番茄果胶裂解酶基因SlPL参与调控裂果机制研究

doi:10.16420/j.issn.0513-353x.2023-0816

摘要/Abstract

摘要：

以裂果率差异极显著的番茄为材料，分析了SlPL（Solyc03g111690）基因的表达差异，进一步利用基因遗传转化对其进行功能验证。结果表明，SlPL在易裂番茄‘NT189’中的表达量显著高于耐裂番茄‘NT91’；在番茄果实中的表达量显著高于根、茎、叶、花等器官，且在果实转色期和红熟期表达量较高；通过灌水处理和ABA处理诱导果实开裂，发现在相同处理时期，易裂果材料果实中SlPL表达量总体显著高于耐裂果材料。通过遗传转化获得SlPL过表达（OEPL）和敲除（pl）的株系，与野生型相比，OEPL更易裂果，且果实硬度显著降低，pl果实硬度升高。OEPL果实中原果胶含量显著低于野生型，水溶性果胶含量显著高于野生型，pl果实中原果胶和总果胶含量显著高于野生型；OEPL果实中果胶裂解酶活性显著高于野生型，pl果实中果胶裂解酶活性显著低于野生型。基因表达分析发现，OEPL果实中细胞壁代谢相关基因SlPG2、SlPME2.1、SlCel2、SlGH9C5和乙烯合成途径相关基因SlACS4、SlACO1的相对表达量显著高于野生型，pl果实中则相反。pl果实中乙烯响应因子SlERF2的相对表达量显著高于野生型。果皮显微结构观察发现，与野生型相比，OEPL表皮层细胞和薄壁细胞排列稀疏，而pl果皮细胞排列更为紧密。

关键词: 番茄, 裂果, SlPL, 功能验证

Abstract:

Tomato with highly significant differences in fruit cracking rate were used as the materials to analyze the expression differences of the SlPL（Solyc03g111690）gene，which was further functionally validated using genetic transformation. The results showed that the expression of SlPL was significantly higher in crack-susceptible tomato‘NT189’than in crack-resistant tomato‘NT91’；The expression of SlPL in tomato fruit was significantly higher than that in other organs such as roots，stems，leaves and flowers，and the expression was higher in the break ripen and red ripen stages of the fruit. Fruit cracking was further induced by irrigation and ABA treatments，and it was found that the expression of SlPL was overall significantly higher in the fruit of crack-susceptible materials than that of crack-resistant ones in the same treatment period. SlPL overexpression（OEPL）and knockout（pl）lines were then obtained by genetic transformation. OEPL was more susceptible to fruit cracking and had significantly lower fruit firmness and pl had higher fruit firmness than the wild type. OEPL fruit had significantly lower pro-pectin content than the wild type，and significantly higher water-soluble pectin content than the wild type，while pl fruit had significantly higher pro-pectin and total pectin content than the wild type. Pectin cleavage enzyme activity was significantly higher in OEPL fruits than in wild type，and pectin cleavage enzyme activity was significantly lower in pl fruits than in wild type. Gene expression analysis revealed that the relative expression of cell wall metabolism-related genes SlPG2，SlPME2.1，SlCel2，SlGH9C5，and ethylene synthesis pathway-related genes SlACS4 and SlACO1 was significantly higher in OEPL fruits than in wild type，and the opposite was true for pl fruits. pl fruits showed a significantly higher relative expression of the ethylene-responsive factor SlERF2 than in wild type. Observations of the pericarp microstructure revealed that the cells of the epidermal layer and thin-walled cells were more sparsely arranged in OEPL compared with wild type，and those cells were tightly arranged in pl.

Key words: tomato, fruit cracking, SlPL, functional identification

仲钊江, 吴震, 周蓉, 朱为民, 杨学东, 于筱薇, 徐艳, 高扬杨, 蒋芳玲. 番茄果胶裂解酶基因SlPL参与调控裂果机制研究[J]. 园艺学报, 2024, 51(2): 295-308.

