利用水稻C0t-1 DNA和基因组DNA对栽培稻、药用野生稻和疣粒野生稻基因组的比较分析

【目的】研究中高度重复序列在稻属不同物种基因组进化中的作用。【方法】用栽培稻C0t-1 DNA和基因组DNA(gDNA)作为探针，分别对栽培稻、药用野生稻和疣粒野生稻进行荧光原位杂交(FISH)和比较基因组杂交(CGH)。【结果】C0t-1 DNA覆盖栽培稻、药用野生稻和疣粒野生稻基因组比例(%)和大小(Mb)分别为47.10±0.16，38.61±0.13，44.38±0.13和212.33±1.21，269.42±0.89以及532.56±1.68。栽培稻gDNA在药用野生稻和疣粒野生稻基因组中的覆盖率约为91.0%和93.6%，含量分别约为634 Mb和1123 Mb，各有365 Mb和591 Mb不属于源自栽培稻基因组的中高度重复序列，未被栽培稻gDNA所覆盖的部分，分别为64 Mb和78 Mb左右。此外，以C0t-1 DNA的组成为依据，对这3个种核型进行了同源性聚类。【结论】稻属中度和高度重复序列和功能基因一样，在不同种中也存在着高度同源性和保守性，并在进化过程中得以保存下来。药用野生稻和疣粒野生稻基因组增大的重要原因之一，可能是基因组中度和高度重复序列加倍的结果，药用野生稻这种序列扩增相对疣粒野生稻要缓和得多。另外，这两个野生种在长期进化过程中，由于存在加倍、重排和基因选择性丢失等现象，形成了具有自己种的特异性的基因组成分。     

栽培稻、斑点野生稻、药用野生稻基因组比较分析

以栽培稻总DNA为探针,对栽培稻(AA)自身、斑点野生稻(BB)以及药用野生稻(CC)体细胞染色体进行基因组荧光原位杂交(GISH),并以斑点野生稻总DNA为探针,对自身和药用野生稻体细胞染色体进行基因组荧光原位杂交,以此研究A、B、C 3个基因组型之间的关系.结果显示,A、B、C基因组之间都存在较高的同源性,其中AA与CC之间的信号最强,BB基因组与AA基因组次之,BB基因组与CC基因组的信号最弱.说明A、B、C 3个基因组之间的亲缘关系,A与C最近,B与C最远.     

利用C基因组对高杆野生稻和宽叶野生稻基因组的比较分析

1材料与方法1．1植物材料和染色体制片药用野生稻稻株1589由广东省国家野生稻圃提供,宽叶野生稻IRW6和高杆野生稻IRW41由华南农业大学卢永根院士提供,试验材料情况见表1。染色体制片分别参照Yan等和Ren等的方法。     

利用C基因组对高杆野生稻和宽叶野生稻基因组的比较分析(英文)

[Objective] Genomic in situ hybridization (GISH) was used to study the relationship between the two CCDD genomes of Oryza alta and Oryza latifolia. [Method] Total DNA of Oryza officinalis (C-genome) was used as a probe for genomic in situ hybridization on metaphase chromosomes from Oryza alta and Oryza latifolia, respectively. [Result] Under certain post-hybridization washing stringencies, C- and D-genome could be distinguished in CCDD genome type; there were huge differences in some CC chromosomes of Oryza alta, Oryza latifolia, and Oryza officinalis. The genome of Oryza latifolia was more original. [Conclusion] Comparative analysis of the Oryza species with identical genome type may facilitate to elucidate the possible approaches to plant genome evolution and species evolution.     

利用C基因组对高杆野生稻和宽叶野生稻基因组的比较分析

[目的]采用基因组原位杂交（GISH）技术研究稻属2种CCDD基因组型的野生稻宽叶野生稻和高杆野生稻基因组之间的关系。[方法]利用药用野生稻C基因组总DNA为探针,分别对高杆野生稻和宽叶野生稻中期染色体进行基因组原住杂交。[结果]杂交结果显示,在一定的洗脱严谨度下,可以把CCDD染色体组中的C、D基因组染色体分开,并且发现高杆野生稻的CCDD基因组中的某些属于CC基因组的染色体与宽叶野生稻和药用野生稻中的CC基因组染色体存在较大差异,宽叶野生稻的基因组更加原始。[结论]对稻属中有相同基因组型的种进行比较分析,将有助于深入阐明植物基因组进化和物种进化及可能的途径。     

利用栽培稻基因组DNA对非洲野生稻进行染色体荧光原位杂交分析

以地高辛标记的栽培稻基因组(基因组为AA)DNA为探针,对非洲野生稻(基因组为BBCC)的体细胞染色体进行荧光原位杂交分析,研究AA染色体组和BBCC染色体组之间的关系,同时对杂交后的染色体进行同源染色体配对。结果表明:栽培稻A基因组和非洲野生稻基因组有较高的同源性,其中高度重复DNA序列在栽培稻和非洲野生稻间具有保守性。     

