摘要
近年来,木材形成的分子机制研究已成为植物分子生物学研究热点之一,而针对木材性状控制分子基础的研究很少。基因芯片分析技术具有高通量、高灵敏度和高可信度等特点,是植物功能基因组学研究的有力工具之一。本研究从美洲黑杨(Populus deltoides)主干不同高度存在木材性状差异入手,通过构建杨树未成熟木质部cDNA文库,制作cDNA表达芯片,用于研究主干不同高度处基因表达差异,筛选出树木材性相关候选基因;克隆部分候选基因的全长序列,包括基因组上全长序列,分析其在杨树基因组上的分布情况;在此基础上,构建适合杨树基因功能研究的pRNAi载体和部分候选基因的RNAi、反义表达载体,对杨树组培苗进行遗传转化、转基因鉴定和候选基因表达分析,主要研究结果如下:
(1)成年美洲黑杨母树主干不同高度木材密度和微纤丝角(microfibril angle)大小分布存在差异,木材密度(wood density)随高度呈下降趋势,而微纤丝角呈先下降后上升趋势;杨树主干由下而上,木材微纤丝角大小与密度之间相关性不大。
(2)利用SMART技术构建了美洲黑杨及美洲黑杨×青杨(Populus deltoides×Populus cathayana) F2子代未成熟木质部全长cDNA文库。从美洲黑杨和美洲黑杨×青杨F2子代未成熟木质部文库中分别随机挑取10000和5000个单克隆用于芯片探针制备,获得11181个单一cDNA探针,制作cDNA表达芯片,用于杨树主干不同高度处基因表达分析。筛选出差异表达点274个,并进行序列测定;得到125条单一序列,与已知功能同源的序列62条,占49.6%;其中,抵抗环境胁迫相关的基因占11%,参与基因功能调控4%,参与合成细胞结构组份有关的基因和转录因子各占6%,与信号传导有关的基因占8%,代谢和次生代谢有关的基因占8%;余下63条序列中,有60条序列在杨树ESTs数据库找到同源序列,有3条序列在EST数据库无同源序列,为美洲黑杨特异表达的序列;
(3)芯片实验发现,组成细胞组分的核糖体rRNA在美洲黑杨主干高密度部位(基部)表达量高;而组成染色体组蛋白H2B1在低密度部位(4 m,6 m)表达量高;丙苯氨酸解氨酶和4-香豆酸-CoA连接酶与木材密度之间的相关系数绝对值分别为0.69和0.95,推测两种酶的表达与木材密度相关;植物Remorin蛋白与维纤丝角分布的相关系数为0.92,表现出很高的相关性;转录因子中,锌指蛋白、细胞延伸因子、CCR4相关因子、DNA/RNA结合蛋白与木材密度的相关性高,其表达可能影响木材密度的形成;乙烯应答元件结合因子的不同基因表达各不相同,乙烯应答元件结合因子4、乙烯应答元件结合因子5和乙烯应答元件结合蛋白与木材密度相关性好,但乙烯应答元件结合蛋白3的表达与木材密度相关性差;控制细胞生长发育或组织器官形成有关的基因中细胞周期有关蛋白,微管组织结构形成有关基因,细胞生长/分化有关基因与木材密度相关性较大,可能与木材密度形成有关。从测序结果分析来看木材密度及微纤丝角大小分布与一些调控有关的基因表达水平间具很高的相关性。这一结果表明,控制木材性状的主要是一些调控基因,而非直接行使功能的基因。
(4)采用RACE技术克隆ZF-RNG、CytoB和EXO全长cDNA;利用拟基因组文库染色体步行法(VGL-Walking)从杨树基因组上获得ZF-RNG全长序列和CytoB 5’端序列。经Spidey软件分析,ZF-RNG基因包含有9个内含子和10个外显子; Blast软件分析表明,ZF-RNG基因位于杨树基因组LG_VI 16611332-16616623物理位置,并在基因组LG_XVIII上具部分同源序列; CytoB基因在染色体上为双拷贝,分别位于杨树基因组数据簇LG_IX物理位置的8914871- 8915817和LG_I物理位置的17937655-17938359。
(5)对目标基因在未成熟木质部、叶片组织、花芽和叶芽中的表达状况分析, ZF-RNG基因在花芽中表达量最高,未成熟木质部组织次之,叶片组织和叶芽中表达量相差无几,结果表明该基因与发育相关;EXO基因在叶片中表达量最高,其它部位间表达量相差无几;CytoB基因在花芽和叶芽组织中表达量最高,在叶片组织中表达量低,而未成熟木质部组织中表达量一般。
(6)克隆了ZF-RNG基因内含子Intron 9片断,利用pUC19克隆载体和pBI121植物表达载体,构建pRNAi载体;利用pRNAi载体、pBI121 Hind III、EcoRI酶切载体片段和CytoB扩增的CDS片段,构建成CytoB基因反义表达载体pAntiCytoB和RNAi表达载体pRNAi-CytoB2EX;采用农杆菌介导法,对中国山杨(Populus davidiana)组培苗25#进行遗传转化,获得再生植株;经PCR验证,获得34个CytoB RNAi转化子和7个CytoB反义转化子;随机对其中2个CytoB RNAi转化子和2个CytoB反义转化子进行RT-PCR检测,其中标号为A24的CytoB RNAi转化子的表达量降低到1/5以下,而其它三个转化子基因表达不发生变化。
Exploration of how wood develops and forms has been a focus in research field of woody plant molecular biology in recent years, but the study of what genes and how they control wood properties is far from attaching importance to scientists. Micro-array technique, with its advantages of high throughput, sensitivity and reliability over other tools developed for investigating genes expression pattern, is competent for assaying thousands of genes in a short time at functional genome era. The cDNA micro-array, prepared from two cDNA libraries of developing xylem tissues of poplar, was used to assay gene expression pattern in immature xylem tissues at different height of main stem of Populus deltoides (15 years old), which was confirmed having distinct wood properties (micro-fibril angle, woody density) along the main stem, and the dots with various expression profile between chips were screened out and the single-clones related to these dots were subjected to 5’sequencing. After 5’sequencing of these single-clones, the bio-informatics tools were employed to identify the candidate genes which may influent the wood properties of poplar. A versatile RNAi vector, adapted to poplar functional genome research, has been constructed with the intron of the candidate gene, which obtained from PCR amplification of poplar genomic DNA according to sequences comparison results between the full length cDNA sequence and the genome sequence of ZF-RNG gene cloned from poplar specimens. Two constructs of RNAi and anti-sense of candidate gene were formed with partial CDS of CytoB gene, and were transformed into the poplar plantlet. The transformed plants were screened out by PCR technique and the target gene expression level assayed by RT-PCR. The results show as follow:
(1) The difference of wood density and micro-fibril angle exist along main stem of Populus deltoids stock tree. From down to up of stem, wood density decreases, and micro-fibril angle decreases at first and then rises. The relation between wood density and micro-fibril angle along stem is low.
