灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

doi:10.3969/j.issn.1004-1524.2022.06.03

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1124-1132.DOI: 10.3969/j.issn.1004-1524.2022.06.03

灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

李文辰(), 刘鑫, 齐泽铮, 于璐, 王芳()

齐齐哈尔大学生命科学与农林学院,黑龙江齐齐哈尔 161006

收稿日期:2021-08-21 出版日期:2022-06-25 发布日期:2022-06-30
通讯作者: 王芳
作者简介:*王芳,E-mail： wangfangnd@hotmail.com
李文辰（1998—）,男,辽宁阜新人,硕士研究生,研究方向为植物抗病性。E-mail： liwenchen2021@163.com
基金资助:
国家自然科学基金(31801725);黑龙江省教育厅基本科研业务费科研项目(135309361);齐齐哈尔大学研究生创新科研项目

Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

LI Wenchen(), LIU Xin, QI Zezheng, YU Lu, WANG Fang()

College of Life Sciences and Agroforestry, Qiqihar University, Qiqihar 161006, Heilongjiang, China

Received:2021-08-21 Online:2022-06-25 Published:2022-06-30
Contact: WANG Fang

摘要/Abstract

摘要：

为明确大豆U-box型E3泛素连接酶介导植物抗病性的机理,以抗胞囊线虫品种灰皮支黑豆为材料,利用RT-PCR技术克隆GmPUB24基因的蛋白质编码区序列（coding sequence,CDS）,对该基因进行生物信息学分析,并接种大豆胞囊线虫,进行诱导表达分析。结果表明,GmPUB24基因CDS总长1 254 bp,编码417个氨基酸,分子量为46.78 ku。蛋白质二级结构分析显示,GmPUB24编码的蛋白质含有α螺旋、无规则卷曲、延伸链、β折叠,α螺旋占比最高为58.9%,为亲水蛋白质且无跨膜结构域,无信号肽;蛋白质系统进化树表明,其与野生大豆亲缘性最高,亚细胞定位显示其定位于细胞质中。GmPUB24基因上游1 500 bp启动子区含有CGTCA-motif、TGACG-motif等响应抗病通路的作用元件,以及ABRE、WUN-motif、TATA-box等响应非生物胁迫的作用元件。实时荧光定量PCR（qRT-RCR）结果显示,GmPUB24在接种大豆胞囊线虫1~3 d持续上调表达,接种3 d时根部表达量为未接种线虫样本的6.14倍,表明GmPUB24基因可被大豆胞囊线虫诱导表达,可能参与大豆抵御胞囊线虫的过程。研究结果为阐明大豆U-box家族基因在抗大豆胞囊线虫病中的调控机制奠定了基础。

关键词: 大豆, 克隆, 启动子, 生物信息学, 表达

Abstract:

In order to clarify the mechanism of soybean U-box E3 ubiquitin ligase mediating plant disease resistance, coding sequence (CDS) of GmPUB24 gene from resistant cultivar Huipizhi black bean was cloned by RT-PCR. Bioinformatics and expression analysis of this gene after inoculation with soybean cyst nematode were carried out. The results showed that the length of CDS was 1 254 bp, encoded a total of 417 amino acids with a molecular weight of 46.78 ku. Protein secondary structure analysis showed that the protein encoded by GmPUB24 contained alpha helix, random coil, extended chain and beta turn, and the alpha helix accounted for the highest proportion of 58.9%. Protein encoded by GmPUB24 gene was a hydrophilic protein without transmembrane domain and signal peptide. Protein phylogenetic tree displayed that it had the highest affinity with wild soybean. Subcellular location predicted that it localized in the cytoplasm. Promoter analysis of 1 500 bp upstream of GmPUB24 gene showed that it contained CGTCA-motif, TGACG-motif and other elements that respond to disease resistance pathways, as well as ABRE, WUN-motif, TATA-box and other corresponding abiotic stress elements. Quantitative real-time PCR (qRT-RCR) results showed that the expression of GmPUB24 was induced by soybean cyst nematode, and the highest expression level in roots was 6.14 times compared with the uninoculated sample on the third day, indicating that GmPUB24 gene was induced by soybean cyst nematode and might be involved in the process of soybean defense against cyst nematode. These results provided a basis for clarifying the regulatory mechanism of soybean U-box family gene in resistance to soybean cyst nematode disease.

Key words: soybean, cloning, promoter, bioinformatics, expression

中图分类号:

李文辰, 刘鑫, 齐泽铮, 于璐, 王芳. 灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析[J]. 浙江农业学报, 2022, 34(6): 1124-1132.

