【作者】 卢秉国；

【导师】 陈伟； 吕柳新；

【作者基本信息】 福建农林大学 ， 果树学， 2009， 博士

【摘要】 龙眼(Dimocarpus Longan Lour)属无患子科(Sapindaceae )龙眼属,是我国南方诸省较重要而有特色的一种热带亚热带木本果树。由于龙眼合子胚遗传背景复杂,童期长,阻碍对其胚胎发育相关基因的研究。本文以龙眼子叶胚为材料,建立cDNA-AFLP技术体系,分析龙眼胚胎发育相关的基因差异表达,为研究龙眼胚胎发育奠定基础。建立了RACE体系,克隆了看家基因3-磷酸甘油醛脱氢酶基因(GAPDH),并作为后续试验的内参;以这个RACE体系克隆了一系列与龙眼胚胎发育相关的基因,同时对这些基因在胚胎发育中的表达特性也进行了研究。1利用cDNA-AFLP技术分析龙眼子叶胚的发育分别以小量法和大量法提取‘红核子’龙眼谢花后35天子叶胚(早期子叶胚)和谢花后50天子叶胚(晚期子叶胚)的RNA,经过合成双链cDNA、酶切、加接头、预扩增和选择性扩增一系列步骤,建立适宜于龙眼子叶胚的cDNA-AFLP分析体系,得到了条带清晰、对应良好、信号强度基本一致、分布均匀的cDNA-AFLP指纹图谱,对41个差异片段回收克隆和序列分析,发现其中21个与已知功能基因具有高度同源性,这些基因的功能主要涉及侧生器官的离轴极性、糖酵解、能量代谢、离子转运、细胞壁伸长、细胞周期调控、RNA转录、RNA翻译和调控、蛋白磷酸化调控和降解、信号转导等;另外20个片段与已知基因的同源性较低或未发现同源性。随机挑选7个进行RACE扩增,RT-PCR检测其表达情况。2龙眼子叶胚GAPDH基因全长cDNA克隆及序列分析根据Super SMARTTM PCR cDNA Synthesis Kit特点,设计两端锚定引物,同时根据与已知的GAPDH基因高度同源的EST序列设计巢式引物,成功地扩增出该基因的3′端和5′端,说明这种RACE方法适合用于扩增龙眼子叶胚的基因。序列分析表明,该基因的cDNA全长为1395bp,包括一个长1008bp,编码336个氨基酸的开放阅读框,其核苷酸和氨基酸序列与其他物种的GAPDH基因具有高度同源性。GAPDH基因是组成型表达,可以作为内参基因。3龙眼子叶胚离轴极性基因YABBY2的克隆和序列分析采用巢式的RACE方法,获得与拟南芥YABBY2同源的基因,共获得三个全长YAB2-1,YAB2-2,YAB2-3和三个片段YAB2-4,YAB2-5,YAB2-6。生物信息学分析表明这几个基因有不同的3′端非编码区和5′端非编码区,有高度一致的开放阅读框,在YAB2-1,YAB2-2,YAB2-3的5′端非编码区发现疑似的IRES序列。半定量RT-PCR分析表明YAB2-3在早期子叶胚显著地表达,在晚期子叶胚中微量表达;YAB2-1和YAB2-2在早期和晚期胚都显著表达,但早期表达量大;YAB2-4是微量表达的基因,在晚期胚胎表达量较大。4龙眼胚胎FVE、Remorin、CCR4-NOT等基因克隆和序列分析采用巢式的RACE方法,成功地扩增龙眼的FVE、Remorin、CCR4-NOT的全长序列,获得硫转运蛋白基因的5′端序列、脱水应答蛋白相关基因3′端序列和几丁质酶基因3′端序列。FVE基因具有WD40superfamily结构域,表达量极其显著,早期表达量较大;Remorin基因在早期胚中显著地表达,在晚期胚微弱地表达;CCR4-NOT基因在早期和晚期胚都显著表达,早期胚胎表达量较大;硫转运蛋白基因是微量表达的基因,在早期胚胎中表达量较大;几丁质酶基因也是微量表达的基因,在晚期胚胎表达量较大。脱水应答蛋白的相关基因在早期胚显著表达,但晚期胚微量表达。更多还原

