Isolation and Expression of Peroxidase POD1 Gene from Cucurbita pepo
-
摘要: 通过西葫芦转录组测序，筛选得到1条功能注释为过氧化物酶（peroxidase，POD）的基因全长序列（c47620.graph_c0）。分析发现，该序列全长1 245 bp，包含1个996 bp的开放读码框（open reading frame，ORF），预测编码331个氨基酸，理论分子量（molecular weight，Mw）为35.70 kDa，等电点（isoelectric point，pI）为5.40，编码的蛋白质与黄瓜、甜瓜和苦瓜等常见瓜果类蔬菜同源蛋白质的相似性在84%、84%和81%，显示其高度的保守性，基因命名为CpPOD1，GenBank登录号为MF988285。荧光定量PCR分析结果表明，CpPOD1基因具有组织表达特异性，在叶片中表达丰度最高，其次为根，在花中的表达最低。CpPOD1基因在低温弱光条件下，其表达量随时间增加而显著上调。初步推测，CpPOD1基因在西葫芦低温弱光胁迫下存在一定上调表达，猜测可能参与调控下游基因对低温弱光胁迫的响应有关。Abstract: A functional annotation as peroxidase (POD) gene (i.e., c47620.graph_c0) was screened from the transcriptomic sequencing of Cucurbita pepo. It was found that thesequence length was 1 245 bp, including a 996bp open reading frame (ORF) encoded a protein of 331 amino acids. The protein had a predicted molecular weight of 35.70 kDa and a hypothetical isoelectric point of 5.40. The similarity of the proteins to the homologous proteins of cucumber, melon and balsam pear were 84%, 84% and 81%, respectively, witha highly conservative nature. It was designated as CpPOD1 gene with a GenBank accession code of MF988285. The RTqPCR revealed the geneto betissue-specific, andits expression in C. pepo highest in the leaves, followed by roots and flowers. Under low temperature and dim light, the CpPOD1 expression was up-regulated over time. It suggested that the gene expression correlatedwith the low temperature/dim light stress as C. pepo regulated the downstream responses. This study could lead to further understanding of the function of CpPOD1 gene inzucchinion cold-and-low-lighttolerance and the regulation mechanism providing evidence and theoretical basis for improved breeding of C. pepo.
-
Key words:
- Cucurbita pepo /
- POD /
- RNA-Seq /
- qRT-PCR
-
图 1 西葫芦低温弱光处理后植株
注：A为正常处理；B为弱光处理；C为低温处理；D为低温弱光处理。
Figure 1. C. pepo plant after low temperature and dim light treatment
图 2 CpPOD1全长cDNA序列及氨基酸序列
Figure 2. cDNA full sequence and corresponding amino acid sequence of CpPOD1
图 3 西葫芦CpPOD1与其他物种中的同源蛋白的多序列比对
注：XP_011653567.1为甜瓜; NP_001295845.1为黄瓜; XP_022153752.1为苦瓜; ABF68751.1为西葫芦; AKN08989.1为丝瓜。
Figure 3. Multiple sequence pairs of CpPOD1 in C. pepo and homologous proteins in other species
图 4 西葫芦CpPOD1与其他同源蛋白序列的系统进化树
Figure 4. Phylogenetic tree of CpPOD1 in C. pepo and other homologous sequences
图 6 西葫芦在不同处理条件下CpPOD1表达模式
Figure 6. CpPOD1 of C. pepo under treatments as analyzed by qPCR
图 7 在不同处理时间下的西葫芦酶活性含量变化
注：图中不同小写字母表示差异达显著水平(P＜0.05)。
Figure 7. Enzyme activities of C. pepo under treatments for varied time periods
表 1 西葫芦不同光照和温度处理组合
Table 1. Combined light and temperature treatments on C. pepo
处理 温度(白天/黑夜)/℃ 光强/lx 1(对照) 25/15 21000 2(弱光) 25/15 4200 3(低温) 15/3 21000 4(低温弱光) 15/3 4200 
下载: 导出CSV
表 2 不同植物来源POD的氨基酸组成成分及理化性质分析
Table 2. The comparison of composition and physical and chemical characterization of amino acid of POD from different plant species
