番茄花柄脱落相关基因表达谱分析及多聚半乳糖醛酸酶性质研究
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摘要
器官脱落是指植物体的部分器官(如花、叶、果等)脱离母体的过程。成熟果实与种子等器官的正常脱落是植物适应环境、进行正常生长生育的需要,而由于生理与胁迫等引起的花果脱落等,却是影响作物产量与品质的重要因素。因此,研究植物器官脱落机理及调控技术具有重要的理论与实践意义。本研究以番茄离体花柄为试材,应用基因芯片分析技术,研究了番茄离体花柄自然脱落与乙烯诱导脱落过程中相关基因的表达差异；明确了多聚半乳糖醛酸酶等基因的表达与脱落过程密切相关；并采用免疫蛋白定位技术对多聚半乳糖醛酸酶进行了组织定位,提取纯化了脱落相关多聚半乳糖醛酸酶,研究了酶的性质与活性调控。主要结果如下：
1.采用Agilent 4x44K microarrays番茄基因表达芯片,获得了自然脱落与乙烯诱导脱落过程中的基因表达谱。以2倍差异表达为筛选标准,筛选出17469个差异表达探针。根据各基因在脱落过程中的表达趋势,聚类分析归纳为8个基因表达组。
2.分析了促进番茄花柄脱落的有关乙烯合成、受体及信号转导基因在脱落过程中的表达情况,结果表明,与乙烯合成及信号传导相关基因SAM、ACO、EGY3、EIN2、ER1、ER33、ER6、ERF1、ERF7、ETR2等在脱落中上调表达,而ETR4、ER49、ER66、EGY1、ERF11、ERF9、ERF3等下调表达；乙烯处理进一步促进了RTE1、TCTR2、ERF4、Never-ripe、ETR1、ETR6、ERS1、ER24等的上调表达,加剧了ER43、ERF2、TCTRlv、ATEBP、ER21、ATERF-1、ERF5、ER5等的下调表达。
3.分析了脱落过程中生长素及其信号传导基因的表达情况,结果表明,AFB5、ARF10、ARF3、ARF6、AUX1、IAA14、LAX3、PIN3、TIR1等上调表达,而ARF1、Axil、IAA8、PIN4.SAR3等下调表达;乙烯处理进一步促进了SAUR-likeprotein、ARGOS、IAA1等的上调表达,加剧了ARF4、ARF8、ATAUX2-11、CTD1、IAA16、IAA29、IAA3、IAA7、IAA9、SARI等的下调表达。
4.对番茄花柄脱落相关的细胞壁降解酶和蛋白的基因表达差异表明,在番茄花柄脱落过程中,与细胞壁降解相关的酶和蛋白主要包括：多聚半乳糖醛酸酶、纤维素酶、果胶酯酶、几丁质酶、木葡聚糖转葡糖苷酶、扩展蛋白与伸展蛋白等均有大量基因探针随着脱落过程而呈显著上调表达,说明这些酶和蛋白在番茄花柄脱落中起重要作用。
5.采用蛋白免疫荧光定位技术分析了番茄脱落过程中多聚半乳糖醛酸酶在离区的组织定位。番茄花柄离区发生脱落前,在离区的皮层组织存在少量的多聚半乳糖醛酸酶蛋白,髓部极少有该酶的存在。随着脱落的启动,酶蛋白首先在皮层组织大量表达,且沿着离层形成方向向髓组织细胞扩展。在脱落过程中,多聚半乳糖醛酸酶在离区的近轴端与远轴端均有显著表达。至脱落后期,在整个离区组织中多聚半乳糖醛酸酶大量表达。
6.采用蛋白免疫胶体金定位技术分析了番茄花柄脱落过程中多聚半乳糖醛酸酶在离区亚细胞水平的定位。番茄花柄离区发生脱落前,酶蛋白主要位于细胞壁、细胞膜及邻近的细胞质部位,在细胞核及液泡中无表达。随着脱落的启动,细胞中多聚半乳糖醛酸酶蛋白显著增多,尤其是在细胞壁及膜系统上。至脱落后期,在整个离区组织中大量表达,除细胞壁、细胞膜外,在细胞核、液泡及细胞质中也大量存在。另外,在脱落过程中,多聚半乳糖醛酸酶在离区近轴端与远轴端的细胞中均有显著表达。
7.建立了番茄花柄脱落相关多聚半乳糖醛酸酶提取纯化技术,即：以50 mmol·L-1乙酸缓冲液(0.1mol·L-1NaCl,1 mmol·L-1DTT, pH=5.5)为提取缓冲液,对脱落相关的多聚半乳糖醛酸酶进行粗提,低温真空浓缩后,经Sephadex G-75凝胶过滤层析(流速0.2 mL·min-1,上样量3.5 mL)和CM Sepharose CL-6B阳离子离子交换层析(pH=5.5乙酸缓冲液,流速为0.3 mL·min-1),纯化得到分子量约为30.2kD的多聚半乳糖醛酸酶。纯化倍数为30.85倍,回收率为52.58%。
8.明确了纯化多聚半乳糖醛酸酶的基本性质。该酶具有较好的热稳定性,最适反应温度为40℃,最适反应pH为5.0,以多聚半乳糖醛酸钠为反应底物的Km值为26.14mg·mL-1。1mmol·L-1的Ca2+、Gu2+、Ba2+、Co2+、Mn2+可抑制多聚半乳糖醛酸酶活性,而1 mmol·L-1的Mg2+、K+、Fe2+、Zn2+、Fe3+则促进多聚半乳糖醛酸酶活性。
9.明确了植物生长调节剂对纯化多聚半乳糖醛酸酶活性的调控作用。在一定浓度下,IAA、GA与ZT对纯化的多聚半乳糖醛酸酶活性具有一定抑制作用,而ABA对酶的活性有一定促进作用,但作用效果均不明显,说明这些植物生长调控剂对酶活性的直接调控作用较小。
通过上述研究,系统分析了脱落过程中相关基因的表达情况,总结了脱落的分子机制。对器官脱落中起重要作用的多聚半乳糖醛酸酶进行了定位,提出该酶在脱落过程中首先于皮层细胞表达,随着脱落进程沿着离层形成方向向髓组织细胞扩展,且在离区的近轴端与远轴端均有表达。提取纯化了脱落相关多聚半乳糖醛酸酶,明确了酶的基本性质与活性调控,为进一步调控脱落过程奠定了基础。
Abscission, in plant sciences it most commonly refers to the process by which a plant drops one or more of its parts, such as leaves, fruits, flowers or seeds. A plant will abscise a part either to discard a member that is no longer necessary, such as a leaf during autumn, or a means of plant defense, or a flower following fertilisation, or for the purposes of reproduction, however, in agricultural production the physiology disorder and bio-stress caused flower and fruit dropping resulted in seriously yield and economic loss. Study of the abscission may have great value in understanding the theory of abscission and provide proper guidance for preventing flower abscission. This paper reports the tomato pedicel abscise-relative genes screened by Agilent microarrays, on basis of this mainly study the character of key abscission gene polygalacturonase and its distribution in cell level during abscission by immunolocalization. The results are as follows:
1. Application of Agilent 4x44K microarrays constructed gene expression profiles of tomato pedicel ethylene induced and natural abscission, screening 17469 differentially expressed probe and clustered into 8 groups according to expression patterns.
2. Ethylene biosynthesis and signal pathway cluster:SAM, ACO, EGY3, EIN2, ER1, ER33, ER6, ERF1, ERF7 and ETR2 were up-regulated expression, while ETR4, ER49, ER66, EGY1, ERF11, ERF9 and ERF3 significant down-regulated. Ethylene treatment up-regulated the expression of RTE1, TCTR2, ERF4, Never-ripe, ETR1, ETR6, ERS1, ER24, and ERF1-1, but down-regulated ER43, ERF2, TCTRlv, ATEBP, ER21, ATERF-1, ERF5 and ER5.
3. Auxin signal pathway cluster:AFB5, ARF10, ARF3, ARF6, AUX1, IAA14, LAX3, PIN3 and TIR1were up-regulated, while ARF1, Axil, IAA8, PIN4 and SAR3 down-regulated. Ethylene treatment up-regulated the expression of SAUR-like protein, ARGOS and IAA1, but down-regulated ARF4, ARF8, ATAUX2-11, CTD1, IAA16, IAA29, IAA3, IAA7, IAA9 and SARI.
4. Cell wall-degrading enzymes and proteins cluster: PG (polygalacturonase), CEL (cellulose), PE (pectin esterase), CHIT (chitinase), XET (xyloglucan endotransglycosylase), EXT (extension) and EXP (expansin). Most of them show up-regulated during separated process which implicated play important roles in abscission.
5. Before tomato flower pedicel separation, PG mainly distributed in cortex of AZ (abscission zone), little observed in and central parenchymatous region. As the abscission initial, PG showed abundant accumulative in cortical tissue and transmitted into central parenchymatous region. PG was significant increased in cell, especially in cell wall and membrane systems. In the late stage of abscission, PG showed abundant accumulative in whole AZ region. Moreover, there is no observed of PG appearance in distal and proximal part throughout abscission.
6. Before tomato flower pedicel separation, PG mainly distributed in AZ (abscission zone) cell wall, cell membrane and cytoplasm of adjacent parts, no observed in nucleus and the vacuole. As the abscission initial, PG was significant increased in cell, especially in cell wall and membrane systems. In the late stage of abscission, PG showed abundant accumulative in whole AZ region, besides in cell wall and membrane systems, cytoplasm, nucleus and the vacuole was also great amount observed.
7. The optimum extraction condition for polygalacturonase from the crude enzyme extracts of tomato pecdicel is 50 mmol·L-1 acetate buffer solution (pH5.5) with 0.1 M NaCl and 1 mM DTT. Then enzyme extracts which concentrated in low temperature are purified by Sephadex G-75 column equilibrated with flow rate of 0.2 mL·min-1 and sample volume of 3.5 mL. Then the active fraction of polygalacturonase which concentrated in low temperature are further absorbed onto a CM sepharose CL-6B ionic exchange chromatographic with acetate buffer at pH5.5 and flow rate of 0.3 mL·min-1. The purified polygalacturonase of tomato pecdicel molecular mass is 30.2 kD,30.85-fold purification and 52.58% recovery.
8. The pH optimum for activity of purified polygalacturonase is determined to be 5.0, temperature optima to be 40℃, and Km 26.14 mg·ml-1. The enzyme activity was inhibited by 1 mmol·L-1 Ca2+, Gu2+, Ba2+, Co2+ and Mn2+ and activated by 1 mmol·L-1Mg2+, K+, Fe2+ Zn2+ and Fe3+.
9. The effect of plant phytohormones on the activity of polygalacturonase was also investigated. The result indicated that GA and IAA could inhibit the activity of PG with the optimum concentration of 0.3 mg·mL-1 and 0.1 mg·mL-1 respectively. ABA could activate the activity of PG with the optimum concentration of 0.3 mg·mL-1. Low concentration of ZT could activate the activity of PG, however, high concentration ZT inhibit the activity of PG.
According to systematic analysis of gene expression profiles, summarized the molecular mechanism of ethylene induced abscission. On basis of this, screened the key abscission gene polygalacturonase and made out PG distribution first accumulative in cortical tissue and transmitted into central parenchymatous region. Moreover, there is no observed of PG appearance in distal and proximal part throughout abscission. We also purified polygalacturonaseis, its enzyme character. The results will be helpful for understanding the theory of abscission and provide proper guidance for preventing flower abscission.
