P119驱动amiRNA介导的PL基因沉默对草莓果实硬度的影响
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摘要
果胶裂解酶基因(Pectate lyase,PL)是调控果实软化的重要靶点。本研究测定了红颜草莓果实在发育过程中果胶裂解酶活性变化和硬度变化规律,并通过人工小干扰RNA(artificial micro-interfering RNA interference,amiRNA)技术,以拟南芥miR390a前体序列作为沉默PL基因的骨架,在番茄果实特异性启动子P119的驱动下,通过农杆菌介导转入草莓果实中瞬时表达,通过RT-PCR分析草莓瞬时转染后PL基因的表达量,并检测了PL沉默对果实硬度的影响。结果表明,随着草莓果实发育的推进,果胶裂解酶活性呈逐渐上升趋势,与果实硬度呈负相关;构建了基于拟南芥miR390a为骨架的amiRNA靶向栽培草莓中的PL基因;在番茄果实特异性启动子P119驱动下,miRNA前体可以在草莓果实中瞬时表达;草莓果实中总PL基因表达量下降达34. 5%;基因沉默组果实硬度比对照组更高,且沉默对果实颜色发育进程无明显影响。该结果表明:果胶裂解酶主要在果实发育后期调节细胞壁的解离,采用amiRNA沉默PL基因可以延缓草莓果实软化进程。
Pectate lyase( PL) is one of the most profitable candidate enzymes for improving the fruit firmness and delaying the softening process in strawberry( Fragaria × ananassa Duch.) by molecular modifying. The role of PL in strawberry fruit softening was studied by amiRNA-mediated silencing. The changes of pectate lyase activity and fruit firmness were determined across the fruit development of cultivated strawberry ‘Benihoppe'.Candidate PLs were submitted toWMD3( http://wmd3. weigelword. org) for miRNA target site screening( target Fragaria × ananassa PUT v183,minimum number of included targets: one). Those candidate miRNA target sites were then manually checked,ensuring that the target loci exist in all PL transcripts. This target sequence was introduced into the Arabidopsis thaliana miR390 a precursor sequence as the backbone. The validated construct was transformed into the Agrobacterium GV3101. Transient expression in strawberry fruits was conducted by agroinfilltration. Expression level of the PL genes was monitored by RT-PCR. Fruit firmness was examined too. With the development of strawberry fruit,the PL activity gradually increased,especially the stages after turning red. The PL activity increased sharply and reached the maximum in the full red period. In contrast,fruit firmness gradually decreased during fruit development especially during the process from big green to color transition,and then the rate of hardness reduction slowed. The P119 promoter from tomato was able to drive amiRNA gene expression,which can reduce the expression of PL gene in strawberry fruit by 34. 5%. The knock down of PL gene does not interfere with the accumulation of anthocyanins in strawberry,and the silencing group had higher fruit hardness than the control group. In a comprehensive analysis, PL regulates the dissociation of cell wall mainly in the late fruit developmental stage. Obviously,from our results,there are also other factors that affect the softening of strawberry. The p119 fruit-specific promoter has transcriptional activity in silencing PL gene of strawberry in the fruit,which lays a foundation with the application in future molecular modification. To this end, stable transformation is underway. Therefore, PL regulates plants cell wall dissociation at the late development stages of strawberry. Silencing the gene with P119 using amiRNA can delay fruit softening process.