LI Wenchen, LIU Xin, QI Zezheng, YU Lu, WANG Fang. Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1124-1132.

图/表 9

图1 GmPUB24基因菌液PCR的琼脂糖凝胶电泳结果 M,DL2000 分子量标记物;1~3,菌液样品的3次重复。

Fig.1 Agarose gel electrophoresis results of bacterial liquid PCR of GmPUB24 geneM, DL2000 marker; 1-3, 3 replicates of bacterial fluid samples.

图2 GmPUB24基因编码的蛋白质U-box结构域

Fig. 2 U-box domain of protein encoded by GmPUB24 gene

图3 GmPUB24编码蛋白质的二级结构红色,延伸链;蓝色,α-螺旋;绿色,β折叠;紫色,无规则卷曲。

Fig. 3 Secondary structure of protein encoded by GmPUB24 Red, Extended strand; Blue, Alpha helix; Green, Beta turn; Purple, Random coil.

图4 GmPUB24蛋白质的亲水性与疏水性以0为界限,正值代表疏水氨基酸,负值代表亲水氨基酸。

Fig. 4 Hydrophilicity and hydrophobicity of GmPUB24 protein Taking 0 as the limit, positive values represented hydrophobic amino acids and negative values represented hydrophilic amino acids.

图5 灰皮支黑豆GmPUB24与其他植物相关蛋白的U-box结构域同源比对黑色阴影为氨基酸具有100%一致性,粉色表示一致性在75%以上,蓝色表示一致性在50%-75%之间,白色表示一致性在50%以下。KAG4974237,野生大豆;XP_003539150,栽培大豆;XP_027911296,豇豆;XP_014523587,小绿豆;XP_027348689,红豆;KYP39063,木豆;XP_004506267,鹰嘴豆;OAP04810,拟南芥;XP_004229574,番茄;XP_015635733,水稻;PWZ26684,玉米。下同。

Fig. 5 Homologous alignment of U-box domains of Huipizhi black bean GmPUB24 with other plant associated proteins Black shading meant amino acids with 100% identity, pink meant more than 75% identity, blue meant 50%-75% identity, and white means less than 50% identity. KAG4974237, Glycine soja; XP_003539150, Glycine max; XP_027911296, Vigna unguiculata; XP_014523587, Vigna radiata var. Radiata; XP_027348689, Abrus precatorius; KYP39063, Cajanus cajan; XP_004506267, Cicer arietinum; OAP04810, Arabidopsis thaliana; XP_004229574, Solanum lycopersicum; XP_015635733, Oryza sativa; PWZ26684, Zea mays. The same as below.

图6 灰皮支黑豆GmPUB24蛋白质与其他植物同源蛋白的系统进化树采用邻接法,自举检验1 000次;图中标尺为遗传距离。

Fig. 6 Phylogenetic tree of GmPUB24 protein from Huipizhi black bean and other plant homologous proteinsNeighbor Joining method with 1 000 bootstrap replicates. The scale bar represented the genetic distance.

表1 GmPUB24启动子顺式元件分析

Table 1 Analysis of cis element of GmPUB24 promoter

名称 Name	数量 Number	序列 Sequence	功能 Function
ABRE	3	ACGTG	参与脱落酸反应 Involved in the abscisic acid reaction
Box 4	7	ATTAAT	参与光反应有关的保守DNA Conserved DNA elements involved in photo reaction
Box II	2	CCACGTGGC	参与光响应Components involved in light response
CAAT-box	20	CCAAT	启动子和增强子区常见的顺式作用元件 Involved in promoter and enhancer regions
CAT-box	1	GCCACT	参与分生组织相关表达 Involved in the expression of meristems
CGTCA-motif	3	CGTCA	参与茉莉酸反应 Involved in the jasmonic acid reaction
TATA-box	35	ATATAT	参与核心启动子30位点转录 Transcription element involved in the 30 position of the core promoter
TGACG-motif	3	TGACG	参与茉莉酸反应 Involved in the jasmonic acid reaction
Unnamed-1	4	GAATTTAATTAA	60s蛋白质活性位点 Involved in 60s protein active site
WUN-motif	1	AAATTTCCT	参与植物伤口反应 Elements involved in plant wound response
ARE	1	AAACCA	无氧诱导 Necessary for anaerobic induction
AT-rich element	1	ATAGAAATCAA	结合蛋白（ATBP-1）的结合位点 Binding site element of binding protein (ATBP-1)
G-box	3	GCCACGTGGA	参与光响应Involved in light response
O2-site	1	GATGATGTGG	参与玉米醇溶蛋白代谢调节 Involved in the regulation of zein metabolism

图7 GmPUB24与TUA4 PCR电泳产物检测 M, DL2000 分子量标记物; 1, TUA4; 2, GmPUB24.

