南果梨成熟相关PG基因筛选及表达分析
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摘要
南果梨是典型的呼吸跃变型果实,其成熟过程中果实乙烯生成速度迅速升高,果实软化硬度下降,严重影响了其货架期和贮藏时间。多聚半乳糖醛酸酶(polygalacturonase, PG)是一类重要的细胞壁水解酶,参与果实的软化过程。但南果梨PG基因的表达特性以及能否受乙烯的调控仍不清楚。以南果梨试材,结合梨基因组数据,筛选南果梨中的PG基因并对其表达特性进行分析;同时考察乙烯和1-MCP(1-methyl cyclopropene,一种乙烯受体抑制剂)处理之后各个基因的表达情况,旨在筛选得到与南果梨果实成熟相关的PG基因。研究结果将为延长南果梨的贮藏期提供一定的理论依据。试验结果表明:南果梨中共筛选出8个PG基因,并命名为PuPG1～PuPG8;PuPG6只在果实发育早期表达,随着果实的发育其表达量逐渐降低,果实成熟后PuPG6表达终止;PuPG2和PuPG5在果实发育期表达量逐渐升高,花后120d达到最大值,果实成熟后表达量逐渐降低;PuPG8在花后30d表达量最高,随着果实的发育其表达量逐渐降低;PuPG1、PuPG4和PuPG7在南果梨果实发育时期几乎不表达,主要在采收后开始表达,随着贮藏时间的增长表达量逐渐升高,并在贮藏15d时达到最大值;同时乙烯也能够诱导PuPG1、PuPG4和PuPG7基因的表达,而在1-MCP处理的果实中检测不到3个基因的表达。PuPG1、PuPG4和PuPG7的表达趋势与南果梨果实乙烯生成速度及硬度变化趋势一致,推测其可能是参与调控南果梨果实软化的关键基因。
The Nan guo pear(Pyrusussuriensis) fruit is typically climacteric, and ethylene is the main factor controlling the ripening of climacteric fruit. During ripening, the fruits become yellow and produce large amounts of ethylene, accompanied by rapid softening, which influence shelf life and storage time seriously. Polygalacturonase(PG) is one type of cell wall hydrolase and is responsible for the degradation of pectin in the cell wall. But the expression patterns of PG genes in Nanguo pear and whether they could be regulated by ethylene is still unclear. Here we screened the PG genes in Nanguo pear by using the pear genome and analyzed their expression profiles. Meanwhile we detected the expression patterns of these genes in the ETH and 1-MCP(1-methyl cyclopropene, an ethylene receptor inhibitor)treated fruits aiming to obtain the PG gene related to Nanguo pear ripening. Our results will provide some theoretical basis for prolonging the shelf time of Nanguo pear. The results showed that there exist eight PG genes in pear genome and named them PuPG1 to PuPG8, respectively. PuPG6 only expressed in fruit development stage and decreased gradually with fruit development. The expression level of PuPG2 and PuPG5 increased gradually during fruit development and reached the highest expression level at 120 days after full bloom, and decreased gradually after harvest; PuPG8 showed the highest expression level at 30 days after full bloom and decreased gradually during fruit development.PuPG1, PuPG4 and PuPG7 mainly expressed in post-harvest stage and its expression level increased with the storage time being,reaching the highest point at the 15 days after harvest. Meanwhile, the expression of these three genes could induced by ethylene treatment and inhibited by 1-MCP treatment. The expression trends of PuPG1, PuPG4, and PuPG7 were consistent with the fruit firmness and ethylene production rate,which indicated that they might be the key PG genes involved in the regulation of fruit softening of Nanguo pear.
The Nan guo pear(Pyrusussuriensis) fruit is typically climacteric, and ethylene is the main factor controlling the ripening of climacteric fruit. During ripening, the fruits become yellow and produce large amounts of ethylene, accompanied by rapid softening, which influence shelf life and storage time seriously. Polygalacturonase(PG) is one type of cell wall hydrolase and is responsible for the degradation of pectin in the cell wall. But the expression patterns of PG genes in Nanguo pear and whether they could be regulated by ethylene is still unclear. Here we screened the PG genes in Nanguo pear by using the pear genome and analyzed their expression profiles. Meanwhile we detected the expression patterns of these genes in the ETH and 1-MCP(1-methyl cyclopropene, an ethylene receptor inhibitor)treated fruits aiming to obtain the PG gene related to Nanguo pear ripening. Our results will provide some theoretical basis for prolonging the shelf time of Nanguo pear. The results showed that there exist eight PG genes in pear genome and named them PuPG1 to PuPG8, respectively. PuPG6 only expressed in fruit development stage and decreased gradually with fruit development. The expression level of PuPG2 and PuPG5 increased gradually during fruit development and reached the highest expression level at 120 days after full bloom, and decreased gradually after harvest; PuPG8 showed the highest expression level at 30 days after full bloom and decreased gradually during fruit development.PuPG1, PuPG4 and PuPG7 mainly expressed in post-harvest stage and its expression level increased with the storage time being,reaching the highest point at the 15 days after harvest. Meanwhile, the expression of these three genes could induced by ethylene treatment and inhibited by 1-MCP treatment. The expression trends of PuPG1, PuPG4, and PuPG7 were consistent with the fruit firmness and ethylene production rate,which indicated that they might be the key PG genes involved in the regulation of fruit softening of Nanguo pear.
