Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (2): 312-326.doi: 10.3864/j.issn.0578-1752.2019.02.010

• HORTICULTURE • Previous Articles     Next Articles

Cloning of Adβgal-1 and Adβgal-2 Genes and Their Roles During Fruit Softening of Kiwifruit

FENG Xin,LAI RuiLian,GAO MinXia,CHEN WenGuang,WU RuJian,CHEN YiTing()   

  1. Fruit Research Institute, Fujian Academy of Agricultural Sciences/Research Centre for Engineering Technology of Fujian Deciduous Fruits, Fuzhou 350013
  • Received:2018-07-01 Accepted:2018-11-12 Online:2019-01-16 Published:2019-01-21
  • Contact: YiTing CHEN E-mail:fujiankiwifruit@163.com

RichHTML

12

PDF

228

Abstract:

【Objective】 Beta-galactosidase is one type of glycosidases involving in cell wall degradation, which plays an important role in plant growth and fruit ripening and softening. In this paper, the identification and expression analysis of two β-galactosidase genes in kiwifruit, as well as their functions during fruit softening, were carried out to provide new insights for the mechanism of kiwifruit softening.【Method】 Actinidia chinensis var. deliciosa ‘Miliang-1’ was used as material to amplify the β-galactosidase genes by RT-PCR, then their characteristics, functional domains, phylogenetic relationships, gene structures and miRNAs were analyzed by using online databases. qRCR was applied to analyze their expression patterns in different tissues, different stages of softening, different storage temperatures, and ABA treatment. Enzymes activities were also analyzed to clarify the function of these two β-galactosidase genes during fruit softening.【Results】Two β-galactosidase genes (Adβgal-1 and Adβgal-2) were obtained from kiwifruit. Their accession numbers were MH319788 and MH319789, respectively. Adβgal-1 had an open reading frame (ORF) of 2 280 bp and encoded 759 amino acids, while the ORF of Adβgal-2 was 2 025 bp encoding 674 amino acids. Protein domain analysis showed that both Adβgal-1 and Adβgal-2 harbored a functional domain (Glyco_hydro_35) of plant glycoside hydrolase family 35. Gene organization analysis revealed that Adβgal-1 had 18 exons and 17 introns, while Adβgal-2 harbored 17 exons and 16 introns. Moreover, Adβgal-1 and Adβgal-2 were clustered in different evolutionary branching in the phylogenetic tree, which was due to the great difference in their sequences. This implied that Adβgal-1 and Adβgal-2 might originate from different ancestral genes. qPCR analysis showed that Adβgal-1 and Adβgal-2 were expressed in all tested tissues (root, stem, leaf, flower, young fruit and ripe fruit) although their expression levels were different. The expression levels of both Adβgal-1 and Adβgal-2 were increased at the start stage of fruit softening, and the amount of gene expression continued to increase as the firmness of fruit decreased. When stored at 25℃, both the Adβgal-1 and Adβgal-2 were upregulated at 3 d, and the amount of gene expression continued to increase as time over. When stored at 4℃, the expression of Adβgal-1 was inhabited, while that of Adβgal-2 was like wavy. ABA induced the expression of Adβgal-1 and Adβgal-2.【Conclusion】 Both Adβgal-1 and Adβgal-2 participated in the process of fruit softening of kiwifruit. The effect of storage temperatures and ABA on fruit softening might be achieved by changing the expression of β-galactosidase genes.

Key words: Actinidia chinensis, beta-galactosidase genes, fruit softening, enzyme activity, gene expression

Fig. 1

The softening stages of kiwifruit S1-S7: Ripe fruits with high hardness (control), start stage of softening, stage I of softening, stage II of softening, stage III of softening, late stage of softening, stage of soft and decay, bar: 1 cm. The same as bellow"

Fig. 2

The physiological and biochemical indexes of kiwifruit during fruit softening ** indicate statistically significant differences at P<0.01. The same as below"

Fig. 3

Sequence alignment of the ORF regions from Adβgal-1 and Ach04g123061"

