【作者】 王保明；

【导师】 谭晓风；

【作者基本信息】 中南林业科技大学 ， 森林培育， 2012， 博士

【摘要】 油茶(Camellia oleifera Abel)是中国原产且最为重要的木本食用油料树种。提高油茶种子含油率是今后很长一段时间油茶育种的重大目标和技术难关,而且通过常规育种技术难以实现。为了实现这个宏大目标,加速油茶品种改良进程,一个可行的方法就是关注参与脂肪酸和油脂合成的关键酶,克隆控制油茶种子油含量的关键基因,并通过分子育种的方法对现有品种实施遗传改良,达到提高油茶种子油含量的目的。异质型乙酰辅酶A羧化酶是各种脂肪酸从头合成和油脂合成的限速酶和关键酶,它由生物素羧化酶(BC)、生物素羧基载体蛋白(BCCP)、α－羧基转移酶(α-CT)和β－羧基转移酶(β-CT)4个亚基组成。本研究以‘华硕’、‘华金’、‘华鑫’、‘衡东号31’、‘衡东65’和‘衡东17号’等6个品种以及‘华硕’的嫩叶、茎段、花芽、雄蕊、雌蕊、子房、4月份幼果、7月份幼果以及成熟种子为材料,从油茶中克隆了ACCase4个亚基的cDNA序列和基因组序列,通过多重RT-PCR与实时荧光定量PCR研究了它们在油茶种子不同发育阶段及不同组织器官中的表达规律,并在拟南芥中研究它们的过量表达和RNA干扰对油脂合成的影响。本研究的主要结果如下：1.油茶ACCase4个亚基基因的cDNA克隆。以‘华硕’成熟种子总RNA反转录的cDNA第一链为模板,采用简并RT-PCR、5’和3’RACE等技术克隆了油茶ACCase4个亚基的cDNA,分别命名为Co-accB(编码BCCP)、Co-accC(编码BC)、Co-accA(编码α-CT)和Co-accD(编码β-CT)。(1)油茶ACCase亚基基因accB的全长cDNA为1153bP,编码272个氨基酸。并具有前体肽以及与其它三个亚基结合形成ACCase复合体的成熟肽,其前体肽的剪切位点可能位于Ser-64与Ala-65之间；它含有生物素(酰)化模块CIIEAMNEE及生物素化位点Lys。(2)油茶BC亚基基因accC的全长cDNA大小为1638bP,含有1602bP的开放读码框,编码533个氨基酸。它具有前体肽以及与其它三个亚基结合形成ACCase的成熟肽。其前体肽剪切位点可能位于Arg46-Va147之间。并具有BC-1、ATP结合位点、BC-2以及生物素酰化位点四个亚结构域。(3)油茶ACCase亚基基因accA的部分cDNA序列大小为1293bP,编码430个氨基酸。它具有保守的氨基酸序列,并含有乙酰辅酶A结合域。(4)在克隆ACCaseβ-CT亚基基因accD时,获得了一个长度为2574bP的序列,该序列包括1,5－二磷酸核酮糖羧化酶／加氧酶大亚基基因rbcL (ribulose1,5-bisphosphate carboxylase/oxygenase large subunit)(部分序列)、rbcL-accD以及accD(全长cDNA序列)三部分。其中,accD的ORF为1530bP,编码510个氨基酸。它含有一个锌指结构和五个保守序列。在其ORF推导的氨基酸序列中含有乙酰辅酶A、羧基生物素结合位点以及位于它们之后的羧基转移酶催化位点等结构域。rbcL、rbcL-accD基因间隔及accD的核苷酸序列与二十多种山茶属植物对应叶绿体基因的核苷酸序列具有高度一致性,这反映了它们具有较近的亲缘关系。油茶ACCase这4个亚基基因推导的氨基酸序列与棉花(Gossypium hirsutum)、花生(Arachis hypogaea)和大豆(Glycine max)等油料植物以及大肠杆菌ACCase对应亚基基因的氨基酸序列具有较高的一致性,属于异质型ACCase基因；它们具有完成ACCase羧化乙酰CoA两个半反应以及亚基间相互结合的结构域；核编码基因accA、accB和accC前体肽剪切后的成熟肽与位于质体的p-CT亚基结合,最终形成具有ACCase活性的多亚基异质型复合体。2.油茶ACCase BC和p-CT亚基基因基因组DNA的克隆。以油茶‘华硕’叶片总DNA为模板,根据accC和accD的cDNA序列分别设计引物,采用降落PCR技术,获得了包括它们基因编码区(分别为1602bP和1533bp)在内的基因组序列。其中,accC的基因组序列长度为1638bp；rbcL、rbcL-accD以及accD的基因组序列大小为2222bp；与它们的对应cDNA序列比较后确认这两个基因均没有内含子。accD上游启动区为约529bP,没有PEP启动子位点,含有NEP型启动子元件,其5’端UTR部分含有-10启动子元件、CAAT BOX1、YACT、napA E-box、 GATA box和DOFCOREZM等位点,并可能与psal,ycf4、cemA和petA等形成操纵子,在NEP型启动子控制下以多顺反子形式转录。在油茶基因组中存在于叶绿体或质体的p-CT亚基基因和存在于核基因组中的BC亚基基因可能作为‘小基因组’,在基因组进化过程中内含子逐渐被淘汰。所获得的accD基因组序列与cDNA序列存在着核苷酸差异,这可能与accD基因家族有关,也可能与基因组序列转录过程的RNA编辑现象有关。获得油茶ACCase两个BC和β-CT亚基基因组序列为油脂合成及油脂含量影响等研究提供了基因材料和理论依据。3.油茶ACCase4个亚基基因转录水平上的表达模式研究。以‘华硕’等6个油茶优良品种的成熟种子以及‘华硕’嫩叶等不同组织器官和不同发育阶段的种子为材料,分别以它们的cDNA第一链为模板,从GAPDH、Actin1和UBC30等参照基因中选取GAPDH做为参照基因；在优化GAPDH与分别油茶ACCase4个亚基基因accA、accB、accC和accD同管扩增最佳反应体系和条件的基础上,研究了油茶ACCase4个亚基基因的表达模式,荧光定量PCR进一步证实和补充了实验结果。(1)油茶accB基因在‘华硕’等6个不同含油率种子中的表达差异不十分明显；荧光定量PCR揭示油茶accB基因在‘衡东17号’、‘衡东65号’和‘华硕’转录水平较高。油茶accB基因在油茶‘华硕’雌蕊和成熟种子中的转录水平较高。(2)油茶accC基因在‘华硕’等6个不同含油率种子中的转录水平差异较为明显,在‘衡东17号’和‘衡东65’中较多。它的转录水平与各品种含油率之间的相关性不十分明显。(3)油茶accA基因在油茶‘华金’表达量较多,在‘衡东31号’、‘衡东17号’和‘华鑫’种子中转录水平相似。其表达量与各品种含油率之间的相关性不明显。它在油茶‘华硕’成熟种子中的表达量最多,而在其它组织及种子发育过程中表达量较少。(4)油茶accD基因在‘华硕’等等不同含油率种子中转录存在着较大差异,在‘华鑫’中表达量最大。它的转录水平与部分油茶品种含油率之间有一定的相关性。它在油茶‘华硕’的叶片表达量和发育的种子中转录水平较高。在油茶种子发育过程中,这4个ACCase基因转录量增多的趋势符合种子由花期到成熟,油脂合成量由小到多而进入高峰期,对脂肪酸需求逐渐增多；种子成熟后,油脂合成逐渐完成,对脂肪酸需求下降的要求。表明这4个ACCase基因转录与脂肪酸形成关系密切。我们的研究结果支持‘异质型ACCase的表达量对其活性和种子含油量至关重要,是种子含油率高低标志这个结论’以及’ACCase在决定油料作物含油率具有重要作用’这个假说。油茶异质型ACCase各亚基基因协调表达,在开花前(后)和种子成熟前后出现了两个转录高峰,只是前者不明显,后者却十分明显。另外,这4个基因在油茶组织器官中呈现出相似的转录水平,这与Ke等的结论相一致。本研究为我们从转录水平检测油茶ACCase亚基基因表达提供一种直观的方法。4. ACCase亚基基因accC的过量表达和accA/accB/accC/accD的RNA干扰研究。构建了pC AMBIA1304-35S-accC植物表达载体,农杆菌介导转入哥伦比亚野生型拟南芥中,获得accC过量表达的转拟南芥T1和T2代抗性植株。利用Gateway技术,分别构建了异质型ACCase多亚基基因的干扰载体pJawohRNAi18-accA/accB/accC/accD,农杆菌介导转入哥伦比亚野生型拟南芥中分别了获得它们的T1代转拟南芥抗性植株。这为今后研究异质型ACCase基因在种子特异启动子作用下分别与4个亚基的两两组合融合、1个和3个组合融合或4个亚基融合构建ACCase过量表达载体,然后转到植物植株中检测它们的表达效果等研究提供理论准备和物质基础。综上所述,本研究获得油茶ACCase4个亚基基因的cDNA序列和2个亚基基因组序列,分析了油茶ACCase4个亚基基因的结构特点、表达模式以及它们在脂肪酸和油脂代谢途径中的作用,为油茶品种评价和丰产栽培措施的调控提供了一种直观的检测方法。这将对今后提高油茶种子含油率,增加油茶单位面积产量和总产量以及油茶一些丰产栽培措施的应用提供理论依据,因而具有重要的理论价值和重大的应用价值。更多还原