ZHONG Zhaojiang, WU Zhen, ZHOU Rong, ZHU Weimin, YANG Xuedong, YU Xiaowei, XU Yan, GAO Yangyang, JIANG Fangling. Study on the Regulatory Mechanism of SlPL Gene Affecting Tomato Fruit Cracking[J]. Acta Horticulturae Sinica, 2024, 51(2): 295-308.

图/表 10

表1 本研究中所用的PCR引物

Table 1 PCR primers used in this study

用途 Purpose	名称 Name	序列（5′-3′） Sequence
基因克隆引物 Primer for genes clone	pbi121-SlPL-F	gagaacacgggggactctagaATGGGCACTTCCTCTGTTTTTC
基因克隆引物 Primer for genes clone	pbi121-SlPL-R	ataagggactgaccacccgggGCAACGAGAACCCTTTTTACAGTT
扩增引物 Amplification primers	CRISPR-SlPL-F	ACGAAGGAACAAGGGACCCA
扩增引物 Amplification primers	CRISPR-SlPL-R	CGGGTGGAGAACTGTTTCGG
测序引物 Sequencing primers	CRISPR-SlPL-F	CAGATAGGAACAAGGAGT
测序引物 Sequencing primers	CRISPR-SlPL-R	GGTGGAAATGCTATGGTG
	35S-F	TCCCACTGAATCAAAGGC
qPCR引物 Primer for qPCR	Actin-F	CTCTACATACTTGAGAGGTGCC
qPCR引物 Primer for qPCR	Actin-R	AGACGAGGAGAAAACATCACAA
	SlPL-F	GGAAATCCAATCGACGATTGTT
	SlPL-R	CGTTTTTCCCAAATCCAATTGC
	SlCel2-F	TATCAAATGGCGTAGAGACTCC
	SlCel2-R	ACATTATCTCCGGCATCGTAAT
	SlPG2-F	CCAAAGGAATAGTATTCTCCTTCTC
	SlPG2-R	GTTTTTCCATCACCCTTAGCTC
	SlPME2.1-F	ATGCTACCATCATTACTGGGAG
	SlPME2.1-R	GATAAGCATCGATACGACAACG
	SlGH9C2-F	CCAGAAGTACTACCGATCTGTC
	SlGH9C2-R	TCAGTCATAGACCAACCAGTTC
	SlACO1-F	CATACAGACGCAGGAGGCATCA
	SlACO1-R	TAGAGTGGCGCATGGGAGGAA
	SlACS4-F	CTCCTCAAATGGGGAGTACG
	SlACS4-R	TTTTGTTTGCTCGCACTACG
	SlERF2-F	CGACCTATGGCCGACTGATT
	SlERF2-R	GGTCACGAATTTCAGCAGCC

表2 番茄遗传转化过程中使用的培养基配方

Table 2 Formulation of media used in the genetic transformation of tomato

用途 Purpose	基础培养基 Culture medium	蔗糖/ （g · L^-1） Sucrose	琼脂粉/ （g · L^-1） Agaric	反式玉米素/ （mg · L^-1） trans-ZT	IAA/ （mg · L^-1）	IBA /（mg · L^-1）	AS / （mg · L^-1）	特美汀/ （mg · L^-1）Trimethoprim	卡那霉素/ （mg · L^-1）Kanamycin
播种 Seeding	1/2MS	30	7.5	0	0	0	0	0	0
预培养 Pre-culture	MS	30	9	2	0.1	0	0	0	0
悬浮培养 Slave culture	MS	30	0	0	0	0	100	0	0
共培养 Co-culture	MS	30	9	2	0.1	0	100	0	0
筛选培养1 Screen 1	MS	30	9	2	0.1	0	0	200	50
筛选培养2 Screen 2	MS	30	9	0.2	0.1	0	0	400	50
生根 Rooting	1/2MS	30	7.5	0	0	2	0	0	50

图1 番茄SlPL启动子顺式作用元件分析

Fig. 1 Analysis of cis-acting elements of SlPL promoter in tomatoes

图2 耐裂果番茄‘NT91’和易裂果番茄‘NT189’SlPL的表达模式使用SPSS-Statistics（版本25.0，IBM，美国）进行单因素方差分析，不同字母表示样品间差异显著（P < 0.05）。下同。

Fig. 2 Expression patterns of SlPL in crack-resistant tomato‘NT91’and crack-susceptible tomato‘NT189’ One-way ANOVA was performed using SPSS-statistics（version 25.0，IBM，USA），with different letters indicating significant differences between samples（P < 0.05）. The same below.

