乙烯诱导柑橘果实脱落的转录基因组学研究及乙烯诱导基因的克隆和鉴定
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摘要
果实脱落是影响柑橘产量的重要因素之一。乙烯调节脱落过程中细胞分裂、伸长、细胞壁水解等多种细胞学、生物化学进程,是主要的脱落激素。乙烯生物合成途径已经阐明。研究指出,柑橘叶片和果实中都存在两种乙烯生成系统,柑橘叶片在采摘后(老叶3-5天、新叶6-7天)乙烯合成表现为系统Ⅰ,老叶采后5天、新叶采后7天则表现出乙烯自动催化等典型的类似跃变型果实的乙烯生成系统Ⅱ的特征；柑橘作为一种非跃变型的果实,成熟果实中只有系统Ⅰ存在,外源乙烯能促进成熟相关的色素变化加快呼吸进程；但在幼果中表现出乙烯生成系统Ⅱ类似的特征。为探寻乙烯在柑橘成熟果实脱落过程中的作用机理,本研究进行了乙烯诱导下柑橘果实离层的表达谱分析,柑橘PRP、ERF和半胱氨酸蛋白酶基因全长cDNA克隆、表达分析和柑橘PRP、ERF、半胱氨酸蛋白酶基因RNAi载体的构建及转化等研究。
1乙烯诱导下柑橘果实离层的表达谱分析
利用Citrus Genome Array研究了在外源乙烯(20μL/L)诱导下柑橘果实离层的基因表达谱,在30395个达到探针对阀值(8对)要求的探针组中,检测到20639探针组对应的基因表达。应用GCOS软件系统进行数据管理、RMA进行数据预处理、随机方差模型的单因素方差分析法分析实验组与对照组间的差异基因之后,发现在FDR(false-discovery rate)≤0.02水平上乙烯处理4h、24h后分别有569和356条基因表达量的变化倍数≥4。其中,4h乙烯处理后表达量上调的有243条,处理24h后上调表达的有176条；而对应时间内表达量下调的则分别有326条和190条。分析了随机挑选的16条基因,所得结果与芯片数据高度一致。根据基因功能分类,发现这些乙烯应答基因涉及协迫应答、生物或非生物刺激、蛋白质代谢、物质转运、信号转导和转录等多种生物学过程。经过基因本体(Gene Ontology,GO)分析,差异表达基因中,发现富含羟脯氨酸蛋白-4(PRP4, proline-rich protein 4)基因、乙烯响应因子1(ERF1, ethylene response factor 1)基因和半胱氨酸蛋白酶(CysP,cysteine proteinase)基因高度受到乙烯调控,它们在柑橘果实脱落中所起的作用值得关注,因而被挑选出来进一步分析。
2柑橘CsPRP4、CsERFl、CsUnknow和CsCysP基因全长cDNA克隆和表达分析
根据HarvEst Citrus (Version 1.25)数据库提供的CsPRP4、CsERFl、CsUnknow和CsCysP序列信息设计了PCR引物,采用RT-PCR和SMART RACE技术和克隆操作,获得了这些基因的全长cDNA序列。
序列分析结果显示,这4个基因的全长cDNA长度分别是1251bp、1453bp、1545bp和1452bp,开放阅读框(ORF)的长度分别是585bp、810bp、1101bp和1050bp,分别编码194、269、365和347个氨基酸,相应的推测分子量分别是20.79KD、29.25KD、41.6KD和38.4KD。蛋白理化性质分析表明CsPRP4和CsERF1性质稳定,而CsUnknow和CsCysP不稳定。
生物信息学和基因差异表达分析表明：CsPRP4是受外源乙烯诱导表达量上升最强烈的编码细胞壁蛋白的基因,在叶片和果实离层均表达,受乙烯诱导后的叶片和果实离层表达存在差异；CsERF1是AP2超基因家族成员,具转录因子活性,在乙烯诱导下表达量大量增加,CsERF作为乙烯信号转导途径的中间组分,具有结合GCC-box的活性,通过与乙烯应答基因启动子中的GCC-box结合调控下游基因表达,可能是最终导致脱落的关键因子之一；CsUnknow基因,定位于内膜系统,具有GCC-box结合域,受乙烯强烈诱导,功能未知；CsCysP蛋白,是重要的水解蛋白酶之一,具有肽链内切酶活性,是一种跨膜蛋白,定位于液泡膜上。外源乙烯作用下,CsCysP在柑橘果实离层中受到强烈抑制,推测其负调控下游的脱落相关基因,受其作用的某些蛋白质的积累可能是脱落过程所必须的。
3柑橘CsPRP4、CsERF1和CsCysP基因RNAi载体的构建及转化
采用RT-PCR方法扩增柑橘目的基因PRP4、ERF1和CysP的cDNA片段,连接到TA克隆载体。双元载体pFGC5941和目的基因的克隆经限制性内切酶两次双酶切,将目的基因片段按正、反向插入到pFGC5941查耳酮合成酶内含子的两端,构成反向重复序列,即RNA干涉载体。通过菌液PCR、酶切检验或测序等证实正、反向基因片段均已正确插入到载体中。研究中保留了采用该法构建的全长正向片段与pFGC5941相连接后得到的产物,以用于基因的过量表达研究。采用根癌农杆菌介导的转基因法得到了大量抗性芽,其中部分已经嫁接成活,为下一步研究在干扰和过量表达这些基因后的表型打下了基础。
4主要结论