1.采用Agilent 4x44K microarrays番茄基因表达芯片,获得了自然脱落与乙烯诱导脱落过程中的基因表达谱。以2倍差异表达为筛选标准,筛选出17469个差异表达探针。根据各基因在脱落过程中的表达趋势,聚类分析归纳为8个基因表达组。
2.分析了促进番茄花柄脱落的有关乙烯合成、受体及信号转导基因在脱落过程中的表达情况,结果表明,与乙烯合成及信号传导相关基因SAM、ACO、EGY3、EIN2、ER1、ER33、ER6、ERF1、ERF7、ETR2等在脱落中上调表达,而ETR4、ER49、ER66、EGY1、ERF11、ERF9、ERF3等下调表达；乙烯处理进一步促进了RTE1、TCTR2、ERF4、Never-ripe、ETR1、ETR6、ERS1、ER24等的上调表达,加剧了ER43、ERF2、TCTRlv、ATEBP、ER21、ATERF-1、ERF5、ER5等的下调表达。
3.分析了脱落过程中生长素及其信号传导基因的表达情况,结果表明,AFB5、ARF10、ARF3、ARF6、AUX1、IAA14、LAX3、PIN3、TIR1等上调表达,而ARF1、Axil、IAA8、PIN4.SAR3等下调表达;乙烯处理进一步促进了SAUR-likeprotein、ARGOS、IAA1等的上调表达,加剧了ARF4、ARF8、ATAUX2-11、CTD1、IAA16、IAA29、IAA3、IAA7、IAA9、SARI等的下调表达。
4.对番茄花柄脱落相关的细胞壁降解酶和蛋白的基因表达差异表明,在番茄花柄脱落过程中,与细胞壁降解相关的酶和蛋白主要包括：多聚半乳糖醛酸酶、纤维素酶、果胶酯酶、几丁质酶、木葡聚糖转葡糖苷酶、扩展蛋白与伸展蛋白等均有大量基因探针随着脱落过程而呈显著上调表达,说明这些酶和蛋白在番茄花柄脱落中起重要作用。
5.采用蛋白免疫荧光定位技术分析了番茄脱落过程中多聚半乳糖醛酸酶在离区的组织定位。番茄花柄离区发生脱落前,在离区的皮层组织存在少量的多聚半乳糖醛酸酶蛋白,髓部极少有该酶的存在。随着脱落的启动,酶蛋白首先在皮层组织大量表达,且沿着离层形成方向向髓组织细胞扩展。在脱落过程中,多聚半乳糖醛酸酶在离区的近轴端与远轴端均有显著表达。至脱落后期,在整个离区组织中多聚半乳糖醛酸酶大量表达。
6.采用蛋白免疫胶体金定位技术分析了番茄花柄脱落过程中多聚半乳糖醛酸酶在离区亚细胞水平的定位。番茄花柄离区发生脱落前,酶蛋白主要位于细胞壁、细胞膜及邻近的细胞质部位,在细胞核及液泡中无表达。随着脱落的启动,细胞中多聚半乳糖醛酸酶蛋白显著增多,尤其是在细胞壁及膜系统上。至脱落后期,在整个离区组织中大量表达,除细胞壁、细胞膜外,在细胞核、液泡及细胞质中也大量存在。另外,在脱落过程中,多聚半乳糖醛酸酶在离区近轴端与远轴端的细胞中均有显著表达。
7.建立了番茄花柄脱落相关多聚半乳糖醛酸酶提取纯化技术,即：以50 mmol·L-1乙酸缓冲液(0.1mol·L-1NaCl,1 mmol·L-1DTT, pH=5.5)为提取缓冲液,对脱落相关的多聚半乳糖醛酸酶进行粗提,低温真空浓缩后,经Sephadex G-75凝胶过滤层析(流速0.2 mL·min-1,上样量3.5 mL)和CM Sepharose CL-6B阳离子离子交换层析(pH=5.5乙酸缓冲液,流速为0.3 mL·min-1),纯化得到分子量约为30.2kD的多聚半乳糖醛酸酶。纯化倍数为30.85倍,回收率为52.58%。
8.明确了纯化多聚半乳糖醛酸酶的基本性质。该酶具有较好的热稳定性,最适反应温度为40℃,最适反应pH为5.0,以多聚半乳糖醛酸钠为反应底物的Km值为26.14mg·mL-1。1mmol·L-1的Ca2+、Gu2+、Ba2+、Co2+、Mn2+可抑制多聚半乳糖醛酸酶活性,而1 mmol·L-1的Mg2+、K+、Fe2+、Zn2+、Fe3+则促进多聚半乳糖醛酸酶活性。
9.明确了植物生长调节剂对纯化多聚半乳糖醛酸酶活性的调控作用。在一定浓度下,IAA、GA与ZT对纯化的多聚半乳糖醛酸酶活性具有一定抑制作用,而ABA对酶的活性有一定促进作用,但作用效果均不明显,说明这些植物生长调控剂对酶活性的直接调控作用较小。
通过上述研究,系统分析了脱落过程中相关基因的表达情况,总结了脱落的分子机制。对器官脱落中起重要作用的多聚半乳糖醛酸酶进行了定位,提出该酶在脱落过程中首先于皮层细胞表达,随着脱落进程沿着离层形成方向向髓组织细胞扩展,且在离区的近轴端与远轴端均有表达。提取纯化了脱落相关多聚半乳糖醛酸酶,明确了酶的基本性质与活性调控,为进一步调控脱落过程奠定了基础。
Abscission, in plant sciences it most commonly refers to the process by which a plant drops one or more of its parts, such as leaves, fruits, flowers or seeds. A plant will abscise a part either to discard a member that is no longer necessary, such as a leaf during autumn, or a means of plant defense, or a flower following fertilisation, or for the purposes of reproduction, however, in agricultural production the physiology disorder and bio-stress caused flower and fruit dropping resulted in seriously yield and economic loss. Study of the abscission may have great value in understanding the theory of abscission and provide proper guidance for preventing flower abscission. This paper reports the tomato pedicel abscise-relative genes screened by Agilent microarrays, on basis of this mainly study the character of key abscission gene polygalacturonase and its distribution in cell level during abscission by immunolocalization. The results are as follows:
1. Application of Agilent 4x44K microarrays constructed gene expression profiles of tomato pedicel ethylene induced and natural abscission, screening 17469 differentially expressed probe and clustered into 8 groups according to expression patterns.
