紫鸭跖草CpBURP的克隆、表达及生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2020-1572

摘要/Abstract

摘要：

BURP蛋白在植物生长发育、抵抗非生物胁迫中具有重要的作用。克隆CpBURP,并分析其在紫鸭跖草中的作用,为后期紫鸭跖草的BURP基因的深入研究奠定基础。根据紫鸭跖草转录组数据库进行筛选,并克隆CpBURP,通过对其进行生物信息学分析及表达分析。结果表明,1 383 bp的CpBURP编码含460个氨基酸的蛋白质,CpBURP蛋白属于亲水蛋白,含有一个BURP保守结构域和50个磷酸化位点,不含跨膜结构和信号肽区域。预测CpBURP蛋白作用于细胞质和过氧化物酶体。系统进化树显示,CpBURP蛋白与13种植物的BURP蛋白被分为6个亚类,CpBURP蛋白与甘蓝型油菜的BURP蛋白亲缘性最近。qRT-PCR结果显示,CpBURP在根中的表达量高于茎和叶,在Cu²⁺胁迫条件下,根中CpBURP的表达量明显高于对照组。CpBURP可能在紫鸭跖草的Cu²⁺胁迫应答反应中具有重要作用。

关键词: 紫鸭跖草, CpBURP, 基因克隆, 生物信息学, 表达分析

Abstract:

BURP protein plays an important role in the growth of plant and resistance to abiotic stress. Cloning a CpBURP and analyzing its role in Commelina purpurea may lay a foundation for further deeply studying the gene BURP in C. purpurea. Screening was performed based on the C. purpurea transcriptome database and a CpBURP was cloned,and then its bioinformatics and expressions were analyzed. The result showed the CpBURP of 1 383 bp encoded a protein with 460 amino acids. The hydrophilic CpBURP protein contained a BURP conserved domain and 50 phosphorylation sites,but no transmembrane structure and signal peptide region. It is predicted that CpBURP protein acted on the cytoplasm and peroxisomes. Phylogenetic tree analysis revealed that CpBURP protein and 13 plant BURP proteins were grouped into 6 subcategories,the CpBURP protein had the closest affinity to the BURP protein in Brassica napus. The results of qRT-PCR demonstrated that the expression of CpBURP in the root was higher than that in the stem and leaf. Under the Cu²⁺ stress,the expression of CpBURP in the roots was significantly higher than that in the control group. CpBURP may play an important role in the response of C. purpurea to Cu²⁺ stress.

Key words: Commelina purpurea, CpBURP, gene cloning, bioinformatics, expression analysis

彭国颖, 卢山, 黄超, 杨坤, 万玮, 黄长干. 紫鸭跖草CpBURP的克隆、表达及生物信息学分析[J]. 生物技术通报, 2021, 37(9): 125-131.

PENG Guo-ying, LU Shan, HUANG Chao, YANG Kun, WAN Wei, HUANG Chang-gan. Cloning,Expression and Bioinformatics Analyses of CpBURP from Commelina purpurea[J]. Biotechnology Bulletin, 2021, 37(9): 125-131.

图/表 10

图1 qRT-PCR扩增程序

Fig.1 Amplification procedure of qRT-PCR

图2 CpBURP的扩增和PCR检测 A：CpBURP的PCR扩增;B：CpBURP的菌液PCR检测;M：DNA Marker DL2 000;1-2：blank;3-6：CpBURP基因

Fig.2 Amplification and PCR detection of CpBURP A: PCR amplification of CpBURP; B: PCR detection of CpBURP bacterial fluid; M: DNA Marker DL2 000; 1-2: blank; 3-6: CpBURP gene

图3 CpBURP核苷酸序列及其编码的氨基酸序列

Fig. 3 CpBURP nucleotide sequence and its encoded amino acid sequence

图4 CpBURP蛋白的亲疏水性分析亲/疏水性分别用负/正分值表示

Fig.4 Hydrophilic and hydrophobic analysis of CpBURP protein The hydrophilic/hydrophobic values are expressed by negative/positive scores respectively

图5 CpBURP蛋白的保守结构域

Fig.5 Conserved domain of CpBURP protein

图6 CpBURP蛋白的磷酸化位点

Fig. 6 Phosphorylation sites of CpBURP protein

图7 CpBURP蛋白的二、三级结构分析 A：二级结构;B：三级结构

Fig.7 Analysis of the secondary and tertiary structure of CpBURP protein A: Secondary structure. B: Tertiary structure