Fig. 7 Electrophoresis detection of GmPUB24 and TUA4 PCR products M, DL2000 marker; 1, TUA4; 2, GmPUB24.

图8 大豆GmPUB24基因在大豆胞囊线虫侵染不同时间的表达量 **代表P ≤ 0.01,***代表P ≤ 0.001,ns代表不显著。

Fig. 8 Expression of soybean GmPUB24 gene at different time of soybean cyst nematode infection ** represented P ≤ 0.01, *** represented P ≤ 0.001, ns represented the difference was not significant.

参考文献 30

[1]	PENG D L, JIANG R, PENG H, et al. Soybean cyst Nematodes: a destructive threat to soybean production in China[J]. Phytopathology Research, 2021, 3: 19. DOI URL
[2]	CALDWELL B E, BRIM C A, ROSS J P. Inheritance of resistance of soybeans to the cyst nematode, Heterodera glycines[J]. Agronomy Journal, 1960, 52(11): 635-636. DOI URL
[3]	MATSON A L, WILLIAMS L F. Evidence of a fourth gene for resistance to the soybean cyst nematode[J]. Crop Science, 1965, 5: 477. DOI URL
[4]	RAO-ARELLI A P, ANAND S C, WRATHER A J. Soybean resistance to soybean cyst nematode race 3 is conditioned by an additional dominant gene[J]. Crop Science, 1992, 32(4): 862-864. DOI URL
[5]	GUO B, SLEPER D A, NGUYEN H T, et al. Quantitative trait loci underlying resistance to three soybean cyst nematode populations in soybean PI 404198A[J]. Crop Science, 2006, 46(1): 224-233. DOI URL
[6]	LIU X H, LIU S M, JAMAI A, et al. Soybean cyst nematode resistance in soybean is independent of the Rhg4 locus LRR-RLK gene[J]. Functional & Integrative Genomics, 2011, 11(4): 539-549.
[7]	COOK D E, LEE T G, GUO X L, et al. Copy number variation of multiple genes at Rhg1 mediates nematode resistance in soybean[J]. Science, 2012, 338(6111): 1206-1209. DOI URL
[8]	LIU S, KANDOTH P K, LAKHSSASSI N, et al. The soybean GmSNAP18 gene underlies two types of resistance to soybean cyst nematode[J]. Nature Communications, 2017, 8: 14822. DOI URL
[9]	LIU S, KANDOTH P K, WARREN S D, et al. A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens[J]. Nature, 2012, 492(7428): 256-260. DOI URL
[10]	PRUITT R N, GUST A A, NURNBERGER T. Plant immunity unified[J]. Nature Plants, 2021, 7(4): 382-383. DOI URL
[11]	NAVEED Z A, WEI X Y, CHEN J J, et al. The PTI to ETI continuum in Phytophthora-plant interactions[J]. Frontiers in Plant Science, 2020, 11: 593905. DOI URL
[12]	PRUNEDA J N, LITTLEFIELD P J, SOSS S E, et al. Structure of an E3: E2-Ub complex reveals an allosteric mechanism shared among RING/U-box ligases[J]. Molecular Cell, 2012, 47(6): 933-942. DOI URL
[13]	VIERSTRA R D. The ubiquitin-26S proteasome system at the Nexus of plant biology[J]. Nature Reviews Molecular Cell Biology, 2009, 10(6): 385-397. DOI URL
[14]	HE Q, MCLELLAN H, BOEVINK P C, et al. U-box E 3 ubiquitin ligase PUB17 acts in the nucleus to promote specific immune pathways triggered by Phytophthora infestans[J]. Journal of Experimental Botany, 2015, 66(11): 3189-3199. DOI URL
[15]	TRUJILLO M, ICHIMURA K, CASAIS C, et al. Negative regulation of PAMP-triggered immunity by an E3 ubiquitin ligase triplet in Arabidopsis[J]. Current Biology, 2008, 18(18): 1396-1401. DOI URL
[16]	雷苏炜. 拟南芥磷脂结合蛋白PUIP4调控抗病性与发育的功能研究[D]. 长沙: 湖南农业大学, 2017.
	LEI S W. Functions of copine protein PUIP4 regulated disease resistance and development[D]. Changsha: Hunan Agricultural University, 2017. (in Chinese with English abstract)