引文
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[2]寇晓红,朱本忠,罗云波,等.番茄果实中乙烯与多聚半乳糖醛酸酶的关系[J].植物生理与分子生物学学报,2004,30(6):675-680.
[3]陈儒钢,杨瑞平,巩振辉,等.辣椒多聚半乳糖醛酸酶基因CaPG的克隆与序列分析[J].西北植物学报,2010,30(10):1941-1945.
[4] MONTGOMERY J,POLLARD V,DEIKMAN J,et al.Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp[J].The Plant Cell,1993,5:1049-1062.
[5] SITRIT Y,BENNETT AB.Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene:a re-examination[J].Plant Physiology,1998,116:1145-1150.
[6]阚娟,金昌海,汪志君,等.β-半乳糖苷酶及多聚半乳糖醛酸酶对桃果实成熟软化的影响[J].扬州大学学报,2006,27(3):76-80.
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[11] BRUMMELL DA,HALL BD,BENNETT AB. Antisense suppression of tomato endo-1,4-b-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening[J].Plant Molecular Biology,1999,40:615-622.
[12] BERGEY DR,OROZCO-CARDENAS M,de MOURA DS,et al.A wound-and systemin-inducible polygalacturonase in tomato leaves[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(4):1756.
[13]张弢.荠菜多聚半乳糖醛酸酶基因CbPG的分子克隆、序列分析和功能预测[J].华北农学报,2012,27(5):12-17.
[14]吴建阳,刘丽琴,石胜友,等.龙眼多聚半乳糖醛酸酶基因DlPG1表达模式与幼果脱落关系研究[J].中国南方果树,2017,46(3):14-19.
[15] ZHU H,DARDICK CD,BEERSEP,et al.Transcriptomics of shading-induced and NAA-induced abscission in apple(Malus domestica)reveals a shared pathway involving reduced photosynthesis,alterations in carbohydrate transport and signaling and hormone crosstalk[J].BMC Plant Biology,2011,11:138.
[16] TAN DM,LI T,WANG A.Apple 1-aminocyclopropane-1-carboxylic acid synthase genes,MdACS1 and MdACS3a,are expressed in different systems of ethylene biosynthesis[J].Plant Molecular Biology Reporter,2013,31:204-209.
[17] HUANG GH,LI T,LI XY,et al.Comparative transcriptome analysis of climacteric fruit of Chinese pear(Pyrus ussuriensis)reveals new insightsinto fruit ripening[J].PLOS ONE,2014,9(9):e107562.
[18]魏建梅,齐秀东,张海娥,等.京白梨果实采后PG、糖苷酶和LOX活性变化及其基因表达特性[J].园艺学报,2012,39(1):31-39.
[19] ZHOU H W,SONEGO L,BEN-ARIE R,et al.Analysis of cell wall components in juice of flavor top nectarines during normal ripening and woolliness[J].Journal of the American Society for Horticultural Science,1999,124(4):424-429.
[20]王刚,贾展慧,张计育,等.槜李果实软化相关基因PsPG的克隆与表达分析[J].植物资源与环境学报,2017,26(4):60-66.
[21]高晓铭,李艳梅,赵要尊,等.多聚半乳糖醛酸酶基因表达与果实成熟[J].河北农业科学,2016,20:51-54.
[22] WANG Z Y,Macrae E A,Wright M A,et al.Polygalacturonase gene expression in kiwifruit:relationship to fruit softening and ethylene production[J].Plant Molecular Biology,2000,42:317-328.
[23]黄森,张院民,王建芳,等.乙烯吸收剂处理对柿果实采后生理效应的影响[J].西北农业学报,2006,15(6):140-143.
[24]以下三条是序号的变化]张伟.桃成熟过程中的活性物质变化及PG、ACO基因的克隆表达与遗传转化[D].兰州:甘肃农业大学,2013.
[25]周宏伟,吴耕西.长把梨贮藏中多聚半乳糖醛酸酶与果胶甲醋酶的作用[J].山东农业大学学报,1992,23(1):67-70.
[26] MCMURCHIE E J,MCGLASSON W B,EAKS I L.Treatment of fruit with propylene gives information about the biogenesis of ethylene[J].Nature,1972,237(5352):235-236.
[2]寇晓红,朱本忠,罗云波,等.番茄果实中乙烯与多聚半乳糖醛酸酶的关系[J].植物生理与分子生物学学报,2004,30(6):675-680.
[3]陈儒钢,杨瑞平,巩振辉,等.辣椒多聚半乳糖醛酸酶基因CaPG的克隆与序列分析[J].西北植物学报,2010,30(10):1941-1945.
