麻风树JcERF22基因的克隆与功能分析

doi:10.3969/j.issn.1004-1524.20231186

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (10): 2219-2228.DOI: 10.3969/j.issn.1004-1524.20231186

麻风树JcERF22基因的克隆与功能分析

唐跃辉(), 陈淑颖, 何文琼, 王涵瑾, 包欣欣, 贾赛男, 王瑶瑶, 陈宇阳, 杨同文

周口师范学院生命科学与农学学院,河南周口 466001

收稿日期:2023-10-13 出版日期:2024-10-25 发布日期:2024-10-30
作者简介:唐跃辉(1985—),男,河南许昌人,博士,副教授,研究方向为植物逆境胁迫。E-mail:yhtang2005@163.com
基金资助:
河南省高等学校重点科研项目(21A180028);河南省大学生创新创业训练计划(202310478039);河南省大学生创新创业训练计划(202310478037)

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

摘要/Abstract

摘要：

AP2/ERF转录因子在植物生长发育和响应非生物胁迫过程中起重要的调控作用,克隆麻风树AP2/ERF基因JcERF22,分析其对非生物胁迫的响应,鉴定其在调控拟南芥抗逆胁迫中的作用,为麻风树耐逆品种的培育提供依据。以麻风树为材料,利用逆转录PCR(RT-PCR)技术克隆麻风树JcERF22基因,利用生物信息学方法分析JcERF22基因序列,使用实时荧光定量PCR(qRT-PCR)技术分析非生物胁迫下JcERF22基因的表达模式,利用花粉浸染法构建JcERF22转基因拟南芥植株,并分析转基因植株在正常生长、缺磷和盐胁迫下的表型。结果显示,克隆的JcERF22基因编码区(CDS)长度为1074 bp,编码357个氨基酸。亚细胞定位结果显示,JcERF22蛋白定位于细胞核。在缺磷和盐胁迫条件下,麻风树叶片中JcERF22基因的表达量均显著降低。正常条件下,过表达JcERF22基因不影响拟南芥地上部分的生长发育,但增加了拟南芥的根毛长度和根毛数量;缺磷胁迫下,JcERF22过表达拟南芥叶片中的花青素含量显著低于野生型,花青素合成相关基因(CHS、DFR、F3H、LDOX、FLS1、UF3GT)的表达量也均显著低于野生型;盐胁迫下,JcERF22过表达拟南芥的叶片白化严重,鲜重显著低于野生型,非生物胁迫相关基因(AtHKT1;1、AtP5CS1)的表达量显著低于野生型。综上,JcERF22基因在麻风树响应缺磷和盐胁迫中发挥重要作用。

关键词: 麻风树, JcERF22基因, AP2/ERF转录因子, 缺磷, 盐胁迫

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

中图分类号:

唐跃辉, 陈淑颖, 何文琼, 王涵瑾, 包欣欣, 贾赛男, 王瑶瑶, 陈宇阳, 杨同文. 麻风树JcERF22基因的克隆与功能分析[J]. 浙江农业学报, 2024, 36(10): 2219-2228.

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.

图/表 10

表1 实时荧光定量PCR引物

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

图1 JcERF22及其同源蛋白氨基酸序列分析 AtWIND1(AT1G78080)和AtWIND2(AT1G22190)来自拟南芥,ZmRAP2.4(NP_001333350)来自玉米,RcRAP2.4(XP_002533146)来自蓖麻,JcERF22(XP_012093083)来自麻风树。

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.

图2 JcERF22基因及其植物表达载体双酶切电泳图 A,JcERF22基因电泳图;B,JcERF22基因植物表达载体双酶切结果。

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.

图3 JcERF22蛋白的亚细胞定位

Fig.3 Subcellular localization of JcERF22 protein

图4 麻风树叶片中JcERF22基因在缺磷和盐胁迫下的表达 *和**分别表示与对照相比在P<0.05和P<0.01水平差异显著。

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.

图5 JcERF22过表达拟南芥的表型 A,JcERF22过表达植株和野生型在正常生长条件下的表型;B,JcERF22基因在拟南芥中的表达;C,4 d的野生型和JcERF22过表达植株的根毛。WT,野生型;OE1、OE2、OE3为JcERF22过表达植株。

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.

图6 JcERF22过表达拟南芥在缺磷胁迫条件下的表型和生理分析 A,野生型和JcERF22过表达植株在正常生长和缺磷胁迫条件下的表型;B,花青素含量;C,类胡萝卜素含量。数据以鲜重计。**表示在缺磷胁迫条件下JcERF22过表达植株中花青素含量与野生型相比差异显著(P<0.01)。WT,野生型;OE1、OE2、OE3为JcERF22过表达植株。

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.

图7 拟南芥叶片中花青素相关基因的表达 **和*表示在缺磷胁迫条件下花青素相关基因在转基因植株中的表达与野生型相比差异显著(**P<0.01,*P<0.05)。WT,野生型;OE1、OE2、OE3为JcERF22过表达植株。

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.

图8 JcERF22过表达拟南芥在盐胁迫条件下的表型和鲜重 A,野生型和JcERF22过表达植株在盐胁迫条件下的表型;B,盐胁迫条件下野生型和JcERF22过表达植株的鲜重。**表示在盐胁迫条件下JcERF22过表达植株的鲜重与野生型相比差异显著(P<0.01)。WT,野生型;OE1、OE2、OE3为JcERF22过表达植株。

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.

图9 盐胁迫下拟南芥叶片中非生物胁迫相关基因的表达 **表示在盐胁迫条件下非生物胁迫相关基因在转基因植株中的表达与野生型相比差异显著(P<0.01)。WT,野生型;OE1、OE2、OE3为JcERF22过表达植株。

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.

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麻风树JcERF22基因的克隆与功能分析

Cloning and functional analysis of JcERF22 gene from Jatropha curcas

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