棉花芽黄突变体10个叶绿体蛋白编码基因RNA编辑位点的测定及分析

 RNA编辑是陆生植物叶绿体转录后基因表达调控的一种方式。已有研究表明植物黄化或白化可能与叶绿体RNA编辑有关。本实验采用PCR,RT-PCR及测序的方法,确定棉花芽黄突变体V1子叶与真叶的10个叶绿体蛋白编码基因中各有34个编辑位点,有6个位点存在编辑效率的差异。将这些编辑位点与柯字310比较发现,芽黄突变体多5个编辑位点。运用生物信息学软件对34个编辑位点进行分析,结果表明accD-109, clpP-187, ndhB-50, ndhB-196, ndhD-128, ndhD-225等13个位点会影响相应蛋白二级或三级结构,表明上述编辑位点的改变可能对其蛋白的正确组装及功能的发挥起重要作用。     

Genetic analysis of ion-beam induced extremely late heading mutants in rice

Two extremely late heading mutants were induced by ion beam irradiation in rice cultivar ‘Taichung 65’: KGM26 and KGM27. The F₂ populations from the cross between the two mutants and Taichung 65 showed clear 3 early: 1 late segregation, suggesting control of late heading by a recessive gene. The genes identified in KGM26 and KGM27 were respectively designated as FLT1 and FLT2. The two genes were mapped using the crosses between the two mutants and an Indica cultivar ‘Kasalath’. FLT1 was located on the distal end of the short arm of chromosome 8. FLT2 was located around the centromere of chromosome 9. FLT1 might share the same locus as EHD3 because their chromosomal location is overlapping. FLT2 is inferred to be a new gene because no gene with a comparable effect to that of this gene was mapped near the centromere of chromosome 9. In crosses with Kasalath, homozygotes of late heading mutant genes showed a large variation of days to heading, suggesting that other genes affected late heading mutant genes.     

禾本科植物叶绿体基因组结构的系统进化研究

为了探讨叶绿体基因组结构变化与系统发生的关系,以烟草为参照,系统比较了禾本科植物叶绿体基因组结构的进化。结果发现：（1）大单拷贝序列上产生3个倒置;（2）3个基因accD、ycf1、ycf2退化;（3）clpP、rpoC1内含子序列丢失;（4）IR与SC的边界变化大都发生于科的水平,但也存在一定的层次性。结构变化的系统发生与叶绿体基因内含子构建的系统进化树相一致。叶绿体基因组的结构变化可以用于构建相关植物的系统发生。     

12C重离子束辐照对油菜(Brassica napus)的影响

重离子束是一种新型辐照源，它对油菜的影响以往研究较少。本文报道了30 Gy、50 Gy和80 Gy ¹²C重离子束辐照对油菜M₁和M₂生育期、植物学性状、品质性状，根尖和花粉母细胞的染色体行为和DNA分子多态性等方面的影响。结果表明：50 Gy和80 Gy辐照处理可引起油菜生育期提早，生长繁茂，部分植株发生变异，出现瘤状根、矮茎、淡绿匙形叶，多雌蕊花，双生角果和黄籽株等。80 Gy辐照处理使油菜种子含油量有不同程度提高，并出现油酸含量高于70%以上植株。 30~80 Gy辐照处理，使根尖染色体和花粉母细胞染色体产生畸变，畸变类型有微核、小核、异常四分体、染色体桥、落后染色体、断片等，其中以微核细胞最多，且辐照剂量愈大畸变率愈高。50 Gy和80 Gy辐照处理对油菜DNA分子有影响，RAPD分析表明，用40个引物对处理后油菜进行扩增，共扩增出43个DNA片段，表明不同处理植株存在一定多态性。     

Fine mapping and photosynthetic characteristics of the lower chlorophyll b 1 mutant in rice (Oryza sativa L.)