[4] MONTGOMERY J,POLLARD V,DEIKMAN J,et al.Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp[J].The Plant Cell,1993,5:1049-1062.
[5] SITRIT Y,BENNETT AB.Regulation of tomato fruit polygalacturonase mRNA accumulation by ethylene:a re-examination[J].Plant Physiology,1998,116:1145-1150.
[6]阚娟,金昌海,汪志君,等.β-半乳糖苷酶及多聚半乳糖醛酸酶对桃果实成熟软化的影响[J].扬州大学学报,2006,27(3):76-80.
[7] HADFIELD KA,BENNETT AB.Polygalacturonases:many genes in search of a function[J].Plant Physiology,1998,117:337-343.
[8] SEKINE D,MUNEMURA I,GAO M,et al. Cloning of cDNAs encoding cell-wall hydrolases from pear(Pyrus communis)fruit and their involvement in fruit softening and development of melting texture[J].Physiologia Plantarum,2006,126:163-174.
[9] ATKINSON R G,SCHRODER R,HALLETT I C,et al. Overexpression of polygalacturonase in transgenic apple trees leads to a range of novel phenotypes involving changes in cell adhesion[J].Plant Physiology,2012,129:122-133.
[10] WANG Z Y,MACRAE E A,WRIGHT M A,et al. Polygalacturonase gene expression in kiwifruit:relationship to fruit softening and ethylene production[J].Plant Molecular Biology,2000,42:317-328.
[11] BRUMMELL DA,HALL BD,BENNETT AB. Antisense suppression of tomato endo-1,4-b-glucanase Cel2 mRNA accumulation increases the force required to break fruit abscission zones but does not affect fruit softening[J].Plant Molecular Biology,1999,40:615-622.
[12] BERGEY DR,OROZCO-CARDENAS M,de MOURA DS,et al.A wound-and systemin-inducible polygalacturonase in tomato leaves[J].Proceedings of the National Academy of Sciences of the United States of America,1999,96(4):1756.
[13]张弢.荠菜多聚半乳糖醛酸酶基因CbPG的分子克隆、序列分析和功能预测[J].华北农学报,2012,27(5):12-17.
[14]吴建阳,刘丽琴,石胜友,等.龙眼多聚半乳糖醛酸酶基因DlPG1表达模式与幼果脱落关系研究[J].中国南方果树,2017,46(3):14-19.
[15] ZHU H,DARDICK CD,BEERSEP,et al.Transcriptomics of shading-induced and NAA-induced abscission in apple(Malus domestica)reveals a shared pathway involving reduced photosynthesis,alterations in carbohydrate transport and signaling and hormone crosstalk[J].BMC Plant Biology,2011,11:138.
[16] TAN DM,LI T,WANG A.Apple 1-aminocyclopropane-1-carboxylic acid synthase genes,MdACS1 and MdACS3a,are expressed in different systems of ethylene biosynthesis[J].Plant Molecular Biology Reporter,2013,31:204-209.
[17] HUANG GH,LI T,LI XY,et al.Comparative transcriptome analysis of climacteric fruit of Chinese pear(Pyrus ussuriensis)reveals new insightsinto fruit ripening[J].PLOS ONE,2014,9(9):e107562.
[18]魏建梅,齐秀东,张海娥,等.京白梨果实采后PG、糖苷酶和LOX活性变化及其基因表达特性[J].园艺学报,2012,39(1):31-39.
[19] ZHOU H W,SONEGO L,BEN-ARIE R,et al.Analysis of cell wall components in juice of flavor top nectarines during normal ripening and woolliness[J].Journal of the American Society for Horticultural Science,1999,124(4):424-429.
[20]王刚,贾展慧,张计育,等.槜李果实软化相关基因PsPG的克隆与表达分析[J].植物资源与环境学报,2017,26(4):60-66.
[21]高晓铭,李艳梅,赵要尊,等.多聚半乳糖醛酸酶基因表达与果实成熟[J].河北农业科学,2016,20:51-54.
[22] WANG Z Y,Macrae E A,Wright M A,et al.Polygalacturonase gene expression in kiwifruit:relationship to fruit softening and ethylene production[J].Plant Molecular Biology,2000,42:317-328.
[23]黄森,张院民,王建芳,等.乙烯吸收剂处理对柿果实采后生理效应的影响[J].西北农业学报,2006,15(6):140-143.
[24]以下三条是序号的变化]张伟.桃成熟过程中的活性物质变化及PG、ACO基因的克隆表达与遗传转化[D].兰州:甘肃农业大学,2013.
[25]周宏伟,吴耕西.长把梨贮藏中多聚半乳糖醛酸酶与果胶甲醋酶的作用[J].山东农业大学学报,1992,23(1):67-70.
[26] MCMURCHIE E J,MCGLASSON W B,EAKS I L.Treatment of fruit with propylene gives information about the biogenesis of ethylene[J].Nature,1972,237(5352):235-236.