[17]	倪雪梅. 晚疫病菌诱导的两个马铃薯泛素连接酶基因的克隆与功能分析[D]. 武汉: 华中农业大学, 2009.
	NI X M. dentification and cloning of two ubiquitin ligase genes induced by Phytophthora infestans in potato[D]. Wuhan: Huazhong Agricultural University, 2009. (in Chinese with English abstract)
[18]	DURNER J, SHAH J, KLESSIG D F. Salicylic acid and disease resistance in plants[J]. Trends in Plant Science, 1997, 2(7): 266-274. DOI URL
[19]	ZHANG C Y, SONG L, CHOUDHARY M K, et al. Genome-wide analysis of genes encoding core components of the ubiquitin system in soybean (Glycine max) reveals a potential role for ubiquitination in host immunity against soybean cyst nematode[J]. BMC Plant Biology, 2018, 18(1): 149. DOI URL
[20]	王芳. 小粒黑豆抗胞囊线虫SSH-cDNA文库构建及重要基因表达分析[D]. 沈阳: 沈阳农业大学, 2012.
	WANG F. Construction of SSH-cDNA library against cyst nematode in small black bean and analysis of important gene expression[D]. Shenyang: Shenyang Agricultural University, 2012. (in Chinese with English abstract)
[21]	KALWA U, LEGNER C, WLEZIEN E, et al. New methods of removing debris and high-throughput counting of cyst nematode eggs extracted from field soil[J]. PLoS One, 2019, 14(10): e0223386. DOI URL
[22]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method[J]. Methods, 2001, 25(4): 402-408. DOI URL
[23]	YEE D, GORING D R. The diversity of plant U-box E3 ubiquitin ligases: from upstream activators to downstream target substrates[J]. Journal of Experimental Botany, 2009, 60(4): 1109-1121. DOI URL
[24]	PONTIER D, BALAGUE C, BEZOMBES-MARION I, et al. Identification of a novel pathogen-responsive element in the promoter of the tobacco gene HSR203J, a molecular marker of the hypersensitive response[J]. The Plant Journal, 2001, 26(5): 495-507. DOI URL
[25]	BARCALA M, GARCIA A, CABRERA J, et al. Early transcriptomic events in microdissected Arabidopsis nematode-induced giant cells[J]. The Plant Journal, 2010, 61(4): 698-712. DOI URL
[26]	张海文, 谢丙炎, 卢向阳, 等. 拟南芥防卫基因PDF1.2启动子中GCC盒是应答茉莉素反应必要的顺式作用元件[J]. 科学通报, 2004, 49(23): 2444-2448.
	ZHANG H W, XIE B Y, LU X Y, et al. The GCC box in the Arabidopsis defense gene PDF1.2 promoter is an essential cis-acting element in response to the jasmin response[J]. Chinese Science Bulletin, 2004, 49(23): 2444-2448. (in Chinese)
[27]	JIN X F, XIONG A S, PENG R H, et al. OsAREB1, an ABRE-binding protein responding to ABA and glucose, has multiple functions in Arabidopsis[J]. BMB Reports, 2010, 43(1): 34-39. DOI URL
[28]	LIU J, XIA W R, HU Y P, et al. Cloning and analysis of MeCWINV6 promoter from biofuel plant cassava (Manihot esculenta Crantz)[J]. Advanced Materials Research, 2014, 986/987: 25-29. DOI URL
[29]	KLINK V P, OVERALL C C, ALKHAROUF N W, et al. A time-course comparative microarray analysis of an incompatible and compatible response by Glycine max (soybean) to Heterodera glycines (soybean cyst nematode) infection[J]. Planta, 2007, 226(6): 1423-1447. DOI URL
[30]	王晗, 金贺, 王旭东, 等. 大豆胞囊线虫侵染后GmC4H、GmLac55和GmLac85的表达模式分析[J]. 沈阳农业大学学报, 2021, 52(3): 336-342.
	WANG H, JIN H, WANG X D, et al. Expression patterns of GmC4H, GmLac55 and GmLac85 after soybean cyst nematode infection[J]. Journal of Shenyang Agricultural University, 2021, 52(3): 336-342. (in Chinese with English abstract)

