【作者】 赖呈纯；

【导师】 赖钟雄；

【作者基本信息】 福建农林大学 ， 果树学， 2010， 博士

【摘要】 本研究以龙眼品种‘红核子’(Dimocarpus longan Lour. cv. Honghezi)胚性愈伤组织(EC)为试验材料,通过龙眼体胚发生精细同步化调控,获得龙眼体胚发生早期5个阶段的胚性培养物,在此基础上进行如下研究:①建立龙眼体胚发生早期胚性培养物高分辨率双向电泳技术体系;②进行龙眼体胚发生早期各阶段胚性培养物蛋白质分离,获得高分辨率的蛋白质双向电泳(2D)图谱,并用2D分析软件ImageMaster 2D Platinum 6.0(GE Healthcare)进行蛋白质差异表达分析;③对龙眼体胚发生早期部分差异表达蛋白进行质谱鉴定及功能分析;④根据鉴定结果采用同源或设计简并引物克隆部分与龙眼体胚发生相关的基因,并采用实时荧光定量PCR(qRT-PCR)技术分析其在龙眼体胚发生过程中的基因转录水平表达变化。主要研究结果如下:1建立龙眼体胚发生早期胚性培养物高分辨率双向电泳技术在前人建立的龙眼体胚同步化调控的条件和方法的基础上,通过调控培养基中2,4-D的浓度结合显微镜观察,获得高度同步化的龙眼体胚发生早期5个不同阶段的胚性培养物,即松散型胚性愈伤组织(FEC)、胚性愈伤II(EC II)、不完全胚性紧实结构(ICpEC)、胚性紧实球形结构(CpECGE)、球形胚(GE)。在此基础上,采用固相pH梯度凝胶双向电泳技术,进行龙眼体胚发生早期胚性培养物蛋白质分离技术研究。结果表明,龙眼体胚发生早期胚性培养物蛋白质等电点主要集中在3.5-7.0之间,在胶条长度不变的情况下,以pH范围为4-7的IPG胶条,蛋白质分离的效果佳。采用固相pH梯度凝胶双向电泳技术可以大大提高龙眼胚性培养物蛋白质2D图谱的分辨率,第一向采用13 cm的IPG胶条所获得的蛋白质双向电泳图谱显示的蛋白点是传统双向电泳技术的2.5倍左右,并且许多低丰度蛋白得以显现。随IPG胶条长度的增加,能够进一步提高龙眼胚性培养物蛋白质2D电泳图谱的分辨率,采用18 cm的IPG胶条比13 cm的胶条可增加2倍左右的蛋白点数,但是采用18 cm的IPG胶条,第一向等电聚焦的运行参数需做相应的改变,并结合第二向SDS-PAGE高质量凝胶,才能获得最佳的龙眼胚性培养物蛋白质2D电泳图谱。2龙眼体胚发生早期各阶段胚性培养物蛋白质双向电泳分析①龙眼体胚发生早期各阶段可溶性蛋白的变化。采用考马斯亮蓝G-250法对龙眼体胚发生早期各阶段胚性培养物可溶性蛋白含量进行测定。结果表明,在松散型胚性愈伤组织阶段可溶性蛋白维持较低水平,随着体胚发育的启动和进行,可溶性蛋白含量迅速增加,胚性愈伤II含量最高,之后各阶段维持一个较高水平,但略有下降。龙眼松散型胚性愈伤组织阶段可溶性蛋白含量明显少于其他阶段,差异极显著(P<0.01),而其他各阶段的差异不大。这显示了龙眼体胚发生早期蛋白总量的变化与体胚的发育有关。②龙眼体胚发生早期各阶段蛋白质2D图谱获取及蛋白点数目的变化。龙眼体胚发生早期能检测到的平均蛋白点数经历了一个先大幅减少后大幅度增加又大幅度减少的过程。其中以第4阶段CpECGE最多,有1798个蛋白点;第2阶段ECII最少,为1203个;其余各阶段蛋白点数分别是FEC阶段为1461个,ICpEC为1582个,GE阶段为1499个。表明在龙眼体胚发生早期,表达的蛋白经历了一个急剧变化的过程,可能与龙眼体胚发生早期相关基因的特异表达有关。③龙眼体胚发生早期各阶段蛋白质等电点(pI)和分子量(MW)变化。龙眼体胚发生早期的蛋白pI值主要集中在5～7之间,且后面3个阶段的蛋白点数一般都比前面2个阶段来得多,但不论哪个范围pI值蛋白点数最高时一般是在CpECGE阶段,ECII阶段除pI值6～7的蛋白点比FEC多外,其它范围都是各阶段最低的,GE阶段蛋白点数都呈下降趋势;不同分子量范围的蛋白点数变化情况显示,ECII阶段除了分子量20.1 kD到30 kD之间的蛋白点数比FEC阶段多外,其它分子量范围的蛋白点数都是整个早期过程最低的,而CpECGE阶段的蛋白点数目除MW≥66 kD外,在其它分子量范围的蛋白点数都是各个阶段中最多的。这表明在龙眼体胚发生早期ECII阶段和CpECGE阶段可能是体胚早期形态建成的2个关键阶段。而GE阶段除了分子量大于66 KD的蛋白质点数比CpECGE阶段略有增加外,其它分子量范围或pI范围的蛋白点数目都呈现一个下降的趋势,可能与龙眼早期体胚发育到GE阶段前期的许多体胚发生相关基因表达关闭有关。④龙眼体胚发生早期各阶段部分差异表达蛋白分析。选取了体胚发生早期表达具有差异性的148个蛋白点进行分析。结果表明,这些蛋白质中有10个蛋白点009、044、047、060、075、090、093、094、138、148是阶段性特异表达的,蛋白点009只出现在CpECGE和GE阶段,044在GE不表达,047在ICpEC阶段不表达,060只在FEC、ICpEC和CpECGE表达,075只在ECII阶段表达,090、093、138在ECII阶段不表达,094只在ICpEC和CpECGE中表达,148在FEC、CpECGE和GE阶段表达。但大部分的蛋白是阶段差异性表达的,蛋白表达有自己的变化规律,其中较有规律的是002、010、024、043和078蛋白随龙眼体胚发育的进行被下调,008、028、030、35、36、120蛋白被上调,032、033、038、039、050、055、071、089、091、119和121在FEC中表达量大,ECII表达量下降,而后随体胚发育逐渐升高,到GE阶段最高,其它的蛋白点在龙眼体胚发生早期过程中呈不规律变化。这种蛋白质差异表达变化表明,在龙眼体胚发生早期过程中,一个庞大的蛋白质群体协同作用,推动龙眼体胚向前发育。3龙眼体胚发生早期各阶段部分差异表达蛋白的质谱鉴定和相关蛋白的功能选取龙眼体胚发生早期差异表达的118个蛋白进行质谱鉴定,其中有45个蛋白点得到可信的匹配,蛋白质鉴定的成功率约为37%。按其功能可以大致可分成8类,其中蛋白是和能量与糖代谢有关的蛋白有10个,占22%;信号转导和细胞凋亡有关的蛋白有3个,占7%;与调控相关的蛋白有5个,占11%;与胁迫反应和防御相关的蛋白有12个,占27%;细胞结构蛋白有2个,占4%;与蛋白质合成、折叠、组装和分解相关的蛋白有3个,占7%;氨基酸代谢相关蛋白有1个,占2%;功能未知蛋白有9个,占20%。在成功鉴定的45个蛋白中,有4个点被鉴定为烯醇化酶(enolase),点067和068被鉴定为同一个蛋白(桤木烯醇化酶),069和070也被鉴定为同一个蛋白(大豆烯醇化酶),它们分布在胶上的不同位置,其中067、068、069分子量相近,但是等电点却不一样,而点070与其它3点差距较大,这很可能它们是同一个蛋白的不同形式,存在不同的翻译后修饰;133和139蛋白点被鉴定为磷酸丙糖异构酶,它们也在2D图谱上不同位置。