【Abstract】 Tea-oil tree(Camellia oleifera Abel), one of most important ligneous edible trees, is originated in China. To improve the seed oil rate is increasingly becoming the significant objective of C. oleifera breeding. To boost the seed oil rate is a long-term objective and technical difficulty. Moreover, it is difficult to reach the aim though traditional breeding. In order to realize the great goal, and to accelerated the C. oleifera improvement as well, one possible method is to look up a key enzyme involved in lipid biosynthesis (fatty acid biosynthesis), and to carry out the molecular biology research of C. oleifera seed’s fatty acid biosynthesis so as to discover the fundamentals of lipid biosynthesis. The heteromeric form acetyl coenzyme A (CoA) carboxylase (ACCase) in plant organs of seeds catalyzes the formation of malonyl CoA from acetyl CoA, and is a rate-limiting step and a key enzyme in de novo fatty acid biosynthesis. The four components that constitute heteromeric ACCase are biotin carboxyl carrier protein (BCCP), biotin carboxylase (BC), the α-and β-subunits of carboxyltransferase (α-and β-CT), encoded by accC, accB, accA and accD genes, respectively. In this research, the four cDNAs and genomic DNAs sequences of C oleifera ACCase were cloned. Then, their expression patterns were analyzed in’Huashuo’ and other five C. oleifera species, as well as in different tissues and developing stages of ’Huashuo’ seeds. Finnally, studies of overexpression and RNA interference of them were carried out in Arabidopsis thaliana, respectively. Major research results are as follows:1. Isolation and cloning of the four cDNAs of C. oleifera ACCase subunits. Using the single-stranded cDNA generated from total extracted RNA of’Huashuo’seeds as template, the cDNAs of four ACCase subunits were cloned by the degenerate PCR, rapid amplification of cDNA ends (RACE) and touch-down PCR, and designated as Co-accB, Co-accC, Co-accD and Co-accA, respectively.(1) The Co-accB (encoded BCCP) was1153bp with an816-bp open reading frame (ORF), encoded272amino acid residues; it had the precursor and mature protein, and its plastid-processing site for C. oleifera BCCP occurs between residues Ser64-Ala65. It contained the biotinylation motif CIIEAMKLMNEIE harboring the biotinyl-Lys residue.(2) The Co-accC (encoded BC) was1638bp with a1602-bp ORF, encoded533amino acid residues. The subcellular localization of the Co-accC protein was in plastid, and the plastid-processing site for C. oleifera BC was postulated to occur between residues Arg46and Val47. It comprised four conserved motifs BC-1, ATP-binding site, BC-2and Biotin carbxylation site.(3) The partial Co-accA (encoded a-CT) was1293bp, and its amino acid residues155-190are acetyl-CoA binding domains.