灰枣扩展蛋白基因ZjEXPA8的克隆及序列分析

doi:10.6048/j.issn.1001-4330.2023.09.017

新疆农业科学 ›› 2023, Vol. 60 ›› Issue (9): 2223-2230.DOI: 10.6048/j.issn.1001-4330.2023.09.017

• 园艺特产·贮藏保鲜加工·林业 • 上一篇下一篇

灰枣扩展蛋白基因ZjEXPA8的克隆及序列分析

靳娟(), 苏比娜·肖克来提, 阿布都卡尤木·阿依麦提, 杨磊, 郝庆, 樊丁宇()

新疆农业科学院园艺作物研究所/农业部新疆地区果树科学观测实验站,乌鲁木齐 830091

收稿日期:2022-11-29 出版日期:2023-09-20 发布日期:2023-09-19
通信作者: 樊丁宇(1983-),男,山西忻州人,研究员,研究方向为果树栽培与生理,(E-mail)fandingyu2413@163.com
作者简介:靳娟(1990-),女,江苏徐州人,助理研究员,研究方向为果树品质调控及逆境分子生物学, (E-mail)475063956@qq.com
基金资助:
国家自然科学基金项目(32160684);新疆红枣产业技术体系专项资金项目([2020-2021]XJCYTX-01);现代农业产业技术体系专项资金(CARS-30)

Cloning and sequence analysis of expansin gene ZjEXPA8 in Huizao

JIN Juan(), Subina Xiaokelaiti, Abudoukayoumu Ayimaiti, YANG Lei, HAO Qing, FAN Dingyu()

Scientific Observing and Experimental Station of Pomology (Xinjiang),Ministry of Agriculture and Rural Affairs of the P.R.C. / Research Institute of Horticultural Crops, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China

Received:2022-11-29 Online:2023-09-20 Published:2023-09-19
Correspondence author: FAN Dingyu (1983-), male, Shanxi Province, researcher, research direction: fruit tree cultivation and physiology,(E-mail)fandingyu2413@163.com
Supported by:
National Natural Science Foundation of China(32160684);Xinjiang Jujube Research System([2020-2021]XJCYTX-01);Earmarked Fund for CARS(CARS-30)

摘要/Abstract

摘要：

【目的】克隆灰枣扩展蛋白基因ZjEXPA8,分析其序列特征,为研究该基因在灰枣果实发育过程中的作用和功能奠定基础。【方法】以不同发育形态的灰枣花和幼果为试材,根据灰枣ZjEXPA8基因(LOC107414006)序列,应用Primer Premier 5.0软件设计引物,RT-PCR技术克隆ZjEXPA8基因,利用生物信息学方法分析ZjEXPA8核苷酸序列。【结果】ZjEXPA8基因的开放阅读框为762 bp(基因登录号为NP_001310794.1),编码253个氨基酸,预测蛋白分子量为 26.91 kDa,等电点为8.32,不稳定系数为30.09,脂肪指数为66.72,总亲水性平均数为-0.107,属于稳定亲水蛋白。ZjEXPA8蛋白主要含有无规则卷曲。ZjEXPA8蛋白的不具有跨膜结构域,含有DPBB_1和Pollen_allerg_1两个保守结构域。ZjEXPA8蛋白与大豆(Glycine max,XP_003523276.1,GmEXPA8)的相似性最高,为93.13%。ZjEXPA8基因与橡胶(Hevea brasiliensis,XP_021684783.1,HbEXPA8)和拟南芥(Arabidopsis thaliana,NP_181593.1,AtEXPA8)的亲缘关系最近。灰枣ZjEXPA8基因在枣花发育不同时期均有表达,蕾黄期表达量最高,萼片展平期次之,该基因可能主要在蕾黄期和萼片展平期发挥作用。【结论】ZjEXPA8基因编码的蛋白与大豆的同源性最高。系统进化树分析发现灰枣ZjEXPA8与橡胶HbEXPA8和拟南芥AtEXPA8亲缘关系较近。ZjEXPA8基因在灰枣花蕾黄期表达量最高。

关键词: 灰枣; ZjEXPA8; 基因克隆; 序列分析

Abstract:

