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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).

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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."

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