Chlorophylls absorb and transfer light energy to the photosynthetic system. Consequently, chlorophyll content is strongly related to crop biomass and yield. We isolated a rice spontaneous mutant, lower chlorophyll b 1 (lcb1), from a recombinant inbred line population. Under normal growth conditions, lcb1 plants produced yellow leaves with decreased total chlorophyll and chlorophyll b contents, but normal chlorophyll a content. Photosynthetic and fluorescence parameters differed between wild‐type and lcb1 plants. Compared with wild type, lcb1 had a higher electron transfer rate, a lower photochemical quenching coefficient and significantly reduced grain number, biomass and yield. A recessive nuclear gene controlled the mutant trait. Through map‐based cloning, we located the LCB1 gene in a 117.4‐kb region on the short arm of chromosome 3, close to the centromere, in a region containing 15 predicted candidate genes. None of these genes was directly related to chlorophylls or the chloroplast; therefore, lcb1 may be a mutation of a novel gene. These results will be useful for further research on the molecular mechanisms controlling biogenesis and chloroplast biochemical processes.     

烟草ATP合酶F_0部分4个亚基基因转录本编辑位点分析

RNA编辑是高等植物线粒体基因转录后表达调控的一种重要方式。为探究ATP合酶F_0部分的4个亚基基因与植物雄性不育性的关系,本研究以3个烟草雄性不育系(MS中烟90、MS云烟85和MS K326)及其同型保持系为供试材料,比较分析atp6、atp9、orf25和orf B线粒体基因转录本的编辑位点。结果表明,orf25和orf B基因转录本在不育系和保持系中发生的编辑位点是一致的。atp6基因在不育系中未发生编辑,在保持系中共有6处发生了RNA编辑。与保持系相比,atp9基因在不育系中除8处共同的C→T编辑外,还缺少2个C→T的特异编辑位点,其中1个导致氨基酸类型的改变。推测不育胞质因缺少特异的线粒体基因转录本编辑而导致烟草的细胞质雄性不育。     

Comparison of RAPD, STS, ISSR and AFLP molecular methods used for assessment of genetic diversity in hop (Humulus lupulus L.)

CoxI genomic and cDNA sequences from gymnosperms and angiosperms were used to study the effects of RNA editing on gene evolution and phylogenyre construction. In six gymnosperms harboring edited coxI gene the number of nucleotide substitutions at 1^st, 2^nd and 3^rd codon positions was similar. In contrast, in angiosperms, the number of nucleotide substitutions at 1^st and 2^nd codon positions was much lower than at the 3^rd. The coxI gene in long-lived gymnosperms evolved much faster than in annual angiosperms. This accelerated rate of nucleotide substitution in gymnosperms is due to accumulation of T-C substitutions at edited sites that can randomly appear at all three codon positions. Editing predominantly occurred at 1^st and 2^nd codon positions as a result of selection against non synonymous T-C substitutions and other types of mutations. The tree topologies for the investigated species based on genomic DNA data were in concordance with their taxonomic positions. The trees based on polymorphic edited sites agreed with trees derived from complete sequence information. This indicates that edited sites are reliable sources of phylogenetic information especially for species that contain many edited sites. However, the fast evolution rate of coxI gene in gymnosperms has caused the long branches in the phylogenetic trees. The inclusion of the species with a processed paralog i.e., edited form of the coxI gene, affected the topology of phylogenetic trees, especially when the taxon with a processed paralog was closely related to the other species analyzed. This revised version was published online in July 2006 with corrections to the Cover Date.     

黏类小麦细胞质雄性不育线粒体atp6基因转录本编辑位点

以黏类小麦细胞质雄性不育系ms(Kots)-90-110(A)及其近等可育基因系BC₅F₂为材料,采用克隆测序与PCR产物直接测序方法,对黏类小麦线粒体atp6基因在花药发育各阶段的RNA编辑进行了分析。结果表明,小麦atp6基因保守区DNA序列在供试材料不育系及其近等可育基因系中完全一致,且与普通小麦和提莫菲维小麦atp6基因序列同源性为99%。两种方法测序分析atp6基因转录本保守区RNA编辑的结果规律相似。atp6基因共有15个编辑位点,其中13个发生在密码子的第一和第二位点上,这些位点的编辑都使氨基酸种类发生了变化;有2个发生在密码子的第三位点上,不引起氨基酸种类的变化;其中第6和第7位点是共转录的。随着花药发育时期的推移,各位点的编辑频率逐渐增高。不育系与其近等可育基因系相比,在引入核恢复基因后,各位点的编辑频率明显提高。编辑不充分的转录产物可能会影响线粒体功能的正常发挥,表明黏类小麦细胞质雄性不育与线粒体atp6基因转录本保守区的编辑有一定的相关性。     

Identification and fine mapping of a candidate gene for oil yellow leaf 2 conferring yellow leaf phenotype in maize

Leaf colour is an important agronomic trait for studying molecular mechanisms in chlorophyll biosynthesis and chloroplast development. Here, a novel mutant oil yellow leaf 2 (Oy2) with a typical yellow leaf phenotype at the seedling stage was identified from the mutant population derived from the maize inbred line RP125. Compared with wild type, Oy2 mutant displays decreased chlorophyll content, reduced photosynthetic capacity and impaired chloroplast structure, which is likely controlled by a single recessive gene. The Oy2 locus was then delimited into a 117 kb region on chromosome 5 harbouring four genes, amongst which the gene Zm00001d013013, encoding a magnesium chelatase subunit D, was identified as the only candidate gene associated with Oy2 mutant phenotype. Moreover, the expression levels of candidate gene Oy2 and genes associated with chlorophyll biosynthesis and photosynthesis were tested by RNA‐seq and qRT‐PCR, implying that the causal gene Oy2 playing a critical role in chlorophyll synthesis. Taken together, we propose that the causal gene Oy2 highly associated with the yellow leaf phenotype may be helpful in elucidating photosynthetic pigments biosynthesis and chloroplast development in maize.     

60Co-γ辐照对三七叶片叶绿体和线粒体超微结构的影响

应用透射电子显微镜观察了经不同剂量⁶⁰Co-γ射线辐照的三七叶片叶绿体和线粒体超微结构,研究了⁶⁰Co-γ辐照对三七叶片叶绿体和线粒体超微结构的影响,旨在为三七辐照育种提供理论依据。结果表明：（1）未受辐照情况下,三七叶片细胞形态规则,细胞内叶绿体数量多,基粒片层、基质片层多且有序,光合作用能力强;线粒体形态饱满,双层膜完整,管状内嵴清晰可见,基质浓厚。（2）当辐照剂量为10 Gy时,叶绿体和线粒体超微结构发生变化,但受影响程度较轻。叶绿体被膜轻微破坏,内部基粒片层和基质片层结构基本不受影响,淀粉粒增大增多;多数线粒体结构完整,数量略微增加,可以看出,线粒体对⁶⁰Co-γ射线辐照的耐受性比叶绿体强。（3）随着辐照剂量的增加,叶绿体和线粒体超微结构受损伤程度随之增强。当辐照剂量达到40 Gy时,叶绿体大量解体,空泡大量出现,并连成大的空洞,结果必然影响叶绿素含量和光反应的正常进行,从而显著减弱光合作用强度;部分线粒体内部降解严重甚至已完全空泡化,线粒体数目明显减少,线粒体结构和功能遭到不可逆的严重损伤,细胞衰老严重。上述结果表明,⁶⁰Co-γ射线辐照对三七叶片叶绿体和线粒体超微结构产生不同程度的损伤,但适当的辐照可增加线粒体数量。以上结论可以为不同目的的三七育种提供理论依据,并为选择最适辐照诱变育种剂量提供理论参考。     

棉花叶绿体基因组的研究进展

近年来,随着分子生物学技术的应用与发展,对棉花叶绿体基因组也有了新认识。本文概述了棉花叶绿体基因组的研究进展,从棉花叶绿体基因组的图谱、功能基因的克隆及研究、叶绿体RNA编辑以及叶绿体转化等方面进行了介绍和评述,并对其在基础研究与应用研究中的前景进行了展望。     