(4) When cloning the accD encoded β-CTsubunit of C. oleifera ACCase, a2574- bp fragment including the partial rbcL, rbcL-accD intergenic spacer and the full-length accD gene was obtained. The ORF of accD was1530bp, encoding510amino acids. It contained a zinc-binding domain and five conserved regions. The putative binding sites for Acetyl-CoA and carboxybiotin were located at amino acid residues332-346and351-368, respectively, and followed by the putative catalytic site of carboxyl-transferase at amino acid residues379-392. The nucleotide sequences of rbcL, rbcL-accD and accD shared high identity with those of rbcL, rbcL-accD and accD from other20Theacae genera species, indicating that there existed the close genetic relationship among them. The amino acid sequence of four genes for C. oleifra ACCase subunits shared some identities with thoese of the corresponding genes for ACCase subunits from G. hirsutum, A. hypogaea and G. max of oil plants, as well as from E. coli, suggesting that they belonged to the heteromeric ACCase genes; moreover, they possess the binding sites of the two-half reactions and structure domains of interaction among ACCase subunits. Their precursors of three nuclear-encoded genes aceA, accB and accC are processed and binded (3-CT to form the heteromeric ACCase with activity after the plastid processing.2. Isolation and cloning of the genomic DNA sequences of BC and β-CT subunits of the C. oleifera ACCase. According to the corresponding cDNA sequence of accC, the primers were designed to clone the genomic DNA sequences using the total DNA of ’Huashuo’ leaves by the touch-down PCR. The1638-bp genomic DNA for accC was obtained, including the1602-bp ORF encoden533aa. Furthermore, the2222-bp genomic DNA sequence including rbcL, rbcL-accD intergenic and accD was isolated, and it contained the1530-bp ORF of accD. Compared with their cDNAs, the genomic DNA sequences of accC and accD were verified to be intronless. The up-stream region of the2222-bp sequence was about529bp in the length. It contained no PEP prometer sites, while had NEP prometer sites,-10PEHVPSBD, CAAT BOX1, YACT and napA E-box, etc. Genomic DNAs of BC (encoded by nuclear) and β-CT (encoded by plasid) in C. oleifera are probably considered as’small genomics’, and their introns might be gradually loss. There are some differences between accD genomic DNA and cDNA, the reason might be corelated with gene family, RNA editing as well. The acquired genomic DNA sequences of accC and accD could provide the materials and theoretical basis for lipid biosynthesis and seed-oil ratio research.3. The studies of the expression pattern of the four genes for C. oleifera ACCase subunits. Total RNAs were extracted from C. oleifera matured seeds of’Huashuo’and other five C. oleifera species, and different tissues and developing stages of seeds of ’Huashuo’, and used to generate the corresponding single-stranded cDNAs. Then, the reference gene GAPDH were selected from references genes of GAPDH, Actinl and UBC30, et al. According to optimized multiplex RT-PCR conditions, the expression patterns of the four ACCase genes were analyzed, and the reliability of this data was confirmed by real-time PCR analysis.(1) The expression level of accB was the highest in the pistil of ’Huashuo’, next is in the8-month-old young fruits. It had a similar expression patterns in tissues of flower bud, stamen, ovary and leaf. There were no distinct differences in six varieties of C. oleifera matured seeds. There were the high expression levels in ’Hengdong17’and in’Hengdong65’.