图8 CpBURP的跨膜结构和信号肽分析 A：跨膜结构;B：信号肽预测

Fig.8 Transmembrane structure and signal peptide predic-tion of CpBURP A: Transmembrane structure; B: signal peptide prediction

图9 不同物种BURP蛋白与CpBURP蛋白的系统进化树分析

Fig.9 Phylogenetic tree analysis of BURP proteins and CpBURP protein of different species

图10 CpBURP在Cu2+胁迫下的表达变化

Fig.10 Expression changes of CpBURP under Cu2+ stress

参考文献 23

[1]	Rufus LC, Minnie M, Yin ML, et al. Phytoremediation of soil metals[J]. Curr Opin Biotechnol, 1997, 8(3):279-284. doi: 10.1016/S0958-1669(97)80004-3 URL
[2]	张杰. 超积累植物东南景天Cd耐性和积累的分子机制[D]. 杭州:浙江大学, 2015.
	Zhang J. Molecular mechanisms of Cd tolerance and accumulation in superaccumulative plant Sedum alfredii[D]. Hangzhou:Zhejiang University, 2015.
[3]	吴贤豪, 李建新, 王永川, 等. 超积累植物热解中重金属迁移及渗滤特性研究[J]. 环境科学学报, 2017, 37(7):2707-2712.
	Wu XH, Li JX, Wang YC, et al. Study on the migration and percolation characteristics of heavy metals in over-accumulation plant pyrolysis[J]. Journal of Environmental Science, 2017, 37(7):2707-2712.
[4]	刘美青, 江荣风, 赵方杰. Zn对Zn超积累植物遏蓝菜(Thlaspi caerulescens L.)[J]. 超氧化物歧化酶, 2006(S2):465-470.
	Liu MQ, Jiang RF, Zhao FJ. Effects of Zinc amendment on superoxide dismutase activity of zinc hyperaccumulator Thlaspi caerulescens L.[J]. Journal of Agro-Environmental Science, 2006(S2):465-470.
[5]	Farha A, Akhtar I. Accumulation of metals, antioxidant activity, growth and yield attributes of mustard(Brassica juncea L.)grown on soil amendments with fly ash together with inorganic nitrogen fertilizer[J]. Acta Physiologiae Plantarum, 2020, 42(9):285-344.
[6]	刘丽杰, 刘凯, 孙玉婷, 等. 车前草对重金属铜和镍的积累及生理响应[J]. 甘肃农业大学学报, 2020, 55(5):171-179.
	Liu LJ, Liu K, Sun YT, et al. Accumulation copper and nickel(Ⅱ)ions by Herba plantaginis and its physiological responses[J]. Journal of Gansu Agricultural University, 2020, 55(5):171-179.
[7]	黄长干. 紫鸭跖草对铜的积累规律及在铜胁迫下的生理反应研究[D]. 长沙:湖南农业大学, 2007.
	Huang CG. Study on accumulation law of Commelina purpurea copper and its physiological response under copper stress[D]. Changsha:Hunan Agricultural University, 2007.
[8]	Yu SW, Zhang LD, Zuo KJ, et al. Isolation and characterization of a BURP domain-containing gene BnBDC1 from Brassica napus involved in abiotic and biotic stress[J]. Physiologia Plantarum, 2004, 122(2):210-218. doi: 10.1111/ppl.2004.122.issue-2 URL
[9]	章燕如, 俞可可, 龚秀, 等. 青花菜BoBURP1基因的克隆与表达分析[J]. 浙江农业学报, 2016, 28(9):1501-1507.
	Zhang YR, Yu KK, Gong X, et al. Cloning and expression analysis of BoBURP1 gene from Brassica oleracea var. italica[J]. Journal of Zhejiang Agriculture, 2016, 28(9):1501-1507.
[10]	Sun HR, Wei HL, Wang HT, et al. Genome-wide identification and expression analysis of the BURP domain-containing genes in Gossypium hirsutum[J]. BMC Genomics, 2019, 20(1):558. doi: 10.1186/s12864-019-5948-y URL
[11]	Kim AB, María JG, Janice MD, et al. Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis[J]. Plant Molecular Biology, 1994, 26(6):1711-1723. doi: 10.1007/BF00019486 URL