(2) Two cDNA libraries, which sampled from developing xylem tissues of Populus deltoids and F2 progeny of Populus deltoids×Populus cathayana, were constructed. After that, cDNA expression micro-arrays were manufactured with 11181 unitary probes of cDNA fragment which amplified from single clones of the two libraries with 10000 and 5000 clones respectively. A total of 125 singlets were obtained after analysis of 274 Clones sequences, which were confirmed having different expression patterns along the main stem of poplar by cDNA expression micro-arrays. 62 of single-pass sequences, occupying 49.6% of total singlets, show similarity to previously described sequences in Genbank. About 11% of the recognized genes participate in pathway of resisting environment stress. 4% of genes are involved in controlling other genes expression. Genes, that synthesize cell components and belong to transcript factor, hold 6% of recognized genes respectively. Genes which felled into the group of metabolism or secondary metabolism and the pathway of signal transduction occupy 8% respectively. The 63 remaining sequences were subjected to homological gene search in poplar ESTs database and annotated with matched gene function. After that, 3 sequences could not be found similar ESTs in poplar database and were presumed specific expression genes of Populus deltoids.
(3) Genes involved in constructing rRNA components expressed more in wood tissues showing high density than that of low density. However, H2B1 gene expresses in high level with wood tissues of low density. PAL and 4CL are related to wood density trait with the correlation coefficient of 0.69 and 0.95 respectively. The plant gene, Remorin, shows high correlation between its expression profiles and micro-fibril angle distribution along main stem. Among the transcript factor genes, zinc finger RING protein (including CCCH, C3HC4 and PIF1), elongation factor, CCR4-associated factor, DNA/RNA biding proteins, ethylene responsive element binding factor 4, ethylene responsive element binding factor 5 and ethylene-responsive transciptional coactivator-like protein (ERT) share high correlation coefficient with wood density distribution and are presumed their expression pattern influence formation of wood density. But, the ethylene responsive element binding factor 3 has low correlation coefficient with wood density distribution. The genes, including cell cycle-related protein (EXO), vascular tissue pattern formation (Q058H06 [POPLAR.104]) and cell growth / morphogenesis (P058D03), may affect the wood density. According to the results of micro-array experiment, genes that control wood properties may be regulatory genes rather than functional genes which participate in process of cell components synthesis directly.
(4) Full length cDNA fragments of ZF-RNG, CytoB and EXO were cloned by RACE technique. The Visual Genome Library Walking experiment was employed to clone full length genes of ZF-RNG and CytoB on chromosome, and the Spidey software was used to assay the splicing pattern of ZF-RNG. After that, the genome locations of ZF-RNG and CytoB were searched by blastn algorithm with poplar genomic database. The result shows that ZF-RNG gene, located on LG_VI ranged from 16611332 to 16616623, consists of 9 introns and 10 exons, and has partial homological sequences on LG_XVIII. However, there are two copies of CytoB gene on poplar genome, located on LG_IX ranged from 8914871 to 8915817 and on LG_I ranged from 16611332 to 16616623.
(5) The expression profiles of candidate genes were detected in developing xylem, leaf, alabastrum and leaf bud tissues by RT-PCR technique. The high expression level in floral bud, media expression level in developing xylem, and low expression level in leaf and leaf bud tissues of ZF-RNG gene suggest that this gene relate to plant development. EXO gene transcript is more abundant in leaf, and has similar levels in other parts. The transcript of CytoB gene is more abundant in alabastrum and leaf bud tissues, and is rare in leaf tissues.
(6) A RNAi vector, named pRNAi, had been constructed with pUC19 plasmid,“35S-GUS-Nos”fragment of pBI121 and the intron9 of ZF-RNG gene which was cloned from poplar genomic DNA . Two constructs of CytoB, which were anti-sense construct pAntiCytoB and RNA interference construct pRNAi-CytoB2EX, were constructed with pRNAi vector, vector fragment of pBI121 digested with Hind III and EcoRI, and partial CDS of CytoB. The constructs were transformed into plantlets of Populus davidiana via agrobacterium tumefaciens-mediated transformation and 34 plants transformed with pRNAi-CytoB2EX and 7 plants transformed with pAntiCytoB were screen out by PCR. Four transformed plants, two of each from transformants of pRNAi-CytoB2EX and pAntiCytoB respectively, were selected randomly for gene expression level assaying of CytoB by RT-PCR. The expression level of one pRNAi-CytoB2EX transformant, labeled with A24, decreased to one fifth that of controls.
引文
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