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题名: 麻竹组培苗开花相关基因的分离和进化发育生物学研究
作者: 陈永燕
学位类别: 博士
答辩日期: 2003
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 李德铢
关键词: 麻竹 ; 组培提前开花 ; MADS-box基因家族 ; 进化发育生物学
学位专业: 植物学
中文摘要: 被子植物开花现象的产生是植物界进化过程中的一个划时代事件。花的研究是植物进化、发育和遗传学的核心问题之一。目前的研究主要集中在模式植物如拟南芥、金鱼草、水稻等。竹子开花现象则是植物学界的一个难解之谜。本文以麻竹组培诱导开花的组培苗为材料,首次对竹子的花发育进行了分子生物学水平的研究。对麻竹开花组培苗中分离到的18个MADS-box基因进行T进化发育生物学〔evo-dovo genetics〕研究,主要研究结果如下:1.麻竹组培苗开花的诱导以麻竹种子为起始材料,在含0.2mg/1BA的MS培养基上萌芽,然后转入含2 mg/lBA的MS培养基诱导出芽丛。诱导开花的基本培养基为MS培养基,其中大量元素浓度降为3/4,另有三种有机成分终浓度较标准Ms培养基高,即vBI为sml,vB6为lml,烟酸为lml。在含6m岁IBA的MS培养基中可在较短时间内诱导出小穗,但在含高浓度BA的培养基上诱导的小穗大多发育不良,此时可以降低BA的浓度到3ml,并可适当加入0.5-1m幼的KT和10%的椰乳,以改善花的质量。通过调节Ms培养基中细胞分裂素的浓度,可以灵活调节该组培体系在营养生长和生殖生长之间的转换。对照实验表明,尽管BA和KT都是细胞分裂素,但它们在诱导麻竹开花过程中的作用并不相同。BA促进组培苗向生殖生长转化,而KT则有助于改善花的质量以及诱导组培苗向营养生长逆转。2.麻竹小穗cDNA文库的构建以麻竹各时期的混合小穗为材料,构建了麻竹小穗的cDNA文库。文库滴度达到了106pfu/ml。随机挑取1玉个菌斑的PcR扩增结果表明,cDNA片段长度在800-2200bp之间,大多数在15。。bp左右。说明cDNA文库中的克隆基本都是全长的基因。3.SsH方法分离小穗特异表达基因片段初探为分离麻竹小穗中特异表达的基因,采用SSH方法,以麻竹叶的cDNA为驱动样品(driver),小穗的cDNA为实验样品(tester),获得了可能在小穗中特异表达的三个EST片段。经blast在线分析没有发现GenBank里有和它们相似程度较高的基因存在。对该基因片段的分析需要进行进一步的研究。4.麻竹MADS-box基因的分离以麻竹组培诱导开花的幼嫩小穗为材料,提取其mRNA并逆转录为cDNA。根据水稻的MADS-box基因的保守序列设计了特异引物,采用RACE方法分离到18个MADS-box基因克隆的cDNA全长。对这18个墓因的系统学分析表明,它们可分为明显的五个支,即DIM03支,包括5个克隆;DIM06支,包括7个克隆;DIM07支,包括2个克降:DIM32支,包括2个克隆;DIM19支,包括2个克隆。且各分支的靴带支持率都为万00%。在这五支MADS-box基因中,DIM03支、DIM06支和DIM32支属于AGL2-基因亚族:DIM19支属于SQUA-基因亚族;DIM07支属于AGL6一基因亚族。根据225条植物MADS-box基因序列构建的NJ树,可以判断各分支基因与模式植物MADS-box中功能已知的哪些基因亲缘关系最近,并进一步根据序列相似程度推测其可能的功能。其中,与DIMo3支基因关系最近,同时又有表达模式和功能分析报道的有水稻的osMADss和玉米的zMM6。OsMADs8的转基因实验表明它具有使植物开花时间提前的功能。ZMM6是参与调控玉米小穗数目的一个很重要的功能基因。DIM03与OsMADSS的cDNA序列一致性达到了893%,与ZMM6则达到了79.4%,因此推测DIM03可能是这两个基因在麻竹中的直系同源基因。而与DIMO6支基因关系最近,同时又有表达模式和功能分析报道的有水稻的OsMADSI,玉米的ZMMS和ZMM14。转基因实验表明OsMADSI参与了花诱导和开花时间调控。野生型水稻的小穗一般只长一个花序,但在osmadsl强突变体中,小穗中会长出新的花序。此外,采用反义RNA技术的转基因实验表明ZMMS基因的功能减弱后,会导致每个小穗中小花数目的增加,说明ZMMS基因参与了小穗中小花数目的调控。麻竹的DIM06基因与OsMADSl的cDNA序列一致性达到了83.5%,与ZMMS和ZMM14则分别达到了79.4%和81.2%。因此作者认为DIM06支基因是这三个基因在麻竹中的直系同源基因,很可能也参与调控麻竹的开花时间,或者在小穗的结构形成方面具有重要的功能。与DIMM32支基因在系统树中关系最近且有功能分析报道的基因为水稻的OsMADS5。转基因实验表明OsMADSS也参与了花诱导和开花时间调控。DIM32支与OsMADSS的cDNA序列一致性达到了86.9%。因此作者推测这-支基因是OsMADS5的直系同源基因,很可能也在调控麻竹的开花时间方面具有重要的功能。5. MADS-box基因在植物中的进化结合从麻竹中分离到的MADS-box基因,对植物中MADS-bOx基因的系统发育和进化机理进行了研究。采用225个植物以DS-box基因构建的NJ系统树表明,大致可以分为8个大支,即A.AGL19-支;B.真双子叶SOUA-支;C.AGL15-支;D.单子叶SOUA-支:E.AGLZ-支;E AGL6-支;GAP3/PI-支;H.AG-支。各分支间的关系不清楚,呈平行关系。该结果和Johansen et al(2002)基本一致。为探讨SQUA-类、AGL2-类和AGL6类基因之间的关系,我们用三类基因的序列构建了J系统树,但它们之间依然是平行的关系,因此后来的分析中还是将这三类基因分开来进行。基于NJ树对SQUA-类、AGL2和AGL6-类基因的相对速率检测结果表明,单子叶植物SQUA-支和真双子叶植物SQUAA-支之间存在极显著差异。为证明这种差异是否为真差异,根据推导的氰基酸排序后的矩阵,对cDNA序列矩阵中对应于氨基酸矩阵中的gaP的核甘酸删除后,再次检测两支间的相对-速率,结果仍然存在极显著差异。目结果表明真双子叶植物SQUA-支基因的相对进化速率显著高于单子叶植物支。为探讨植物中SQUA-类基因的进化机理,采用似然比检验方法和PAML软件中的branch模型,对单子叶植物和真双子叶植物最近共同祖先中的SQUA基因发生基因重复后,分别在环境的选择压力下形成的适应性进化机理进行了分析。结果表明,sQUA-基因发生基因重复后,在真双子叶植物中的非同义置换的比例较在单子叶植物中高。为进一步检测真双子叶植物祖先谱系中影响单个位点的正选择,我们对同一套数据用队ML软件里的branch-site模型进行了分析。在该模型下的参数估计结果表明,真双子叶SQUA一支的ω=1.16,基因中28.5%的密码子处于正选择。比较branch-site模型和site-specific模型的似然比检验26InL=198.65。说明branch-site模型显然是更符合的。因此,似然比检验为正选择压力导致真双子叶植物SQUA支基因的高度分化提供了有力的证据。单子叶植物花被的分化程度远较真双子叶植物低。SQUA-基因属于花发育ABc模型中的A类基因,单独决定花尊的形成,也参与决定花瓣的形成。本文所获得的真双子叶植物SQUA-类基因进化速率远远高于单子叶植物SQUA-类基因,以及似然比检验结果都为单子叶植物在第一轮花器官的分化程度上远远低于真双子叶植物提供了新的分子进化生物学的证据。