本实验在转录水平上大规模地研究了外源乙烯诱导下柑橘果实离层的基因表达谱的变化,获得了大量潜在的参与脱落进程的基因及其表达数据。在全面综合分析这些数据的基础上,基本明确了参与脱落过程的生理生化过程,加深了对乙烯在诱导柑橘果实脱落过程中基因调节等分子机理的认识。
克隆获得了4条在柑橘果实脱落过程中受到强烈调控的基因CsPRP4、CsERFl、CsUnknow和CsCysP的全长cDNA。对这些基因进行了生物信息学分析,结合表达分析,推测了其在脱落过程中可能的作用。
构建了CsPRP4、CsERF1、CsUnknow和CsCysP基因的过量表达载体和RNAi载体。通过根癌农杆菌介导的遗传转化,获得了大量抗性再生芽,部分芽已高接成活,为下一步揭示这些基因的功能奠定了基础。
Abscission is an important determinant of citrus yield. Ethylene is a major abscission hormone that regulates many biological processes involved in abscission such as cell division, cell expansion and cell wall decomposition. The biosynthesis of ethylene has been elucidated. Previous researches indicated that two ethylene production systems, systemⅠandⅡ, exist in both citrus fruit and leaves. During the first phase (3～5d in mature leaves and 6～7d in young leaves, after detachment)systemⅠis operating. During the second phase, which started 5d and 7d, respectively, in young leaves and mature leaves after harvest, systemⅡis initiated and ethylene evolution becomes autocatalytic and climacteric-like, which is typical in systemⅡ. Mature citrus fruits, which are generally considered as non-climacteric, only exhibit systemⅠ-like behavior, and respond to exogenous ethylene by undergoing ripening-related pigment changes and accelerated respiration. Young citrus fruitlets exhibit a climacteric-like rise in ethylene production after detachment, indicating an autocatalytic systemⅡ-like ethylene biosynthesis.
To explore the mechanism of ethylene action in the abscission of mature fruit, changes in transcriptome in the abscission zone of mature fruit during ethylene-induced abscission were studied. Several highly ethylene-regulated genes, CsPRP4、CsERF1、CsUnknow and CsCysP, were full-length cloned. Bio-informatics researches, together with quantitative RT-PCR studies on their expression changes, were performed on these genes to probe their possible roles in ethylene-induced abscission. Construction of overexpression and RNAi vectors was conducted for these genes, and transformation was done via agrobacterium-mediated transformation using the constructs and sweet orange explants. Regenerated antibiotic buds were obtained and some of them were successfully grafted.
