青花菜SKIP基因的克隆与表达分析

doi:10.3969/j.issn.1004-1524.2022.05.11

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (5): 966-973.DOI: 10.3969/j.issn.1004-1524.2022.05.11

青花菜SKIP基因的克隆与表达分析

韦海忠¹(), 潘丽芹¹, 汤紫依², 田盛野², 何海叶², 尹龙飞², 郑德伟², 张慧娟², 蒋明²^,^*()

1.台州科技职业学院,浙江台州 318020
2.台州学院生命科学学院,浙江台州 318000

收稿日期:2021-12-17 出版日期:2022-05-25 发布日期:2022-06-06
通讯作者: 蒋明
作者简介:* 蒋明,E-mail: jiangming1973@139.com
韦海忠(1968—),男,浙江东阳人,硕士,副教授,主要从事植物生理生化研究。E-mail: hzwei910@126.com
基金资助:
台州市科技计划(1901ny08);浙江省自然科学基金(LY19C150004)

Isolation and expression analysis of a SKIP gene from Brassica oleracea var. italica

WEI Haizhong¹(), PAN Liqin¹, TANG Ziyi², TIAN Shengye², HE Haiye², YIN Longfei², ZHENG Dewei², ZHANG Huijuan², JIANG Ming²^,^*()

1. Taizhou Vocational College of Science and Technology, Taizhou 318020, Zhejiang, China
2. College of Life Science, Taizhou University, Taizhou 318000, Zhejiang, China

Received:2021-12-17 Online:2022-05-25 Published:2022-06-06
Contact: JIANG Ming

摘要/Abstract

摘要：

SKIP(SKI-interacting protein)在植物的生长、发育和逆境响应中起着重要作用。以青花菜为材料,在克隆SKIP基因的基础上,利用生物信息学方法进行序列分析,并通过实时荧光定量PCR研究其在生物(寄生霜霉菌、野油菜黄单胞菌)、非生物(NaCl、PEG6000)胁迫下的表达模式。结果表明,青花菜SKIP基因全长为1 836 bp,没有内含子;BoiSKIP编码611个氨基酸,具1个SNW/SKI结构域,位于推导蛋白质序列的188-347处;该蛋白质的分子量为69.22 ku,理论等电点为9.15,平均亲水性指数为-1.12,为亲水性蛋白质。系统发育分析结果表明,芸薹属植物的SKIP聚为一组,支持率达100%,BoiSKIP与甘蓝SKIP的关系最近,它们处于同一分支。基因表达分析结果表明,BoiSKIP的表达受NaCl和PEG6000的诱导,表达量分别在诱导24、12 h达到最大;BoiSKIP受寄生霜霉菌和野油菜黄单胞菌的诱导,分别在处理24 h和72 h达到最大值。青花菜SKIP基因的克隆与表达分析,为后续开展该基因在抗逆反应中的功能研究奠定了基础。

关键词: 青花菜, 系统发育分析, 霜霉病, 黑腐病

Abstract:

SKIP (SKI-interacting protein) plays an important role in plant growth, development and stress responses. SKIP gene from Brassica oleracea var. italic was isolated and its sequence was analyzed by using bioinformatic tools. Besides, its expression patterns under biotic stresses (Hyaloperonospora parasitica and Xanthomonas campestris pv. campestris) and abiotic stresses (NaCl and PEG6000) were obtained by quantitative real-time PCR. The results indicated that broccoli SKIP was 1 836 bp in length with no intron identified. BoiSKIP encoded 611 amino acids and contained one SNW/SKI domain, which was located at 188-347 of the deduced protein sequences. The molecular weight and theoretical isoelectric point were 69.22 ku and 9.15, respectively, with a grand average of hydropathy index of -1.12, and BoiSKIP was identified as a hydrophilic protein. Phylogenetic analysis indicated that SKIPs from Brassica plants were grouped to the same clade, with a support rate of 100%. The closest relationship was found between BoiSKIP and cabbage SKIP, and they were gathered in the same branch. Expression analysis results indicated that BoiSKIP was induced by both NaCl and PEG6000, and the highest expression levels were observed at 24 h and 12 h, respectively. BoiSKIP was also induced by both H. parasitica and X. campestris pv. campestris, and the highest expression levels were found at 24 h and 72 h, respectively. Isolation and expression analysis of broccoli SKIP gene provided an insight into future study about its function on stress resistance responses.

Key words: Brassica oleracea var. italica, phylogenetic analysis, downy mildew, black rot

中图分类号:

S635

韦海忠, 潘丽芹, 汤紫依, 田盛野, 何海叶, 尹龙飞, 郑德伟, 张慧娟, 蒋明. 青花菜SKIP基因的克隆与表达分析[J]. 浙江农业学报, 2022, 34(5): 966-973.

WEI Haizhong, PAN Liqin, TANG Ziyi, TIAN Shengye, HE Haiye, YIN Longfei, ZHENG Dewei, ZHANG Huijuan, JIANG Ming. Isolation and expression analysis of a SKIP gene from Brassica oleracea var. italica[J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 966-973.

图/表 6

图1 青花菜BoiSKIP基因序列及其推导的蛋白质序列高亮部分为SNW/SKI结构域。

Fig.1 Sequence of broccoli BoiSKIP gene and its deduced protein The SNW/SKI domain was highlighted.

图2 BoiSKIP的亲水性/疏水性预测

Fig.2 Hydrophobic/hydrophilic character predication of BoiSKIP

图3 不同植物SKIP同源蛋白的SNW/SKI结构域比对 XP_013688403,甘蓝型油菜;XP_018456548,萝卜;CAA7013451,Microthlaspi erraticum;KAF2606023,Brassica cretica;OAP19162,拟南芥;RID56290,芜菁;XP_006301006,荠菜;XP_006390116,山嵛菜;XP_010471904,亚麻荠;XP_013591577,甘蓝;XP_020890221,琴叶拟南芥;XP_031736674,黄瓜。

Fig.3 Comparisons of SNW/SKI domains of SKIP from different plants XP_013688403, Brassica napus; XP_018456548, Raphanus sativus; CAA7013451, Microthlaspi erraticum; KAF2606023, Brassica cretica; OAP19162, Arabidopsis thaliana; RID56290, Brassica rapa; XP_006301006, Capsella rubella; XP_006390116, Eutrema salsugineum; XP_010471904, Camelina sativa; XP_013591577, Brassica oleracea var. oleracea; XP_020890221, A. lyrata subsp. lyrata; XP_031736674, Cucumis sativus.

图4 青花菜BoiSKIP及其同源序列的系统发育树

Fig.4 Phylogenetic tree of BoiSKIP and its homologous sequences

图5 BoiSKIP基因在非生物胁迫下的表达

Fig.5 Expression of BoiSKIP under abiotic stresses

图6 BoiSKIP基因在病原菌诱导下的表达 A,寄生霜霉菌处理;B,野油菜黄单胞菌处理。

Fig.6 Expression of BoiSKIP challenged by pathogens A, Hyaloperonospora parasitica ; B, Xanthomonas campestris pv. campestris.

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青花菜SKIP基因的克隆与表达分析

Isolation and expression analysis of a SKIP gene from Brassica oleracea var. italica

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