2. Ethylene biosynthesis and signal pathway cluster:SAM, ACO, EGY3, EIN2, ER1, ER33, ER6, ERF1, ERF7 and ETR2 were up-regulated expression, while ETR4, ER49, ER66, EGY1, ERF11, ERF9 and ERF3 significant down-regulated. Ethylene treatment up-regulated the expression of RTE1, TCTR2, ERF4, Never-ripe, ETR1, ETR6, ERS1, ER24, and ERF1-1, but down-regulated ER43, ERF2, TCTRlv, ATEBP, ER21, ATERF-1, ERF5 and ER5.
3. Auxin signal pathway cluster:AFB5, ARF10, ARF3, ARF6, AUX1, IAA14, LAX3, PIN3 and TIR1were up-regulated, while ARF1, Axil, IAA8, PIN4 and SAR3 down-regulated. Ethylene treatment up-regulated the expression of SAUR-like protein, ARGOS and IAA1, but down-regulated ARF4, ARF8, ATAUX2-11, CTD1, IAA16, IAA29, IAA3, IAA7, IAA9 and SARI.
4. Cell wall-degrading enzymes and proteins cluster: PG (polygalacturonase), CEL (cellulose), PE (pectin esterase), CHIT (chitinase), XET (xyloglucan endotransglycosylase), EXT (extension) and EXP (expansin). Most of them show up-regulated during separated process which implicated play important roles in abscission.
5. Before tomato flower pedicel separation, PG mainly distributed in cortex of AZ (abscission zone), little observed in and central parenchymatous region. As the abscission initial, PG showed abundant accumulative in cortical tissue and transmitted into central parenchymatous region. PG was significant increased in cell, especially in cell wall and membrane systems. In the late stage of abscission, PG showed abundant accumulative in whole AZ region. Moreover, there is no observed of PG appearance in distal and proximal part throughout abscission.
6. Before tomato flower pedicel separation, PG mainly distributed in AZ (abscission zone) cell wall, cell membrane and cytoplasm of adjacent parts, no observed in nucleus and the vacuole. As the abscission initial, PG was significant increased in cell, especially in cell wall and membrane systems. In the late stage of abscission, PG showed abundant accumulative in whole AZ region, besides in cell wall and membrane systems, cytoplasm, nucleus and the vacuole was also great amount observed.
7. The optimum extraction condition for polygalacturonase from the crude enzyme extracts of tomato pecdicel is 50 mmol·L-1 acetate buffer solution (pH5.5) with 0.1 M NaCl and 1 mM DTT. Then enzyme extracts which concentrated in low temperature are purified by Sephadex G-75 column equilibrated with flow rate of 0.2 mL·min-1 and sample volume of 3.5 mL. Then the active fraction of polygalacturonase which concentrated in low temperature are further absorbed onto a CM sepharose CL-6B ionic exchange chromatographic with acetate buffer at pH5.5 and flow rate of 0.3 mL·min-1. The purified polygalacturonase of tomato pecdicel molecular mass is 30.2 kD,30.85-fold purification and 52.58% recovery.
8. The pH optimum for activity of purified polygalacturonase is determined to be 5.0, temperature optima to be 40℃, and Km 26.14 mg·ml-1. The enzyme activity was inhibited by 1 mmol·L-1 Ca2+, Gu2+, Ba2+, Co2+ and Mn2+ and activated by 1 mmol·L-1Mg2+, K+, Fe2+ Zn2+ and Fe3+.
9. The effect of plant phytohormones on the activity of polygalacturonase was also investigated. The result indicated that GA and IAA could inhibit the activity of PG with the optimum concentration of 0.3 mg·mL-1 and 0.1 mg·mL-1 respectively. ABA could activate the activity of PG with the optimum concentration of 0.3 mg·mL-1. Low concentration of ZT could activate the activity of PG, however, high concentration ZT inhibit the activity of PG.
According to systematic analysis of gene expression profiles, summarized the molecular mechanism of ethylene induced abscission. On basis of this, screened the key abscission gene polygalacturonase and made out PG distribution first accumulative in cortical tissue and transmitted into central parenchymatous region. Moreover, there is no observed of PG appearance in distal and proximal part throughout abscission. We also purified polygalacturonaseis, its enzyme character. The results will be helpful for understanding the theory of abscission and provide proper guidance for preventing flower abscission.
引文
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