图3 番茄野生型（WT）‘Micro Tom’、SlPL过表达（OEPL）和敲除（pl）株系的表型和果实横径及硬度

Fig. 3 Phenotypes and fruit diameter across and firmness of wild-type（WT）tomato‘Micro Tom’，SlPL overexpression（OEPL）and knockout（pl）lines

图4 番茄野生型（WT）‘Micro Tom’、SlPL过表达（OEPL）和敲除（pl）株系果实果胶含量及果胶裂解酶活性

Fig. 4 Fruit pectin content and pectin cleavage enzyme activity of wild-type（WT）tomato‘Micro Tom’，SlPL overexpression（OEPL）and knockout（pl）lines

图5 番茄野生型（WT）‘Micro Tom’、SlPL过表达（OEPL）和敲除（pl）株系果实中细胞壁代谢相关基因的相对表达量

Fig. 5 Relative expression of cell wall metabolism-related genes in fruits of wild-type（WT）tomato‘Micro Tom’，SlPL overexpression（OEPL）and knockout（pl）lines

图6 番茄野生型（WT）‘Micro Tom’、SlPL过表达（OEPL）和敲除（pl）株系果实中乙烯合成相关基因和乙烯响应因子的相对表达量

Fig. 6 Relative expression of ethylene synthesis-related genes and ethylene response factors in fruits of wild-type（WT）tomato‘Micro Tom’，SlPL overexpression（OEPL）and knockout（pl）lines

图7 耐裂果番茄‘NT91’和易裂果番茄‘NT189’果皮横切面组织结构

Fig. 7 Histological structure of pericarp cross sections of crack-resistant tomato‘NT91’and crack-susceptible tomato‘NT189’

图8 番茄野生型（WT）‘Micro Tom’、SlPL过表达（OEPL）和敲除（pl）株系果实果皮横切面组织结构

Fig. 8 Histological structure of fruit pericarp cross sections of wild-type（WT）tomato‘Micro Tom’，SlPL overexpression（OEPL）and knockout（pl）lines

