欧石楠体细胞胚发生过程中的DNA甲基化

利用甲基化敏感扩增多态性（MSAP）方法,对欧石楠大田苗、胚性愈伤组织和再生苗的DNA甲基化进行了研究。从64对选扩增引物中筛选出19对,共扩增得到506条带,统计显示,大田苗、胚性愈伤组织和再生苗的全基因组DNA甲基化水平分别为31．42％、27．86％和29．05％,3种试材发生甲基化变异的有175条带,变异率为34．58％。体细胞胚诱导形成胚性愈伤组织过程中,甲基化水平降低,而在再生苗中有所恢复,与大田苗接近。在外侧胞嘧啶甲基化水平上,胚性愈伤组织的甲基化水平有所增加,且在再生苗中可部分维持。另外,在175条变异带中,再生苗恢复到大田苗DNA甲基化模式的有62条,占总变异条带的35．43％,而与胚性愈伤组织维持相同DNA甲基化模式的有59条,占33．71％。回收部分甲基化变异条带,最终得到8条有效的基因组DNA序列。BLASTnI：对分析表明,在欧石楠基因组中,包括抗性基因、蛋白激酶、质体基因等在内的多种DNA序列均存在DNA基化修饰现象。     

机械伤害处理橡胶树萌条树皮的DNA甲基化分析

该研究采用甲基化敏感扩增多态性技术,分析了机械伤害处理橡胶树萌条树皮的DNA甲基化的变化。结果显示：（1）与对照相比,伤害后0.5和2 h,DNA甲基化水平略有上升;伤害后48 h的DNA甲基化水平出现了较大幅度的下降。（2）甲基化变化类型分析表明,在伤害2 h主要发生了DNA的甲基化;伤害后48 h,主要发生了DNA的去甲基化。（3）差异甲基化位点的回收、测序及注释表明,ATP合酶F1亚基1、磷酸核糖胺 甘氨酸类连接酶、冷激结构域蛋白3、光系统II 47 kD 蛋白、E3泛素蛋白连接酶RING1、NADH泛醌氧化还原酶和一些假定蛋白参与了伤害的响应。（4）经重亚硫酸盐测序验证,ATP合酶F1亚基1和NADH泛醌氧化还原酶的CCGG位点发生了去甲基化。研究推断DNA的甲基化可能参与了橡胶树萌条对机械伤害的响应。     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对15种柑橘胚性愈伤组织进行体细胞胚诱导,发现逆境处理有利于体细胞胚发生,并可以恢复部分品种的体细胞胚发生能力.对具有和失去体细胞胚发生能力的两种纽荷尔脐橙( Citrus sinensis Osb.)愈伤组织进行随机扩增多态性DNA (RAPD) 分析没有检测到带型的差异,而对它们的甲基化敏感扩增多态性 (MSAP) 进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低.     

逆境处理和DNA甲基化影响柑橘体细胞胚发生

对 1 5种柑橘胚性愈伤组织进行体细胞胚诱导 ,发现逆境处理有利于体细胞胚发生 ,并可以恢复部分品种的体细胞胚发生能力。对具有和失去体细胞胚发生能力的两种纽荷尔脐橙 (CitrussinensisOsb .)愈伤组织进行随机扩增多态性DNA (RAPD)分析没有检测到带型的差异 ,而对它们的甲基化敏感扩增多态性 (MSAP)进行分析则发现两种愈伤组织间具有明显的DNA甲基化差异 ,具体细胞胚发生能力的愈伤组织的甲基化水平较失去体细胞胚发生能力的低     

枸杞体细胞胚发生中DNA代谢动态的立体计量

本文以宁夏枸杞无菌苗叶片为材料,离体培养,并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数学图像处理软件计量由光学底片经Ａ／Ｄ转换成的数学图像中的ＤＮＡ大分子,对枸杞体细胞胚发生过程中ＤＮＡ分子的代谢动态进行量化分析。结果表明：在整个体细胞胚发和过程中ＤＮＡ代谢呈现动态变化。非胚性细胞与胚性细胞期的量化值分别为１．８２％和１．９１％;在二细胞胚、四细胞胚、多细胞胚时期ＤＮＡ缓慢增长,随着胚性愈     

镉胁迫下萝卜基因组DNA甲基化敏感扩增多态性分析

应用甲基化敏感扩增多态性(MSAP)技术分析了重金属镉(cd)胁迫处理后萝卜基因组DNA甲基化程度的变化。结果表明,经50、250和500mg/L CdCl_2处理后,MSAP比率分别为37%、43%和51%,均高于对照(34%);全甲基化率(双链C~mCGG)分别为23%、25%和27%,而其对照为22%,表明重金属CdCl_2胁迫后,某些位点发生了重新甲基化。萝卜叶片DNA中总甲基化水平的增加与CdCl_2处理浓度呈显著正相关。甲基化变异可分为重新甲基化、去甲基化、不定类型以及与对照相同的甲基化模式等类型,Cd胁迫处理引起的植株基因组DNA甲基化程度的提高主要是重新甲基化。     