英文摘要: The occurrence of flowering phenomena of angiosperms was a landmark during the plant evolutional process. Studies on flowering became one of the core issues on plant evolution, development and genetics. However, much more attention was paied on the model plants such as Arabidopsis, Antirrhinum, Oryza, and so on. It is still a riddle that the bamboo flowering in the plant. This thesis studied the bamboo flowering development at molecular level for the first time with the materials of in vitro flowering cultures of Dendrocalamus latiflorus. Some eve-devo genetics research was done on eighteen MADS-box genes isolated from Dendrocalamus latiflorus. Main results were given as follows. 1. in vitro flowering After the dry seeds of D. latiflorus were disinfected, they were cultured on a modified MS based gelled medium contain 0.2mg/l BA to germinate. Then transferred to MS medium contain 2mg/l BA to develop fascicular shoots. The MS medium which induced precocious flowering contains NH4NO3) KNO3, MgSO4-7H2O, KH2PO4 and CaCl2-2H2O lowered to 3/4 times of normal concentrations in which VB| was changed to 5mg I*1 while VB6 was changed to lmg I"1 and Niacine (nicotinic acid) to lmg I"1. The cultures on MS medium with 6mg/l BA would be flowering in short time, but most of spikelets are hypoplastic. When these cultures were transferred to medium with 3mg/l BA, 0.5-lmg/l KT and 10% coconut water, the quality of spikelets could be improved. So flowering of tissue cultures could be free controlled by adjusting consentrations of BA and/or KT. Our results also indicated that the functions of BA and KT in the procedure of artificial induced flowering were different although both of them belong to cytokinin family. BA is the only and absolutely essential cytokinin inducing in vitro flowering in bamboos. On the contrary, KT is helpful to improve the quality of bamboo spikelets and even conduce to reversed vegetative growth. 2. cDNA libraby construction By using mixed spikelets of D. latiflorus in various stages as materials, a cDNA library was constructed with the Clontech SMART cDNA library construction kit. The capacity of cDNA library is about 106 pfu/ml. The sizes of cDNA inserts from twelve random clones were from 0.8kb to 2.2kb by PCR identification. These data indicated that most of cDNA clone in this library are full-length genes. 3. SSH results SSH (suppression subtractive hybridization) was used to isolate specific expression genes in spikelets of Dendrocalamus latiflorus. In this study we got three EST fragments expressed in spikelets specifically treated leafy cDNA as driver and spikelets as tester. When we made an online search in GenBank with blast software, none of them was found as its homologus gene. We suggest that further research should be carried out as for these three ESTs. 4. MADS-box genes