水稻(Oryza sativa L.)核基因组存在叶绿体psbA基因的同源片段

序列比较说明,重复DNA顺序pRRD9与水稻叶绿体基因组中编码QB蛋白的psbA基因存在高度的同源。用pRRD9亚克隆片段pRRD9R和片段pRRD9L对水稻的叶绿体和核DNA进行Southern杂交分析,揭示了psbA基因同源片段在某个进化时期由叶绿体基因组转移到水稻核基因组,而且两者在水稻进化过程中的变异程度存在明显的差异。利用它们对野生稻和栽培稻总DNA的Southern杂交分析,显示亚洲栽培稻与AA基因组型的野生稻有较近的亲缘关系,以及在部分野生稻产生特异的杂交带谱,说明它可以作为一种分子探针来研究水稻的进化问题。     

叶绿体中蛋白质合成的调控

高等植物和绿藻的叶绿体有自己的遗传系统.烟草、欧龙亚草和水稻叶绿体DNA的全部核苷酸序列含有130个基因密码.除叶绿体rRNA和30tRNA的基因外,已发现至少有70个密码子的40个ORF和已确定的40个及未定的11个叶绿体蛋白质基因.在一些植物种中,rRNA基因组附近的某些基因组出现二个反向重复片段.人们把质体基因分成三类:(1)编码光合系统的蛋白质基因.光合作用中蛋白质的合成     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子, 并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明, 除加入的SD序列外, 扩增片段与水稻（Oryza sativa）叶绿体基因组DNA序列16S启动子相应区域同源性为100%。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子, 并以烟草叶绿体trnH-psbA和trnK为同源片段构建了烟草叶绿体表达载体pR16S。 用基因枪转化烟草, 转化植株经Southern、Northern检测及后代遗传学分析, 发现16S启动子具有启动活性, 融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

水稻叶绿体16S启动子克隆改造、载体构建及转化研究

利用PCR方法从水稻叶绿体基因组DNA中分离16S启动子,并在其下游加入rbcL基因SD序列,以增强该启动子的翻译能力;序列分析表明,除加入的SD序列外,扩增片段与水稻(Oryza sativa)叶绿体基因组DNA序列16S启动子相应区域同源性为100％。将16S启动子与bar基因和gfp基因的融合基因连接,以psbA基因的3′序列为终止子,并以烟草叶绿体trnH—psbA和trnK为同源片段构建了烟草叶绿体表达载体pRl6S。用基因枪转化烟草,转化植株经Southern、Northern检测及后代遗传学分析,发现：16S启动子具有启动活性,融合基因已在烟草叶绿体中稳定整合并遵循母系遗传规律。     

叶绿体DNA的限制性内切酶谱分析在植物系统分类学中的应用

业已证明,叶绿体DNA为一共轭闭合环状分子。绝大多数植物叶绿体DNA均含有一对反向重复序列。对于所有已检测过的植物,叶绿体基因组所编码的基因数量、基因组成和基因排列是基本一致的。这表明叶绿体DNA具有进化上的保守性。由于其进化上的保守性,叶绿体DNA常为研究层次较高的分类单位间(属间、科间、目间、纲间等)进化关系和进化历史提供较准确的信息。1976年,Vedel等首先建立了叶绿体DNA的内切酶谱分析方法,并将其应用于植物系统分类学中。Vedel等用限制酶ECoRI酶切从豌豆     

小麦和水稻叶绿体基因组进化中核苷酸替代的种属特异性

以玉米叶绿体基因组为参照序列,采用三序列比较法系统分析了小麦和水稻分化过程中叶绿体基因组核苷酸替代的发生方式.结果表明,小麦中存在(A+T)/(G+C)替代偏差,水稻则无,该差异对小麦和水稻分化后叶绿体基因组G+C含量产生不同的影响,替代使小麦叶绿体基因组G+C含量降低、水稻叶绿体基因组G+C含量表现增加.无论在编码区、非编码区,还是不同功能基因区,小麦叶绿体基因组转换与颠换的比值都显著低于水稻.小麦和水稻叶绿体基因组进化中核苷酸替代呈现种属特异性.     

甘薯叶绿体rbcL基因的克隆与序列分析

根据烟草、水稻和菠菜叶绿体的全基因组序列设计引物,以甘薯的叶绿体基因组DNA为模板,PCR扩增包舍甘暑叶绿体rbcL完整基因(GenBank登录号为AY942199)在内的一段序列.序列分析表明:此片段的全长为1 627 bp,包括1 443bp的编码区序列在内.推测编码480个氨基酸,同时构建了此片段的限制性酶切图谱.相似性比较显示,此基因编码区序列与烟草、菠菜、小麦、水稻、玉米、矮牵牛、紫花苜蓿、拟南芥、莨菪、葡萄以及甜菜的rbcL基因核苷酸的同源性为85%～98%,氨基酸的同源性为92%～95%.     

Shotgun法测序制备高纯度水稻基因组DNA的方法探讨(英)

在用散弹 (shotgun)法测定水稻 (OryzasativaL .ssp .indica)基因组全序列的过程中 ,叶绿体和线粒体DNA的污染问题非常严峻 .应用脉冲场电泳 (PFGE)技术对水稻基因组DNA进行纯化 ,结果表明它能够有效去除叶绿体和线粒体DNA ,使其污染率从 3%降低到 0 2 % .同时 ,比较了水稻绿苗和黄化苗的DNA得率 ,以及HB法和NIB法制备大分子质量(HMW)DNA的异同 .最后提出一套制备水稻基因组DNA的方法 ,包括黄化苗培养 ;细胞核的分离、包埋和裂解 ;脉冲场电泳纯化、回收聚集在低熔点 (LMP)胶中的水稻HMWDNA .用该方法所得的水稻基因组DNA ,纯度高 (无叶绿体和线粒体DNA污染 )、基因组完整 (机械剪切和降解少 )、回收率高 (操作过程中DNA损失少 ) .另外 ,首次报道在融化的低熔点(LMP)胶中对水稻HMWDNA于 38℃进行超声波处理 ,能够获得用于shotgun文库和梯度文库构建所需要的各种DNA片段(1 5～ 3kb ,3～ 12kb) ,并且效果优于在TE中进行操作     

水稻（Oryza sativa L.）核基因组存在叶绿体psbA基因的同源 …

序列比较说明、重复ＤＮＡ顺序ｐＲＲＤ９＾＊与水稻叶绿体基因组中编码ＱＢ蛋白的ｐｓｂＡ基因存在高度的同源。用ｐＲＢＤ９亚克隆片段ｐＲＲＤ９Ｒ和片段ｐＲＲＤ９Ｌ对水稻的叶绿体和核ＤＮＡ进行Ｓｏｕｔｈｅｒｎ杂交分析,揭示了ｐｓｂＡ基因同源片段在某个进化时期由叶绿体基因组转移到水稻核基因组,而且两者在水稻进化过程中的变异程度存在明显的差异。     

水稻EPSP合酶cDNA克隆、序列分析及其拷贝数测定

根据本室分离的水稻EPSP合酶基因的基因组序列设计一对引物 ,利用RT_PCR方法首次从水稻 (Oryzasati vaL .subsp .indica)叶片的RNA中扩增获得了水稻编码EPSP合酶的全长为 15 85bp的cDNA片段 ,它含有一个完整的开放读码框 ,编码 5 11个氨基酸 ,包括 44 4个氨基酸组成的成熟肽序列以及N端的 6 7个氨基酸组成的叶绿体转运肽序列。成熟肽氨基酸序列对比表明 ,除真菌来源的EPSP合酶变异较大外 ,其他来源的EPSP合酶同源性较高 ,均在 5 1%以上。而叶绿体转运肽氨基酸序列同源性较低。Southern杂交表明水稻EPSP合酶基因在水稻基因组中以单拷贝形式存在。RT_PCR分析表明 ,水稻EPSP合酶基因在根、未成熟种子和叶片中均有转录表达 ,在叶片中表达量最高     

Structural changes in the plastid DNA of rice (Oryza sativa L.) during tissue culture

To investigate the rearrangement of the plastid genome during tissue culture, DNA from rice callus lines, which had been derived individually from single protoplasts isolated from seed or pollen callus (protoclones), was analyzed by Southern hybridization with rice chloroplast DNA (ctDNA) clones as probes. Among 44 long-term cultured protoclones, maintained for 4, 8 or 11 years, 28 contained plastid DNA (ptDNA) from which portions had been deleted. The ptDNA of all protoclones that had been maintained for 11 years had a deletion that covered a large region of the plastid genome. The deletions could be classified into 15 types from their respective sizes and positions. By contrast, no deletions were found in the ptDNA of 38 protoclones that had been maintained for only 1 month. These results indicate that long-term culture causes deletions in the plastid genome. Detailed hybridization experiments revealed that plastid genomes with deletions in several protoclones were organized as head-to-head or tail-to-tail structures. Furthermore, ptDNAs retained during long-term culture all had a common terminus at one end, where extensive rearrangement is known to have occurred during the speciation of rice and tobacco. Morphological analysis revealed the accumulation of starch granules in plastids and amyloplasts in protoclones in which the plastid genome had undergone deletion. Our observations indicated that novel structural changes in the plastid genome and morphological changes in the plastid had occurred in rice cells during long-term tissue culture. Moreover, the morphological changes in plastids were associated with deletions in the plastid genome.     