1. Profiling genes involved in ethylene-induced abscission in mature citrus fruit
Ethylene responsive genes in mature Valencia orange fruit abscission zones were analyzed by using Affymetrix GeneChip citrus genome array. Fruit were treated with 20μL/L of ethylene for 0,4 and 24h and RNA was extracted from abscission zones and subjected to genechip analysis. GCOS software was used to analyze the original genechip hybridization data. RMA (robust multiarray analysis) was used to filtrate the unreliable dada. Of the 30395 total probe sets that exceed the probe pair threshold on the microarray,20639 detected changed expression of their corresponding genes.569 and 326 genes were identified to be ethylene-responsive as shown by more than 4-fold changes in their expression levels after 4 and 24 h of ethylene treatment at FDR (false-discovery rate)≤0.02 level. Of these genes,243 and 176 were up-regulated, whereas 326 and 190 were down-regulated, respectively, after 4 and 24h of ethylene treatment. Results from relative quantitative RT-PCR analysis performed on 16 selected genes were highly agreeable with those observed from microarray analysis. Classification based on their functions showed that these ethylene-responsive genes are related to a variety of different processes including responses to stress, responses to abiotic or biotic stimulus, protein metabolism, transport, signal transduction and transcription. The most significant changes in expression were found for genes encoding proline-rich protein 4, ethylene response factor 1 and cysteine proteinase, and their possible involvement in abscission was further studied.
2. Cloning and Expression Analysis of CsPRP4、CsERF1、CsUnknow and CsCysP genes
Primers were designed from sequences deposited in HarvEst Citrus (Version 1.25) database, and the full-length cDNAs of CsPRP4、CsERF、CsUnknow and CsCysP genes were cloned by using methods of RT-PCR and SMART-RACE.
Sequence analysis revealed that the full-length cDNAs of CsPRP4, CsERF, CsUnknow and CsCysP are 1251bp、1453bp、1545bp and1452bp respectively, and their corresponding ORFs are 585bp、810bp、1101bp and 1050bp in length which would encode 194,269,365 and 347 amino acids with calculated molecular masses of 20.79KD,29.25KD,41.6KD and 38.4KD, respectively. Protein physiochemistry analysis suggested that CsPRP4 and CsERF1 would be stable while CsUnknow and CsCysP would be unstable.
Bioinformatics and expression analysis showed that CsPRP4 was probably the most highly induced cell-wall gene and expressed differentially between leaves and fruit abscission zones when induced by exogenous ethylene. CsERF1 is a member of the AP2 supperfamily possessing transcription activator activity. ERF1, with GCC-box binding activity and as a component of the ethylene signal transduction pathway, plays an important role in modulating the expression of genes downstream of it by binding the GCC boxes located in the promoters of many ethylene responsive genes, and may be one of the key factors that eventually cause abscission. The function of CsUnknow encoded protein is unknown, but its expression was highly induced by ethylene. The sequence of CsUnknow suggested that it probably located in endomembrane system and possessed GCC-box binding motif. CsCysP, a transmembrane protein located in vacuolar membrane, is one of the important hydrolytic proteases with endopeptidase activity. The expression of CsCysP was strongly repressed by exogenous ethylene treatment, suggesting that it negatively regulates downstream gene(s) and that the accumulation of proteins targeted by CsCysP may be required in abscission. 3. Construction of CsPRP4、CsERF1 and CsCPA RNAi vectors and transformation
By using RT-PCR, the targeted cDNA sequences of PRP4、ERF1 and CysP were amplified from citrus and T/A cloned. Binary vector pFGC594 and target sequences were sequentially digested twice with endonucleases and then the target sequences were inserted in sense and antisense directions, respectively, into the two sides of the chalcone synthase gene intron that was carried in the vector to form inverted repeats. Various methods such as PCR by using bacteria containing the constructs, digestion of the isolated plasmids with endonucleases, and sequencing verified that all the constructs were correct. The sense constructs used in the construction of RNAi expression vectors were used as over-expression constructs. Antibiotic resistant buds were regenerated after transformation of citrus explants via agrobacterium-mediated transformation, and some of the buds have been successfully grafted onto rootstock seedlings, which should have paved a way towards the elucidation of the genes' functions.
4. Conclusion remarks
In this work, a large-scale study on the changes in transcriptome profiles of ethylene-induced genes during fruit abscission was carried out and a large quantity of data about the genes involved in abscission and their expression changes were generated. Comprehensive analysis of the data provided information of the biological pathways involved in abscission, and deepened our understanding of the molecular mechanism on gene regulation during ethylene-induced abscission.