【Objective】 To clone a expansin gene ZjEXPA8 and analyze its sequence characteristics analyzed in the hope of laying a foundation for further study on the role and function of the gene in the fruit development of Huizao.【Methods】 In this study, the flower and young fruit of Huizao were used as the tested materials.According to the sequence of ZjEXPA8 gene (LOC107414006), Primer Premier 5.0 software was used to design primers, and RT-PCR was used to clone ZjEXPA8 gene.In addition, Bioinformatics method was used to analyze the nucleotide sequence of ZjEXPA8 gene.【Results】 ZjEXPA8 contained a 762 bp open reading frame (gene accession number was NP_001310794.1), which encoded 253 amino acids.The predicted molecular weight of the protein was 26.91 kDa, the isoelectric point was 8.32, the instability coefficient was 30.09, and the fat index was 66.72.The mean total hydrophilicity was -0.107, which belonged to stable hydrophilic protein.The main component of the secondary and tertiary structure of ZjEXPA8 protein was irregular curls.The structure analysis of ZjEXPA8 protein showed that the protein did not have transmembrane domain, but contained two conserved domains of DPBB_1 and Pollen_allerg_1.Homologous sequence alignment analysis showed that ZjEXPA8 protein exhibited the highest similarity with soybean (Glycine Max, XP_003523277.1, GmEXPA8) (93.13%).Phylogenetic tree analysis showed that ZjEXPA8 gene was most closely related to Hevea Brasiliensis (XP_021684783.1, HbEXPA8) and Arabidopsis thaliana (NP_181593.1, AtEXPA8).Real-time quantitative PCR revealed that ZjEXPA8 gene expressed in different flower development stages of Huizao with the highest expression level in bud yellow stage and the second in sepal spread stage, suggesting that ZjEXPA8 gene may mainly play its role in bud yellowing and sepal flattening stage.【Conclusion】 The protein encoded by ZjEXPA8 exhibits the highest homology with soybean.Phylogenetic tree analysis shows that ZjEXPA8 is closely related to Hevea Brasiliensis (HbEXPA8) and Arabidopsis thaliana (AtEXPA8).

Key words: Huizao; ZjEXPA8; gene cloning; sequence analysis

中图分类号:

S665.1

靳娟, 苏比娜·肖克来提, 阿布都卡尤木·阿依麦提, 杨磊, 郝庆, 樊丁宇. 灰枣扩展蛋白基因ZjEXPA8的克隆及序列分析[J]. 新疆农业科学, 2023, 60(9): 2223-2230.

JIN Juan, Subina Xiaokelaiti, Abudoukayoumu Ayimaiti, YANG Lei, HAO Qing, FAN Dingyu. Cloning and sequence analysis of expansin gene ZjEXPA8 in Huizao[J]. Xinjiang Agricultural Sciences, 2023, 60(9): 2223-2230.

图/表 10

图1 灰枣ZjEXPA8基因的克隆注:M.DL2000 DNA Marker;1, 2: ZjEXPA8

Fig.1 Gene cloning of ZjEXPA8 gene from Huizao Note:M.DL2000 DNA Marker; 1, 2: ZjEXPA8

图2 灰枣ZjEXPA8基因的核苷酸和氨基酸序列

Fig.2 Nucleotide and amino acid sequence of ZjEXPA8 gene in huizao

表1 ZjEXPA8编码蛋白的氨基酸组成

Tab.1 The amino acids composition of ZjEXPA8 encoded protein

氨基酸的种类 Types of amino acid	氨基酸的数目 Number of amino acids	占氨基酸总数的百分比 Percent of total amino acids (%)	氨基酸的种类 Types of amino acid	氨基酸的数目 Number of amino acids	占氨基酸总数的百分比 Percent of total amino acids (%)
丙氨酸Ala	18	7.1	亮氨酸Leu	20	7.9
精氨酸Arg	6	2.4	赖氨酸Lys	7	2.8
天冬酰胺Asn	21	8.3	甲硫氨酸Met	4	1.6
天冬氨酸Asp	8	3.2	苯丙氨酸Phe	14	5.5
半胱氨酸Cys	10	4.0	脯氨酸Pro	10	4.0
谷氨酰胺Gln	13	5.1	丝氨酸Ser	25	9.9
谷氨酸Glu	35	13.8	苏氨酸Thr	17	6.7
甘氨酸Gly	34	7.3	色氨酸Trp	7	2.8
组氨酸His	4	1.6	酪氨酸Tyr	10	4.0
异亮氨酸Ile	9	3.6	缬氨酸Val	13	5.1

图3 ZjEXPA8编码蛋白亲水/疏水性的预测

Fig.3 Prediction of hydrophobicity and rophilicity of ZjEXPA8 encoded protein

图4 ZjEXPA8蛋白的二级结构预测注: 红色: 延伸链; 蓝色: α-螺旋; 紫色:无规则卷曲; 绿色:β-转角

Fig.4 The predicted secondary structure of ZjEXPA8 Note: Red: Extended strand; Blue: Alpha helix; Purple: Random coil; Green: Beta turn

图5 ZjEXPA8蛋白的三级结构预测

Fig.5 The predicted tertiary structure of ZjEXPA8

图6 ZjEXPA8蛋白质保守结构域预测

Fig.6 Prediction of protein conserved domain of ZjEXPA8

图7 ZjEXPA8的氨基酸序列与其他植物EXPA8氨基酸序列比对

Fig.7 Sequence alignment of ZjEXPA8 amino acid with EXPA8 in other plants

图8 ZjEXPA8蛋白序列的系统进化

Fig.8 Phylogenetic analysis of ZjEXPA8 protein

图9 ZjEXPA8在枣花和幼果中基因表达量注:L.蕾黄期;H.萼片展平期;S.花丝萎蔫期;G.子房膨大期;Y.幼果期

Fig.9 ZjEXPA8 gene expression in jujube flower and young fruit Note: L.Bud yellow stage; H.Sepal spread stage; S.Filament wilting stage; G.Ovary inflation stage; Y.Young fruit stage

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灰枣扩展蛋白基因ZjEXPA8的克隆及序列分析

Cloning and sequence analysis of expansin gene ZjEXPA8 in Huizao

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