梨Ⅲ型聚酮合成酶家族的比较基因组学研究及表达模式分析

植物Ⅲ型聚酮合成酶(PKS Ⅲ)在植物次生代谢产物生物合成中起着非常重要的作用。本研究从7种蔷薇科果树中共鉴定出57个PKS家族成员,随后进行种间比较基因组学分析。基因结构和保守基序分析表明,57个PKS多数由两个外显子和一个内含子组成,都具有PKS特有的保守结构域Chal-sti-synt-N和Chal-sti-synt-C。进化分析显示,57个PKS可明显分为4个亚家族,其中I亚家族成员数目最多。染色体定位及基因复制事件表明,PKS不均匀地分布在2~5条染色体上,且PKS家族进化过程主要受纯化选择作用。荧光定量分析发现,PbPKS4和PbPKS5表达模式与‘砀山酥梨’(Pyrus bretschneideri cv.‘Dangshan Su’)不同发育时期果实中木质素及石细胞含量变化趋势类似,因此推测这2个基因在梨果实木质素合成及石细胞发育过程中发挥一定作用。本研究为今后研究蔷薇科果树PKS家族建立了基础,也为从分子水平改善梨果实品质提供了理论依据。     

亚洲棉ARF基因家族全基因组鉴定与分析

生长素响应因子(ARF)是植物中可以特异性结合生长素应答基因启动子的转录因子,在植物的生长发育中起着重要的调控作用。为了更好的了解亚洲棉中ARF基因家族的种类和数量,本文利用生物信息学的方法,分析了亚洲棉基因组中ARF基因的种类、进化关系、物理定位以及基因的结构和保守基序。结果表明在亚洲棉基因组中发现并初步确定了29个ARF基因,命名为Ga ARF,通过基因定位发现除在2、3、5、11号染色体上没有ARF基因外,其他染色体上都有ARF基因存在;而且在10号染色体上还存在着基因簇;进化树分析表明,亚洲棉部分ARF蛋白和已知功能的拟南芥聚类到一起;表达模式分析显示,不同组织不同处理条件下ARF基因的表达存在差异。     

砀山酥梨果实苯丙氨酸解氨酶基因cDNA克隆及序列分析

为了研究梨石细胞形成与木质素合成的关系,以砀山酥梨果实为材料,通过基于同源性的RT—PCR方法,克隆木质素合成途径中的关键酶苯丙氨酸解氨酶（PAL）基因。同时利用基因数据库资料对克隆的PAL序列进行比较分析。结果表明,从砀山酥梨果实中克隆的PAL基因eDNA片段长度为585bp,推断其编码195个氨基酸序列。该序列包含与其它植物PAL相同的活性催化位点,经序列分析和同源性比对,该氨基酸序列与其它植物PAL氨基酸序列的同源性在90％以上。系统进化树分析表明,砀山酥梨PAL氨基酸序列与蔷薇科的甜樱桃PAL氨基酸序列聚类关系最近。     

砀山酥梨石细胞发育过程中PPO酶学特性及其cDNA片段克隆

以砀山酥梨果实为材料,研究了影响石细胞形成的木质素合成酶-多酚氧化酶(polyphenol oxidase,PPO)的酶学特性,并对PPO基因进行了克隆.结果表明,在砀山酥梨果实发育的过程中,PPO活性在花后27 d和47 d出现高峰,早于木质素含量高峰(花后47 d、61 d)和石细胞团含量高峰(花后51 d),且P...     

毛果杨GATA基因家族全基因组水平鉴定及表达分析

GATA转录因子基因家族在植物生长发育、细胞分化以及响应环境变化中具有重要作用。然而,目前在木本植物中尚无该基因家族全基因组水平的分析报导。本项研究从基因组水平对毛果杨GATA家族成员的数量、基因结构、染色体定位、系统进化、编码蛋白的理化特征和保守基序等信息进行了系统分析,结果表明,毛果杨GATA家族包含39个基因,共分布于15条染色体上,其中5号染色体上含有6个基因,9号、13号和19号染色体含有基因数量为1,其余染色上无基因分布。该家族各基因的结构与编码蛋白的基本特性均存在一定异性,可分成4个亚族。qRT-PCR研究表明,GATA家族各基因在不同发育阶段的茎部表达量存在明显差异,且盐胁迫对各基因的表达特性影响显著。以上结果表明,毛果杨GATA家族基因在复制后,基因的结构与功能产生了明显分化,其中部分基因在毛果杨次生生长与盐胁迫响应中可能具有重要作用。本项研究为全面解析毛果杨GATA家族各成员在其生长发育与盐胁迫响应中的生物学功能奠定了基础。     