以上情况表明在龙眼体胚发生早期基础能量代谢很旺盛,是龙眼体胚发生发育的基础,但它们有的表达量在龙眼体胚发生早期过程中变化比较大,可能这些基础代谢的蛋白还有其它功能。点041、066和100分别为ATP合酶的不同亚基,其中041和066是定位在线粒体的,而100是液泡型的,这说明在龙眼体胚发生过程中,能量代谢很旺盛。点111、112、113、114、116、117被鉴定为同一蛋白(烟草过氧化物酶),点115被鉴定为亚麻过氧化物酶,从2D胶上可以看出他们在胶上的不同位置,有的分子量相近也有等电点相近的,可能它们是同一个蛋白的不同形式,翻译后不同修饰。在龙眼体胚发生早期成功鉴定的蛋白中,与氧化胁迫反应相关的蛋白比例最多,表明在龙眼体胚发育的过程中,氧化胁迫是一个普遍的生理现象,在龙眼体胚发生的触发和体胚的发育中有重要功能。从成功鉴定但功能未知的蛋白来看,它们的分子量都在30 kD以下,说明是一些小分子量的蛋白,可能是调控体胚发育顺利进行的一些关键的调控蛋白。4龙眼体胚发生早期部分相关蛋白基因克隆及其在体胚发生过程中的表达分析①克隆了龙眼胚性愈伤组织线粒体ATP合酶β亚基基因cDNA序列,该序列全长2099 bp,包含1个由1677 bp核苷酸组成的ORF,编码一558个氨基酸的蛋白。龙眼线粒体ATP合酶β亚基基因转录水平随龙眼体胚的发育呈动态变化,其中以球形胚阶段的转录水平最高,但该基因在体胚各阶段中蛋白表达水平却较恒定,这种情况说明从胚性愈伤组织到球形胚阶段其mRNA转录水平的提高,有利于保持ATP合酶的稳定,从而为复杂的体胚形态建成提供充足的能量。②克隆了龙眼胚性愈伤组织2个类型的磷酸丙糖异构酶基因(TPI)。TPI基因类型I基因cDNA的全长序列,为1084 bp,其最大开放框从第73个碱基开始,到837个碱基终止,共765个碱基编码254个氨基酸。TPI基因类型II基因cDNA的全长序列,为1113 bp,其最大开放框从第157个碱基开始,到921个碱基终止,共765个碱基编码254个氨基酸。在2个类型TPI基因的保守区域设计引物进行实时荧光定量PCR分析,结果表明龙眼TPI基因在FEC阶段的转录水平比较低,但到了ECII阶段转录水平急剧升高,这种情况一直持续到胚性紧实球形结构阶段,到球形胚阶段转录水平大幅下调,之后到子叶胚阶段都维持在一个较低水平。表明TPI在龙眼体胚发生过程中除维持基础代谢外,有其它的生物学功能。③分离了龙眼胚性培养物DlUP-3基因,cDNA的全长序列有1681 bp,其最大开放框从第391个碱基开始,到1407个碱基终止,共1017个碱基编码338个氨基酸。该基因与其它植物的同类未知蛋白或蓖麻推定的GTP -binding protein在核苷酸序列和推导的氨基酸序列方面均有着比较高的相似性。该在龙眼松散胚性愈伤组织阶段转录水平极低,而在球形胚阶段异常高,推测该基因是龙眼体胚特异表达的基因,并且是在体胚发生早期阶段特异表达的,它在龙眼球形胚的形成过程中扮演重要的角色或是决定性的因素。④分离了龙眼胚性培养物DlUP-4基因,cDNA的全长序列为1061 bp,其最大开放框从第112个碱基开始,到846个碱基终止,共735个碱基编码244个氨基酸。该基因与其它植物的同类未知蛋白或拟南芥的60S ribosomal protein L7在核苷酸序列和推导的氨基酸序列方面均有着比较高的相似性。从龙眼体胚DlUP-4基因mRNA转录水平变化可知其在龙眼体胚发生过程中具有阶段表达的特点,可能参与部分体胚特异表达蛋白质的合成。⑤分离了龙眼胚性培养物DlUP-5基因,cDNA的全长序列为887 bp,其最大开放框从第7个碱基开始,到687个碱基终止,共681个碱基编码226个氨基酸。该基因与其它植物的同类未知蛋白或蓖麻推定的Frigida在核苷酸序列和推导的氨基酸序列方面均有着比较高的相似性。从龙眼体胚DlUP-5基因mRNA转录水平变化,推测其在龙眼愈伤组织胚胎发生能力维持方面可能起到关键作用,并且其在龙眼体胚发生的早期和体胚成熟期都有其特殊的功能。⑥2个龙眼胚性培养物未知功能蛋白基因的克隆,DlUP-6基因和DlUP-7基因是根据质谱鉴定成功且在其它植物上也为未知功能蛋白的基因,目前已经得到保守区和3’部分cDNA序列,与质谱肽段比较后,初步确定其为质谱鉴定成功的、为2D凝胶上显示的未知功能蛋白。更多还原

【Abstract】 In this experiment, five early-different-stage embryogenic culutures obtained after synchronization regulation from the embryogenic callus (EC) were used as the materials for the following studies in longan (Dimocarpus longan Lour. cv. Honghezi):①establishment of the high-resolution two-dimensional electrophoresis technology system for analysis of the proteins from early-different-stage embryogenic cultures in longan;②separation and preparation of proteins from 5 early-different-stage embryogenetic culutures by two-dimensional electrophoresis, and the analysis of protein differential expression using the soft of ImageMaster 2D Platinum 6.0( GE Healthcare;③identification of parts of differential expression proteins from 5 early-different-stage embryogenetic culutures by mass spectrometry and analyses of their functions;④cloning of parts of early embryogenic cultures related protein genes with homologous cloning or degenerate primers according to the protein identified results, and detection of transcriptional expressions of these genes during somatic embryogenesis of longan by real-time fluorescence