Fig. 4

Sequence alignment of the ORF regions from Adβgal-2 and Ach09g294421"

Fig. 5

Multiple sequence alignments of the Adβgal and other plant beta-galactosidases Identical conserved residues are shaded in black, while similar residues are shaded in gray. The conserved motif of glycoside hydrolase family 35 is boxed. Ccβgal：克萊门柚Citrus clementina，XP006434246；Siβgal：芝麻Sesamum indicum，XP011069386；Muβgal：香蕉Musa acuminata，XP018676452；Ppβgal：碧桃Prunus persica XP007199661；Mnβgal：川桑Morus notabilis，XP010107841；Jrβgal：核桃Juglans regia，XP018840337"

Fig. 6

Phylogenetic tree of the beta-galactosidase proteins from various plant species Numbers at the nodes represent the reliability percent of bootstrap values on 1000 replications. The bar shows branch length"

Fig. 7

Genomic structure of the Adβgal genes A: The extron and intron organization of Adβgal genes. B: The length of extrons and introns of Adβgal genes. The length of introns was in brackets. The unit of length (bp)"

Fig. 8

Relative expression of the Adβgal genes in different tissues"

Fig. 9

Relative expression of the Adβgal genes during fruit softening"

Fig. 10

Relative expression of the Adβgal genes under different storage temperatures indicates statistically significant differences at P<0.05"

Fig. 11

Relative expression of the Adβgal genes after ABA treatment"