应用PRINS技术定位黄鳝Hox基因的研究

Hox基因是近年来发现的支持基因组复制进化理论的最有力的证据。该研究应用引物原位延伸 (PRINS)技术 ,在黄鳝有丝分裂染色体标本上开展Hox基因的定位研究。研究结果表明 ,在黄鳝染色体组中可能存在有 6个Hox基因簇 ,这些基因簇分别位于黄鳝第 1、2、3、6、8和 10号染色体 ,相对着丝粒距离分别为 2 8 2 4± 2 88、4 5 5±1 39、13 89± 2 0 3、74 32± 1.86、38 0 3± 2 .4 1和 5 8 18± 2 0 5。该研究定位黄鳝Hox基因将有助于挖掘黄鳝染色体的来源和进化特征 ,并为基因组复制进化理论提供黄鳝这一特化物种的细胞遗传学证据。     

植物细胞遗传图及其应用

细胞遗传图（cytogenetic map）综合了来自遗传图（genetic map）和细胞学图（cytological map）两方面的信息,它既能反映基因或DNA标记之间在染色体上的真实距离,又能显示它们与染色体的细胞学结构间确切的位置关系。构建植物细胞遗传图的宗旨是将遗传图上的诸多标记与其在染色体的具体位置联系起来。目前主要有两种方法用于细胞遗传图的构建。较广泛使用的一种方法是借助染色体断点来确定遗传标记在染色体上的位置,另一种方法是利用荧光原位杂交（FISH）直接把DNA序列定位到染色体上。此外,利用RN-cM图也可以把遗传标记定位于粗线期染色体。从细胞遗传图可以看出,染色体两臂的远端有较高的基因密度和重组频率。细胞遗传图在比较近缘植物基因组的同线性、揭示植物的进化关系、研究基因定位克隆等方面都有重要意义.     

‘砀山酥梨’及其褐皮芽变果皮中多胺类物质含量和相关基因表达分析

为研究多胺类物质在‘砀山酥梨’芽变品系‘锈酥’果皮褐色形成中的作用,以‘砀山酥梨’和‘锈酥’花后不同时期果皮为试材,采用HPLC方法测定果皮中多胺类物质的含量,利用RACE技术克隆多胺合成过程中的关键基因,通过Protparam网站与MEGA5.0软件分析相关基因蛋白的理化性质及其系统进化,用荧光定量qRT-PCR方法分析不同时期果皮中相关基因的相对表达量。结果表明:(1)除花后50和150d外,其它时期‘砀山酥梨’果皮中腐胺含量均显著高于‘锈酥’,花后50d、75d、150d时,‘锈酥’果皮中的亚精胺、精胺含量显著高于‘砀山酥梨’果皮。(2)克隆出多胺合成的基因ADC、SPDS和SPMS(GenBank登录号分别为KM923903、KM923905和KM923906),预测ADC和SPMS均为水溶性蛋白、SPDS为疏水性蛋白,它们与苹果的亲缘关系最近。(3)荧光定量PCR结果显示,花后50d,多胺合成中ADC、SPDS和SPMS在‘锈酥’果皮中表达量均显著高于‘砀山酥梨’。研究推测,‘锈酥’果皮中多胺代谢及相关基因的上调表达可能与‘锈酥’的果皮褐色形成有关。     

拟南芥和水稻金属蛋白酶ftsH基因家族的基因组比较分析

FtsH(Filamentation temperature-sensitive H)是一种广泛存在于原核生物和真核生物中的ATP依赖型金属蛋白酶。同源性分析表明,在拟南芥和水稻基因组中分别有12个和9个ftsH基因。ftsH基因在染色体上的分布有明显的偏爱性,如拟南芥的1、2、5号染色体和水稻的1、5号染色体。亚细胞定位分析表明,所有FtsH蛋白均定位于叶绿体或线粒体中。系统进化分析表明,21个FtsH蛋白成员可分为8个类群,其中AtFtsH12在水稻中没有发现种间同源物。每个类群成员的蛋白序列高度保守,种内同源物显示出大于80%的相似性,而种间同源物的相似性也大于70%。类群内的同源基因并非平行进化产生的,拟南芥基因组中进化出AtftsH1/5、AtftsH2/8、AtftsH3/10和AtftsH7/9共4个同源基因对,而水稻基因组中只有OsftsH3/8和OsftsH4/5两个同源基因对。每一类群中的成员在基因外显子-内含子边界分布上表现出高度保守性,在蛋白功能结构域的可变残基上具有偏爱性,而内含子在碱基组成和序列长度上表现出广泛的变异。拟南芥和水稻ftsH基因家族的比较分析为其他物种ftsH基因的特...     

番茄AS2基因家族的系统进化分析

AS2基因家族是一类直接参与转录调控并广泛存在于植物中的转录因子家族,能够调控植物生长发育和抗逆境胁迫过程。本研究通过分析番茄基因组,鉴定了番茄中具有特定结构域的AS2基因,并利用生物信息学手段对番茄AS2基因的系统进化、染色体定位以及基因结构等信息进行了分析。系统进化分析共鉴定了45个番茄AS2基因家族成员,分成5个亚类。亚细胞定位分析表明,该家族基因主要分布在细胞核上。染色体定位揭示了AS2基因分布于10条染色体上,基因主要以片段复制的方式进行扩增。本研究为进一步探究番茄和其他物种中AS2基因功能奠定了基础。     

不同基因型梨木质素代谢对石细胞含量和果实口感的影响

以五种基因型梨果实为材料,对石细胞团的大小、分布进行解剖学观察,并测定石细胞、木质素含量和木质素相关合成酶PAL、POD、PPO活性,探讨不同基因型梨果实木质素代谢对石细胞含量及口感的影响.结果表明,不同基因型梨木质素含量高时,石细胞含量也高,石细胞团相对较大,分布较密集,口感差.各基因型的梨木质素、石细胞含量和大小为...     

