Cloning and sequence analysis of expansin gene ZjEXPA8 in Huizao

doi:10.6048/j.issn.1001-4330.2023.09.017

Abstract

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

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

CLC Number:

S665.1

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.

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.

Figures/Tables 10

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

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

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

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

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

Fig.5 The predicted tertiary structure of ZjEXPA8

Fig.6 Prediction of protein conserved domain of ZjEXPA8

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

Fig.8 Phylogenetic analysis of ZjEXPA8 protein

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

References 17

[1]	Guo M X, Zhang Z R, Cheng Y W, et al. Comparative population genomics dissects the genetic basis of seven domestication traits in jujube[J]. Horticulture Research, 2020, 7:89. DOI PMID
[2]	丁蒙蒙, 林敏娟, 王振磊. 不同枣品种花粉萌发及花粉管生长特性的研究[J]. 塔里木大学学报, 2016, 28(3): 20-24.
	DING Mengmeng, LIN Minjuan, WANG Zhenlei. Pollen germination and pollen tube growth of different cultivars of Chinese jujube[J]. Journal of Tarim University, 2016, 28(3): 20-24.
[3]	Dingyu F, Lei Y, Juan J, et al. Hormonal and transcriptomic analysis reveal gibberellin-induced fruit set in jujube (Ziziphus jujuba Mill.)[J]. Plant Molecular Biology Reporter, https://doi.org/10.1007/s11105-021-01308-4.
[4]	张奇艳, 雷忠萍, 宋银, 等. 陆地棉扩展蛋白基因的鉴定与特征分析[J]. 中国农业科学, 2019, 52(21):3713-3732. DOI
	ZHANG Qiyan, LEI Zhongping, SONG Yin, et al. Identification and characterization of the expansin gene family in upland cotton (Gossypium hirsutum)[J]. Scientia Agricultura Sinica, 2019, 52(21):3713-3732.
[5]	Li Y, Jones L, Mcqueen-Mason S. Expansins and cell growth[J]. Current Opinion in Plant Biology, 2003, 6(6):603-610. DOI PMID
[6]	Cosgrove D J. Plant expansins: Diversity and interactions with plant cell walls[J]. Current Opinion in Plant Biology, 2015, 25:162-172. DOI PMID
[7]	蔡兆琴, 吴强, 陈慧, 等. 甘薯扩展蛋白基因IbEXPA4的克隆和表达分析[J]. 农业生物技术学报, 2021, 29(10):1914-1925.
	CAI Zhaoqin, WU Qiang, CHEN Hui, et al. Cloning and expression analysis of expansin gene IbEXPA4 in sweet potato (Ipomoea batatas)[J]. Journal of Agricultural Biotechnology, 2021, 29(10):1914-1925.
[8]	Bajwa K S, Shahid A A, Rao A Q, et al. Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton[J]. Frontiers in Plant Science, 2015, 6:838.
[9]	赵湾湾, 冯力, 胡绍彬, 等. 番木瓜果实软化相关CpEXPA2基因的克隆与表达分析[J]. 果树学报, 2018, 35(7):785-793.
	ZHAO Wanwan, FENG Li, HU S haobin, et al. Cloning and expression analysis of CpEXPA2 gene related to softening of papaya fruit[J]. Journal of Fruit Science, 2018, 35(7):785-793.
[10]	刘孟军, 王玖瑞, 刘平, 等. 中国枣生产与科研成就及前沿进展[J]. 园艺学报, 2015, 42(9):1683-1698. DOI
	LIU Mengjun, WANG Jiurui, LIU Ping, et al. Historical achievements and frontier advances in the production and research of Chinese Jujube (Ziziphus jujuba) in China[J]. Acta Horticulturae Sinica, 2015, 42(9):1683-1698. DOI
[11]	新疆维吾尔自治区统计局. 新疆统计年鉴[J]. 北京: 中国统计出版社, 2020.
	Statistics Bureau of Xinjiang Uyghur Autonomous Region. Xinjiang Statistical Yearbook[J]. Beijing: China Statistics Press, 2020.
[12]	吕丽敏, 左东云, 王省芬, 等. 陆地棉纤维发育相关基因GhEXPs 的分析及表达研究[J]. 棉花学报, 2021, 33(3):280-290.
	LYU Limin, ZUO Dongyun, WANG Shengfen, et al. Analysis and expression of GhEXPs related to fiber development in Gossypium hirsutum L.[J]. Cotton Science, 2021, 33(3):280-290.
[13]	An Yan, Minjie Wu, Limei Yan, et al. AtEXP2 is involved in seed germination and abiotic stress response in Arabidopsis[J]. PLoS ONE, 2014, 9(1): e85208. DOI URL
[14]	Kuluev B R, Knyazev A V, Mikhaylova E V, et al. The role of expansin genes PtrEXPA3 and PnEXPA3 in the regulation of leaf growth in poplar[J]. Russian Journal of Genetics, 2017, 53(6):651-660. DOI URL
[15]	Ren H, Wen L Z, Guo Y H, et al. Expressional and functional verification of the involvement of CmEXPA4 in chrysanthemum root development[J]. Journal of Plant Growth Regulation, 2019, 38:1375-1386. DOI
[16]	Saito T, Tuan P A, Katsumi-Horigane A, et al. Development of flower buds in the japanese pear (Pyrus pyrifolia) from late autumn to early spring[J]. Tree Physiology, 2015, 35(6):653-662. DOI PMID
[17]	杨绍兰, 陈妙金, 张波, 等. 乙酰水杨酸调控猕猴桃果实后熟软化进程中Ad-EXP1 基因表达[J]. 果树学报, 2007, 24(6):778-782.
	YANG Shaolan, CHEN Miaojin, ZHANG Bo, et al. Expression pattern and regulation of Ad-EXP1 gene in kiwifruit treated with ASA[J]. Journal of Fruit Science, 2007, 24(6):778-782.