[1] MCATEE P A, RICHARDSON A C, NIEUWENHUIZEN N J, GUNASEELAN K, HOONG L, CHEN X, ATKINSON R G, BURDON J N, DAVID K M, SCHAFFER R J . The hybrid non-ethylene and ethylene ripening response in kiwifruit (Actinidia chinensis) is associated with differential regulation of MADS-box transcription factors. BMC Plant Biology, 2015,15(1):304.
[2] 黄文俊, 钟彩虹 . 猕猴桃果实采后生理研究进展. 植物科学学报, 2017,35(4):622-630.
HUANG W J, ZHONG C H . Research advances in the postharvest physiology of kiwifruit. Plant Science Journal, 2017,35(4):622-630. (in Chinese)
[3] 李东, 谭书明, 邓毅, 张程榕, 蒋想 . 猕猴桃采前处理保鲜技术研究进展. 食品工业, 2015,36(8):251-255.
LI D, TAN S M, DENG Y, ZHANG C R, JIANG X . Advances in preharvest preservation methods for kiwifruit. Food Industry, 2015,36(8):251-255. (in Chinese)
[4] 朱明月, 沈文涛, 周鹏 . 果实成熟软化机理研究进展. 分子植物育种, 2005,3(3):421-426.
ZHU M Y, SHEN W T, ZHOU P . Research advances on mechanism of fruit ripening and softening. Molecular Plant Breeding, 2005,3(3):421-426. (in Chinese)
[5] 田爱梅, 刘金龙, 曹家树 . 植物β-半乳糖苷酶. 中国细胞生物学学报, 2014,36(5):703-707.
TIAN A M, LIU J L, CAO J S . Beta galactosidase in plants. Chinese Journal of Cell Biology, 2014,36(5):703-707. (in Chinese)
[6] 陆胜民, 席玙芳, 张耀洲 . 梅果采后软化与细胞壁组分及其降解酶活性的变化. 中国农业科学, 2003,36(5):595-598.
LU S M, XI Y F, ZHANG Y Z . Softening of green mume flesh and changes of cell wall components and activities of their degrading enzymes during the postharvest period. Scientia Agricultura Sinica, 2003,36(5):595-598. (in Chinese)
[7] DAWSON D M, MELTON L D, WATKINS C B . Cell wall changes in nectarines ( Prunus persica): Solubilization and depolymerization of pectic and neutral polymers during ripening and in mealy fruit. Plant Physiology, 1992,100:1203-1210.
[8] LIDSTER P D, MCRAE K B, DICK A J . Effects of a vacuum infusion of a partially purified beta-galactosidase inhibitor on apple quality. HortScience, 1985,20:80-82.
[9] 庄军平, 苏菁, 陈维信 . 香蕉果实β-半乳糖苷酶基因cDNA克隆及序列分析. 西北植物学报, 2006,26(1):18-22.
ZHUANG J P, SUN J, CHEN W X . cDNA cloning and sequencing of β-galactosidase genes in banana ( Musa spp.) fruit. Acta Botanica Boreali-occidentalia Sinica, 2006,26(1):18-22. (in Chinese)
[10] 周厚成, 李刚, 赵霞, 郭蔼光 . 草莓β-半乳糖苷酶基因FaTβgal的克隆与表达分析. 西北植物学报, 2015,35(12):2385-2390.
ZHOU H C, LI G, ZHAO X, GUO A G . Cloning and expression analysis of FaTβgal of β-galactosidase family from Fragaria ananassa fruit. Acta Botanica Boreali-occidentalia Sinica, 2015,35(12):2385-2390. (in Chinese)
[11] 吕燕荣, 任小林, 周会玲 . 枣果实β-半乳糖苷酶基因的克隆及表达分析. 西北植物学报, 2011,31(7):1318-1325.
LÜ Y R, REN X L, ZHOU H L . Cloning and expressing analysis of β-galactosidase gene in jujube fruit. Acta Botanica Boreali- occidentalia Sinica, 2011,31(7):1318-1325. (in Chinese)
[12] 高山, 陈桂信, 许端祥, 林碧英, 林义章, 潘东明 . 苦瓜果实β-半乳糖苷酶基因的克隆、表达及亚细胞定位. 植物遗传资源学报, 2013,14(6):1124-1129.
GAO S, CHEN G X, XU D X, LIN B Y, LIN Y Z, PAN D M . Cloing, expression and subcellular localization of β-galactosidase gene from Momordica charantia L. fruit. Journal of Plant Genetic Resources, 2013,14(6):1124-1129. (in Chinese)
[13] 卞伟华, 江昌俊, 王朝霞 . 桃叶片β-半乳糖苷酶基因全长cDNA克隆及原核表达. 园艺学报, 2006,33(4):721-725.
BIAN W H, JIANG C J, WANG Z C . Cloning and expression of β-galactosidase gene cDNA in peach. Acta Horticulturae Sinica, 2006,33(4):721-725. (in Chinese)