[12]	Vokkaliga TH, Le VS, Christiane S, et al. AtRD22 and AtUSPL1, members of the plant-specific BURP domain family involved in Arabidopsis thaliana drought tolerance[J]. PLoS One, 2018, 9(10):e110065. doi: 10.1371/journal.pone.0110065 URL
[13]	José TM, Felipe A, Carmen E, et al. Inspection of the grapevine BURP superfamily highlights an expansion of RD22 genes with distinctive expression features in berry development and ABA-mediated stress responses[J]. PLoS One, 2017, 9(10):e110372. doi: 10.1371/journal.pone.0110372 URL
[14]	钟活权. Cu²⁺胁迫下拟南芥AtRD22的表达及作用分析[D]. 深圳:深圳大学, 2017.
	Zhong HQ. Expression and Function analysis of AtRD22 in Arabidopsis thaliana under Cu²⁺ stress[D]. Shenzhen:Shenzhen University, 2017.
[15]	Ding XP, Hou X, Xie KB, et al. Genome-wide identification of BURP domain-containing genes in rice reveals a gene family with diverse structures and responses to abiotic stresses[J]. Planta, 2009, 230(1):149-163. doi: 10.1007/s00425-009-0929-z URL
[16]	饶俊, 郑新欣, 胡英考. BURP蛋白家族研究进展[J]. 生物技术通报, 2009(7):8-11.
	Rao J, Zheng XX, Hu YK. Advances in the study of BURP protein family[J]. Biotechnology Bulletin, 2009(7):8-11.
[17]	Le S, Jens T, Twan R, et al. The BURP domain protein AtUSPL1 of Arabidopsis thaliana is destined to the protein storage vacuoles and overexpression of the cognate gene distorts seed development[J]. Plant Molecular Biology, 2009, 71(4/5):319-329. doi: 10.1007/s11103-009-9526-6 URL
[18]	米子岚, 钟活权, 江年琼, 等. BURP蛋白家族与植物对非生物胁迫的响应[J]. 中国细胞生物学学报, 2015, 37(9):1302-1308.
	Mi ZL, Zhong HQ, Jiang NQ, et al. BURP protein family and plant response to abiotic stress[J]. Chinese Journal of Cell Biology, 2015, 37(9):1302-1308.
[19]	米子岚. 拟南芥AtRD22蛋白与重金属离子的相互作用研究[D]. 深圳:深圳大学, 2016.
	Mi ZL. Interaction between Arabidopsis thaliana AtRD22 protein and heavy metal ions[D]. Shenzhen:Shenzhen University, 2016.
[20]	杨振红, 王芳, 王舰. 马铃薯StWRKY40基因的序列及表达模式分析[J]. 分子植物育种, 2020, 18(22):7283-7292.
	Yang ZH, Wang F, Wang J. Sequence and expression pattern analysis of StWRKY40 gene in potato[J]. Molecular Plant Breeding, 202, 18(22):7283-7292.
[21]	张永福, 莫丽玲, 徐金会, 等. 葡萄耐铝毒基因MATE的克隆与表达分析[J]. 江西农业大学学报, 2020, 42(5):906-914.
	Zhang YF, Mo LL, Xu JH, et al. Cloning and expression analysis of MATE, a gene resistant to aluminum toxicity in grape[J]. Journal of Jiangxi Agricultural University, 2020, 42(5):906-914.
[22]	张亚楠, 蔺银鼎, 孟林, 等. 马蔺重金属ATP酶基因HMA2的RNAi载体构建及其遗传转化[J]. 草业科学, 2017, 34(5):988-996.
	Zhang YN, Lin YD, Meng L, et al. Construction of RNAi expression vector of heavy metal ATPases gene HMA2 and genetic transformation in Iris lactea[J]. Science of Grass Industry, 2017, 34(5):988-996.
[23]	黄佳欢, 刘关君, 陈瑾元, 等. 小黑杨PnCCH10基因的克隆、功能初步鉴定及遗传转化[J]. 植物生理学报, 2019, 55(11):1625-1637.
	Huang JH, Liu GJ, Chen JY, et al. Cloning, preliminary function identification and genetic transformation of PnCCH10 gene in Populus simonii×Populus nigra[J]. Journal of Plant Physiology, 2019, 55(11):1625-1637.