参考文献 40

[1]	Abu-Goukh A B A, Bashir H A. 2003. Changes in pectic enzymes and cellulase activity during guava fruit ripening. Food Chemistry, 83 (2):213-218.
[2]	Balbontín C, Ayala H, Bastias R M, Tapia G, Ellena M, Torres C, Yuri J A, Quero-García J, Ríos J C, Silva H. 2013. Cracking in sweet cherries:a comprehensive review from a physiological molecular and genomic perspective. Chilean Journal of Agricultural Research, 73 (1):66-72.
[3]	Brüeggenwirth M, Knoche M. 2016. Mechanical properties of skins of sweet cherry fruit of differing susceptibilities to cracking. Journal of the American Society for Horticultural Science, 141 (2):162-168.
[4]	Cao Jian-kang, Jiang Wei-bo, Zhao Yu-mei. 2007. Guidelines for postharvest physiological and biochemical experiments on fruits and vegetables. Beijing: China Light Industry Press. (in Chinese)
	曹建康, 姜微波, 赵玉梅. 2007. 果蔬采后生理生化实验指导. 北京: 中国轻工业出版社.
[5]	Capel C, Yuste-Lisbona F J, López-Casado G, Angosto T, Cuartero J, Lozano R, Capel J. 2017. Multi-environment QTL mapping reveals genetic architecture of fruit cracking in a tomato RIL Solanum lycopersicum × S. pimpinellifolium population. Theoretical and Applied Genetics, 130 (1):213-222.
[6]	Chen Bin, Wu Zhen, Wen Jun-qin, Lin Hao-wei, Yu Lu, Xue Ling-zi, Zhou Rong, Jiang Fang-ling. 2021. QTL Mapping and candidate genes analysis of irregular fruit cracking in tomato. Acta Horticulturae Sinica, 48 (7):1329-1339. (in Chinese) doi: 10.16420/j.issn.0513-353x.2020-1032
	陈斌, 吴震, 文军琴, 林昊维, 于璐, 薛灵姿, 周蓉, 蒋芳玲. 2021. 番茄不规则裂果性状的QTL定位及候选基因分析. 园艺学报, 48 (7):1329-1339. doi: 10.16420/j.issn.0513-353x.2020-1032
[7]	Chen Le-tian, Wang Hui-ting, Han Jing-luan, Luan Ying. 2019. Research progress and perspective pectin lysase. Journal of South China Agricultural University, 40 (5):71-77. (in Chinese)
	陈乐天, 王慧婷, 韩靖鸾, 栾莹. 2019. 植物果胶裂解酶的研究现状及展望. 华南农业大学学报, 40 (5):71-77.
[8]	Correia S, Schouten R, Silva A P, Goncalves B. 2018. Sweet cherry fruit cracking mechanisms and prevention strategies:a review. Scientia Horticulturae, 240:369-377.
[9]	de Freitas S T, Shackel K A, Mitcham E J. 2011. Abscisic acid triggers whole-plant and fruit-specific mechanisms to increase fruit calcium uptake and prevent blossom end rot development in tomato fruit. Journal of Experimental Botany, 62 (8):2645-2656. doi: 10.1093/jxb/erq430 pmid: 21282326
[10]	Deng H, Chen Y, Liu Z Y, Liu Z Q, Shu P, Wang R C, Hao Y W, Su D, Pirrello J, Liu Y S, Li Z, Grierson D, Giovannoni J J, Bouzayen M, Liu M C. 2022. SlERF.F12 modulates the transition to ripening in tomato fruit by recruiting the co-repressor TOPLESS and histone deacetylases to repress key ripening genes. Plant Cell, 34:1250-1272.
[11]	Emmons C L, Scott J W. 1997. Environmental and physiological effects on cuticle cracking in tomato. Journal of American Society Horticultural Science, 122:797-801.
[12]	Hahn F. 2011. Fuzzy controller decreases tomato cracking in greenhouses. Computers and Electronics in Agriculture, 77:21-27.
[13]	Jiang F L, Lopez A, Jeon S, de Freitas S T, Yu Q H, Wu Z, Labavitch J M, Tian S K, Powell A L T, Mitcham E. 2019. Disassembly of the fruit cell wall by the ripening-associated polygalacturonase and expansin influences tomato cracking. Horticulture Research, 6:17. doi: 10.1038/s41438-018-0105-3 pmid: 30729007