[14] ZHANG H M, LIU J Y . Molecular cloning and characterization of a β-galactosidase gene expressed preferentially in cotton fibers. Journal of Integrative Plant Biology, 2005,47(2):223-232.
doi: 10.1111/jipb.2005.47.issue-2
[15] 何玮毅, 陈晓静, 申艳红, 卢秉国 . 番木瓜β-Gal基因的克隆分析与植物表达载体构建. 热带作物学报, 2010,31(2):217-223.
HE W Y, CHEN X J, SHEN Y H, LU B G . Isolation of a papaya β-Gal gene and construction of plant expresson vector. Chinese Journal of Tropical Crops, 2010,31(2):217-223. (in Chinese)
[16] BAN Q, HAN Y, MENG K, HOU Y, HE Y, RAO J . Characterization of β-galactosidase genes involved in persimmon growth and fruit ripening and in response to propylene and 1-methylcyclopropene. Journal of Plant Growth Regulation, 2016,35(4):1025-1035.
doi: 10.1007/s00344-016-9601-6
[17] TATEISHI A, NAGASHIMA K . Differential expression of members of the beta-galactosidase gene family during Japanese pear( Pyrus pyrifolia L.) fruit growth and on-tree ripening. Journal of the American Society for Horticultural Science, 2005,130(6):819-829.
[18] PANIAGUA C, BLANCO-PORTALES R, BARCELO-MUNOZ M, CARCIA-GAGO J A, WALDRON K W, QUESADA M A, MUNOZ-BLANCO J, MERCADO J A . Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening. Journal of Experimental Botany, 2016,67(3):619-631.
[19] ROSS G S, REDGWELL R J, MACRAE E A . Kiwifruit β-galactosidase: Isolation and activity against specific fruit cell-wall polysaccharides. Planta, 1993,189(4):499-506.
[20] 陈昆松, 张上隆, >Ross Gavin S . β-半乳糖苷酶基因在猕猴桃果实成熟过程的表达. 植物生理学报, 2000,26(2):117-122.
CHEN K S, ZHANG S L, Ross G S . Expression pattern of β-galactosidase gene in ripening Actinidia chinensis fruit. Acta Phytophysologica Sinica, 2000,26(2):117-122. (in Chinese)
[21] HUANG S X, DING J, DENG D, TANG W, SUN H, LIU D, ZHANG L, NIU X, ZHANG X, MENG M, YU J, LIU J, HAN Y, SHI W, ZHANG D, CAO S, WEI Z, CUI Y, XIA Y, ZENG H, BAO K, LIN L, MIN Y, ZHANG H, MIAO M, TANG X, ZHU Y, SUI Y, LI G, SUN H, YUE J, SUN J, LIU F, ZHOU L, LEI L, ZHENG X, LIU M, HUANG L, SONG J, XU C, LI J, YE K, ZHONG S, LU B, HE G, XIAO F, WANG H, ZHENG H, FEI Z, LIU Y . Draft genome of the kiwifruit Actinidia chinensis. Nature communications, 2013,4. doi: 10.1038/ ncomms3640.
[22] 张慧琴, 鸣谢, 肖金平, 周利秋, 宋根华 . 猕猴桃实时荧光定量PCR分析中内参基因的筛选. 浙江农业学报, 2015,27(4):567-573.
ZHANG H Q, XIE M, XIAO J P, ZHOU L Q, SONG G H . Screening of reference genes for real-time quantitative PCR in kiwifruit. Acta Agruiculturae Zhenjiangensis, 2015,27(4):567-573. (in Chinese)
[23] DAI X, ZHAO P X . psRNATarget: A plant small RNA target analysis server. Nucleic Acids Research, 2011,39(suppl 2):W155-W159.
doi: 10.1093/nar/gkr319
[24] LIU J H, GAO M H, LV M L, CAO J S . Structure, evolution, and expression of the β-galactosidase gene family in Brassica campestris ssp. chinensis. Plant Molecular Biology Reporter, 2013,31(6):1249-1260.
[25] TATEISHI A, SHIBA H, OGIHARA J, ISOBE K, NOMURA K, WATANABE K, INOUE H . Differential expression and ethylene regulation of β-galactosidase genes and isozymes isolated from avocado (Persea americana Mill.) fruit. Postharvest Biology and Technology, 2007,45(1):56-65.