Pectate lyase( PL) is one of the most profitable candidate enzymes for improving the fruit firmness and delaying the softening process in strawberry( Fragaria × ananassa Duch.) by molecular modifying. The role of PL in strawberry fruit softening was studied by amiRNA-mediated silencing. The changes of pectate lyase activity and fruit firmness were determined across the fruit development of cultivated strawberry ‘Benihoppe'.Candidate PLs were submitted toWMD3( http://wmd3. weigelword. org) for miRNA target site screening( target Fragaria × ananassa PUT v183,minimum number of included targets: one). Those candidate miRNA target sites were then manually checked,ensuring that the target loci exist in all PL transcripts. This target sequence was introduced into the Arabidopsis thaliana miR390 a precursor sequence as the backbone. The validated construct was transformed into the Agrobacterium GV3101. Transient expression in strawberry fruits was conducted by agroinfilltration. Expression level of the PL genes was monitored by RT-PCR. Fruit firmness was examined too. With the development of strawberry fruit,the PL activity gradually increased,especially the stages after turning red. The PL activity increased sharply and reached the maximum in the full red period. In contrast,fruit firmness gradually decreased during fruit development especially during the process from big green to color transition,and then the rate of hardness reduction slowed. The P119 promoter from tomato was able to drive amiRNA gene expression,which can reduce the expression of PL gene in strawberry fruit by 34. 5%. The knock down of PL gene does not interfere with the accumulation of anthocyanins in strawberry,and the silencing group had higher fruit hardness than the control group. In a comprehensive analysis, PL regulates the dissociation of cell wall mainly in the late fruit developmental stage. Obviously,from our results,there are also other factors that affect the softening of strawberry. The p119 fruit-specific promoter has transcriptional activity in silencing PL gene of strawberry in the fruit,which lays a foundation with the application in future molecular modification. To this end, stable transformation is underway. Therefore, PL regulates plants cell wall dissociation at the late development stages of strawberry. Silencing the gene with P119 using amiRNA can delay fruit softening process.
引文
1.邓明琴,雷家军.中国果树志---草莓卷[M].北京:中国林业出版社,2005:20-32.Deng M Q,Lei J J.Chinese fruit tree-strawberry[M].Beijing:China Forestry Publishing House,2005:20-32.
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3.Tucker G A,Mitchell J.Cell walls,structure,utilisation and manipulation[M].//Griersond.Biosynthesis and manipulation of plant products.Dordrecht:Springer,1993:55-103.
4.Wong D.Enzymatic deconstruction of backbone structures of the ramified regions in pectins[J].The Protein Journal,2008,27(1):30-42.
5.Ridley B L,O’neill M A,Mohnen D.Pectins:structure,biosynthesis,and oligogalacturonide-related signaling[J].Phytochemistry,2001,57(6):929-967.
6.Uluisik S,Chapman H N,Smith R,et al.Genetic improvement of tomato by targeted control of fruit softening[J].Nature Biotechnology,2016,34(9):950-954.
7.Youssef S M,Jiménez-Bermúdez S,Bellido M L,et al.Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry Pectate lyase gene[J].Scientia Horticulturae,2009,119(2):120-125.
8.Lara I,Garciía P,Vendrell M.Modifications in cell wall composition after cold storage of calcium-treated strawberry(Fragaria×ananassa Duch.)fruit[J].Postharvest Biology and Technology,2004,34(3):331-339.
9.Wang D D,Yeats T H,Uluisik S,et al.Fruit softening:revisiting the role of pectin[J].Trends in Plant Science,2018,23(4):302-310.
10.Collmer A,Keen N T.The role of pectic enzymes in plant pathogenesis[J].Annual Review of Phytopathology,1986,24:383-409.
11.Jiménez-Bermúdez S,Redondo-Nevadoj,Mu1oz-Blancov,et al.Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene[J].Plant Physiology,2002,128(2):751-759.
12.Medina-Escobar N,Cárdenas J,Moyano E,et al.Cloning,molecular characterization and expression pattern of a strawberry ripening-specific c DNA with sequence homology to pectate lyase from higher plants[J].Plant Molecular Biology,1997,34(6):867-877.
13.Davuluri G R,Van Tuinen A,Fraser P D,et al.Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes[J].Nature Biotechnology,2005,23(7):890-895.
14.Hoffmann T,Kalinowski G,Schwab W.RNAi-induced silencing of gene expression in strawberry fruit(Fragaria×ananassa)by agroinfiltration:a rapid assay for gene function analysis[J].The Plant Journal,2006,48(5):818-826.
15.Han Y,Dang R H,Li J X,et al.SUCROSE NONFER-MENTING1-RELATED PROTEIN KINASE2.6,an ortholog of OPEN STOMATA1,is a negative regulator of strawberry fruit development and ripening[J].Plant Physiology,2015,167(3):915-965.
16.Fait A,Hanhineva K,Beleggia R,et al.Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development[J].Plant Physiology,2008,148(2):730-750.
17.Payasi A,Misra P C,Sanwal G G.Effect of phytohormones on pectate lyase activity in ripening Musa acuminata[J].Plant Physiology and Biochemistry,2004,42(11):861-865.
18.Chen Q,Yu H W,Wang X R,et al.An alternative cetyltrimethylammonium bromide-basedprotocol for RNA isolation from blackberry(Rubus L.)[J].Genetics and Molecular Research,2012,11(2):1773-1782.