利用45S rDNA探针对宽叶野生稻和高秆野生稻基因组的FISH分析

利用45S rDNA作为探针,通过荧光原位杂交(FISH)技术对同样含有CCDD基因组的高秆野生稻(Oryza alta)和宽叶野生稻(O.latifolia)进行rDNA的荧光原位杂交定位分析和核型分析。结果显示:宽叶野生稻中45S rDNA信号分布于多条染色体上,位点数目为10~16;高秆野生稻中有6个信号点,分布于3对同源染色体上,其中2对信号位点位于染色体短臂,1对位于染色体长臂。研究结果表明,高秆野生稻45S rDNA在基因组中位点数目稳定,宽叶野生稻中45S rDNA位点数在不同个体中呈现一定的动态变化,显示这2种野生稻基因组存在一定差异;核型分析结果也表明二者基因组存在较大的差异。由此推测,高秆野生稻分化较早而趋向稳定,宽叶野生稻可能形成较晚,还处于进化过程之中。鉴于二者在基因组结构上的明显差异和进化上的不平衡性,建议把这2种野生稻划分为不同野生稻种,可能会更加符合二者的进化特性。同时,讨论了45S rDNA在染色体中分布特点与机制。     

唐菖蒲蛋白磷酸酶GhPP2C1基因功能及其对ABA的响应

为探究唐菖蒲GhPP2C1（Protein phosphatase 2C1）的功能及对脱落酸（Abscisic acid,ABA）的响应,以唐菖蒲‘Rose Supreme’为试材,利用同源克隆的方法获得ABA信号转导过程中的关键蛋白磷酸酶基因GhPP2C1,利用实时荧光定量分析、亚细胞定位和启动子β-葡萄糖苷酸酶（GUS）染色等方法检测其对ABA的响应情况;对其启动子序列进行响应元件分析;在拟南芥中异源过表达GhPP2C1基因,对转基因株系进行ABA敏感性测定、株型分析及下游基因表达情况分析。结果表明：1）GhPP2C1的表达在ABA处理的诱导下显著上调。2）GhPP2C1定位于细胞质中,可被ABA处理诱导入核。3）GhPP2C1基因启动子包含ABA、赤霉素（Gibberellins,GAs）、干旱和低温相关响应元件。在烟草叶片和拟南芥根尖中,GhPP2C1启动子活性可被ABA激活。4）在拟南芥中异源过表达GhPP2C1基因导致ABA信号通路中下游基因的表达量显著下降并出现分枝数、株高和种荚数量明显增加的表型。综上,推测唐菖蒲GhPP2C1可能通过减弱ABA信号传导,促进种子休眠解除,且在植株营养生长过程中也发挥作用。     

STK1基因对山梨醇高渗胁迫下玉米大斑病菌菌丝生长和发育的调控

【目的】明确STK1基因在山梨醇高渗胁迫条件下对玉米大斑病菌菌丝生长和发育的调控作用。【方法】将野生型菌株(WT)与STK1基因敲除突变体(KO)分别接种在不同山梨醇浓度的葡萄糖-蛋白胨(DPA)培养基上,对比其生长速度、菌落形态、菌丝形态的差异变化;并通过油红O染液进行菌丝细胞脂肪染色,观察脂类物质沉积的形态学变化。【结果】山梨醇高渗胁迫显著抑制了野生型菌株(WT)的菌落生长速度,但1.0 mol/L山梨醇对STK1基因敲除KO菌株的生长有显著的促进作用,这种促进作用随着山梨醇浓度的增加而迅速降低,山梨醇浓度增加至1.5~2.0mol/L时,菌落生长速度受到显著抑制,并且抑制程度显著高于WT菌株。显微观察菌丝细胞内容物,发现山梨醇高渗胁迫后,KO菌株的颗粒状物明显少于WT菌株。利用油红O染色观察菌丝细胞的脂类物质沉积情况,发现在山梨醇高渗胁迫条件下,KO菌株的脂滴数量远远少于WT菌株。【结论】山梨醇胁迫具有与盐胁迫不同的渗透调节机制,STK1基因可能通过调节脂类物质的合成及分布调控对山梨醇高渗透胁迫的调节。     