quantified PCR (qRT-PCR). The main results were summarized as follows:1. Establishment of high-resolution two-dimensional electrophoresis technology for the analysis of the proteins from early embryogenic cultures in longanThe synchronization of embryogenic cultures was carried out by regulating the concentration of 2, 4-D on the medium and microscopic observation as reported previously. Five early-different-stage embryogenetic culutures of longan were obtained including the friable-embryogenic callus (FEC), embryogenic callus II (ECII), incomplete compact pro-embryogenic cultures (ICpEC), compact pro-embryogenic cultures globular embryos (CpECGE) and globular embryos (GE). Immobilized pH gradients (IPG) two-dimensional electrophoresis was developed to investigate the separation of proteins from early embryogenic cultures of longan. The results indicated that the isoelectric points (pI) of proteins mainly focused on the pH value between 3.5 and 7.0. The better result was obtained under the condition of the pH value between 4 and 7 using same length IPG strips. The resolution was improved by the IPG two-dimensional electrophoresis. The numbers of protein spots separated using IPG two-dimensional electrophoresis with 13 cm strips were two and a half times of that using traditional two-dimensional electrophoresis, and many low abundance proteins could be detected. The resolution of proteins could be further improved with increasing of strip lengths. The numbers of protein spots detected in 18 cm gels strips was about two times of that in 13 cm gels strips. But a perfect 2-D map could be obtained by adjusting of isoelectric focusing parameters and with high quality gels of SDS-PAGE.2. 2-DE analysis of embryogenic cultures at early different stages in longan①The variation of soluble protein amounts of embryogenic cultures at early different stages during longan somatic embryogenesis. The soluble protein contents of embryogenic cultures at early different stages during longan somatic embryogenesis were detected by method of Coomassie brilliant blue G-250. The results showed that the soluble protein contents increased with the initiation of longan somatic embryogenesis. They were low in FEC stage and reached highest in ECII stage, then decreased slightly and remained at a higher level than that of FEC at subsequential stages. The soluble protein contents at FEC stage were extremely significant (P<0.01) lower than that of other stages, but there was little differences at other stages. These results suggested that the variation of soluble protein contents of different embryogenic cultures at early stages during longan somatic embryogenesis be related to development of somatic embryos.