19.Lin Y X,Jiang L Y,Chen Q,et al.Comparative transcriptome profiling analysis of red-and white-fleshed strawberry(Fragaria×ananassa)provides new insight into the regulation of the anthocyanin pathway[J].Plant and Cell Physiology,2018,59(9):1844-1859.
20.Chen S Y,Tang Y M,Hu Y Y,et al.Fa TT12-1,a multidrug and toxin extrusion(MATE)member involved in proanthocyanidin transport in strawberry fruits[J].Scientia Horticulturae,2018,231:158-165.
21.Braybrook S A,J9nsson H.Shifting foundations:the mechanical cell wall and development[J].Current Opinion in Plant Biology,2016,29:115-120.
22.周厚成.草莓果实成熟软化相关基因的研究[D].杨凌:西北农林科技大学,2014.Zhou H C.Research on genes related to fruit ripening and softening of strawberry[D].Yangling:Northwest A&F U-niversity,2014.
23.PoséS,Paniagua C,Cifuentes M,et al.Insights into the effects of polygalacturonase Fa PG1 gene silencing on pectin matrix disassembly,enhanced tissue integrity,and firmness in ripe strawberry fruits[J].Journal of Experimental Botany,2013,64(12):3803-3815.
24.Quesada M A,Blanco-Portales R,PoséS,et al.Antisense down-regulation of the Fa PG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening[J].Plant Physiology,2009,150(2):1022-1032.
25.Tieman D M,Harriman R W,Ramamohan G,et al.An antisense pectin methylesterasegene alters pectin chemistry and soluble solids in tomato fruit[J].The Plant Cell,1992,4(6):667-679.
26.周鹤莹,张玮,张卿,等.森林草莓‘Ruegen’果胶裂解酶基因的克隆及荧光定量表达分析[J].园艺学报,2015,34(3):455-461.Zhou H Y,Zhang W,Zhang Q,et al.The cloning and quantitative expression analysis of pectate lyase gene in Fragaria vesca[J].Acta Horticulturae Sinica,2015,34(3):455-461.
27.陈品文.amiRNA介导的Fa CRYs基因对草莓花青素苷合成的影响研究[D].成都:四川农业大学,2016.Chen P W.Effect of amiRNA-mediated Fa CRYs genes on anthocyanin biosynthesis in strawberry[D].Chengdu:Sichuan Agricultural University,2016.
2.阮金兰,陈静波,赵晓亚,等.草莓的化学成分研究[J].中国中药杂志,2001,26(9):610-612.Ruan J L,Chen J B,Zhao X Y,et al.Studies on chemical constituents of Fragaria ananassa Duch[J].China Journal of Chinese Materia Medica,2001,26(9):610-612.
3.Tucker G A,Mitchell J.Cell walls,structure,utilisation and manipulation[M].//Griersond.Biosynthesis and manipulation of plant products.Dordrecht:Springer,1993:55-103.
4.Wong D.Enzymatic deconstruction of backbone structures of the ramified regions in pectins[J].The Protein Journal,2008,27(1):30-42.
5.Ridley B L,O’neill M A,Mohnen D.Pectins:structure,biosynthesis,and oligogalacturonide-related signaling[J].Phytochemistry,2001,57(6):929-967.
6.Uluisik S,Chapman H N,Smith R,et al.Genetic improvement of tomato by targeted control of fruit softening[J].Nature Biotechnology,2016,34(9):950-954.
7.Youssef S M,Jiménez-Bermúdez S,Bellido M L,et al.Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry Pectate lyase gene[J].Scientia Horticulturae,2009,119(2):120-125.
8.Lara I,Garciía P,Vendrell M.Modifications in cell wall composition after cold storage of calcium-treated strawberry(Fragaria×ananassa Duch.)fruit[J].Postharvest Biology and Technology,2004,34(3):331-339.
9.Wang D D,Yeats T H,Uluisik S,et al.Fruit softening:revisiting the role of pectin[J].Trends in Plant Science,2018,23(4):302-310.
10.Collmer A,Keen N T.The role of pectic enzymes in plant pathogenesis[J].Annual Review of Phytopathology,1986,24:383-409.
