Cloning and functional analysis of JcERF22 gene from Jatropha curcas

doi:10.3969/j.issn.1004-1524.20231186

Acta Agriculturae Zhejiangensis ›› 2024, Vol. 36 ›› Issue (10): 2219-2228.DOI: 10.3969/j.issn.1004-1524.20231186

• Horticultural Science • Previous Articles Next Articles

Cloning and functional analysis of JcERF22 gene from Jatropha curcas

TANG Yuehui(), CHEN Shuying, HE Wenqiong, WANG Hanjin, BAO Xinxin, JIA Sainan, WANG Yaoyao, CHEN Yuyang, YANG Tongwen

College of Life Science and Agronomy, Zhoukou Normal University, Zhoukou 466001, Henan, China

Received:2023-10-13 Online:2024-10-25 Published:2024-10-30

Abstract

Abstract:

AP2/ERF transcription factors play an important role in growth, development and responding to abiotic stress processes of plant. The object of this study was to isolate a Jatropha curcas AP2/ERF gene JcERF22, to analyze its expression response to abiotic stress, and to identify its role in regulating abiotic stress in transgenic Arabidopsis thaliana, so as to provide the molecular basis for the cultivation of stress-tolerant varieties of J. curcas. In this study, the JcERF22 gene was cloned from J. curcas using reverse transcription PCR (RT-PCR) technique. The sequence of JcERF22 gene was analyzed by bioinformatics. The expression pattern of JcERF22 gene under abiotic stress was analyzed using real time fluorogenic quantitative PCR (qRT-PCR). JcERF22 overexpressing A. thaliana plants were constructed using the pollen dip method, and the phenotypes of the transgenic plants were analyzed under normal growth, phosphorus deficiency, and salt stress. The results showed that the coding sequence (CDS) length of JcERF22 gene was 1 074 bp, encoding 357 amino acids. Subcellular localization analysis showed that JcERF22 protein was located in the nucleus. The expression level of JcERF22 gene in the leaves of J. curcas was significantly decreased under phosphorus deficiency and salt stress. Under normal conditions, overexpression of JcERF22 gene did not affect the growth and development of A. thaliana, but increased the length and number of root hairs. Under phosphorus deficiency stress, the anthocyanin content in the leaves of JcERF22 overexpressing A. thaliana was significantly lower than that of wild type, and the expression levels of anthocyanin synthesis related genes (CHS, DFR, F3H, LDOX, FLS1, UF3GT) were also significantly lower than those of wild type. Under salt stress, the leaves of JcERF22 overexpressing A. thaliana were seriously albino, and the fresh weight was significantly lower than that of the wild type, and the expression levels of abiotic stress-related genes (AtHKT1;1, AtP5CS1 ) were significantly lower than those of wild type. In summary, the JcERF22 gene plays an important role in the response of J. curcas to phosphorus deficiency and salt stress.

Key words: Jatropha curcas, JcERF22 gene, AP2/ERF transcription factor, phosphorus deficiency, salt stress

CLC Number:

TANG Yuehui, CHEN Shuying, HE Wenqiong, WANG Hanjin, BAO Xinxin, JIA Sainan, WANG Yaoyao, CHEN Yuyang, YANG Tongwen. Cloning and functional analysis of JcERF22 gene from Jatropha curcas[J]. Acta Agriculturae Zhejiangensis, 2024, 36(10): 2219-2228.

Figures/Tables 10

Table 1 Primers for real-time fluorescent quantitative PCR

基因Gene	正向引物Forward primer(5’→3’)	反向引物Reverse primer (5’→3’)
JcERF22	TTGAAAGGTGAACTGAAAAGGGA	GTGAAGAAGAACCAGCCAATGAT
UF3GT	ATCGAATGAATCGTCAAGCATGA	GAGGGATAGAGATGGTGTGGAAAG
CHS	AAGTTGGTCTCACCTTCCATCTCCT	TGTCGCCCTCATCTTCTCTTCCTT
DFR	TGGTCGGTCCATTCATCACAACG	CTTGGCGGCTGCTTGTTCGTATA
LDOX	TCTACGAGGGCAAATGGGTCACT	TCCGGCAACGGCTTAAGAACAATC
F3H	AGTGACGGAGGAGTATAGTGAGAGG	TGTAGCAGCAAGGTAATGGTTCCAG
FLS1	ATGCGTCAATTACAGATTCTGGCCT	TTCTTTCAACGCATCACGCTTTAAC
AtHKT1;1	GGCTGCAAAGACGCGAGTT	AGACGAGGGGTAAAGAATCCA
AtP5CS1	AGGCTTGCACTGTTGAAGTTGTAG	CTTCGTGATCCTCTGTCACAATG
JcActin	TAATGGTCCCTCTGGATGTG	AGAAAAGAAAAGAAAAAAGCAGC
AtActin2	GCACCCTGTTCTTCTTACCG	AACCCTCGTAGATTGGCACA

Fig.1 Amino acid sequence analysis of JcERF22 and its homologous proteins AtWIND1 (AT1G78080) and AtWIND2 (AT1G22190) were from Arabidopsis thaliana, ZmRAP2.4 (NP_001333350) was from Zea mays, RcRAP2.4 (XP_002533146) was from Ricinus communis, and JcERF22 (XP_012093083) was from Jatropha curcas.

Fig.2 Electrophoretogram of JcERF22 gene and double enzyme digestion result of its plant expression vector A, Electrophoretogram of JcERF22 gene; B, Double enzyme digestion result of JcERF22 gene plant expression vector.

Fig.3 Subcellular localization of JcERF22 protein

Fig.4 Expression of JcERF22 gene under phosphorus deficiency and salt stress in Jatropha curcas leaves * and ** indicated significant differences at P<0.05 and P<0.01 levels compared with the control, respectively.

Fig.5 Phenotype of JcERF22 overexpressing Arabidopsis A, Phenotype of JcERF22 overexpressing plants and wild type under normal growth conditions; B, Expression of JcERF22 gene in Arabidopsis plants; C, Phenotype of root hair of wild type and JcERF22 overexpressing plants at 4 days. WT, Wild type; OE1, OE2 and OE3, JcERF22 overexpressing plants.

Fig.6 Phenotypical and physiological characterization of JcERF22 overexpressing Arabidopsis under phosphorus deficiency stress A, Phenotype of wild-type and JcERF22 overexpressing plants under normal growth and phosphorus deficiency stress; B, Anthocyanin content; C, Carotenoid content. Data was detected based on fresh weight. ** indicated that the anthocyanin content of JcERF22 overexpressing plants under phosphorus deficiency stress was significantly different from that of wild type. WT, Wild type; OE1, OE2 and OE3, JcERF22 overexpressing plants.

Fig.7 Expression of anthocyanin-related genes in Arabidopsis leaves ** and * indicate that the expression of anthocyanin-related genes in transgenic plants was significantly different from that in the wild type under phosphorus deficiency stress (**P<0.01, *P<0.05). WT, Wild type; OE1, OE2 and OE3, JcERF22 overexpressing plants.

Fig.8 Phenotype and fresh weight of JcERF22 overexpressing Arabidopsis plants under salt stress A, Phenotype of wild-type and JcERF22 overexpressing plants under salt stress; B: Fresh weight of wild-type and JcERF22 overexpressing plants under salt stress. ** indicated that the fresh weight of JcERF22 overexpressing plants was significantly different from that of wild type under salt stress (P<0.01). WT, Wild type; OE1, OE2 and OE3, JcERF22 overexpressing plants.

Fig.9 Expression of abiotic stress-related gene in Arabidopsis leaves under salt stress ** indicated that the expression of abiotic stress-related genes in transgenic plants under salt stress was significantly different from that of wild type (P<0.01). WT, Wild type; OE1, OE2 and OE3, JcERF22 overexpressing plants.

References 20

[1]	JIANG S L, TANG Y Q, FAN R, et al. Response of Carex breviculmis to phosphorus deficiency and drought stress[J]. Frontiers in Plant Science, 2023, 14: 1203924.
[2]	RAMAIAH M, JAIN A, RAGHOTHAMA K G. Ethylene Response Factor070 regulates root development and phosphate starvation-mediated responses[J]. Plant Physiology, 2014, 164(3): 1484-1498.
[3]	MURAKAMI H, KAKUTANI N, KUROYANAGI Y, et al. MYB-like transcription factor NoPSR1 is crucial for membrane lipid remodeling under phosphate starvation in the oleaginous microalga Nannochloropsis oceanica[J]. FEBS Letters, 2020, 594(20): 3384-3394.
[4]	刘光瑞, 宗渊, 李云, 等. 当归转录因子AsMYB44的克隆与功能研究[J]. 浙江农业学报, 2023, 35(6): 1253-1264.
	LIU G R, ZONG Y, LI Y, et al. Cloning and functional research of MYB transcription factor AsMYB44 from Angelica sinensis[J]. Acta Agriculturae Zhejiangensis, 2023, 35(6): 1253-1264. (in Chinese with English abstract)
[5]	李小兰, 张瑞, 郝兰兰, 等. 桃NAC家族基因生物信息学分析及其响应低温胁迫的表达特征[J]. 浙江农业学报, 2022, 34(4): 766-780.
	LI X L, ZHANG R, HAO L L, et al. Bioinformatics analysis of peach NAC gene family and its expression characteristics in response to low temperature stress[J]. Acta Agriculturae Zhejiangensis, 2022, 34(4): 766-780. (in Chinese with English abstract)
[6]	JARAMBASA T, REGON P, JYOTI S Y, et al. Genome-wide identification and expression analysis of the Pisum sativum(L.) APETALA2/ethylene-responsive factor (AP2/ERF) gene family reveals functions in drought and cold stresses[J]. Genetica, 2023, 151(3): 225-239.
[7]	QI W W, SUN F, WANG Q J, et al. Rice ethylene-response AP2/ERF factor OsEATB restricts internode elongation by down-regulating a gibberellin biosynthetic gene[J]. Plant Physiology, 2011, 157(1): 216-228.
[8]	HUANG Y P, LIU L, HU H T, et al. Arabidopsis ERF012 is a versatile regulator of plant growth, development and abiotic stress responses[J]. International Journal of Molecular Sciences, 2022, 23(12): 6841.
[9]	QI Y T, YANG Z, SUN X Y, et al. Heterologous overexpression of StERF3 triggers cell death in Nicotiana benthamiana[J]. Plant Science, 2022, 315: 111149.
[10]	LEE P F, ZHAN Y X, WANG J C, et al. The AtERF19 gene regulates meristem activity and flower organ size in plants[J]. The Plant Journal, 2023, 114(6): 1338-1352.
[11]	ZHU J T, WEI X N, YIN C S, et al. ZmEREB57 regulates OPDA synthesis and enhances salt stress tolerance through two distinct signalling pathways in Zea mays[J]. Plant, Cell & Environment, 2023, 46(9): 2867-2883.
[12]	CHARFEDDINE M, CHIAB N, CHARFEDDINE S, et al. Heat,drought, and combined stress effect on transgenic potato plants overexpressing the StERF94 transcription factor[J]. Journal of Plant Research, 2023, 136(4): 549-562.
[13]	MAGHULY F, LAIMER M. Jatropha curcas, a biofuel crop: functional genomics for understanding metabolic pathways and genetic improvement[J]. Biotechnology Journal, 2013, 8(10): 1172-1182.
[14]	TANG Y H, QIN S S, GUO Y L, et al. Genome-wide analysis of the AP2/ERF gene family in physic nut and overexpression of the JcERF011 gene in rice increased its sensitivity to salinity stress[J]. PLoS One, 2016, 11(3): e0150879.
[15]	AN J P, LI R, QU F J, et al. R2R3-MYB transcription factor MdMYB23 is involved in the cold tolerance and proanthocyanidin accumulation in apple[J]. The Plant Journal, 2018, 96(3): 562-577.
[16]	CLOUGH S J, BENT A F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal, 1998, 16(6): 735-743.
[17]	YAN S S, CHEN N, HUANG Z J, et al. Anthocyanin fruit encodes an R2R3-MYB transcription factor, SlAN2-like, activating the transcription of SlMYBATV to fine-tune anthocyanin content in tomato fruit[J]. The New Phytologist, 2020, 225(5): 2048-2063.
[18]	CHEN Z H, NIMMO G A, JENKINS G I, et al. BHLH32 modulates several biochemical and morphological processes that respond to Pi starvation in Arabidopsis[J]. The Biochemical Journal, 2007, 405(1): 191-198.
[19]	AN D, CHEN J G, GAO Y Q, et al. AtHKT1 drives adaptation of Arabidopsis thaliana to salinity by reducing floral sodium content[J]. PLoS Genetics, 2017, 13(10): e1007086.
[20]	LV W T, LIN B, ZHANG M, et al. Proline accumulation is inhibitory to Arabidopsis seedlings during heat stress[J]. Plant Physiology, 2011, 156(4): 1921-1933.

Cloning and functional analysis of JcERF22 gene from Jatropha curcas

RichHTML

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 10

References 20

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	MA Zhonghua, WU Na, CHEN Juan, ZHAO Cong, YAN Chenghong, LIU Jili. Effects of salt stress and phosphorus supply on physiological characteristics of switchgrass seedlings [J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1205-1216.
[2]	LI Liyan, TAN Haixia, LI Jing, WANG Lianlong, DU Yinghui, XU Zhiwen. Screening of salt-tolerant growth-promoting Bacillus strains and their effect on oat growth under salt stress [J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1268-1276.
[3]	LIU Chen, XU Haobo, SI Yuyang, LI Yapeng, GUO Yuting, DU Changxia. Research progress on regulation mechanism of plant response to salt stress based on transcriptomics [J]. Acta Agriculturae Zhejiangensis, 2022, 34(4): 870-878.
[4]	YANG Xinxia, TANG Mansheng, ZHANG Bin. Identification of soybean PP2C family genes and transcriptome analysis in response to salt stress [J]. Acta Agriculturae Zhejiangensis, 2022, 34(2): 207-220.
[5]	LIU Tao, CHEN Hairong, WANG Chengzhong, REN Li, ZHANG Di. Physiology of stress resistance of Agapanthus praecox under drought and salt stress [J]. Acta Agriculturae Zhejiangensis, 2022, 34(12): 2669-2681.
[6]	MENG Na, XUE Hui, WEI Ming, WEI Shenghua. Ion characteristics on chloride channel blocker ameliorating salt injury to Glycine max [J]. Acta Agriculturae Zhejiangensis, 2022, 34(10): 2095-2104.
[7]	ZHOU Beining, MAO Lian, HUA Zhuangzhuang, LU Jianguo. Effects of alkaline salt stress on growth and ion allocation of Sinocalycanthus chinensis [J]. Acta Agriculturae Zhejiangensis, 2022, 34(1): 79-88.
[8]	YANG Xinxia, ZHANG Bin. Identification of soybean LAZ1 gene family and functional analysis of GmLAZ1-9 [J]. Acta Agriculturae Zhejiangensis, 2021, 33(4): 586-594.
[9]	LU Anqiao, ZHANG Fengju, WANG Xueqin, XU Xing. Effects of NaCl and Na₂SO₄ stress on content and distribution of K ⁺ and Na ⁺ of Echinochloa frumentacea seedlings [J]. Acta Agriculturae Zhejiangensis, 2021, 33(3): 396-403.
[10]	ZHAO Hua, REN Qingwen, WANG Xiyu, LI Zhenni, TANG Xiumei, JIANG Lihui, LIU Peng, XING Chenghua. Effects of arbuscular mycorrhizal fungi on antioxidant enzymes activities and photosynthetic characteristics of Solanum lycopersicum L. under salt stress [J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2075-2084.
[11]	MAO Shuang, ZHOU Wanli, YANG Fan, DI Xiaolin, LIN Jixiang, YANG Qingjie. Research progress on mechanism of plant roots response to salt-alkali stress [J]. Acta Agriculturae Zhejiangensis, 2021, 33(10): 1991-2000.
[12]	SONG Xindan, CHEN Binbin, MA Zengling, XU Lili, LIN Lidong, WU Mingjiang. Effects of salinity level on photosynthetic characteristics of Sargassum fusiforme seedlings [J]. , 2020, 32(9): 1634-1644.
[13]	SHI Jing, LIU Dongyang, ZHANG Fenghua. Physiological response and salt tolerance mechanism of cotton seedlings to salt stress [J]. , 2020, 32(7): 1141-1148.
[14]	ZHU Senlin, MEI Zhong, XING Chenghua. Inhibition of phosphorus deficiency on cadmium accumulation in Arabidopsis thaliana [J]. , 2020, 32(5): 804-809.
[15]	BIAN Jianwen, CUI Yan, YANG Songqi, LUO Guanghong, MENG Xiangang. Effects of Chlamydomonas debaryana Gor. and Anabaena azotica Ley. on wheat seedling growth under salt stress [J]. , 2020, 32(10): 1748-1756.