病毒侵染对西伯利亚百合DNA甲基化的影响

采用基于AFLP的甲基化敏感扩增多态性(MSAP)技术,用10对引物对侵染百合花叶病毒和丛簇病毒的西伯利亚百合植株和无毒植株进行DNA甲基化水平和模式分析.结果发现,西伯利亚百合无毒植株和病毒侵染植株的平均甲基化水平分别为40.1%和31.5%;平均全甲基化率分别为13.0%和9.7%;半甲基化率分别为27.1%和21.8%.研究表明,百合DNA甲基化多以半甲基化的形式存在;病毒侵染导致百合植株DNA甲基化水平降低,且对整体甲基化水平、全甲基化水平和半甲基化水平均产生了影响;说明病毒侵染百合后植株出现的症状在一定程度上与DNA甲基化存在关联.     

枸杞体细胞胚发生中DNA代谢动态的立体计量

本文以宁夏枸杞无菌苗叶片为材料,离体培养,并诱导体细胞胚胎发生。根据细胞形态计量学原理,应用数字图像处理软件计量由光学底片经A/D转换成的数字图像中的DNA大分子,对枸杞体细胞胚发生过程中DNA分子的代谢动态进行量化分析。结果表明：在整个体细胞胚发生过程中DNA代谢呈现动态变化。非胚性细胞与胚性细胞期的量化值分别为1.82%和1.91%;在二细胞胚、四细胞胚、多细胞胚时期DNA缓慢增长,随着胚性愈伤组织的发育,DNA的含量在梨形胚时期达到高峰;成熟胚的DNA含量虽有所下降,但仍维持较高水平。因此DNA的合成动态变化与体胚生长发育和细胞增殖密切相关。     

光暗条件下大蒜DNA甲基化差异的初步研究

用甲基敏感扩增多态性(MSAP)技术分析光暗条件下生长的大蒜DNA甲基化水平差异。结果表明,用8对引物组合扩增出343条带,完全一致的带型240条,不一致的103条。在不一致的条带中,光照条件下相同的47条带与黑暗条件下相同的带型有差异,与黑暗相比光照下出现的去甲基化带28条,有56条差异带在2个处理内个体间也有差异。总的趋势是光照引起大蒜DNA去甲基化。     

植物体细胞胚发生过程中基因表达的研究进展

植物体细胞胚胎发生是一个复杂的发育过程,研究者们通过分析植物体细胞胚发生过程中的基因表达或胚性组织和非胚性组织中基因的差异表达,获得了在体细胞胚发生过程不同时期表达的基因,并分析了这些基因在胚胎发生途径中可能的作用。综述了在植物体细胞胚发生过程中细胞周期相关基因、胁迫和激素应答相关基因、信号转导相关基因、晚期胚胎丰富蛋白基因及与体细胞胚发生相关的胞外蛋白基因表达的研究进展。     

Genetic transformation and regeneration of rubber tree (Hevea brasiliensis Muell. Arg) transgenic plants with a constitutive version of an anti-oxidative stress superoxide dismutase gene

Agrobacterium tumefaciens-mediated genetic transformation and the regeneration of transgenic plants was achieved in Hevea brasiliensis. Immature anther-derived calli were used to develop transgenic plants. These calli were co-cultured with A. tumefaciens harboring a plasmid vector containing the H. brasiliensis superoxide dismutase gene (HbSOD) under the control of the CaMV 35S promoter. The -glucuronidase gene (uidA) was used for screening and the neomycin phosphotransferase gene (nptII) was used for selection of the transformed calli. Factors such as co-cultivation time, co-cultivation media and kanamycin concentration were assessed to establish optimal conditions for the selection of transformed callus lines. Transformed calli surviving on medium containing 300 mg l^-1 kanamycin showed a strong GUS-positive reaction. Somatic embryos were then regenerated from these transgenic calli on MS2 medium containing 2.0 mg l^-1 spermine and 0.1 mg l^-1 abscisic acid. Mature embryos were germinated and developed into plantlets on MS4 medium supplemented with 0.2 mg l^-1 gibberellic acid, 0.2 mg l^-1 kinetin (KIN) and 0.1 mg l^-1 indole-3-acetic acid. A transformation frequency of 4% was achieved. The morphology of the transgenic plants was similar to that of untransformed plants. Histochemical GUS assay revealed the expression of the uidA gene in embryos as well as leaves of transgenic plants. The presence of the uidA, nptII and HbSOD genes in the Hevea genome was confirmed by polymerase chain reaction amplification and genomic Southern blot hybridization analyses.Communicated by L. Peña     