Comparative Analysis of Genomes in Oryza sativa, O.officinalis, and O. meyeriana with C0t-1 DNA and Genomic DNA of Cultivated Rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosomes preparations of Oryza sativa, O. officinalis, and O. meyeriana with labeled probes of C0t-1 DNA and genomic DNA from the cultivated rice. The coverage percentage (%) and size (Mb) of C0t-1 DNA in O. sativa, O. officinalis, and O. meyeriana were 47.1 ±0.16, 38.61 ±0.13, 44.38±0.13, and 212.33± 1.21,269.42 ± 0.89, 532.56± 1.68 Mb, respectively. The coverage percentage and size of genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0, 93.6% and 634, 1 123 Mb, respectively, in which 365 and 591 Mb in O. officinalis and O. meyeriana were from O. sativa genomic DNA, but not from repetitive sequences of O. sativa, and the uncoverage genome size in O. officinalis and O. meyeriana were 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C0t-1 DNA in O. sativa, O. officinalis, and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during evolution. The repetitive sequences reduplication may be one of the important causes of the genome enlargement of O. officinalis and O. meyeriana, and O. officinalis genome enlarged more slowly when compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana were formed by reduplication, rearrangement, and gene selective loss during the evolution process.     

Comparative analysis of genomes in Oryza sativa, O. officinalis and O. meyeriana with C 0 t-1 DNA and genomic DNA of cultivated rice

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) were applied to somatic chromosome preparations of Oryza sativa, O. officinalis and O. meyeriana with labeled probes of C ₀ t-1 DNA and genomic DNA from cultivated rice. The coverage percentage (%) and size (Mb) of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana were 47.1 ± 0.16, 38.61 ± 0.13, 44.38 ± 0.13 and 212.33 ± 1.21, 269.42 ± 0.89, 532.56 ± 1.68, respectively. The coverage percentage and size of probe signals with genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0%, 93.6% and 634 Mb, 1 123 Mb respectively, in which there were 365 and 591 Mb in O. officinalis and O. meyeriana which came from O. sativa genomic DNA not from repetitive sequences of O. sativa, and the uncovered genome size in O. officinalis and O. meyeriana was 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of C ₀ t-1 DNA in O. sativa, O. officinalis and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during the evolution process. The repetitive sequence reduplication might be one of the important causes of genome enlargement in O. officinalis and O. meyeriana; the O. officinalis genome enlarged more slowly compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana formed by reduplication, rearrangement and gene selective loss during the evolution process. Translated from Scientia Agricultura Sinica, 2006, 39(6): 1083–1090 [译自: 中国农业科学]     

Comparative karyotypic analysis of A and C genomes in the genus Oryza with C 0 t-1 DNA and RFLP

Fluorescence in situ hybridization (FISH) was applied to somatic chromosomes preparations of Oryza sativa L. (AA), O. glaberrima (AA), and O. officinalis Wall. (CC) with a labeled probe of C ₀ t-1 DNA. Genomic in situ hybridization to its own chromosomes (self-GISH) was conducted in a control experiment. The homologous chromosomes showed similar signal bands probed by C ₀ t-1 DNA, while karyotypic analysis of chromosomes between A genome in the two cultivated species and C genome in O. officinalis were conducted based on the band patterns. The ideograms with C ₀ t-1 DNA signal bands were also built. The nonuniform distribution of hybridization signals of C ₀ t-1 DNA from O. sativa and that on its own chromosome of O. officinalis were observed. However, the similarity and correspondence between C ₀ t-1 DNA signal patterns and genomic DNA signal patterns indicated that the self-GISH signals actually resulted from the hybridization of genomic repetitive sequences to the chromosomes. The restriction fragment length polymorphism (RFLP) marker, R2676, from the chromosome 8 of O. sativa and O. officinalis, was used as a probe to somatic hybrid on chromosomes for comparative karyotypic analysis between O. glaberrima and O. officinalis. The results showed that R2676 was located on the short arm of chromosome 7 in O. officinalis and chromosome 4 in O. glaberrima. The percentage distances from the centromere to hybridization sites were 91.56±5.62 and 86.20±3.17. Our results revealed that the relative length of O. officinalis chromosome 8 does not follow conventional chromosome length in descending order of number. C ₀ t-1 DNA of A genome signals were detected in the end of the short arm of O. officinalis chromosome 8, indicating that the highly and moderately repetitive DNA sequences in this region were considerably similar between C and A genomes. However, the fluorescence intensity on the chromosomes of C ₀ t-1 DNA of A genome was less than that of its own C genome from O. officinalis, which would be one of the causes for the fact that highly and moderately repetitive DNA sequences were amplified in O. officinalis. No homology signal of C ₀ t-1 DNA from O. sativa was detected in the end of the long arm of O. glaberrima, indicating that repetitive DNA sequences of A genome in two cultivated rice were lost in the evolutional history. In this paper, using comparative karyotypic analysis of RFLP combined C ₀ t-1 DNA signal bands, the evolutionary mechanism of genome in genus Oryza was also discussed.     