The full-length cDNAs of 4 ethylene-regulated genes CsPRP4、CsERF1 CsUnknow and CsCysP during ethylene-induced abscission were obtained, and were analyzed with bioinformatics methods. In combination with the expression analysis, the roles of the genes in abscission were assumed.
The overexpression and RNAi expression vectors were constructed for CsPRP4、CsERF1、CsUnknow and CsCysP. Regenerated antibiotic buds were obtained through agrobacterium-mediated transformation, and some of the buds were survived after grafting, which placed a base for further characterization of the genes'functions.
1乙烯诱导下柑橘果实离层的表达谱分析
利用Citrus Genome Array研究了在外源乙烯(20μL/L)诱导下柑橘果实离层的基因表达谱,在30395个达到探针对阀值(8对)要求的探针组中,检测到20639探针组对应的基因表达。应用GCOS软件系统进行数据管理、RMA进行数据预处理、随机方差模型的单因素方差分析法分析实验组与对照组间的差异基因之后,发现在FDR(false-discovery rate)≤0.02水平上乙烯处理4h、24h后分别有569和356条基因表达量的变化倍数≥4。其中,4h乙烯处理后表达量上调的有243条,处理24h后上调表达的有176条；而对应时间内表达量下调的则分别有326条和190条。分析了随机挑选的16条基因,所得结果与芯片数据高度一致。根据基因功能分类,发现这些乙烯应答基因涉及协迫应答、生物或非生物刺激、蛋白质代谢、物质转运、信号转导和转录等多种生物学过程。经过基因本体(Gene Ontology,GO)分析,差异表达基因中,发现富含羟脯氨酸蛋白-4(PRP4, proline-rich protein 4)基因、乙烯响应因子1(ERF1, ethylene response factor 1)基因和半胱氨酸蛋白酶(CysP,cysteine proteinase)基因高度受到乙烯调控,它们在柑橘果实脱落中所起的作用值得关注,因而被挑选出来进一步分析。
2柑橘CsPRP4、CsERFl、CsUnknow和CsCysP基因全长cDNA克隆和表达分析
根据HarvEst Citrus (Version 1.25)数据库提供的CsPRP4、CsERFl、CsUnknow和CsCysP序列信息设计了PCR引物,采用RT-PCR和SMART RACE技术和克隆操作,获得了这些基因的全长cDNA序列。
序列分析结果显示,这4个基因的全长cDNA长度分别是1251bp、1453bp、1545bp和1452bp,开放阅读框(ORF)的长度分别是585bp、810bp、1101bp和1050bp,分别编码194、269、365和347个氨基酸,相应的推测分子量分别是20.79KD、29.25KD、41.6KD和38.4KD。蛋白理化性质分析表明CsPRP4和CsERF1性质稳定,而CsUnknow和CsCysP不稳定。
生物信息学和基因差异表达分析表明：CsPRP4是受外源乙烯诱导表达量上升最强烈的编码细胞壁蛋白的基因,在叶片和果实离层均表达,受乙烯诱导后的叶片和果实离层表达存在差异；CsERF1是AP2超基因家族成员,具转录因子活性,在乙烯诱导下表达量大量增加,CsERF作为乙烯信号转导途径的中间组分,具有结合GCC-box的活性,通过与乙烯应答基因启动子中的GCC-box结合调控下游基因表达,可能是最终导致脱落的关键因子之一；CsUnknow基因,定位于内膜系统,具有GCC-box结合域,受乙烯强烈诱导,功能未知；CsCysP蛋白,是重要的水解蛋白酶之一,具有肽链内切酶活性,是一种跨膜蛋白,定位于液泡膜上。外源乙烯作用下,CsCysP在柑橘果实离层中受到强烈抑制,推测其负调控下游的脱落相关基因,受其作用的某些蛋白质的积累可能是脱落过程所必须的。
3柑橘CsPRP4、CsERF1和CsCysP基因RNAi载体的构建及转化
采用RT-PCR方法扩增柑橘目的基因PRP4、ERF1和CysP的cDNA片段,连接到TA克隆载体。双元载体pFGC5941和目的基因的克隆经限制性内切酶两次双酶切,将目的基因片段按正、反向插入到pFGC5941查耳酮合成酶内含子的两端,构成反向重复序列,即RNA干涉载体。通过菌液PCR、酶切检验或测序等证实正、反向基因片段均已正确插入到载体中。研究中保留了采用该法构建的全长正向片段与pFGC5941相连接后得到的产物,以用于基因的过量表达研究。采用根癌农杆菌介导的转基因法得到了大量抗性芽,其中部分已经嫁接成活,为下一步研究在干扰和过量表达这些基因后的表型打下了基础。
4主要结论
本实验在转录水平上大规模地研究了外源乙烯诱导下柑橘果实离层的基因表达谱的变化,获得了大量潜在的参与脱落进程的基因及其表达数据。在全面综合分析这些数据的基础上,基本明确了参与脱落过程的生理生化过程,加深了对乙烯在诱导柑橘果实脱落过程中基因调节等分子机理的认识。
克隆获得了4条在柑橘果实脱落过程中受到强烈调控的基因CsPRP4、CsERFl、CsUnknow和CsCysP的全长cDNA。对这些基因进行了生物信息学分析,结合表达分析,推测了其在脱落过程中可能的作用。
构建了CsPRP4、CsERF1、CsUnknow和CsCysP基因的过量表达载体和RNAi载体。通过根癌农杆菌介导的遗传转化,获得了大量抗性再生芽,部分芽已高接成活,为下一步揭示这些基因的功能奠定了基础。