[26] MONEO-SANCHEZ M, IZQUIERDO L, MARTIN I, LABRADOR E, DOPICO B . Subcellular location of Arabidopsis thaliana subfamily a1 β-galactosidases and developmental regulation of transcript levels of their coding genes. Plant Physiology and Biochemistry, 2016,109:137-145.
[27] O'Donoghue E M, SOMERFIELD S D, DEROLES S C, SUTHERLAND P W, HALLETT I C, ERRIDGE Z A, BRUMMELL D A, HUNTER D A, . Simultaneous knock-down of six β-galactosidase genes in petunia petals prevents loss of pectic galactan but decreases petal strength. Plant Physiology and Biochemistry, 2017,13:208-221.
[28] 张美芳, 何玲, 张美丽, 郭宇欢 . 猕猴桃鲜果贮藏保鲜研究进展. 食品科学, 2014,35(11):343-347.
doi: 10.7506/spkx1002-6630-201411066
ZHANG M F, HE L, ZHANG M L, GUO Y H . Advances in preservation method for kiwifruit. Food Science, 2014,35(11):343-347. (in Chinese)
doi: 10.7506/spkx1002-6630-201411066
[29] 陈义挺, 赖瑞联, 程春振, 冯新, 屈蒙蒙, 刘萍元, 陈文光, 吴如健 . 猕猴桃果实软化过程中AcPG基因的克隆与表达. 农业生物技术学报, 2017,25(2):205-213.
CHEN Y T, LAI R L, CHENG C Z, FENG X, QU M M, LIU P Y, CHEN W G, WU R J . Cloning and expression of polygalacturonase gene ( AcPG) during fruit softening of kiwifruit( Actinidia chinensis). Journal of Agricultural Biotechnology, 2017,25(2):205-213. (in Chinese)
[30] 徐秋红 . 中国李果实软化相关基因的克隆及表达分析[D]. 南京: 南京农业大学, 2009.
XU Q H . Isolation and expression of genes related to Chinese plum fruit softening[D]. Nanjing: Nanjing Agruicultural University, 2009.
[31] 陈昆松, 李方, 张上隆 . 猕猴桃果实成熟进程中木葡聚糖内糖基转移酶mRNA水平的变化. 植物学报, 1999,41(11):1231-1234.
CHEN K S, LI F, ZHANG S L . The expression pattern of xyloglucan endotransglycosylase gene in fruit ripeing of Actinidia chinensis. Acta Botanica Sinica, 1999,41(11):1231-1234. (in Chinese)
[32] 陈昆松, 李方, 张上隆, Ross Gavin S . ABA和IAA对猕猴桃果实成熟进程的调控. 园艺学报, 1999,26(2):81-86.
CHEN K S, LI F, ZHANG S L, ROSS G S . Role of abscisic acid and indole-3-acetic acid in kiwifruit ripening. Acta Horticulturae Sinica, 1999,26(2):81-86. (in Chinese)
[33] 陈昆松, 张上隆, 吕均良, 陈青俊 . 脱落酸、吲哚乙酸和乙烯在猕猴桃果实后熟软化进程中的变化. 中国农业科学, 1997,30(2):54-57.
CHEN K S, ZHANG S L, LÜ J L, CHEN Q J . Variation of abscisic acid, indole-3-acetic acid and ethylene in kiwifruit during fruit ripening. Scientia Agricultura Sinica, 1997,30(2):54-57. (in Chinese)
[1] GU LiDan,LIU Yang,LI FangXiang,CHENG WeiNing. Cloning of Small Heat Shock Protein Gene Hsp21.9 in Sitodiplosis mosellana and Its Expression Characteristics During Diapause and Under Temperature Stresses [J]. Scientia Agricultura Sinica, 2023, 56(1): 79-89.
[2] ZHANG KeKun,CHEN KeQin,LI WanPing,QIAO HaoRong,ZHANG JunXia,LIU FengZhi,FANG YuLin,WANG HaiBo. Effects of Irrigation Amount on Berry Development and Aroma Components Accumulation of Shine Muscat Grape in Root-Restricted Cultivation [J]. Scientia Agricultura Sinica, 2023, 56(1): 129-143.
[3] ZHANG JiaHua,YANG HengShan,ZHANG YuQin,LI CongFeng,ZHANG RuiFu,TAI JiCheng,ZHOU YangChen. Effects of Different Drip Irrigation Modes on Starch Accumulation and Activities of Starch Synthesis-Related Enzyme of Spring Maize Grain in Northeast China [J]. Scientia Agricultura Sinica, 2022, 55(7): 1332-1345.