isolated from Dendrocalamus latiflorus mRNA in young spikelets of Dendrocalamus latiflorus was isolated, and was reverse -transcribed to sscDNA. Special degenerated primers were designed based on the conserved region of rice MADS-box genes. PCR product was amplified with RACE methods. Finally 18 clones were sequenced with full-length cDNAs. These 18 clones were clustered to five clades based on phylogenetic analysis, i.e. D1M03 clade, including five clones; D1M06 clade, including seven clones; D1M07 clade, including two clones; D1M32 clade, including two clones; and D1M19 clade, including two clones. Bootstrap value of each clade is 100%. Among of them, D1M03, D1M06 and D1M32 clades belong to AGL2 subfamily while D1M07 clade belongs to AGL6 subfamily and D1M19 clade to SQUA subfamily. Based on phylogenetic NJ tree constructed by 225 plant MADS-box genes, we may identify which gene is most similar to each MADS-box gene clade from Dendrocalamus latiflorus. Moreover, we may deduce its function according to its orthologs in model plants. Among those genes that its function have been reported, highest sequence identity happened between D1M03 and OsMADS8 in rice, then between D1M03 and ZMM6 in maize. Transgenic plants expressing the OsMADS8 gene exhibited the phenotype of early flowering and dwarfism. This indicated that OsMADS8 is involved in controlling flowering time. A reduction of ZMM6 gene function in maize plants leads to a moderate increase in the number of spikelets, since triple rather than paired spikelets developed here and there. Sequence identity between D1M03 and OsMADS8 / ZMM6 is 89.3% and 79.4% respectively. It is suggested that the D1M03 clade is their orthologs in Dendrocalamus latiflorus. There is highest sequence identity between D1M06 and OsMADSl in rice, and between D1M06 and ZMM8, ZMM14 in maize. OsMADSl is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. Normally, wild-type rice plants produced one spikelet, but more spikelets produced in osmadsl overexpressing mustants. Moreover, a reduction of ZMM8 gene function in transgenic maize plants leads to an increase in the number of florets per spikelet, indicating that ZMM8 is involved in confering determinacy to the spikelet primordia which generate the floret primordia. Sequence identity between D1M06 and OsMADSl, ZMM8 and ZMM14 is 83.5%, 79.4% and 81.2% respectively. It is suggested that the D1M06 clade is their orthologs in Dendrocalamus latiflorus. Among those genes that its function have been reported, highest sequence identity happened between D1M32 and OsMADS5 in rice. Transgenic plants of OsMADS5 exhibited the phenotype of weak dwarfism and early flowering. This indicate that OsMADSS is involved in controlling flowering time. Sequence identity between D1M32 and OsMADS5 is 86.9%. It is implied that the DIM32 clade is its orthoiog in Dendrocalamus latiflorus. 