黄秋葵纤维素合酶基因家族鉴定及表达分析

doi:10.16420/j.issn.0513-353x.2021-0277

摘要/Abstract

摘要：

为了研究黄秋葵（Hibiscus esculentus L.）果实老化的机理,测定了其发育过程和采后纤维素含量,显微观察了纤维素在细胞结构中的分布。结果表明,黄秋葵果实老化过程中纤维素含量及在细胞壁的分布大量增加,推测纤维素增加是果实老化的主导因素。从黄秋葵果实转录组测序的RNA-seq数据库中筛选得到9条功能注释为CESA基因家族的片段序列,克隆获得黄秋葵纤维素合酶基因家族CESA1、CESA2、CESA3、CESA4、CESA6、CESA7、CESA8等7个基因全长及CESA5、CESA9的片段。通过荧光定量PCR分析这9个基因的表达,结果表明HeCESA1、HeCESA3、HeCESA6表达模式相同且其蛋白具有初生细胞壁特异性CESA的保守序列CQIC和SVICEXWF基序;HeCESA4、HeCESA8、HeCESA7的表达模式基本一致,并且在果实发育过程中和采后常温贮藏中纤维素显著增加时表达量显著上调,且远远高于同一时期其他CESA。由此推测HeCESA4、HeCESA8和HeCESA7在黄秋葵果实衰老过程纤维素合成中起重要作用。

关键词: 黄秋葵, 老化, 纤维素, 纤维素合酶基因

Abstract:

In order to study the aging mechanism of Hibiscus esculentus fruit,the changes of cellulose content were measured during fruit development and postharvest period,and the distribution of cell structures were observed by microscope. The results showed that during the aging process of H. esculentus fruit,the content of cellulose increased greatly. It was suggested that the increase of cellulose was the main factor of fruit aging. Moreover,nine fragments annotated as CESA gene family were screened from the RNA-seq database of H. esculentus fruit,then the full-length cDNA sequences of CESA1,CESA2,CESA3,CESA4,CESA6,CESA7,CESA8 genes and CESA5and CESA9 gene fragments were cloned. Expression of these nine genes of cellulose synthase gene family in H. esculentus was analyzed by fluorescence quantitative PCR. The results showed HeCESA1,HeCESA3 and HeCESA6had the same expression pattern

and conserved sequences CQIC and SVICEXWF motifs of primary cell wall specific CESAs. HeCESA4,HeCESA8 and HeCESA7 had the same expression pattern,and their expression levels were significantly up-regulated during fruit development and postharvest storage when cellulose increased significantly,which was much higher than that of other CESA genes at the same period. It can be inferred that HeCESA4,HeCESA8and HeCESA7 play an important role in cellulose synthesis of H. esculentus fruit during senescence.

Key words: Hibiscus esculentus, aging, cellulose, cellulose synthase gene

中图分类号:

S649

李永平, 陈敏氡, 刘建汀, 曾美娟, 朱海生, 温庆放. 黄秋葵纤维素合酶基因家族鉴定及表达分析[J]. 园艺学报, 2022, 49(1): 73-85.

LI Yongping, CHEN Mindong, LIU Jianting, Zeng Meijuan, ZHU Haisheng, WEN Qingfang. Identification and Expression Analysis of CESA Gene Family in Hibiscus esculentus[J]. Acta Horticulturae Sinica, 2022, 49(1): 73-85.