[4] LAI ChunWang, ZHOU XiaoJuan, CHEN Yan, LIU MengYu, XUE XiaoDong, XIAO XueChen, LIN WenZhong, LAI ZhongXiong, LIN YuLing. Identification of Ethylene Synthesis Pathway Genes in Longan and Its Response to ACC Treatment [J]. Scientia Agricultura Sinica, 2022, 55(3): 558-574.
[5] SHU JingTing,SHAN YanJu,JI GaiGe,ZHANG Ming,TU YunJie,LIU YiFan,JU XiaoJun,SHENG ZhongWei,TANG YanFei,LI Hua,ZOU JianMin. Relationship Between Expression Levels of Guangxi Partridge Chicken m6A Methyltransferase Genes, Myofiber Types and Myogenic Differentiation [J]. Scientia Agricultura Sinica, 2022, 55(3): 589-601.
[6] GUO ShaoLei,XU JianLan,WANG XiaoJun,SU ZiWen,ZHANG BinBin,MA RuiJuan,YU MingLiang. Genome-Wide Identification and Expression Analysis of XTH Gene Family in Peach Fruit During Storage [J]. Scientia Agricultura Sinica, 2022, 55(23): 4702-4716.
[7] ZHU ChangWei,MENG WeiWei,SHI Ke,NIU RunZhi,JIANG GuiYing,SHEN FengMin,LIU Fang,LIU ShiLiang. The Characteristics of Soil Nutrients and Soil Enzyme Activities During Wheat Growth Stage Under Different Tillage Patterns [J]. Scientia Agricultura Sinica, 2022, 55(21): 4237-4251.
[8] ZHANG Chuan,LIU Dong,WANG HongZhang,REN Hao,ZHAO Bin,ZHANG JiWang,REN BaiZhao,LIU CunHui,LIU Peng. Effects of High Temperature Stress in Different Periods on Dry Matter Production and Grain Yield of Summer Maize [J]. Scientia Agricultura Sinica, 2022, 55(19): 3710-3722.
[9] KANG Chen,ZHAO XueFang,LI YaDong,TIAN ZheJuan,WANG Peng,WU ZhiMing. Genome-Wide Identification and Analysis of CC-NBS-LRR Family in Response to Downy Mildew and Powdery Mildew in Cucumis sativus [J]. Scientia Agricultura Sinica, 2022, 55(19): 3751-3766.
[10] YuXia WEN,Jian ZHANG,Qin WANG,Jing WANG,YueHong PEI,ShaoRui TIAN,GuangJin FAN,XiaoZhou MA,XianChao SUN. Cloning, Expression and Anti-TMV Function Analysis of Nicotiana benthamiana NbMBF1c [J]. Scientia Agricultura Sinica, 2022, 55(18): 3543-3555.
[11] XIA QianWei,CHEN Hao,YAO YuTian,DA Da,CHEN Jian,SHI ZhiQi. Effects of ‘Good Quality Standard’ Rice System on Soil Environment of Paddy Field [J]. Scientia Agricultura Sinica, 2022, 55(17): 3343-3354.
[12] HU YaLi,NIE JingZhi,WU Xia,PAN Jiao,CAO Shan,YUE Jiao,LUO DengJie,WANG CaiJin,LI ZengQiang,ZHANG Hui,WU QiJing,CHEN Peng. Effect of Salicylic Acid Priming on Salt Tolerance of Kenaf Seedlings [J]. Scientia Agricultura Sinica, 2022, 55(14): 2696-2708.
[13] JIN MengJiao,LIU Bo,WANG KangKang,ZHANG GuangZhong,QIAN WanQiang,WAN FangHao. Light Energy Utilization and Response of Chlorophyll Synthesis Under Different Light Intensities in Mikania micrantha [J]. Scientia Agricultura Sinica, 2022, 55(12): 2347-2359.
[14] YUAN JingLi,ZHENG HongLi,LIANG XianLi,MEI Jun,YU DongLiang,SUN YuQiang,KE LiPing. Influence of Anthocyanin Biosynthesis on Leaf and Fiber Color of Gossypium hirsutum L. [J]. Scientia Agricultura Sinica, 2021, 54(9): 1846-1855.
[15] SHU JingTing,JI GaiGe,SHAN YanJu,ZHANG Ming,JU XiaoJun,LIU YiFan,TU YunJie,SHENG ZhongWei,TANG YanFei,JIANG HuaLian,ZOU JianMin. Expression Analysis of IGF1-PI3K-Akt-Dependent Pathway Genes in Skeletal Muscle and Liver Tissue of Yellow Feather Broilers [J]. Scientia Agricultura Sinica, 2021, 54(9): 2027-2038.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd