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Acta Agronomica Sinica ›› 2019, Vol. 45 ›› Issue (9): 1311-1318.doi: 10.3724/SP.J.1006.2019.81053

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

Function analysis of 5′ untranslated region introns in drought-resistance gene TaSAP1

CHANG Jian-Zhong1,DONG Chun-Lin1,ZHANG Zheng1,QIAO Lin-Yi2,YANG Rui1,JIANG Dan1,ZHANG Yan-Qin1,YANG Li-Li1,WU Jia-Jie3,JING Rui-Lian4,*()   

  1. 1 Dryland Agriculture Research Center, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
    2 Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
    3 Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong, China;
    4 National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-12-12 Accepted:2019-05-12 Online:2019-09-12 Published:2019-05-17
  • Contact: Rui-Lian JING E-mail:jingruilian@caas.cn
  • Supported by:
    This study was supported by the National Natural Science Foundation of China(31401385);the Key Project of Natural Science Foundation of Shanxi Province(201601D011001);the Optional Project of Advantage Research Group of Shanxi Academy of Agricultural Sciences(YYS1705)

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Abstract:

Stress associated proteins (SAPs), a group of A20/AN1 zinc-finger domain-containing proteins, are mainly involved in responding to abiotic stresses in plant. TaSAP1 involved in the responses of wheat to several abiotic stresses has two introns in the 5' untranslated region intron (5UIs). In this study, the two 5UIs were removed respectively by using overlapping PCR, and the expression vectors were constructed and transformed into Brachypodium distachyon via Agrobacterium-mediated transformation. The functions of TaSAP1 promoter and 5UIs were analyzed according to GUS activity. The activity of TaSAP1 promoter (P1) was up-regulated under the stresses of drought, cold and exogenous abscisic acid (ABA) by 10, 6, and 4 folds, respectively. The 5UIs were absolutely necessary for the promoter activity that lost in the double mutants of 5UIs. Intron-1 deletion led to 2.7-fold decrease (P<0.05) in GUS activity, whereas Intron-2 deletion resulted in that P1 was not able to respond to drought, low temperature and exogenous abscisic acid. These results provide basic information for further study in biological function of TaSAP1 5UIs.

Key words: wheat, 5′ untranslated region introns, promoter, quantitative GUS assay

Fig. 1

Sketch map of deletion mutation of TaSAP1 5UIs"

Fig. 2

Sketch map of overexpression vector construction"

Fig. 3

Sequence and cis-acting elements distribution of TaSAP1 promoter The cis-acting elements are highlighted in red."

Table 1

Main cis-acting elements and predicted functions in the TaSAP1 promoter sequence"

元件名称
Name		 序列
Sequence		 功能
Function		 位置
Location (bp)
ABRE GACACGTGGC/TACGTG ABA响应
cis-acting element involved in abscisic acid responsivenes		 -937 to -926,
-510 to -504
ARE AAACCA 低氧响应
cis-acting regulatory element essential for anaerobic induction		 -1256 to -1249
Box-W1 GGTCAA 真菌诱导响应
Fungal elicitor responsive element		 -2062 to -2055
CGTCA-motif CGTCA 茉莉酸甲酯响应
cis-acting regulatory element involved in MeJA-responsiveness		 -2107 to -2101
CCAAT-box CCGTTG MYB转录因子结合元件
MYB binding site		 -1176 to -1169,
-658 to -651
GC-motif CGGGGGC 低氧特异增强元件
Enhancer-like element involved in anoxic specific inducibility		 -1534 to -1526
LTR CCGAAA 低温响应
cis-acting element involved in low-temperature responsiveness		 -1233 to -1226,
-1207 to -2199
MBS TGACCGA MYB转录因子结合元件
MYB binding site		 -1448 to -1440,
-39 to -32
Skn-1 motif GTCAT 胚乳特异表达元件
cis-acting regulatory element required for endosperm expression		 -1323 to -1317,
-1108 to -1102
TGACG-motif TGACG 茉莉酸甲酯响应
cis-acting regulatory element involved in MeJA-responsiveness		 -1914 to -1908
WUN-motif TCATTGCGAA 伤害响应
Wound-responsive element		 -2103 to -2092

Fig. 4

Deletion mutation of TaSAP1 5UIs and construction of expression vectors M: marker III (Tiangen); A: 1st cycle PCR with primers TaspF1/R1 (lane 1) and TaspF2/R2 (lane 2); B: deletion of intron-1 by 2nd cycle PCR with TaspF1/R2; C: -2I and Δ2I fragments were amplified with primers TspF1/R3 (lane 1) and TspF1/R3 (lane 2), respectively; D: lanes 1 to 4 show the double digestion of P91z-P1, P91z-(-1I), P91z-(-2I), and P91z-Δ2I, respectively."

Fig. 5

Genetic transformation of Brachypodium distachyon A: callus differentiated from immature embryo of Brachypodium distachyon; B: brown embry ogenic callus; C and D: regeneration of transgenic plants; E and F: transgenic plants after transplanting."

Fig. 6

Histochemical staining and quantification of GUS activity in transgenic Brachypodium distachyon A: histochemical GUS staining analysis of transgenic (upper) and non-transgenic (lower) Brachypodium distachyon. The tissues from the left to right are leaf, young panicle, glume, seed, young stem and young root, respectively. B: quantification of GUS activity in transgenic Brachypodium distachyon. * indicate significant difference at P < 0.05 (t-test)."

Fig. 7

Response of promoter and 5UI deletion mutants of TaSAP1 to different stresses A: histochemical GUS staining activity in transgenic Brachypodium distachyon leaves under different stresses. B: quantification of GUS activity in transgenic Brachypodium distachyon under different stresses. NaCl, PEG, 4°C and ABA represents two-week Brachypodium distachyon plant treated with NaCl (250 mmol L-1), PEG-6000 (-0.5 MPa), 4°C and ABA (50 μmol L-1) for 8 h, respectively; CK is the control without stress treatment. * and ** indicate significant difference between a stress treatment and CK at the 0.05 and 0.01 probability levels, respectively."

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