[14]	Huang Yijin, He Jiali, Jiang Lina, Cao Yanhong, Qin Sijun, Lü Deguo. 2022. The physiological and biochemical research progress for the changes of fruit crispy. Acta Horticulturae Sinica, 49 (12):2641-2658. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-0875
	黄译瑾, 何佳丽, 姜李娜, 曹艳红, 秦嗣军, 吕德国. 2022. 果实脆性变化的生理生化研究进展. 园艺学报, 49 (12):2641-2658. doi: 10.16420/j.issn.0513-353x.2022-0875
[15]	Jiang J, Yao L, Yu Y, Lv M L, Miao Y, Cao J S. 2014. PECTATE LYASE-LIKE 10 is associated with pollen wall development in Brassica campestris. Journal of Integrative Plant Biology, 56 (11):1095-1105.
[16]	Khadivi-Khub A. 2015. Physiological and genetic factors influencing fruit cracking. Acta Physiology Plant, 37:1718.
[17]	Li Hong-li, Liu Gang-shuai, Tian Hui-qing, Fu Da-qi. 2021. Fruit cracking:a review. Chinese Journal of Biotechnology, 37 (8):2737-2752. (in Chinese)
	李泓利, 刘港帅, 田慧琴, 傅达奇. 2021. 果实开裂研究进展. 生物工程学报, 37 (8):2737-2752.
[18]	Li Hui-jia. 2016. Genetic analysis and QTL mapping in tomato cracking[M. D. Dissertation]. Harbin: Northeast Agricultural University. (in Chinese)
	李会佳. 2016. 番茄裂果性状的遗传分析及QTL定位[硕士论文]. 哈尔滨: 东北农业大学.
[19]	Li Min. 2013. The study on the mechanisms of ethylene-regulated early ripening apple fruit softening and dehiscence[M. D. Dissertation]. Tai’an: Shandong Agriculture University. (in Chinese)
	李敏. 2013. 乙烯调控早熟苹果果实软化和裂果机理的初步研究[硕士论文]. 泰安: 山东农业大学.
[20]	Liao N Q, Hu Z Y, Li Y Y, Hao J F, Chen S N, Xue Q, Ma Y Y, Zhang K J, Mahmoud A, Ali A, Malangisha G K, Lyu X L, Yang J H, Zhang M F. 2019. Ethylene-responsive factor 4 is associated with the desirable rind hardness trait conferring cracking resistance in fresh fruits of watermelon. Plant Biotechnology Journal, 18 (4):1066-1077.
[21]	Ling Hao-wei. 2022. Identification of key genes associated with fruit cracking in tomato and verification of SlERF2 gene function[M. D. Dissertation]. Nanjing: Nanjing Agricultural University. (in Chinese)
	林昊维. 2022. 番茄裂果关键基因筛选和 SlERF2 基因功能验证[硕士论文]. 南京: 南京农业大学.
[22]	Liu Y D, Tang M F, Liu M C, Su D D, Chen J, Gao Y S, Bouzayen M, Li Z G. 2020. The molecular regulation of ethylene in fruit ripening. Small Methods, 4:1900485.
[23]	Lu Yanqing, Lin Yanjin, Lu Xinkun. 2023. Cell wall material metabolism and the response to high temperature and water deficiency stresses in pericarp are associated with fruit cracking of‘Duwei’pummelo. Acta Horticulturae Sinica, 50 (8):1747-1768. (in Chinese) doi: 10.16420/j.issn.0513-353x.2022-0653
	卢艳清, 林燕金, 卢新坤. 2023. 果皮细胞壁物质代谢及果皮对高温和水分亏缺逆境的响应与‘度尾文旦柚’裂果相关. 园艺学报, 50 (8):1747-1768.
[24]	Marin-Rodriguez M C, Orchard J, Seymourg B. 2002. Pectate lyases,cell wall deg-radation and fruit softening. Journal of Experimental Botany, 53 (377):2115-2119.
[25]	Marin-Rodriguez M C, Smith D L, Manning K, Orchard J, Seymour G B. 2003. Pectate lyase gene expression and enzyme activity in ripening banana fruit. Plant Molecular Biology, 51 (6):851-857.
[26]	Measham P F, Bound S A, Gracie A J, Wilson S J. 2010. Vascular flow of water induces side cracking in sweet cherry(Prunus avium L.). Advances in Horticultural Science, 24 (4):243-248.