图/表 10

参考文献 40

[1]	Appenzeller L, Doblin M, Barreiro R, Wang H, Niu X, Kollipara K, Carrigan L, Tomes D, Chapman M, Dhugga K S. 2004. Cellulose synthesis in maize:isolation and expression analysis of the cellulose synthase(CesA)gene family. Cellulose, 11 (3-4):287-299. doi: 10.1023/B:CELL.0000046417.84715.27 URL
[2]	Bosca S, Barton C J, Taylor N G, Ryden P, Neumetzler L, Pauly M, Roberts K, Seifert G J. 2006. Interactions between mur10/cesa7-dependent secondary cellulose biosynthesis and primary cell wall structure. Plant Physiology, 142:1 353-1 363.
[3]	Burn J E, Hocart C H, Birch R J, Cork C, Williamson R E. 2002. Functional analysis of the cellulose synthase genes CESA1,CESA2 and CESA3 in Arabidopsis. Plant Physiology, 129 (2):797-807. doi: 10.1104/pp.010931 URL
[4]	Cano-Delgado A, Penfield S, Smith C, Catley M, Bevan M. 2003. Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana. Plant J, 34:351-362. doi: 10.1046/j.1365-313X.2003.01729.x URL
[5]	Desprez T, Juraniec M, Crowell E F, Jouy H, Pochylova Z, Parcy F, Hofte H, Gonneau M, Vernhettes S. 2007. Organization of cellulose synthase complexes involved in primary cell wall synthesis in Arabidopsis thaliana. Proceedings of the National Academy of Sciences, 104 (39):15572-15577.
[6]	Ellis C, Karafyllidis I, Wasternack C, Turner J G. 2002. The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Plant Cell, 14:1557-1566. doi: 10.1105/tpc.002022 URL
[7]	Holland N, Holland D, Helentjaris T, Dhugga K S, Cazares X B, Delmer D P, 2000. A comparative analysis of the plant cellulose synthase (CESA)gene family. Plant Physiol, 123 (4):1313-1324. pmid: 10938350
[8]	Hu Liushen, Dong Xiaoying, Li Peihuan, Wang Yongzhang, Liu Chenglian, Yuan Yongbing. 2007. Changes in cell wall component and degrading enzyme activity and their relation to hardness in fruits of peach(Prunus persica L.)before and after ripening. Plant Physiology Communications, 43 (5):837-841. (in Chinese)
	胡留申, 董晓颖, 李培环, 王永章, 刘成连, 原永兵. 2007. 桃果实成熟前后细胞壁成分和降解酶活性的变化及其与果实硬度的关系. 植物生理学通讯, 43 (5):837-841.
[9]	Kaur S, Dhugga K S, Gill K, Singh J. 2016. Novel structural and functional motifs in cellulose synthase(CESA)genes of bread wheat (Triticum aestivum). PLoS ONE, 11 (1):e147046.
[10]	Li Chunxiu. 2005. Cloning and characterization of cellulose synthase from poplar(Populus Tomentosa Carr.)[Ph. D. Dissertation]. Beijing: Chinese Academy of Forestry. (in Chinese)
	李春秀. 2005. 毛白杨纤维素合成酶基因的克隆及功能鉴定[博士论文]. 北京: 中国林业科学研究院.
[11]	Li Feng. 2012. A pilot procedure for paraffin section fabrication of plants. Science & Technology Information,(5):104-105.
	李峰. 2012. 植物石蜡切片制作(paraffin section)的试验流程探讨. 科技信息,(5):104-105. (in Chinese)
[12]	Li S, Lei L, Gu Y. 2013. Functional analysis of complexes with mixed primary and secondary cellulose synthases. Plant Signaling Behavior, 8 (3):e23179. doi: 10.4161/psb.23179 URL
[13]	Li Xianliang. 2014. Functional characterization of cellulose synthase complexes and related proteins in cotton fibers[Ph. D. Dissertation]. Wuhan: Huazhong Agricultural University. (in Chinese)
	李先良. 2014. 棉花纤维素生物合成复合体及相关蛋白功能研究[博士论文]. 武汉: 华中农业大学.