Comparative Analysis of Genomes in Oryza sativa, O. officinalis, and O. meyeriana with Cot- 1 DNA and Genomic DNA of Cultivated Rice

Fluorescence in situ hybridization （FISH） and comparative genomic hybridization （CGH） were applied to somatic chromosomes preparations of Oryza sativa, O. officinalis, and O. meyeriana with labeled probes of Cot-1 DNA and genomic DNA＇from the cultivated rice. The coverage percentage （%） and size （Mb） of Cot-1 DNA in O. sativa, O. officinalis, and O. meyeriana were 47.1 ±0.16, 38.61 ±0.13, 44.38＋_0.13, and 212.33 ± 1.21,269.42 ± 0.89, 532.56± 1.68 Mb, respectively. The coverage percentage and size of genomic DNA from O. sativa in O. officinalis and O. meyeriana were 91.0, 93.6% and 634, 1 123 Mb, respectively, in which 365 and 591 Mb in O. officinalis and O. meyeriana were from O. sativa genomic DNA, but not from repetitive sequences of O. sativa, and the uncoverage genome size in O. officinalis and O. meyeriana were 64 and 78 Mb, respectively. In addition, karyotype analysis was conducted based on the signal bands of Cot-1 DNA in O. sativa, O. officinalis, and O. meyeriana. The results showed that highly and moderately repetitive sequences in Oryza genus were conserved as the functional genes during evolution. The repetitive sequences reduplication may be one of the important causes of the genome enlargement of O. officinalis and O. meyeriana, and O. officinalis genome enlarged more slowly when compared with O. meyeriana. Based on the above results, it is concluded that O. officinalis and O. meyeriana were formed by reduplication, rearrangement, and gene selective loss during the evolution process.     

野生稻细胞核DNA提取的研究

药用野生稻是中国最具利用价值的野生稻资源之一,具有许多优良的性状和有利基因,是改良栽培稻培育新品种的宝贵基因库,而其CC基因组的优良基因有望以大片段形式通过BIBAC等载体转化到栽培稻中.BIBAC文库不仅能作为大片段基因组文库的载体,而且能通过根癌农杆菌介导将克隆片段导入植物基因组直接进行转化,可用于筛选分离基因等研究.如何获得基因组DNA大片段非常关键,本研究采取 琼脂糖包埋基因组的方法获得大片段DNA,并对其中的一些步骤进行了优化,为构建药用野生稻BIBAC文库打下了很好的基础,值得在其它植物类似研究中参考.     

Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis

Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa.It has a number of elite genes for rice breeding in resistance or tolerance.However,breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O.sativa and O.officinalis.Characteristics of the breeding barriers were systemically studied in this paper.When both the diploid (AA,2n=2x=24) and autotetraploid (AAAA,2n=4x=48) cultivated rice were crossed as maternal parents with O.officinalis (CC,2n=2x=24),none F1 hybrid seeds were obtained.The young hybrid ovaries aborted at 13-16 d after pollinations (DAP).By rescuing hybrid embryos,in vitro F1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5％.Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4xx2x.Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale.Almost no normal endosperm cells formed at 3 DAP.Development of a lot of embryos ceased at globular- or pear-shaped stage as well as some degenerated gradually.The hybrid plantlets were both male and female sterility.Due to the abnormal development,a diversity of abnormal embryo sacs formed in hybrids,and hybrid pollen grains were typically abortive.It showed that conflicts ofgenome A and C in hybrid induced abnormal meioses of meiocytes.     

栽培稻与Oryza officinalis相似群野生稻基因组VDAC基因的比较

根据水稻基因组文库日本晴的基因组VDAC基因的序列,设计引物分别扩增6种不同基因组的野生稻(BB、CC、BBCC及CCDD)和2种栽培稻(AA)的基因组DNA的VDAC基因片段.所有分别代表8个VDAC基因的8对引物中,除引物VDAC3外,其它7对引物均能扩增出预期大小的特异条带,其中一些片段是所有试验材料共有的,而另一些则具有明显的基因组或种的特异性.AA、BB、CC基因组均具有VDAC1、VDAC4、VDAC7和VDAC8引物的扩增位点,而DD基因组则不能确定.VDAC6的引物位点是AA基因组所特有.VDAC2引物的扩增位点存在于基因组AA、BB、DD,而CC基因组中则无此位点.而VDAC5则可区分同为CCDD的宽叶野生稻和高秆野生稻.栽培稻种与野生稻种基因组相比能扩增出更多的VDAC基因片段的试验结果,为进一步研究稻属中VDAC基因的起源及进化关系提供了基础.     

云南药用野生稻BIBAC文库的构建及分析

云南药用野生稻具有许多优良性状和有利基因,其CC基因组的优良基因很难通过有性杂交转移到栽培稻(AA基因组)上,但可以通过双元细菌人工染色体(BIBAC)以大片段的形式转化到栽培稻中.利用BIBAC2载体构建了云南药用野生稻基因组DNA文库,该文库包含53 760个克隆,平均插入片段76 kb,保存在140块384孔板中...