[27]	Moctezuma E, Smith D L, Gross K C. 2003. Antisense suppression of a β-galactosidase gene(TBG6)in tomato increases fruit cracking. Journal of Experimental Botany, 54 (390):2025-2033. doi: 10.1093/jxb/erg214 pmid: 12867545
[28]	Peet M M. 1992. Fruit cracking in tomato. HortTechnology, 2 (2):216-223.
[29]	Sawada E. 1931. Studies on the cracking of cherries. I. The cause of cracking. Agriculture and Horticulture, 6:964.
[30]	Song Dong-liang, Shen Jun-hui, Li Lai-geng. 2008. Cellulose synthesis in the cell walls of higher plants. Plant Physiology Communications, 4:791-796. (in Chinese)
	宋东亮, 沈君辉, 李来庚. 2008. 高等植物细胞壁中纤维素的合成. 植物生理学通讯, 4:791-796.
[31]	Wang Ao-xue, Miao Shuang, Chen Xiu-ling, Zhang Yao, Liu Jia-yin. 2019. Study on pericarp tissue senescence of different fruit cracking types of tomato during ripening process. Journal of Northeast Agricultural University, 50 (4):19-28. (in Chinese)
	王傲雪, 苗爽, 陈秀玲, 张瑶, 刘佳音. 2019. 不同裂果类型番茄成熟过程果皮组织衰老研究. 东北农业大学学报, 50 (4):19-28.
[32]	Wang D, Yeats T H, Uluisik S, Rose J K C, Seymour G B. 2018. Fruit softening:revisiting the role of pectin. Trends in Plant Science, 23 (4):302-310.
[33]	Wei J M, Ma F W, Shi S G, Qi X D, Zhu X Q, Yuan J Y. 2010. Changes and postharvest regulation of activity and gene expression of enzymes related to cell wall degradation in ripening apple fruit. Postharvest Biology and Technology, 12 (56):147-154.
[34]	Wieczorek K, Elashry A, Quentin M, Grundler F M, Favery B, Seifert G J, Bohlmann H. 2014. A distinct role of pectate lyases in the formation of feeding structures induced by cyst and root-knot nematodes. Molecular Plant Microbe Interact, 27 (9):901-912.
[35]	Xu De-li, Yang Jing, Bao Ji-you, Xie Ling. 2013. The main reasons and preventive measures of tomato fruit cracking in winter and spring facilities in Huaibei area. Jiangsu Agricultural Sciences, 41 (9):147-148. (in Chinese)
	徐德利, 杨静, 鲍继友, 谢玲. 2013. 淮北地区冬春季设施番茄裂果的主要原因及预防措施. 江苏农业科学, 41 (9):147-148.
[36]	Xue L Z, Sun M T, Wu Z, Yu L, Yu Q H, Tang Y P, Jiang F L. 2020. LncRNA regulates tomato fruit cracking by coordinating gene expression via a hormone-redox-cell wall network. BMC Plant Biology, 20 (1):162. doi: 10.1186/s12870-020-02373-9 pmid: 32293294
[37]	Yang Z E, Wu Z, Zhang C, Hu E M, Zhou R, Jiang F L. 2016. The composition of pericarp,cell aging,and changes in water absorption in two tomato genotypes:mechanism,factors,and potential role in fruit cracking. Acta Physiology Plant, 38 (9):215.
[38]	Zhang C, Cui L W, Liu C H, Fan X C, Fang J G. 2023. Mining candidate genes of grape berry cracking based on high density genetic map. Horticultural Plant Journal, 9 (4):743-753.
[39]	Zhu Yu, Fan Lina, Huang Zejun, Du Yongchen, Guo Yanmei, Li Junming, Liu Lei, Shu Jinshuai, Wang Xiaoxuan. 2020. Fine mapping of fruit cracking-resistant gene Cr3a in tomato. Acta Horticulturae Sinica, 47 (2):275-286. (in Chinese)
	朱玉, 范丽娜, 黄泽军, 杜永臣, 国艳梅, 李君明, 刘磊, 舒金帅, 王孝宣. 2020. 番茄耐裂果基因Cr3a的精细定位. 园艺学报, 47 (2):275-286.
[40]	Zhu Zhen-hua, Jiang Fang-ling, Wen Jun-qin, Shi Xiao-pu, Wu Cui-yun, Liu Min, Wu Zhen. 2021. Identification and eat resistance analysis of SlGRAS4 gene in tomato. Acta Botanica Boreali Occidentalia Sinica, 41 (4):539-548. (in Chinese)
	朱珍花, 蒋芳玲, 文军琴, 石潇瀑, 吴翠云, 刘敏, 吴震. 2021. 番茄SlGRAS4基因特征分析和耐热功能鉴定. 西北植物学报, 41 (4):539-548.