RNA编辑研究进展

RNA编辑是近年来发现的一种转录后水平的修饰方式,通过改变单个核苷酸使得转录的成熟RNA与模板DNA的编码序列不完全一致。随着分子生物学的深入研究,一些新的RNA编辑类型不断被发现,有些编辑类型揭示RNA编辑具有一定的记忆功能。本文从RNA编辑的类型,RNA编辑的机制和生物学意义对RNA编辑进行简单的介绍。     

Development of SNP assays associated with oleic acid QTLs in N00-3350 soybean

Increasing the amount of oleic acid in soybean oil would reduce the need for hydrogenation, a process that creates unhealthy trans fatty acids. A previous study mapped six oleic acid quantitative trait loci (QTLs) from N00-3350 (~550 g kg⁻¹ oleic acid) on soybean chromosomes (Gm) 5, 17, 18, and 19, which corresponds to linkage groups (LG) A1, D2, G, and L, respectively. The objectives of this study were to develop high throughput assays to detect single nucleotide polymorphism (SNP) markers associated with these oleic acid QTLs. Several candidate genes that are putatively responsible for the oleic acid phenotype in N00-3350 were identified by BLAST searches against the soybean genomic assembly using the sequences of fatty acid synthesis genes from soybean and Arabidopsis. Putative accC-2 and KAS I genes were located in the region of the cqOle-001 on Gm05 (LG-A1). A putative KAS I gene was located in the region of the cqOle-003 on Gm18 (LG-G) and a putative accB-1 gene was located in the region of the cqOle-004 on Gm18 (LG-G). These fatty acid candidate genes and sequence-tagged sites were used to identify SNPs between N00-3350 and ‘Boggs’. Based on these SNPs, seven SimpleProbe probes for melting curve analysis were developed to rapidly identify the alleles from the mid-oleic acid soybean line N00-3350 at the desired loci. These assays provide a high-throughput and rapid SNP genotyping method which can be used to accelerate the development of mid-oleic acid content soybean cultivars.     

Effect of gamma and ultraviolet irradiation on adventitious regeneration from in vitro cultured pear leaves

Summary Irradiation of in vitro explants and subsequent adventitious regeneration has been tested for 4 commercially important varieties of pear (Pyrus communis) with the aim to create mutants with a reduced susceptibility to fire-blight (Erwinia amylovora). The effect of gamma and ultraviolet irradiation of leaves on adventitious regeneration ability has been studied. The LD50 (50% decrease of regeneration) after gamma irradiation was genotype-dependent and was between 20 and 50 grays. The curves of regeneration showed a threshold dose underneath which none or a very slight decrease was registered. The decrease might result from cumulative events. After an ultraviolet irradiation as low as 62.5 J/m², the leaves became crumbly and rolled up, and their metabolism seemed to be altered. The LD50 was about 125 J/m² for all varieties and the decrease of regeneration was linear. Histological investigations showed leaves with flattened epidermal cells after ultraviolet irradiation and slack spongy parenchyma after gamma irradiation.Abbreviations Gy grays - J joules - LD lethal dose     

Physical mapping of differences between the chloroplast DNAs of Beta vulgaris and B. webbiana

A physical map of Beta webbiana (a wild species of the section Pro-cumbentes) chloroplast genome was constructed by localizing the cleavage sites of SmaI, PstI, PvuII, XhoI, and HindIII, and the map was then aligned with the map of sugarbeet (B. vulgaris) chloroplast DNA. This alignment shows 27 restriction-site changes and 11 insertions/deletions, most of which occur in the large single-copy region of the genome. A 0.7-kb long mutation, located within an unidentified open reading frame (ORF2280) in the inverted repeat, was also found.     