PbrmiR397a regulates lignification during stone cell development in pear fruit

Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.     

Alterations in the Biosynthesis of Lignin in Transgenic Plants with Chimeric Genes for 4-Coumarate: Coenzyme A Ligase

The introduction of chimeric sense and antisense gene constructsfor 4-coumarate:coenzyme A ligase into tobacco plants causedthe reduction of the 4CL activity in the transgenic plants.In the transgenic plants, the cell walls of the xylem tissuein stems were brown and the molecular structure of lignin inthe colored cell walls was dramatically different from thatin the control plants. Analysis with different types of stainrevealed that levels of cinnamyl aldehyde residues and syringylunits in lignin were depressed in the brownish cell walls. Furthermore,the lignin content in colored tissue was lower than that inthe normal tissue. Our results indicate that 4CL has importantroles in the determination of the composition and the amountof lignin in tobacco plants. (Received December 27, 1995; Accepted July 23, 1996)     

鸭梨ＡＣＣ 氧化酶基因ｃＤＮＡ 片段的克隆及农杆菌介导的反义遗传转化

研究根据ACC氧化酶基因的保守序列设计一对特异性引物,以鸭梨果实为试材,借助RT PCR方法扩增得到一条长度为831bp的鸭梨ACC氧化酶基因cDNA片段,该片段编码276个氨基酸残基,与其它梨品种ACC氧化酶基因序列同源性均在94%以上。将此片段反向插入真核表达载体pBI121的CaMV 35S启动子和NOS终止子之间,构建了鸭梨ACC氧化酶基因的反义表达载体,并在农杆菌LBA4404的介导下实现对鸭梨组培苗的遗传转化。经PCR鉴定证实共有4株鸭梨组培苗中外源基因得到成功转化,Southern杂交显示在这4株转基因鸭梨中除有1株外源基因呈双拷贝外,其余3株中外源基因均以单拷贝形式存在。     

Lignin isolated from primary walls of hybrid aspen cell cultures indicates significant differences in lignin structure between primary and secondary cell wall.

Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.     

基于转录组测序的慈竹笋木质素基因筛选及其表达分析

慈竹（Bambusa emeiensis）纤维素含量丰富,是较好的造纸原料,但竹茎中木质素影响着制浆生产及纸浆质量。目前,对慈竹木质素生物合成机制所知甚少,这限制了遗传调控竹木质素的研究。本文以拟南芥、水稻等植物的已知木质素基因作为查询序列,通过BLASTp和系统进化分析,从10、50、100和150 cm慈竹笋转录组数据中筛选到351个木质素生物合成相关Unigenes,包括51个LAC,37个4CL、26个PAL、34个CCR和25个CAD相关转录子,其数量高于其他已报道的竹类植物。转录丰度和定量基因表达分析发现16个木质素基因,包括2个PAL、5个CCR、3个4CL、2个CADH2和4个LAC,随着笋发育而表达上调,表明其可能与发育性木质素积累相关。     

Nitrogen fertilizer application affects lodging resistance by altering secondary cell wall synthesis in japonica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>)

Stem mechanical strength is an important agricultural quantitative trait that is closely related to lodging resistance in rice, which is known to be reduced by fertilizer with higher levels of nitrogen. To understand the mechanism that regulates stem mechanical strength in response to nitrogen, we analysed stem morphology, anatomy, mechanical properties, cell wall components, and expression of cell wall-related genes, in two varieties of japonica rice, namely, Wuyunjing23 (lodging-resistant variety) and W3668 (lodging-susceptible variety). The results showed that higher nitrogen fertilizer increased the lodging index in both varieties due to a reduction in breaking strength and bending stress, and these changes were larger in W3668. Cellulose content decreased slightly under higher nitrogen fertilizer, whereas lignin content reduced remarkably. Histochemical staining revealed that high nitrogen application decreased lignin deposition in the secondary cell wall of the sclerenchyma cells and vascular bundle cells compared with the low nitrogen treatments, while it did not alter the pattern of cellulose deposition in these cells in both Wuyunjing23 and W3668. In addition, the expression of the genes involved in lignin biosynthesis, OsPAL, OsCoMT, Os4CL3, OsCCR, OsCAD2, OsCAD7, OsCesA4, and OsCesA7, were also down-regulated under higher nitrogen conditions at the early stage of culm growth. These results suggest that the genes involved in lignin biosynthesis are down-regulated by higher nitrogen fertilizer, which causes lignin deficiency in the secondary cell walls and the weakening of mechanical tissue structure. Subsequently, this results in these internodes with reduced mechanical strength and poor lodging resistance.