica Sinica, 2023, 49(12): 3227-3238.
[10] ZHOU Yue, ZHAO Zhi-Hua, ZHANG Hong-Ning, KONG You-Bin. Cloning and functional analysis of the promoter of purple acid phosphatase gene GmPAP14 in soybean [J]. Acta Agronomica Sinica, 2022, 48(3): 590-596.
[11] CHEN Xiang-Qian, JIANG Qi-Yan, SUN Xian-Jun, NIU Feng-Juan, ZHANG Hui-Yuan, HU Zheng, ZHANG Hui. Construction and application of soybean CRISPR/Cas9 multiplex editing vector [J]. Acta Agronomica Sinica, 2022, 48(11): 2706-2714.
[12] ZHAO Wen-Qing, XU Wen-Zheng, YANG Liu-Yan, LIU Yu, ZHOU Zhi-Guo, WANG You-Hua. Different response of cotton leaves to heat stress is closely related to the night starch degradation [J]. Acta Agronomica Sinica, 2021, 47(9): 1680-1689.
[13] SHI Lei, MIAO Li-Juan, HUANG Bing-Yan, GAO Wei, ZHANG Zong-Xin, QI Fei-Yan, LIU Juan, DONG Wen-Zhao, ZHANG Xin-You. Characterization of the promoter and 5'-UTR intron in AhFAD2-1 genes from peanut and their responses to cold stress [J]. Acta Agronomica Sinica, 2021, 47(9): 1703-1711.
[14] XU Nai-Yin, ZHAO Su-Qin, ZHANG Fang, FU Xiao-Qiong, YANG Xiao-Ni, QIAO Yin-Tao, SUN Shi-Xian. Retrospective evaluation of cotton varieties nationally registered for the Northwest Inland cotton growing regions based on GYT biplot analysis [J]. Acta Agronomica Sinica, 2021, 47(4): 660-671.
[15] WANG Xiao-Chun, WANG Lu-Lu, ZHANG Zhi-Yong, QIN Bu-Tan, YU Mei-Qin, WEI Yi-Hao, MA Xin-Ming. Transcription characteristics of wheat glutamine synthetase isoforms and the sequence analysis of their promoters [J]. Acta Agronomica Sinica, 2021, 47(4): 761-769.
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