【Abstract】 Longan (Dimocarpus Longan Lour), which belongs to Sapindaceae Lour, is one of the most important and characteristic tropical and subtropical woody fruit trees in those provinces located in South of China. Due to the complex genetic background of Longan zygotic embryos and long juvenile period, the research on genes related to embryonic development is hindered. In the present study, with the cotyledon embryos as plant materials, the cDNA-AFLP technology system was established and then used to investigate the differentially expressed genes concerning embryonic development of Longan, which aimed to lay the foundation for the research on Longan embryonic development. Secondly, the RACE system was established and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) in Longan was successfully isolated which could serve as a reference gene in the subsequent study. Moreover, with the RACE system established here, a series of genes relevant to Longan embryonic development were cloned and the expression profile of these genes during embryonic development was also demonstrated.1. Differentially expressed analysis in Longan cotyledon embryo by cDNA-AFLPThe‘honghezi’Longan (Dimdcarpus longan Lour.) cotyledon embryo, of which the flowers had faded and flied for more than thirty-five days (early cotyledon embryos) or fifty days (late cotyledon embryos) , was utilized as plant materials to isolate the RNA. Then, the corresponding RNA was used for double-stranded cDNA synthesis, enzyme digest, anchors ligation, pre-amplification and selective amplification. The result of polyacrylamide gel electrophoresis showed that, with strong signal and equal distribution, the amplification bands of cDNA-AFLP were clear and easy to discriminate. Thus, the cDNA-AFLP system suitable for the differential expression analysis was established successfully. Then, 41 differentially expressed fragments were recovered, cloned and sequenced. The corresponding sequences of 20 differentially expressed fragments were found to highly homologous with some cloned genes with known function, the function mainly involved in the abaxial polarity of lateral organ, glycolysis, energy metabolism, ion transport, cell wall elongation, cell cycle regulation, RNA transcription, RNA translation and regulation, protein phosphorylation regulation and protein degradation, signal transduction and so on; while the sequences of the remaining genes had low homologous with function-known genes or even had no homologous with any function-known genes. Finally, seven genes were selected for RACE amplification, and the expression of these genes was also detected with RT-PCR.2. Full-length cDNA sequence cloning and sequence analysis of GAPDH gene in Longan cotyledon embryo According to the features of Super SMARTTM PCR cDNA Synthesis Kit, the primers anchored at both ends were designed, and at the same time in accordance with the sequence of EST which shows highly homologous to cloned GAPDH gene, nested primers were also designed. Then, the 3 ’UTR and 5’ region of GAPDH gene in Longan were successfully amplified. The amplification indicated that the method is suitable for RACE amplification of gene in Longan cotyledon embryo.The result of sequence analysis showed that the full-length cDNA sequence of GAPDH gene in Longan was 1395 bp long, including a 1008 bp long open reading frame (ORF) encoding 336 amino acids. The nucleotide and amino acid sequence of this cloned GAPDH gene were found to be highly homologous with GAPDH genes in other plant species. Through RT-PCR analysis, the expression of this GAPDH gene cloned here was constitutively expressed and could be used as a reference gene.3. Cloning and sequence analysis of abaxial polarity gene YABBY2 in Longan cotyledon embryoUsing the method of nested RACE amplification, the abaxial polarity YABBY2 genes which shared high homology with Arabidopsis abaxial polarity gene YABBY2 were obtained. In total, there were three full-length cDNA sequences YAB2-1, YAB2-2 and YAB2-3 and three partial fragments YAB2-4, YAB2-5 and YAB2-6.Bioinformatics analysis indicated that these genes were highly homologous with each other in the corresponding ORF regions and had different kinds of 3 ’UTR and 5’ non-coding region. In the 5 ’end of YAB2-1, YAB2-2 and YAB2-3, the putative non-coding region of the IRES was found.Semi-quantitative RT-PCR analysis showed that YAB2-3 was significantly expressed in the early cotyledon embryo and only a small amount of expression value existed in the late cotyledon embryo; the expression of both YAB2-1 and YAB2-2 in both early and late cotyledon embryos were obviously; however, YAB2-4, with only trace amounts of expression, had a relatively more amounts of expression in later embryos.4. Cloning and sequence analysis of FVE, Remorin, CCR4-NOT genes in Longan cotyledon embryoThe nested RACE method was used and the full-length cDNA sequences of FVE, Remorin and CCR4-NOT genes were successfully obtained, plus the 5 ’sequence of sulfur transfer protein gene and the 3’ sequences of chitinase gene and dehydration-responsive protein-related gene. The expression of FVE genes was significant and especially high in the early phase; Remorin gene was expressed with a large amount in the early embryo and the expression of this gene in late embryonic expression was only faint; CCR4-NOT gene was highly expressed in both early late embryos and the expression in early embryo was relatively larger; Sulfur transfer protein gene, which was assumed a gene with tiny expression, was expressed at a relatively higher amount in the early embryo; The expression of chitinase gene was also tiny and the expression of this gene in late embryo was larger than that in early embryo; dehydration-responsive protein-related gene was found to be expressed significantly in the early embryo, but its expression in late embryo was only trace.更多还原