②Obtaining of 2-DE gels of the total proteins of five early-different-stage embryogenic cultures in longan and changes of the number of their total proteins. The average numbers of total protein spots decreased sharply at first and then increased greatly, subsequently decreased abruptly again. The spots of total protein were maximum at CpECGE stage with 1798 spots, and it reduced to a minimum with 1203 spots at ECII stage. Total protein numbers at other stages were 1461 (FEC), 1582 (ICpEC) and 1499 (GE) spots. It was concluded that the sharp fluctuation of total protein numbers of different embryogenic cultures may be related to specific expression of genes at early stage during somatic embyogenesis in longan.③Changes of isoelectric point (pI) and molecular weight (MW) of total proteins in different embryogenic cultures at early stage during longan somatic embryogenesis. The results showed that pI of most proteins ranged from 5 to 7. The average numbers of these proteins was more at last three stages than that at the first two stages. And the numbers reached the highest at CpECGE stage under any range of pH values. ECII stage had lowest numbers of proteins than other stages expect under the pI value with 6 to 7. Average number of proteins at ECII stage was lowest than that of other stages, but it had more proteins than FEC stage in the range of MW from 20.1 kD and 30 kD. The numbers of proteins with other MW at this stage were lower than that of other stages. Number of proteins with different MW at CpECG stage was more than that of other stages, except that proteins with MW larger than 66 kD. This implicated that ECII and CpECGE stages were critical for morphogenesis. Number of proteins with different MW decreased at GE stage, except that proteins with MW larger than 66 kD, which increased slightly at this stage. These may be related to shut down the expression of some genes involved in somatic embryogenesis, which was only obligatory at early stages.④Analysis of differentially expressed proteins in different embryogenic cultures at early stage during longan somatic embryogenesis. 148 differentially expressed proteins were selected for followed analysis. The results showed that 10 proteins (spots of 009, 044, 047, 060, 075, 090, 093, 094, 138 and 148) were specific expressed at different stages. Spot of 009 only expressed at CpECGE stage and GE stage. Spot of 044 was absent at GE stage, spot of 047 was absent at ICpEC stage, spot of 060 only expressed at FEC stage, ICpEC stage and CpECGE stage. Spot of 075 only expressed at ECII but spots of 090, 093 and 138 were absent at this stage. Spot of 094 only expressed at ICpEC stage and CpECGE stage. Spot of 148 expressed at FEC stage, CpECGE stage and GE stage. Most of the proteins were differentially expressed in rules, spots of 002, 010, 024, 043 and 078 were down regulated with early development of somatic embryo. Spots of 008, 028, 030, 35, 36 and 120 were opposite. Expression level of spots of 032, 033, 038, 039, 050, 055, 071, 089, 091, 119 and 121 were higher at FEC stage. They decreased at EC II stage, then increased with somatic embryo growth and reached the highest at GE stage. These suggested that the development of somatic embryo during the early stage be modulated by large sets of proteins.3. Identification of some differential expression proteins by mass spectrometry and analysis of their functions118 proteins were subjected to mass spectrometry (MALDI-TOF/TOF) analysis. 