植物 氨基酸个数 相对分子质量/kDa 等电点 氨基酸比例/% 总平均疏水性 酸性 碱性 脂肪族 芳香族 西葫芦Cucurbita pepo 331 35.7 5.40 33 27 288 28 0.039 甜瓜Cucurbita melo 331 35.6 5.71 28 24 305 26 0.088 黄瓜Cucumis sativus 330 35.5 5.51 28 23 294 28 0.102 丝瓜Luffa aegyptiaca 331 36.3 5.54 31 26 307 61 0.057 笋瓜Cucumis maxima 332 35.8 5.27 33 27 300 27 0.067 苦瓜moschata charatia 328 35.2 6.06 28 25 278 28 0.052 南瓜Cucurbit moschata 320 34.6 5.29 33 27 278 28 0.052 毛茛Aquilegia coerulea 327 34.7 5.89 35 28 288 28 0.065
下载: 导出CSV
-
[1] 康恩祥.低温弱光对西葫芦幼苗生长及生理生化特性的影响[D].兰州: 甘肃农业大学, 2006. [2] 姜军剑.水稻光温敏核不育系的生化特性研究[D].延吉: 延边大学, 2009. [3] CHAVES M M, FLEXAS J, PINHEIRO C. Photosynthesis under drought and salt stress:regulation mechanisms from whole plant to cell.[J]. Annals of botany, 2009, 103(4):551-60. doi: 10.1093/aob/mcn125 [4] 王春萍, 黄启中, 雷开荣, 等.低温弱光下辣椒幼苗叶绿素荧光特性及其与品种耐性的关系[J].园艺学报, 2015, 42(9):1798-1806. http://d.old.wanfangdata.com.cn/Periodical/yyxb201509015 [5] 何勇, 符庆功, 朱祝军.低温弱光对辣椒叶片光合作用、叶绿素荧光猝灭及光能分配的影响[J].核农学报, 2013, 27(4):479-486. http://d.old.wanfangdata.com.cn/Periodical/hnxb201304012 [6] KANG H M, SALTVEIT M E. Chilling tolerance of maize, cucumber and rice seedling leaves and roots are differentially affected by salicylic acid[J]. Physiol Plant, 2002, 115(4):571-576. doi: 10.1034/j.1399-3054.2002.1150411.x [7] 侯伟, 孙爱花, 杨福孙, 等.低温弱光对西瓜幼苗光合作用和抗氧化酶活性的影响[J].热带作物学报, 2015, 36(7):1232-1237. doi: 10.3969/j.issn.1000-2561.2015.07.008 [8] AMNA M, SEJIR C, FRANK Van BREUSEGEM, et al. Catalase function in plants:a focus on Arabidopsis mutants as stress-mimic models.[J]. Journal of Experimental Botany, 2010, 61(15):4197-4220. doi: 10.1093/jxb/erq282 [9] MANI R, MEENA B, VALIVITTAN K, et al. Glutathione-S-transferase and catalase activity in different tissues of marine catfish arius arius on exposure to cadmium[J]. International Journal of Pharmacy & Pharmaceutical Sciences, 2014, 6(1):326-332. [10] HU L, YANG Y, JIANG L, et al. Thecatalasegene family in cucumber:genome-wide identification and organization[J]. Genetics & Molecular Biology, 2016, 39(3):408-415. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5004828/ [11] 林如, 薛秋华.唐菖蒲鲜切花瓶插衰老过程中抗氧化酶活性和膜脂过氧化水平初探[J].福建农林大学学报(自然科学版), 2002, 31(3):352-355. http://d.old.wanfangdata.com.cn/Periodical/fjnydxxb200203020 [12] WANG Z, JIN Z Q, WANG J S, et al. Cloning and characterization of an ascorbate peroxidase gene regulated by ethylene and abscisic acid during banana fruit ripening[J]. African Journal of Biotechnology, 2012, 11(11):10089-10097. http://www.academicjournals.org/ajb/abstracts/abs2012/29May/Wang%20et%20al.htm [13] COLLINS M D. Improved method for quantifying capsaicinoids in Capsicum using performance liquid chromatography[J]. Hort Science, 1995, 30(1):310-314. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=31054cbb116fc412602f6830a6b2fa51 [14] 朱海生, 刘建汀, 王彬.