【作者】 杨学文；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2009， 博士

【摘要】 毛竹(Phyllostachys edulis)属于禾本科(Gramineae)竹亚科(Bambusoideae)刚竹属(Phyllostachys),是我国栽培面积最大、经济利用价值最高的竹种。毛竹含有丰富的纤维素,适合作造纸原料。然而,在毛竹制浆造纸工业中,由于原材料富含木质素,必须利用大量的化学品将原料中的木质素与纤维素分离,纤维素用于造纸,而分离的木质素形成造纸废液,造成严重的环境污染,而且脱木质素的化学药品投入及废液的碱回收处理大大增加了造纸成本。通过生物技术手段改变木质素含量或木质素单体组成,定向培育适合工农业生产所需的毛竹新品种,已成为当前毛竹生物技术领域中一项重要的研究课题。本项研究构建了毛竹不同器官全长cDNA文库,在大规模测序和数据分析的基础上,从cDNA文库中分离克隆了编码木质素单体合成途径中几个关键酶的基因,即香豆酸-3-羟基化酶(C3H)、肉桂酰辅酶A还原酶(CCR)、肉桂醇脱氢酶(CAD)的编码基因,以及阿魏酸5-羟基化酶类似基因(F5H-like),着重研究了C3H、F5H-like基因的表达模式,并通过转基因的方法初步研究了F5H-like基因的可能功能,得出以下结论。(1)从毛竹全长cDNA文库中分离得到了5个CCR基因。5个CCR蛋白具有表异构酶(Epimerase)结构域,属于依赖于NAD的表异构酶/脱水酶(NAD dependent epimerase/dehydratase)家族的氧化还原酶;毛竹5个CCR蛋白中有4个没有明显的信号肽和跨膜螺旋结构,推测定位在细胞质中;PheCCR5的N端具有类似信号肽的结构,亚细胞定位预测表明,它可能定位于线粒体中。采用最大似然法对CCR蛋白构建系统进化树,结果表明,CCR蛋白家族起源于真核生物分化之前,高等植物中CCR蛋白的多样性源自于种属特异的基因扩增和多样化事件。(2)从毛竹全长cDNA文库中分离得到了5个CAD基因。毛竹5个CAD蛋白中,有4个具有醇脱氢酶GroES-like(ADH_N)和锌结合脱氢酶(ADH_zinc_N)两个保守功能域,这是大多数CAD蛋白的保守结构域;另外一个具有与CCR蛋白相同的Epimerase结构域,但全蛋白序列与CCR蛋白相似性不高,属于另一类CAD蛋白,即具有Epimerase结构域的CAD蛋白,这类CAD与具有ADH_N和ADH_zinc_N两个保守功能域的CAD蛋白的相似性极低。采用最大似然法对两类CAD蛋白分别构建系统进化树,结果表明,高等植物的具有醇脱氢酶GroES-like和锌结合结构域的CAD蛋白处于2个进化枝上,这种分化可能发生在高等植物产生之前;高等植物产生以后,一些植物的基因组内又发生了基因扩增和多样化事件,产生了多个CAD同源基因。高等植物的具有Epimerase结构域的CAD蛋白,在高等植物产生之后有一个共同的祖先。(3)在毛竹全长cDNA文库中找到1个推定的毛竹C3H基因,命名为PheC3H。其编码蛋白PheC3H分子量为58.38kDa,理论等电点为8.77。PheC3H蛋白与小麦的CYP98A相似性最高,PheC3H应属于CYP98家族A亚家族蛋白。PheC3H的前21个氨基酸残基推定为信号肽区域,成熟蛋白很可能定位在内质网上。采用实时定量PCR研究了PheC3H在毛竹幼苗不同营养器官中的表达,结果表明,PheC3H在3年生茎中表达量最高,其次是一年生叶鞘;在一年生毛竹不同营养器官中的表达量顺序为:叶鞘>根>叶片>茎。(4)采用同源比对的方法,在毛竹全长cDNA文库中找到10个与高等植物F5H基因序列和相应的编码蛋白质序列相似性较高的基因。对其编码蛋白的序列分析表明,这10个基因的编码蛋白质均属于细胞色素P450家族成员,10个基因初步命名为PheCYP-1,…, PheCYP-10。10个基因及其编码蛋白相互之间的相似性不高;10个P450蛋白与高等植物已经报道的F5H蛋白相似性也不高,因此这10个基因可能具有不同的功能。PheCYP-2属于CYP81A亚家族,PheCYP-4属于CYP81家族,PheCYP-5属于CYP706家族一新的亚家族,PheCYP-9可能是一个类黄酮-3’5’-羟基化酶。10个P450蛋白均含有N端信号肽,很可能定位在内质网膜上。(5)采用实时定量PCR研究了10个PheCYP基因在毛竹幼苗不同营养器官中的表达情况,根据相对表达水平,10个PheCYP基因可以分成3组:①高表达基因,有3个:PheCYP-1、PheCYP-2和PheCYP-7,PheCYP-1和PheCYP-2主要在叶片中表达,PheCYP-7主要在笋中表达;②中度表达基因,有4个,包括PheCYP-3、PheCYP-4、PheCYP-6和PheCYP-9,PheCYP-3主要在叶片、茎和根中表达,PheCYP-4主要在叶片中表达,PheCYP-6主要在笋中表达,PheCYP-9主要在叶鞘和茎中表达;③低表达基因,有3个,包括PheCYP-5、PheCYP-8和PheCYP-10,PheCYP-5主要在笋中表达,PheCYP-8主要在笋、根和叶鞘中表达,PheCYP-10主要在叶片中表达。根据已报道的F5H基因及木质素合成相关基因的表达模式推测,这10个基因可能与木质化进程有关。(6)对PheCYP-5、PheCYP-6和PheCYP-10基因构建植物表达载体,转化拟南芥,目前已经得到了3个基因的T1代转基因植株。对转PheCYP-5和PheCYP-10基因的T1代拟南芥花葶的木质部发育情况进行切片观察,结果表明,过表达PheCYP-5和PheCYP-10基因的转基因拟南芥,次生木质部中导管壁的厚度显著增加,进一步证明PheCYP-5和PheCYP-10与次生壁的形成有关。更多还原

【Abstract】 Phyllostachys edulis belongs to Bambusoideae subfamily in Gramineae family, and it has the largest cultivation area and highest values of usage in China. Ph. edulis is rich in fiber and suitable for making paper. However, due to the high level of lignin content in Ph. edulis, a large quantity of chemicals must be used to separate fiber from lignin, with lignin left in the waste, resulting in serious environmental pollution. Moreover, the chemicals for separation, and the re-collection