11.Jiménez-Bermúdez S,Redondo-Nevadoj,Mu1oz-Blancov,et al.Manipulation of strawberry fruit softening by antisense expression of a pectate lyase gene[J].Plant Physiology,2002,128(2):751-759.
12.Medina-Escobar N,Cárdenas J,Moyano E,et al.Cloning,molecular characterization and expression pattern of a strawberry ripening-specific c DNA with sequence homology to pectate lyase from higher plants[J].Plant Molecular Biology,1997,34(6):867-877.
13.Davuluri G R,Van Tuinen A,Fraser P D,et al.Fruit-specific RNAi-mediated suppression of DET1 enhances carotenoid and flavonoid content in tomatoes[J].Nature Biotechnology,2005,23(7):890-895.
14.Hoffmann T,Kalinowski G,Schwab W.RNAi-induced silencing of gene expression in strawberry fruit(Fragaria×ananassa)by agroinfiltration:a rapid assay for gene function analysis[J].The Plant Journal,2006,48(5):818-826.
15.Han Y,Dang R H,Li J X,et al.SUCROSE NONFER-MENTING1-RELATED PROTEIN KINASE2.6,an ortholog of OPEN STOMATA1,is a negative regulator of strawberry fruit development and ripening[J].Plant Physiology,2015,167(3):915-965.
16.Fait A,Hanhineva K,Beleggia R,et al.Reconfiguration of the achene and receptacle metabolic networks during strawberry fruit development[J].Plant Physiology,2008,148(2):730-750.
17.Payasi A,Misra P C,Sanwal G G.Effect of phytohormones on pectate lyase activity in ripening Musa acuminata[J].Plant Physiology and Biochemistry,2004,42(11):861-865.
18.Chen Q,Yu H W,Wang X R,et al.An alternative cetyltrimethylammonium bromide-basedprotocol for RNA isolation from blackberry(Rubus L.)[J].Genetics and Molecular Research,2012,11(2):1773-1782.
19.Lin Y X,Jiang L Y,Chen Q,et al.Comparative transcriptome profiling analysis of red-and white-fleshed strawberry(Fragaria×ananassa)provides new insight into the regulation of the anthocyanin pathway[J].Plant and Cell Physiology,2018,59(9):1844-1859.
20.Chen S Y,Tang Y M,Hu Y Y,et al.Fa TT12-1,a multidrug and toxin extrusion(MATE)member involved in proanthocyanidin transport in strawberry fruits[J].Scientia Horticulturae,2018,231:158-165.
21.Braybrook S A,J9nsson H.Shifting foundations:the mechanical cell wall and development[J].Current Opinion in Plant Biology,2016,29:115-120.
22.周厚成.草莓果实成熟软化相关基因的研究[D].杨凌:西北农林科技大学,2014.Zhou H C.Research on genes related to fruit ripening and softening of strawberry[D].Yangling:Northwest A&F U-niversity,2014.
23.PoséS,Paniagua C,Cifuentes M,et al.Insights into the effects of polygalacturonase Fa PG1 gene silencing on pectin matrix disassembly,enhanced tissue integrity,and firmness in ripe strawberry fruits[J].Journal of Experimental Botany,2013,64(12):3803-3815.
24.Quesada M A,Blanco-Portales R,PoséS,et al.Antisense down-regulation of the Fa PG1 gene reveals an unexpected central role for polygalacturonase in strawberry fruit softening[J].Plant Physiology,2009,150(2):1022-1032.
25.Tieman D M,Harriman R W,Ramamohan G,et al.An antisense pectin methylesterasegene alters pectin chemistry and soluble solids in tomato fruit[J].The Plant Cell,1992,4(6):667-679.
26.周鹤莹,张玮,张卿,等.森林草莓‘Ruegen’果胶裂解酶基因的克隆及荧光定量表达分析[J].园艺学报,2015,34(3):455-461.Zhou H Y,Zhang W,Zhang Q,et al.The cloning and quantitative expression analysis of pectate lyase gene in Fragaria vesca[J].Acta Horticulturae Sinica,2015,34(3):455-461.
27.陈品文.amiRNA介导的Fa CRYs基因对草莓花青素苷合成的影响研究[D].成都:四川农业大学,2016.Chen P W.Effect of amiRNA-mediated Fa CRYs genes on anthocyanin biosynthesis in strawberry[D].Chengdu:Sichuan Agricultural University,2016.
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