5. Evolution of MADS-box genes in plants Combined with MADS-box genes isolated from Dendrocalamus latiflorus, phylogenetic analysis and evolutional mechanism of the MADS-box genes in plants have been made. Phylogenetic NJ tree constructed with 225 plant MADS-box gene sequences showed that this tree could be subdivided eight clades: A. AGL19- clade; B. SQUA- clade in eudicots; C. AGL15- clade; D. SQUA- clade in monocots; E. AGL2- clade; F. AGL5- clade; G. AG- clade. Relationship among clades is still unclear. This result is consistent with Johansen's (2002). Gene sequences in B, D, E and F clades above were conbined to a new data matrix to contruct a phylogenetic NJ tree in order to check relationships among these four clades. But it is a pity that phylogenetic relationship is still unclear among them. So they were subdivided two data matrix to do more analysis. Relative-rate tests (RRTs) were performed based on NJ tree of SQUA- genes and of AGL2-/AGL6- genes respectively. Significant rate difference was detected between eudicots-SQUA clade and monocots-SQUA clade. This rate difference was detected again based on nucleic acid data matrix removed three nucleotides relative to gaps in amino acid data matrix. Significant rate difference was still detected. This result suggested that relative rate in eudicots-SQUA genes is really higher than in monocots-SQUA genes. To understand the mechanisms of evolutionary rate variation in SQUA- genes between the two lineages, likelihood rate tests (LRTs) based on branch model of codon substitution were applied to explore the mechanisms of adaptive evolution of SQUA- genes under selective pressure in ancestral of eudicots and monocots after gene duplication. These programs implemented in PAML package. The results suggest an increase in the nonsynonymous substitution rate in the lineage ancestral to eudicots-SQUA genes after gene duplication. Furthermore, we performed a second analysis on the same data to test for positive selection affecting individual sites along the lineage ancestral to the eudicots-SQUA genes. The branch-site model implemented in the codeml programof the PAML package was used. Parameter estimates under this model suggested that 28.5% of codons in the gene were under positive selection with co2=1.16 along that lineage. The LRTs comparing the branch-site model and the site-specific model gave 251nL=l98.65, indicating that the branch-site model fits the data significantly better. The LRTs thus provides significant evidence for positive pressure driving functional divergence of eudicots-SQUA genes. Perianth differentiation of monocots is much lower than of eudicots. The SQUA-gene belonging to A genes which specify sepals solely and specify petals together with B genes. So our RRTs and LRTs results on SQUA-genes between monocots and eudicots provided a molecular evolutional evidence for perianth differentiated diaparity between monocots and eudicots.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/660
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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麻竹组培苗开花相关基因的分离和进化发育生物学研究.陈永燕[d].中国科学院昆明植物研究所,2003.20-25
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