丝瓜过氧化物酶基因的克隆及其在果实褐变中的表达分析[J].农业生物技术学报, 2018, 26(1):43-52. http://d.old.wanfangdata.com.cn/Periodical/nyswjsxb201801005 [15] 朱海生, 王彬, 陈敏氡.中国南瓜内参基因18S rRNA的克隆及其引物开发[J].西北农林科技大学学报(自然科学版), 2017, 45(3):118-125. http://d.old.wanfangdata.com.cn/Periodical/xbnydxxb201703017 [16] 高俊凤.植物生理学实验指导[M].北京:高等教育出版社, 2006. [17] 范小玉, 张显.油菜素内酯对低温弱光胁迫下西瓜幼苗耐冷性的影响[J].北方园艺, 2012(7):5-8. http://d.old.wanfangdata.com.cn/Periodical/bfyany201207002 [18] JYOTHSNA P, MURTHY S D. A review on effect of senescence in plants and role of phytohormone in delaying senescence[J]. Int J Plant Animal Environ Sci, 2016, 6(1):152-161. [19] WANG Y, TONG Y, CHU H, et al. Effects of different light qualities on seedling growth and chlorophyll fluorescence parameters of Dendrobium officinale[J]. Biologia, 2017, 72(7):735-744. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=7da2ead0bb96d7118a12d39474cf2fd2 [20] KANG E X, LUO J J, QIU H Z, et al. Effects of Low Temperature and Light Stress on Gas Exchange and Chlorophyll Fluorescence of Pumpkin Seedlings[J]. Advanced Materials Research, 2012, 450-451:537-542. doi: 10.4028/www.scientific.net/AMR.450-451 [21] 毛丽萍, 任君, 郭尚, 等.西葫芦幼苗耐低温弱光生理指标分析[J].中国蔬菜, 2009, (18):51-55. http://d.old.wanfangdata.com.cn/Periodical/zgsc200918011 [22] 康恩祥, 陈年来, 安翠香, 等.低温弱光对西葫芦幼苗抗氧化酶活性和质膜透性的影响[J].中国蔬菜, 2007(4):16-19. doi: 10.3969/j.issn.1000-6346.2007.04.005 [23] CAUSIN H F, MARCHETTI C F, PENA L B, et al. Down-regulation of catalase activity contributes to senescence induction in wheat leaves exposed to shading stress.[J]. Biologia Plantarum, 2015; 59(1):154-162. doi: 10.1007/s10535-014-0480-z [24] 左应梅, 张金渝, 杨天梅, 等.温度胁迫对三七光合特性及生理指标的影响[J].南方农业学报, 2017, 48(12):2145-2151. doi: 10.3969/j.issn.2095-1191.2017.12.05 [25] AMAKO K, CHEN G X, ASADA K. Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants[J]. Plant & Cell Physiology, 1994, 35(3):497-504. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=HighWire000002843447 [26] WANG S, YUN K, CHENG J, et al. Effects of supplied light on photosynthetic characteristics and translocation of 14C-assimilates of cucumber growing under solar greenhouse.[J]. Transactions of the Chinese Society of Agricultural Engineering, 2008, 24(9):203-206. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=nygcxb200809041 [27] 唐懿, 余雪娜, 何娟, 等.外源褪黑素对低温弱光胁迫下茄子幼苗生理特性的影响[J].湖南农业大学学报(自然科学版), 2017, 43(3):257-261. http://d.old.wanfangdata.com.cn/Periodical/hunannydx201703006 [28] 韩兰兰.西瓜对低温、弱光及其双重胁迫的响应分析[D].武汉: 华中农业大学, 2016. [29] 杜洪涛, 刘世琦, 蒲高斌.光质对彩色甜椒幼苗生长及叶绿素荧光特性的影响[J].西北农业学报, 2005, 14(1):41-45. doi: 10.3969/j.issn.1004-1389.2005.01.010 [30] 庞金安.黄瓜耐低温弱光生理生化机制及其鉴定指标的研究[D].泰安: 山东农业大学, 1996. [31] 徐心诚.主成分分析法对番茄品种耐弱光性的综合评价[J].中国农学通报, 2012, 28(4):135-140. doi: 10.3969/j.issn.1000-6850.2012.04.025 -
点击查看大图
计量
- 文章访问数: 1126
- HTML全文浏览量: 182
- PDF下载量: 22
- 被引次数: 0