[14]	Liu Yuxiang. 2014. The cDNA cloning and expression analysis of cellulose synthase a gene family of Boehmeria nivea[M. D. Dissertation]. Changsha: Hunan Agricultural University. (in Chinese)
	刘昱翔. 2014. 苎麻CESA基因家族cDNA克隆与表达分析[硕士论文]. 长沙: 湖南农业大学.
[15]	Lu Longjie, Su Nong, Yue sen. 2004. Cabbage-flowering plant-Okra. Vegetables,(10):36. (in Chinese)
	卢隆杰, 苏浓, 岳森. 2004. 菜药花兼用型植物--黄秋葵. 蔬菜,(10):36.
[16]	Pear J, Kawagoe Y, Schreckengost W E, Delmer D P, Stalker D M. 1996. Higher plants contain homologs of bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proc Natl Acad Sci USA, 93:12637-12642. doi: 10.1073/pnas.93.22.12637 URL
[17]	Persson S, Paredez A, Carroll A, Palsdottir H, Doblin M, Poindexter P, Khitrov N, Auer M, Somerville R. 2007. Genetic evidence for three unique components in primary cell-wall cellulose synthase complexes in Arabidopsis. Proceedings of the National Academy of Sciences, 104 (39):15566-15571.
[18]	Ren Jian. 2019. Preliminary study on the fruit aging mechanism of Okra(Hibiscus esculentus L.)[M. D. Dissertation]. Harbin: Northeast Agricultural University. (in Chinese)
	任健. 2019. 秋葵果实老化机理的初步研究[硕士论文]. 哈尔滨: 东北农业大学.
[19]	Ritenour M A, Albrigo L G, Burns J K, Miller W M. 2004. Granulation in Florida citrus. Proc Fla State Hort Soc, 117:358-361.
[20]	Samuga A, Joshi C P. 2002. A new cellulose synthase gene(PtrCES A2)from aspen xylem is orthologous to Arabidopsis AtCESA7(irx3)gene associated with secondary cell wall synthesis. Gene, 296 (1-2):37-44. pmid: 12383501
[21]	Saxena I M, Brown Jr. R M, Fevre M, Geremia R A, Henrissat B. 1995. Multidomain architecture of β-glycosyl transferases:implications for mechanism of action. J Bacteriol, 177:1419-1424. pmid: 7883697
[22]	Scheible W R, Eshed R, Richmond T, Delmer D, Somerville C. 2001. Modifications of cellulose synthase confer resistance to isoxaben and thiazolidinone herbicides in Arabidopsis Ixr1 mutants. Proc Natl Acad Sci USA, 98:10079-10084. doi: 10.1073/pnas.191361598 URL
[23]	Shan Chengying, Ma Shihong, Zhang Weiming. 2012. Utilization and prospect of Okra:a health vegetable. Chinese Wild Plant Resottrces, 31 (2):68-71. (in Chinese)
	单承莺, 马世宏, 张卫明. 2012. 保健蔬菜黄秋葵的应用价值与前景. 中国野生植物资源, 31 (2):68-71.
[24]	She Wenqin, Zhao Xiaoling, Pan Dongming, Lin Hetong. 2008. Relationship between cell wall metabolism and fruit juicy sac granulation during fruit mature stage of pummelo[Citrus grandis(L.)Osbeck“Guanxi-miyou”]. Journ al of Tropical and Subtropicd Botany, 16 (6):545-550. (in Chinese)
	佘文琴, 赵晓玲, 潘东明, 林河通. 2008. 细胞壁代谢与琯溪蜜柚果实成熟过程汁胞粒化的关系. 热带亚热带植物学报, 16 (6):545-550.
[25]	Singh P, Tripathi R D, Singh H N. 1974. Effect of age of picking on the chemical composition of the fruits of Okra. Indian Journal of Agricultural Sciences, 44 (1):22-26.
[26]	Song D L, Shen J H, Li L G. 2010. Characterization of cellulose synthase complexes in Populus xylem differentiation. New Phytologist, 187 (3):777-790. doi: 10.1111/j.1469-8137.2010.03315.x URL
[27]	Soraya D, Mats L, Lars A, Fredrik S, Tuula T T. 2005. The genome sequence of black cottonwood(Populus trichocarpa)reveals 18 conserved cellulose synthase(CESA)genes. Planta, 221 (5):739-746. pmid: 15940463
[28]	Tanaka K, Murata K, Yamazaki M, Onosato K, Miyao A, Hirochika H. 2003. Three distinct rice cellulose synthase catalytic subunit genes required for cellulose synthesis in the secondary wall. Plant Physiol, 133 (1):73-83.
[29]	Taylor N G, Howells R M, Huttly A K, Vickers K, Turner S R. 2003. Interactions among three distinct CESA proteins essential for cellulose synthesis. Proceedings of the National Academy of Sciences,USA, 100:1450-1455.
[30]	Tong Tingting. 2016. Preliminary analysis of the functions of the CESA genes in sacred lotus(Nelumbo nucifera)[M. D. Dissertation]. Guangzhou: South China Agricultural University. (in Chinese)
	仝婷婷. 2016. 几个莲花纤维素合酶基因功能的初步分析[硕士论文]. 广州: 华南农业大学.
[31]	Wang D, Yuan S, Yin L, Zhao J, Guo B, Lan J, Li X. 2012. A missense mutation in the transmembrane domain of CESA9 affects cell wall biosynthesis and plant growth in rice. Plant Sci, 196:117-124. doi: 10.1016/j.plantsci.2012.08.002 URL
[32]	Wang L Q, Guo K, Li Y, Tu Y Y, Hu H Z, Wang B R, Cui X C, Peng L C. 2010. Expression profiling and integrative analysis of the CESA/CSL superfamily in rice. BMC Plant Biology, 10 (1):282. doi: 10.1186/1471-2229-10-282 URL
[33]	Wang Junyao, Zhou Jun, Tang Guping. 2003. A study on the effect of anti-fatigue of okra. Chinese Journal of Modern Applied Pharmacy, 20 (4):316-317. (in Chinese)
	王君耀, 周峻, 汤谷平. 2003. 黄秋葵抗疲劳作用的研究. 中国现代应用药学, 20 (4):316-317.
[34]	Wu Hua, Chen Pingting, Yuan Ling, Xu Yongrong, Chen Longqing. 2011. Study on the technique of making paraffin section of Fern. Hubei Agricultural Sciences, 50 (18):3767-3774. (in Chinese)
	吴华, 陈娉婷, 袁玲, 徐永荣, 陈龙清. 2011. 蕨类植物石蜡切片制作技术探讨. 湖北农业科学, 50 (18):3767-3774.
[35]	Wu Yaoting, Zhang Hengmu, Liu Jinyuan. 2003. Cellulose biosynthesis in developing cotton fibers. Cotton Science, 15 (3):174-179. (in Chinese)
	武耀廷, 张恒木, 刘进元. 2003. 棉纤维细胞发育过程中纤维素的生物合成. 棉花学报, 15 (3):174-179.
[36]	Xu Ruyi, Luo Feng, Yuan Tingqing, Huang Rengong, Li Jinsong. 2011. Effect of picking time on the fruit characters of Okra. Journal of Changjiang Vegetables,(2):18-20. (in Chinese)
	许如意, 罗丰, 袁廷庆, 黄仁功, 李劲松. 2011. 不同采摘期对黄秋葵果实性状和品质的影响. 长江蔬菜,(2):18-20.
[37]	Xiong Bo, Wang Zhihui, Shi Dongdong, Zhang Tingting, Gao Jingfei, Li Qingnan, Cao Shuyan. 2014. Relationship between granulation and materials of cell wall,polyamine in Huangguogan fruit. Acta Agriculturae Boreali Sinica, 29:239-242. (in Chinese)
	熊博, 汪志辉, 石冬冬, 张婷婷, 高婧斐, 李清南, 曹淑燕. 2014. 黄果柑果实粒化与细胞壁物质及多胺的关系. 华北农学报, 29:239-242.
[38]	Xu Changtong. 2009. Preliminary study on sensory and physicochemical evaluation of Olive fruit quality. Fujian Fruits,(4):35-37. (in Chinese)
	许长同. 2009. 橄榄鲜食果品质的感观与理化评价初探. 福建果树,(4):35-37.
[39]	Yue Xiang. 2009. Studies on the analysis of dietary fiber content and physiology and biochemistry characterisics in non-heading Chinese cabbage(Brassica campestris ssp. chinensis Makino)[M. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	岳翔. 2009. 不结球白菜膳食纤维含量分析及理化特性研究[硕士论文]. 南京: 南京农业大学.
[40]	Zhang Xiaorong, Tan Junfeng, Wen Manqing, Miao Zhenyan. 2019. Systematic identification and functional study of CESA family in maize. Journal of Northwest A＆F University(Nat Sci Ed.),47 (2):45-53. (in Chinese)
	张晓榕, 谭俊峰, 温曼晴, 苗震龑. 2019. 玉米CESA家族的系统鉴定及功能研究. 西北农林科技大学学报(自然科学版), 47 (2):45-53.