45 (37%) of them were identified, and they were classified to eight functional classes, which including 10 proteins involved in energy and glucose metabolism (22%), 3 proteins involved in signal transduction and programmed cell death (7%), 5 proteins involved in regulation (11%), 12 proteins involved in stress response and defense (27%), 2 proteins involved in cell structural (4%),3 proteins involved in protein synthesis and processing (7%), 1 proteins involved in amino acid metabolism (2%), and 9 unknown proteins (20%).Among of the identified proteins, 4 proteins were identified as enolases. Spots of 067 and 068 were identified as alnus enlases and spots of 069 and 070 were identified as soybean enlase. But they were located on the different sites in the 2D gels. Spots of 067, 068 and 069 were almost the same in molecular weight but different with pI. Spot of 70 was different from others. These proteins may be the different forms of the same protein which had different post translated modifications. Spots of 133 and 139 were identified as triosephosphate isomerase. These results suggested basal metabolism was activity at early stages of longan somatic embryogenesis, which was the basis of embryo development, but their expression levels were different which implicated that they may have other functions. Spots of 41, 66 and 100 were subunits of ATP synthase, Spots of 41 and 66 loclalized in mitochondria, and spot of 100 was subunit of vacuolar ATP synthase. These suggested that energy metabolism was activity during somatic embryogenesis. Spots of 111, 112, 113, 114, 116 and 117 were identified as tobacco peroxidase, spot of 115 was fiberflax peroxidase. Some of them had the same MW and some of them had the same pI. They may be different post translated modification products of peroxidase. Most of the identified proteins were related to oxidative stress response, which indicated oxidative stress is general during early somatic embryogenesis. They might play an important role in the activation and developments of longan somatic embryos. MW of most unknown proteins was lower than 30 kD, which were critical in regulation of somatic embryogenesis.4. Cloning of several embryogenic cultures associated protein genes at early stage during somatic embryogenesis of longan①The full-length cDNA sequence of longan embryogenic callus mitochondrial F1-ATPase beta subunit gene was 2099 bp. It contained a 1677-nucleotides-long open reading frame (ORF) which encoded protein of 588 amino acid residues. Transcription level of mitochondrial ATP synthaseβsubunit changed with development of somatic embryogenesis in longan and reached highest at GE stage, but its protein expression level remained constantly. The results showed the increase of its transcription level was good for stabilization of this protein, and could supply sufficient energy to the morphogenesis forming.