of fiber using alkali have greatly increased the cost of paper making process. Hence, altering lignin content and/or monolignol composition by biotechnology and directionally cultivating new varieties have become an important research project in current biotechnological field of Ph. edulis.In this research, we constructed four full-length cDNA libraries of different organs of Ph. edulis, and on the basis of mass sequencing and data analysis, several genes encoding the key enzymes in monolignol biosynthetic pathway in Ph. edulis, namely cinnamate 3-hydroxylase (C3H), cinnamoyl CoA reductase (CCR), cinnamyl dehydrogenase (CAD), and ferulate 5-hydroxylase (F5H)-like genes, were isolated from the cDNA libraries. And we made a further research on the expression patterns of C3H and F5H-like genes, and transgene experiments were also carried out with three F5H-like genes, with the purpose of deducing their possible functions. The results are listed as follows.(1) Five CCR genes were isolated from full-length cDNA libraries of Ph. edulis. The five deduced CCR proteins have conserved Epimerase domain, and they belong to NAD dependent epimerase/dehydratase family. Four of the five deduced CCR proteins have no obvious signal peptide or trans-membrane structure, and are predicted to be localized in cytoplasm. PheCCR5, however, a signal peptide-like structure is detected on its N-terminal and might be predicted to be localized in mitochondria. Using maximum likelihood method, the phylogenetic tree was constructed. The result shows that the CCRs appeared before the differentiation of eucaryotes and that lineage-specific expansion and divergence events occurred in some angiosperms, after the divergence from lower land plants. (2) Five CAD genes were isolated from the cDNA libraries. Four of the five deduced PheCAD proteins contain two functional domains of ADH_N and ADH_zinc_N, which are the conserved domains for most CAD proteins. PheCAD4, however, has a domain of Epimerase similar to that of CCR proteins rather than ADH_N and ADH_zinc_N domains, but it has low similarity with CCR proteins. So it is a CAD protein of another group, namely CADs with Epimerase domain. CAD proteins of this group have very low similarity with CADs containing the ADH_N and ADH_zinc_N domains, so we deduce that the two kinds of CAD proteins might be another group of CAD. Using maximum likelihood method, the phylogenetic tree was constructed. It is shown that the angiosperm CADs containing ADH_N and ADH_zinc_N domains are located at two branches, and that such differentiation took place before the appearance of higher plants; after the divergence of angiosperms, lineage-specific expansion and divergence events occurred, resulting in the multiple CADs in the genome. The angiosperm CADs with Epimerase domain shared a common ancestor.