编辑推荐

Metrics

阅读次数

全文

756

HTML			PDF

最新录用	在线预览	正式出版	最新录用	在线预览	正式出版
0	0	8	0	0	748

来源	本网站	其他网站

次数	444	312
比例	59%	41%

摘要

445

最新录用	在线预览	正式出版

0	0	445

	来源	本网站

	次数	445
	比例	100%

灰皮支黑豆GmPUB24基因的生物信息学与胞囊线虫诱导表达分析

Bioinformatics of Huipizhi Black soybean GmPUB24 and expression under Heterodera glycines infection

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 30

相关文章 15

编辑推荐

Metrics

本文评价

[1]	董袁袁, 徐恒, 张华, 张恒, 王伏林, 顾娜娜, 朱英. 水稻种子成熟后期高湿环境下种子休眠相关基因的表达[J]. 浙江农业学报, 2022, 34(6): 1103-1113.
[2]	刘凯, 谢楠, 郭炜, 马恒甲. 三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析[J]. 浙江农业学报, 2022, 34(6): 1162-1174.
[3]	洪森荣, 向琼钰, 谢颖, 熊晨露, 徐晨慧, 徐璐珂, 陈荣华, 蔡红. 怀玉山三叶青烟草病毒增殖蛋白1基因克隆、亚细胞定位和组织表达分析[J]. 浙江农业学报, 2022, 34(6): 1193-1204.
[4]	熊昕宜, 许泽玉, 何念佳, 何俊博, 陈正礼, 黄超, 刘文涛, 罗启慧. 大豆异黄酮干预肥胖大鼠肝氧化应激及炎症反应[J]. 浙江农业学报, 2022, 34(5): 942-948.
[5]	夏煜琪, 孙宇, 刘志鑫, 孙瑞青, 杨楠, 蒲金基, 张贺. 杧果转录因子BES1s家族全基因组鉴定及生物信息学分析[J]. 浙江农业学报, 2022, 34(5): 984-994.
[6]	余艳玲, 罗洪林, 罗辉, 冯鹏霏, 潘传燕, 宋漫玲, 肖蕊, 张永德. 卵形鲳鲹生肌调节因子基因家族的鉴定及在胚胎中的表达[J]. 浙江农业学报, 2022, 34(4): 695-705.
[7]	邓哲宇, 王乙婷, 王颖洁, 胡菜, 吴宇慧, 赵宗仪, 左其生, 张亚妮. 鸡gga-miR-31-5p 启动子真核表达载体的构建及其转录因子结合位点预测[J]. 浙江农业学报, 2022, 34(4): 713-719.
[8]	刘同金, 徐铭婕, 汪精磊, 刘良峰, 崔群香, 包崇来, 王长义. 萝卜ALMT基因家族的鉴定与表达[J]. 浙江农业学报, 2022, 34(4): 746-755.
[9]	樊有存, 张红岩, 杨旭升, 韩芊, 刘玉皎, 武学霞. 蚕豆耐盐相关基因VfHKT1;1的克隆、生物信息学分析及表达特性[J]. 浙江农业学报, 2022, 34(4): 756-765.
[10]	李小兰, 张瑞, 郝兰兰, 王鸿. 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.
[11]	刘晨, 徐浩博, 斯钰阳, 李亚鹏, 郭玉婷, 杜长霞. 基于转录组学的植物响应盐胁迫调控机制研究进展[J]. 浙江农业学报, 2022, 34(4): 870-878.
[12]	丁燕玲, 王鹏飞, 杨朝云, 周小南, 赵志艳, 张岩峰, 史远刚, 康晓龙. 牛miR-144靶基因预测与组织表达分析[J]. 浙江农业学报, 2022, 34(3): 471-479.
[13]	王乾昆, 张小辉, 庞有志, 祁艳霞, 雷莹, 白俊艳, 户运奇, 赵毅威, 苑志文, 王涛. 基于RNA-seq技术挖掘鹌鹑羽色自别雌雄相关基因[J]. 浙江农业学报, 2022, 34(3): 498-506.
[14]	兰国湘, 金思琪, 李星润, 刘喜雨, 李国美, 董新星. 高原雨点鸽与詹森鸽胸肌转录组差异表达基因筛选与功能分析[J]. 浙江农业学报, 2022, 34(3): 507-516.
[15]	杨昕霞, 唐满生, 张斌. 大豆PP2C家族基因鉴定与响应盐胁迫的转录组分析[J]. 浙江农业学报, 2022, 34(2): 207-220.