Evaluation of breeding semidwarf rice by induced mutation and hybridization

Summary Semidwarf indica varieties of rice (Oryza sativa L.) were developed by induced mutation. They generally have a yield capacity equal to that of the semidwarf varieties (T(N)1, IR 8 and others) selected from hybridization. Genetic studies showed that semidwarf genes induced in the mutants and spontaneous ones in existing varieties are at the same locus. These semidwarf mutants, selected in Taiwan and from an American long-grain variety, Centura Patna 231, were compared with their parents, the semidwarf variety Dee-goe-woo-gen, T(N)1 and IR 8. The semidwarf mutants average about 35% shorter than their tall parents. The plant height is reduced by shortening five internodes from the top, while panicle length is not affected. The mutants, Dee-geo-woo-gen, and improved short-statured varieties had similar patterns of internode elongation. The mutants derived from varieties sensitive to photoperiod have lost the sensitivity. One more crop can be grown within a year. All induced and hybrid-derived semidwarf varieties were highly susceptible to bacterial leaf blight when Xanthomonas oryzae was inoculated although some of their parents were moderately resistant. The mutation rate from irradiation of this semidwarf gene was estimated to be 0.1% in I-kung-bau, a native variety of Taiwan. This particular type of mutation has not yet been reported in japonica varieties. However, several promising strains with reduced plant height, earlier maturity and more grain yielding were recently obtained from the California japonica variety, Calrose, by X-irradiation. The merits of mutation breeding in rice are discussed.     

Decay of the Chloroplast Pool of Ascorbate Switches on the Oxidative Burst in UV‐B‐Irradiated Rice

A parallel was drawn between UV‐B irradiation and the oxidative burst in the chloroplast lamellae of rice plants (Oryza sativa L. cv. Safari). After irradiation with a total biological effective UV‐B of 20.825 kJ m^?2 between the 15th and 21st days following germination, it was found that superoxide, hydroxyl radicals and hydrogen peroxide increased significantly in stressed leaves. The levels of ascorbate and the kinetics of superoxide dismutase, glutathione reductase, dehydroascorbate reductase, ascorbate peroxidase and catalase became increasingly inhibited. The developing oxidative burst also diminished photosynthetic pigments and galactolipids accumulation, while the de‐epoxidation state of xanthophylls and lipids peroxidation increased. Following grana disorganisation, the accumulation of chloroplast polypeptides having 72/69, 33/32, 28/26, 22/20 and 18/16 kDa decreased, but the relative proportion of the 49/46 kDa polypeptides increased. The 69/65 kDa polypeptides also diminished on the 15th day. These alterations implicated the failure of both photosystems functioning, but the inhibition of the Hill reactions coupled to the water splitting complex was uppermost. In the 28th day, the chloroplast lamellae of leaves grown after UV‐B exposure revealed a general recovery from oxidative burst and a subsequent higher stacking of thylakoids. It is concluded that UV‐B irradiation becomes lethal when ascorbate oxidation in the xanthophyll cycle limits the antioxidant enzymes kinetics, triggering chloroplast proteolysis and acyl lipid peroxidation because of reactive oxygen species propagation.     

不同水分供给对小麦叶与非叶器官叶绿体结构和功能的影响

在田间不同灌水条件(春季不灌水、春季灌2水和春季灌4水, 每次灌水750 m3 hm^-2)下, 于灌浆后期对小麦叶片与非叶器官(穗颖、穗下节和叶鞘)叶绿体结构进行了电镜观察, 并对不同器官的净光合速率(P_n)和光化学效率(F_v/F_m)进行了测定, 以期探明小麦叶与非叶器官光合结构和功能对水分变化的适应性差异。结果表明, 在充足供水条件(春季灌4水)下, 叶片的叶绿体数目明显多于各非叶器官, 但护颖和外稃叶绿体含有较多的淀粉粒。在灌浆期上层土壤重度水分亏缺(春季不灌水)条件下, 植株各器官叶绿体均出现明显的衰老特征, 但衰老程度存在器官间显著差异, 旗叶叶片叶绿体受损程度明显大于非叶器官。在所测非叶器官中, 外稃叶绿体对干旱胁迫反应较为敏感, 而护颖、穗下节间和旗叶鞘叶绿体结构具有较强的稳定性。水分胁迫下各器官P_n和F_v/F_m值均降低, 叶片降低幅度最大, 且随灌浆进程其光合下降最快, 但穗下节间、旗叶鞘和穗器官较为稳定, 可能与其叶绿体结构的稳定性有关。说明小麦非叶绿色器官光合结构与功能对水分亏缺具有较强的耐性。