②Two longan embryogenic callus triosephosphate isomerase (TPI) genes were cloned. The cDNA full-length of TPI I was 1084 bp and its ORF was 765 bp which started from the 73rd base and ended in the 837th base. It encoded 254 amino acids. The cDNA full-length of TPI II was 1113 bp and its ORF was 765 bp which started from the 157th base and ended in the 921st base. It encoded 254 amino acid residues. A pair of primers were designed according to the conserved sequence of these two TPI genes and used for real-time fluorescence quota PCR analysis. The results indicated their mRNA transcription level was low at FEC stage, then increased sharply at ECII stage and remained stable until CpECGE stage. The level decreased dramatically at GE stage and remained at a lower level from GE stage to cotyledonary stage. These implicated they had other function during somatic embryogenesis in addition to basal metabolism.③DlUP-3 gene was cloned and its cDNA full-length was 1681 bp. Its ORF was 1017 bp which started from the 391st base and ended in the 1407th base and encoded 338 amino acids. Its sequence was highly similar to that of castor-oil plant putative GTP-binding protein. Its transcript level was low at FEC stage and over expressed at GE stage. It might be a specific protein, which played an important role during GE stage forming in longan.④DlUP-4 gene was cloned which cDNA full-length was 1061 bp. Its ORF was 735 bp which started from the 1121st base and ended in the 846th base and encoded 244 amino acids. Its sequence was high similar to other plants and 60S ribosomal protein L7 of Arabidopsis thaliana. It specifically expressed during somatic embryogenesis in longan, and might involved in synthesis of somatic embryos related specific proteins.⑤DlUP-5 gene was cloned which cDNA full-length was 887 bp. Its ORF was 681 bp which started from the 7th base and ended in the 687th base and encoded 226 amino acids. Its sequence was highly similar to that of castor-oil plant putative Frigida. According to its variations of mRNA transcription levels, It might play a critical role in maintaining ability of somatic embryogenesis and a particular role in early somatic embryogenesis and maturation of somatic embryos.⑥Cloning of two unknown protein genes related to early somatic embryogenesis in longan. Conserved regions and 3 ’end cDNA sequences of DlUP-6 and DlUP-7 were obtained. DlUP-6 gene and DlUP-7 gene were the two unknown protein genes in other plants. Furthermore, compared with the peptide sequences information from mass spectrometry, it was primarily proved that the two conserved regions and the two 3 ’end cDNA sequences were the unknown proteins in the 2D maps.更多还原