(2) The expression level of accC was higher in the8-month-old young fruits and in flower buds of’Huashuo’; there were relatively lower levels in tissues of stamen, pistil, ovary and leaf. There were distinct expression differences in the six C. oleifera matured seeds. There were high expression levels in’Hengdong17’and ’Huaxin’, followed by in ’Hengdong31’,’Huajin’,’Huashuo’ and ’Hengdong65’.(3) The expression amount of accA was higher in’Hengdong17’and in ’Huaxin’. Moreover, its expression amount was more in young fruits and matured seeds, while less in leaves and stems.(4) There were distinct expression differences in six C. oleifera matured seeds. There was the highest expression amount in’Huaxin’. Its transcription levels are parti call y corelated with seed-oil ratio of some C. oleifera species. The expression levels of accD were higher in leaves and developing seeds. Taken together, the four genes of heteromeric ACCase are coordinately expressed, and there exsited trends that transcription levels of the four genes are gradually increased, which are in agreement with requirement of the lipid biosynthesis in the development from flowering stage to mature in the growth and development of seeds, and with that of Ke’s research. Their expressions also present two transcription peaks. The former are not abvious, while the later are relative distinct. These suggest that there are close relationships between the transcription levels of the four ACCase genes lipid biosynthesis, and thus C. oleifera ACCase may be the rate-limiting step and a key enzyme. Our results support the conclusion that the expressions of the four ACCase genes are significant to the activity and seed oil ratio, and that ACCase can be used as a marker in the breeding program. Furthermore, our research also provides a visual detection method of the four ACCase genes of C. oleifera from transcription levels.4. Overexpression of accC from C. oleifera and RNA interference (RNAi) expression of accAlaccBlaccClaccD. The overexpression pCambia1304-accC vector was constructed, and introduced into Agrobacterium tumefaciens by chemical poration, and transformed the wild Arabidopsis thaliana. Regenerated T1and T2Arabidopsis transformation lines were obtained, respectively. The interference vectors of pJawohl8-RNAi-Co-accA/Co-accB/Co-accC/Co-accD were constructed via Gateway technology, and introduced into A. tumefaciens, and transformed the wild A. thaliana. Regenerated T1Arabidopsis transformation lines of Co-accB/Co-accC/Co-accD RNAi were obtained, respectively. The rearches will provide the material and theoretical basis basis for overexpression of C. oleifera ACCase genes.In summary, the sequences of four cDNAs genes and two genomic DNAs of C. oleifera ACCase subunits were obtained. Their structure features, expression patterns and their roles in fat acid biosynthesis and lipid biosynthesis were analyzed. It provided a visual detecting method for C. oleifera variety assessment as well as regulatory measures through high yield cultivating techniques. The research will lay the fundamental theoretical basis for improving seed-oil ratio and oil production of per unit area yield and total output, and for applying of some cultivation measures for C. oleifera high yield, and thus possess the significant potential values of exploitation and application.更多还原