Abscission is an important determinant of citrus yield. Ethylene is a major abscission hormone that regulates many biological processes involved in abscission such as cell division, cell expansion and cell wall decomposition. The biosynthesis of ethylene has been elucidated. Previous researches indicated that two ethylene production systems, systemⅠandⅡ, exist in both citrus fruit and leaves. During the first phase (3～5d in mature leaves and 6～7d in young leaves, after detachment)systemⅠis operating. During the second phase, which started 5d and 7d, respectively, in young leaves and mature leaves after harvest, systemⅡis initiated and ethylene evolution becomes autocatalytic and climacteric-like, which is typical in systemⅡ. Mature citrus fruits, which are generally considered as non-climacteric, only exhibit systemⅠ-like behavior, and respond to exogenous ethylene by undergoing ripening-related pigment changes and accelerated respiration. Young citrus fruitlets exhibit a climacteric-like rise in ethylene production after detachment, indicating an autocatalytic systemⅡ-like ethylene biosynthesis.
To explore the mechanism of ethylene action in the abscission of mature fruit, changes in transcriptome in the abscission zone of mature fruit during ethylene-induced abscission were studied. Several highly ethylene-regulated genes, CsPRP4、CsERF1、CsUnknow and CsCysP, were full-length cloned. Bio-informatics researches, together with quantitative RT-PCR studies on their expression changes, were performed on these genes to probe their possible roles in ethylene-induced abscission. Construction of overexpression and RNAi vectors was conducted for these genes, and transformation was done via agrobacterium-mediated transformation using the constructs and sweet orange explants. Regenerated antibiotic buds were obtained and some of them were successfully grafted.
1. Profiling genes involved in ethylene-induced abscission in mature citrus fruit
Ethylene responsive genes in mature Valencia orange fruit abscission zones were analyzed by using Affymetrix GeneChip citrus genome array. Fruit were treated with 20μL/L of ethylene for 0,4 and 24h and RNA was extracted from abscission zones and subjected to genechip analysis. GCOS software was used to analyze the original genechip hybridization data. RMA (robust multiarray analysis) was used to filtrate the unreliable dada. Of the 30395 total probe sets that exceed the probe pair threshold on the microarray,20639 detected changed expression of their corresponding genes.569 and 326 genes were identified to be ethylene-responsive as shown by more than 4-fold changes in their expression levels after 4 and 24 h of ethylene treatment at FDR (false-discovery rate)≤0.02 level. Of these genes,243 and 176 were up-regulated, whereas 326 and 190 were down-regulated, respectively, after 4 and 24h of ethylene treatment. Results from relative quantitative RT-PCR analysis performed on 16 selected genes were highly agreeable with those observed from microarray analysis. Classification based on their functions showed that these ethylene-responsive genes are related to a variety of different processes including responses to stress, responses to abiotic or biotic stimulus, protein metabolism, transport, signal transduction and transcription. The most significant changes in expression were found for genes encoding proline-rich protein 4, ethylene response factor 1 and cysteine proteinase, and their possible involvement in abscission was further studied.