花后天数/d Days post anthesis	纤维素 Cellulose	采后天数/d Days after fruit harvest	纤维素 Cellulose
2	2.40 ± 1.95 d	0	2.74 ± 1.19 e
4	2.69 ± 1.45 c	1	2.83 ± 0.71 d
6	2.74 ± 2.21 c	2	3.07 ± 3.25 c
8	2.96 ± 1.96 b	3	3.21 ± 1.42 b
10	3.54 ± 1.01 a	4	3.36 ± 4.23 a

花后天数/d Days post anthesis	纤维素 Cellulose	采后天数/d Days after fruit harvest	纤维素 Cellulose
2	2.40 ± 1.95 d	0	2.74 ± 1.19 e
4	2.69 ± 1.45 c	1	2.83 ± 0.71 d
6	2.74 ± 2.21 c	2	3.07 ± 3.25 c
8	2.96 ± 1.96 b	3	3.21 ± 1.42 b
10	3.54 ± 1.01 a	4	3.36 ± 4.23 a

蛋白编号 Protein code	氨基酸数 Number of amino acids	分子量/Da Molecular weight	理论等电点 Theoretical pI	脂肪族指数 Aliphatic index	疏水性指数 Grand average of hydropathicity	不稳定性指数 Instability index	亚细胞定位 Subcellular localization	组别 Group
HeCESA1	1 095	121 800.60	6.14	86.98	-0.187	38.32	质膜Plasma membrane	4
HeCESA2	1 134	128 019.45	6.39	88.53	-0.142	43.31	质膜Plasma membrane	2
HeCESA3	956	107 205.54	8.13	90.54	-0.085	35.78	质膜Plasma membrane	5
HeCESA4	1 034	117 311.12	8.28	82.00	-0.229	36.20	质膜Plasma membrane	6
HeCESA5	719	80 874.97	8.80	90.67	-0.019	38.40	质膜Plasma membrane	2
HeCESA6	1 090	123 207.56	7.27	88.95	-0.143	38.66	质膜Plasma membrane	2
HeCESA7	1 043	117 870.50	6.36	82.44	-0.213	41.50	质膜Plasma membrane	3
HeCESA8	974	109 452.91	6.33	85.15	-0.104	38.75	质膜Plasma membrane	1
HeCESA9	901	102 156.53	8.69	90.95	-0.101	38.76	质膜Plasma membrane	2

蛋白编号 Protein code	氨基酸数 Number of amino acids	分子量/Da Molecular weight	理论等电点 Theoretical pI	脂肪族指数 Aliphatic index	疏水性指数 Grand average of hydropathicity	不稳定性指数 Instability index	亚细胞定位 Subcellular localization	组别 Group
HeCESA1	1 095	121 800.60	6.14	86.98	-0.187	38.32	质膜Plasma membrane	4
HeCESA2	1 134	128 019.45	6.39	88.53	-0.142	43.31	质膜Plasma membrane	2
HeCESA3	956	107 205.54	8.13	90.54	-0.085	35.78	质膜Plasma membrane	5
HeCESA4	1 034	117 311.12	8.28	82.00	-0.229	36.20	质膜Plasma membrane	6
HeCESA5	719	80 874.97	8.80	90.67	-0.019	38.40	质膜Plasma membrane	2
HeCESA6	1 090	123 207.56	7.27	88.95	-0.143	38.66	质膜Plasma membrane	2
HeCESA7	1 043	117 870.50	6.36	82.44	-0.213	41.50	质膜Plasma membrane	3
HeCESA8	974	109 452.91	6.33	85.15	-0.104	38.75	质膜Plasma membrane	1
HeCESA9	901	102 156.53	8.69	90.95	-0.101	38.76	质膜Plasma membrane	2

基因 Gene	采后处理 Fruit after harvest	果实发育过程 Fruit development	全部处理 All the treatments	基因 Gene	采后处理 Fruit after harvest	果实发育过程 Fruit development	全部处理 All the treatments
HeCESA1	0.9551^**	0.0411	0.0456	HeCESA6	0.5459	0.5596	0.0018
HeCESA2	0.2166	0.1058	0.0613	HeCESA7	0.9759^**	0.8939^**	0.6995^*
HeCESA3	0.2653	0.0411	0.0196	HeCESA8	0.9297^**	0.7204^*	0.6162^*
HeCESA4	0.9912^**	0.7394^*	0.6232^*	HeCESA9	0.4677	0.4771	0.0046
HeCESA5	0.7558*	0.5842	0.0124