Phylogenetic relationships within Hevea brasiliensis as deduced from a polymorphic mitochondrial DNA region

We have cloned a 4.5-kb mtDNA fragment showing a high RFLP polymorphism between various Hevea genotypes. Subcloning and sequencing of a 1.4-kb segment of this clone allowed us to design PCR amplification primers to isolate homologous mtDNA segments of about 0.9 kb from 23 representative genotypes of Hevea. Complete sequences from 4 genotypes showed between 6.7% and 20.2% of nucleotide diversity, suggesting the presence of a hypervariable, or hotspot, region. A sequence of 345 nucleotides within this region was determined for the 23 genotypes. The phylogenetic relationships inferred from the sequence comparison are in general agreement with the results obtained from mtDNA RFLP analysis, indicating that this polymorphic mtDNA region is a useful molecular marker for phylogenetic analysis within Hevea.     

Mitochondrial DNA polymorphism and phylogenetic relationships inHevea brasiliensis

Using fourteen random mitochondrial DNA probes, we have examined restriction fragment length polymorphism (RFLP) in wild and cultivatedHevea brasiliensis. A total of 395 accessions, including 345 from various prospectings collected in Brazil, Colombia and Peru and 50 cultivated clones, were analyzed. Two other species (H. benthamiana andH. pauciflora) were also included in the study for comparison. The high level of mitochondrial polymorphism allowed us to divide all the accessions analyzed into 212 distinct genotypes. The genetic variability of cultivated clones was limited to four genotypes forming two clusters. In contrast, considerable genetic variation was found in the wild collections. In almost all cases, accessions displaying the same RFLP profile were restricted to the same geographical area (same or neighbor administrative districts). In addition, accessions whose genetic closeness was predicted by RFLP profiles were also clustered according to geographical origin. In a few cases, however, similar RFLP profiles were found for accessions originating from geographically distant districts. This discrepancy can be explained either by seed dispersion (by river) or possibly by similar genetic events occurring independently in different geographical locations. Chloroplast DNA RFLP was also analyzed in 217 accessions, representative of 126 distinct mitochondrial genotypes. Very few differences were found, indicating that the chloroplast genome is more highly conserved than the mitochondrial genome.     

Mash2基因在体细胞克隆牛肺脏中DNA甲基化状态分析

体细胞核移植(体细胞克隆)技术在动物生产、医药工业、治疗性克隆以及对珍稀濒危动物的拯救有重要意义,然而克隆效率低下以及克隆动物发育异常,严重制约了克隆技术的发展和应用．在体细胞核克隆中,供体核来自高度分化了的体细胞,发生在核移植后几小时内供体核的重编程,决定了克隆胚胎的发育能力．印记基因是由等位基因表观遗传修饰的不对称导致的基因表达具有亲本选择性,而DNA甲基化是调控印记的一个主要方式．印记基因Mash2在胚胎发育和器官形成过程中起着非常重要的作用．为了探求核移植过程中Mash2基因DNA 甲基化的表观重编程是否充分,利用亚硫酸氢盐测序法对出生48 h内死亡的体细胞核移植牛和正常对照牛肺脏中Mash2基因的DNA甲基化状态进行分析．结果显示,尽管位于Mash2基因启动子和第一个外显子处的CpG岛在正常牛和克隆牛中甲基化水平都不高(20.04%,5.55%),但克隆组的甲基化水平仍显著低于正常对照组 (P < 0.05)．甲基化模式正常组中9N3有5种不同的形式,9N4仅1种;而克隆组9C3和9C5也分别是1种．推测Mash2基因的异常DNA甲基化很可能是导致克隆牛肺脏发育异常的一个重要原因．     

Polyphenol oxidases from latex of Hevea brasiliensis: purification and characterization

Polyphenol oxidase (PPO) was isolated from the B-serum obtained after repetitive freeze-thawing of the bottom fraction isolated from ultracentrifuged fresh latex. The B-serum was subjected to acetone precipitation and CM-Sepharose chromatography, affording two PPOs, PPO-I and PPO-II, which, upon SDS-PAGE, were 32 and 34 kDa, respectively. Both PPOs possessed the same pI (9.2), optimum pH (7) and optimum temperature (35-45 degrees C). They are stable up to 60 degrees C and active at broad pH ranges from 4-9. The K(m) values of PPO-I for dopamine, L-dopa and catechol as substrates are 2.08, 8.33 and 9.09 mM, while those for PPO-II are 2.12, 4.76 and 7.14 mM, respectively. Among various PPO inhibitors tested, 4-hexylresorcinol was the most potent. Anionic detergents were among the most effective activators of the enzymes, while cationic and nonionic detergents showed little and no effect on the PPO activities, respectively.