2. Cloning and Expression Analysis of CsPRP4、CsERF1、CsUnknow and CsCysP genes
Primers were designed from sequences deposited in HarvEst Citrus (Version 1.25) database, and the full-length cDNAs of CsPRP4、CsERF、CsUnknow and CsCysP genes were cloned by using methods of RT-PCR and SMART-RACE.
Sequence analysis revealed that the full-length cDNAs of CsPRP4, CsERF, CsUnknow and CsCysP are 1251bp、1453bp、1545bp and1452bp respectively, and their corresponding ORFs are 585bp、810bp、1101bp and 1050bp in length which would encode 194,269,365 and 347 amino acids with calculated molecular masses of 20.79KD,29.25KD,41.6KD and 38.4KD, respectively. Protein physiochemistry analysis suggested that CsPRP4 and CsERF1 would be stable while CsUnknow and CsCysP would be unstable.
Bioinformatics and expression analysis showed that CsPRP4 was probably the most highly induced cell-wall gene and expressed differentially between leaves and fruit abscission zones when induced by exogenous ethylene. CsERF1 is a member of the AP2 supperfamily possessing transcription activator activity. ERF1, with GCC-box binding activity and as a component of the ethylene signal transduction pathway, plays an important role in modulating the expression of genes downstream of it by binding the GCC boxes located in the promoters of many ethylene responsive genes, and may be one of the key factors that eventually cause abscission. The function of CsUnknow encoded protein is unknown, but its expression was highly induced by ethylene. The sequence of CsUnknow suggested that it probably located in endomembrane system and possessed GCC-box binding motif. CsCysP, a transmembrane protein located in vacuolar membrane, is one of the important hydrolytic proteases with endopeptidase activity. The expression of CsCysP was strongly repressed by exogenous ethylene treatment, suggesting that it negatively regulates downstream gene(s) and that the accumulation of proteins targeted by CsCysP may be required in abscission. 3. Construction of CsPRP4、CsERF1 and CsCPA RNAi vectors and transformation
By using RT-PCR, the targeted cDNA sequences of PRP4、ERF1 and CysP were amplified from citrus and T/A cloned. Binary vector pFGC594 and target sequences were sequentially digested twice with endonucleases and then the target sequences were inserted in sense and antisense directions, respectively, into the two sides of the chalcone synthase gene intron that was carried in the vector to form inverted repeats. Various methods such as PCR by using bacteria containing the constructs, digestion of the isolated plasmids with endonucleases, and sequencing verified that all the constructs were correct. The sense constructs used in the construction of RNAi expression vectors were used as over-expression constructs. Antibiotic resistant buds were regenerated after transformation of citrus explants via agrobacterium-mediated transformation, and some of the buds have been successfully grafted onto rootstock seedlings, which should have paved a way towards the elucidation of the genes' functions.
4. Conclusion remarks
In this work, a large-scale study on the changes in transcriptome profiles of ethylene-induced genes during fruit abscission was carried out and a large quantity of data about the genes involved in abscission and their expression changes were generated. Comprehensive analysis of the data provided information of the biological pathways involved in abscission, and deepened our understanding of the molecular mechanism on gene regulation during ethylene-induced abscission.
The full-length cDNAs of 4 ethylene-regulated genes CsPRP4、CsERF1 CsUnknow and CsCysP during ethylene-induced abscission were obtained, and were analyzed with bioinformatics methods. In combination with the expression analysis, the roles of the genes in abscission were assumed.
The overexpression and RNAi expression vectors were constructed for CsPRP4、CsERF1、CsUnknow and CsCysP. Regenerated antibiotic buds were obtained through agrobacterium-mediated transformation, and some of the buds were survived after grafting, which placed a base for further characterization of the genes'functions.
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