Intraspecific variation of chloroplast DNA in Dioscorea bulbifera L.

Summary Restriction fragment length polymorphism (RFLP) analysis of chloroplast (ct) DNAs from 15 accessions of Dioscorea bulbifera collected from Africa and Asia was carried out using the Southern hybridization technique. Eight cloned ctDNA fragments of D. bulbifera and D. opposita, which cover 80% of the total chloroplast genome, were used as the probes to detect variation in ctDNA digested with nine restriction endonucleases. Ten variable sites, located in the large and small single-copy regions, were disclosed among the 15 accessions, of which six showed base substitution and four carried length mutation. Positions of the latter mutations were determined on the physical map of ctDNA. Based on these results, chloroplast genomes of the 15 accessions could be classified into nine types. Their phylogenetic relationships are assumed to be as follows: (1) African and Asian chloroplast genomes diverged from each other at the earliest point in time; (2) E-type chloroplast genome, occurring in the south-east edge of the Asian continent, appears to be the most ancient among all the Asian chloroplast genomes; and (3) four chloroplast genomes, found in Asian insular regions, are probably derived independently from the E-type genome. The discrepancy between the taxonomic relationship and the proposed chloroplast genome phylogeny of the present materials is noted.     

OsPPR1, a pentatricopeptide repeat protein of rice is essential for the chloroplast biogenesis

In this paper, we report a novel pentatricopeptide repeat (PPR) protein gene in rice. PPR, a characteristic repeat motif consisted of tandem 35 amino acids, has been found in various biological systems including plant. Sequence analysis revealed that the gene designated OsPPR1 consisted of an open reading frame of 2433 nucleotides encoding 810 amino acids that include 11 PPR motifs. Blast search result indicated that the gene did not align with any of the characterized PPR genes in plant. The OsPPR1 gene was found to contain a putative chloroplast transit peptide in the N-terminal region, suggesting that the gene product targets to the chloroplast. Southern blot hybridization indicated that the OsPPR1 is the member of a gene family within the rice genome. Expression analysis and immunoblot analysis suggested that the OsPPR1 was accumulated mainly in rice leaf. Antisense transgenic strategy was used to suppress the expression of OsPPR1 and the resulted transgenic rice showed the typical phenotypes of chlorophyll-deficient mutants; albinism and lethality. Cytological observation using microscopy revealed that the antisense transgenic plant contained a significant defect in the chloroplast development. Taken together, the results suggest that the OsPPR1 is a nuclear gene of rice, encoding the PPR protein that might play a role in the chloroplast biogenesis. This is the first report on the PPR protein required for the chloroplast biogenesis in rice.     

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.     

Distribution of transfer RNA genes in thePisum sativum chloroplast DNA

Purified chloroplast tRNAs were isolated fromPisum sativum leaves and radioactively labeled at their 3′ end using tRNA nucleotidyl transferase and α³²P-labeled CTP. Pea ctDNA was fragmented using a number of restriction endonucleases and hybridized with thein vitro labeled chloroplast tRNAs by DNA transfer method. Genes for tRNAs have been found to be dispersed throughout the chloroplast genome. A closer analysis of the several hybrid regions using recombinant DNA plasmids have shown that tRNA genes are localized in the chloroplast genome in both single and multiple arrangements. Two dimensional gel electrophoresis of total ct tRNA have identified 36 spots. All of them have been found to hybridize withPisum sativum ctDNA. Using recombinant clones, 30 of the tRNA spots have been mapped inPisum sativum ctDNA.     

Nucleotide sequence of the rice cytochrome f gene and the presence of sequence variation near this gene

The rice (Oryza sativa L. var. Labelle) chloroplast (cp) gene encoding cytochrome f has been isolated and sequenced. The coding region of this rice gene displays 95.1%, 85.3% and 85.2% nucleotide sequence homology with that of wheat, pea and spinach, respectively. To examine the cpDNA sequence variation in rice, cpDNA from Labelle and its parents, Belle Patna and Dawn was compared. Using the cytochrome f gene as the probe for hybridization, we found several differences in the size and number of restriction fragments in the cp genome of three rice varieties. An additional restriction fragment found in the Belle Patna cp suggests that this cp genome is either heterogeneous or contains two copies of cytochrome f gene per cpDNA.