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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (7): 1750-1761.doi: 10.3724/SP.J.1006.2024.34171

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

Functional analysis of flax LuWRI1a in response to drought and salt stresses

LI Wen-Juan(), WANG Li-Min, QI Yan-Ni, ZHAO Wei, XIE Ya-Ping, DANG Zhao, ZHAO Li-Rong, LI Wen, XU Chen-Meng, WANG Yan, ZHANG Jian-Ping*()   

  1. Institute of Crop Research, Gansu Academy of Agricultural Sciences, Lanzhou 730070, Gansu, China
  • Received:2023-10-19 Accepted:2024-01-30 Online:2024-07-12 Published:2024-02-20
  • Contact: *E-mail: zhangjpzw3@gsagr.ac.cn
  • Supported by:
    National Natural Science Foundation of China(31460388);National Natural Science Foundation of China(32360502);China Agriculture Research System(CARS-14-1-05);China Agriculture Research System of MOF and MARA(2022GAAS04);Science and Technology Program of Lanzhou(2023-3-37)

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Abstract:

The AP2/ERF family of transcription factors is involved in the regulation of plant responses to biotic and abiotic stresses. Previously, we cloned LuWRI1a, a WRINKLED1 homologous gene from flax. Protein sequence analysis showed that LuWRI1a contained two AP2 DNA-binding domains and belonged to the AP2/ERF transcription factor family. The cis-acting elements of LuWRI1a were analyzed that pLuWRI1a was found containing multiple abiotic stress elements in response to light, drought, low temperature and hormones. In this study, the flax cultivar Longya 10 and LuWRI1a overexpression transgenic pure lines were used as the experimental materials, and salt stress and drought stress treatments were simulated with 200 mmol L-1 NaCl nutrient solution and 25% PEG nutrient solution. The results showed that the relative plant height, primary root length, lateral root number, and leaf number of transgenic plants were elevated after salt and drought stress treatments. The activities of three antioxidant enzymes were significantly higher than the control, while MDA content was lower. The relative expression levels of the abiotic stress-responsive genes, LuAREB, LuDREB, LuLEA, and LuNCED, were up-regulated. By exploring the biological function of LuWRI1a under adversity stress, it was found that LuWRI1a enhanced flax tolerance by resisting the inhibition of flax growth by salt stress and drought stress, enhancing the scavenging ability of reactive oxygen species, reducing the oxidative damage of membrane lipids, and activating the expression of adversity stress response genes. In summary, LuWRI1a may be a multifunctional gene, which was not only involved in fatty acid synthesis and metabolism pathway, but also may be involved in plant abiotic stress signaling pathway. This study provides a new genetic resource for the improvement of stress-tolerant varieties of flax.

Key words: flax, LuWRI1a, salt stress, drought stress, functional analysis

Table 1

Primers used for PCR"

基因名称
Gene name		 上游引物
Forward primer sequence (5′-3′)		 下游引物
Reverse primer sequence (5′-3′)
GAPDH
bar
LuWRI1a
LuAREB
LuDREB
LuLEA
LuNCED		 CTTTACCCTCAGCAAATCCG
AGTCCAGCTGCCAGAAAC
GATGATCAAGAAGCAGCTG
GCATGGAGAGACTCCAGCAA
TATGACACGGCGGTTTTCCA
GCCCGCAACTGTGAAGAAAG
GCTCCTTACCTCCATGCCTC		 AGGTTCTTCCCGCTCTCAAT
GGTCAACTTCCGTACCGA
CATTGCCACCTCAGCGGCC
GCAGTATCTATCTGCGGCGA
GGATGAGACCCCTCCTTCCT
TCCTGTTCTCGGGTCTGGAA
CCCTCTTGAGATGGTGAGCG

Fig. 1

Cis-acting elements of LuWRI1a gene promoter in flax Different colored boxes indicate different cis-acting elements in the scale at the bottom."

Table 2

Predicted cis-acting elements of LuWRI1a gene promoter"

调控元件
Cis-element		 核心序列
Sequence (5′-3′)		 位置
Position		 功能
Function
ACE CTAACGTATT -1239 光响应顺式作用元件
Cis-acting element involved in light responsiveness
AE-box AGAAACAA +144 光响应模块的组成部分
Part of a module for light response
ARE AAACCA -1057, -1801, +2406 厌氧诱导必需的顺式作用调控元件
Cis-acting regulatory element essential for the anaerobic induction
Box 4 ATTAAT +579, -2178 光响应保守模块的一部分
Part of a conserved DNA module involved in light responsiveness
CGTCA-motif CGTCA +963, +1169 MeJA响应顺式作用元件
Cis-acting regulatory element involved in the MeJA-responsiveness
GT1-motif GGTTAA(T) +17, -679, -680 光响应元件
Light responsive element
Gap-box CAAATGAA(A/G)A +524, +1038 光响应元件的一部分
Part of a light responsive element
LTR CCGAAA +1983 低温响应顺式作用元件
Cis-acting element involved in low-temperature responsiveness
MBS CAACTG +2127 参与干旱诱导的MYB结合位点
MYB binding site involved in drought-inducibility
MBSI aaaAaaC(G/C)GTTA -2215 参与干旱诱导的MYB结合位点
MYB binding site involved in flavonoid biosynthetic genes regulation
MYB CAACCA, TAACCA -16, +391, +681, -759, +1853, +2105, +2123 干旱、盐、ABA 和低温响应
Drought, salt, ABA, and low-temperature responsive
MYC CATTTG +73, +267, -524, -569, -1038, -1363 干旱和ABA响应
Drought and ABA responsive
O2-site GATGATGTGG +350 参与玉米醇溶蛋白新陈代谢调节的顺式调控元件
Cis-acting regulatory element involved in zein metabolism regulation
STRE AGGGG +1024, -1548, -1580, +1604, +2307 由热激、渗透应激、低pH值和营养缺乏激活的元件
Activated by heat shock, osmotic stress, low pH, and nutrient starvation
ERE ATTTTAAA, ATTTCATA +1439, -1905 乙烯响应元件
Ethylene-responsive element
TCA-element CCATCTTTTT +2343 水杨酸响应的顺式作用元件
Cis-acting element involved in salicylic acid responsiveness
TGACG-motif TGACG -963, -1169 MeJA 响应顺式作用元件
Cis-acting regulatory element involved in the MeJA-responsiveness

Fig. 2

Acquisition of transgenic flax A: agrobacterium tumefaciens infected the hypocotyl of flax; B: the adventitious bud formation induced by hypocotyl; C: rooting; D: transplanting of transformed seedlings."

Fig. 3

Molecular identification of transgenic flax A: PCR detection transgenic flax plants LuWRI1a; M: DL2000 DNA marker; 1-24: transgenic plants. B: the relative expression level of LuWRI1a genes; WT: wild type (flax cultivar Longya 10) and LuWRI1a over-expressed Longya 10 (OE-2, OE-20, and OE-22)."

Fig. 4

Performances of WT and transgenic flax under drought and salt stresses Performances of WT and transgenic flax after treatment of 25% PEG-6000 and 250 mmol L-1 NaCl for 14 days. WT: CK (Longya 10); OE-2, OE-20, and OE-22: LuWRI1a transgenic lines."

Fig. 5

Characteristics of transgenic plants under salt and drought stresses * and ** mean significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 6

Detection of enzyme activity of LuWRI1a-overexpressing flax * and ** mean significant difference at the 0.05 and 0.01 probability levels, respectively."

Fig. 7

Relative expression level of stress response genes in transgenic flax under drought and salt stresses * and ** mean significant difference at the 0.05 and 0.01 probability levels, respectively."

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