(3) We got one putative C3H gene from the cDNA library and named it as PheC3H. The molecular mass of its deduced protein is 58.38 kDa with pI 8.77. PheC3H has very high similarity to TraCYP98A protein and it should belong to the subfamily A of CYP98 family. The sequence of the first 21 amino acid residues at N-terminal is predicted to be the signal peptide, and the mature protein might be located on endoplasmic reticulum. Quantitative real-time PCR was used to detect the organ-specific expression in nutritive organs of Ph. edulis seedlings. The highest expression level of PheC3H is in 3-year-old stems followed by leaf sheaths of one-year old seedlings. The relative expression level of PheC3H in one-year-old seedlings is ranged as: leaf sheaths > roots > leaves > stems.(4) Using similarity blast, we found 10 genes having relatively higher similarity to some known F5H genes. Sequence analysis shows that the ten proteins belong to cytochrome P450 superfamily, so we name them as PheCYP-1, PheCYP-2,…, PheCYP-10. The similarities between the ten proteins are not high, and the similarities between the ten proteins and know F5H proteins are not high, either, which indicates that the ten genes might have different functions. According to sequence similarity, PheCYP-2 belongs to CYP81A subfamily, PheCYP-4 belongs to CYP81 family, PheCYP-5 is a new member in CYP706 family, and PheCYP-9 might be a putative flavonoid 3’,5’-hydroxylase. Each of the ten proteins contains a predicted signal peptide sequence at its N-terminal, and they may be located on endoplasmic reticulum.(5) Quantitative real-time PCR was carried out to detect the expression patterns of the ten PheCYPs in different organs of Ph. edulis seedlings. According to the relative expression level, the ten genes can be divided into three groups:①High expression genes, including PheCYP-1、PheCYP-2 and PheCYP-7. PheCYP-1 and PheCYP-2 are mainly expressed in leaves and PheCYP-7 mainly in shoots.②Intermediate expression genes, including PheCYP-3, PheCYP-4, PheCYP-6 and PheCYP-9. PheCYP-3 has relatively higher expression in leaves, stems and roots, PheCYP-4 in leaves, PheCYP-6 in shoots and PheCYP-9 in leaf sheaths and stems.③Low expression genes, including PheCYP-5, PheCYP-8 and PheCYP-10. PheCYP-5 has relative higher expression in shoots, PheCYP-8 in shoots, roots and leaf sheaths, and PheCYP-10 in leaves. According to the documents on known F5H genes and lignin biosynthesis-related genes, we can deduce that the ten genes might have some relationship on lignification process in Ph. edulis.(6) PheCYP-5, PheCYP-6 and PheCYP-10 were cloned into plant expression vector and transformed into Arabidopsis thaliana. We have got the transgenic Arabidopsis T1 generations. Xylem development of PheCYP-5 and PheCYP-10 transgenic Arabisopsis T1 generation were detected by tissue section. Transgenic Arabidopsis plants overexpressing PheCYP-5 or PheCYP-10 have significant increased width of vessel walls in the secondary xylem, indicating that PheCYP-5 and PheCYP-10 may be involved in secondary cell wall development of vessels in xylem.更多还原