Paternal inheritance of plastids in the genus Daucus

Summary The plastid DNAs of the species Daucus carota (ssp. sativus, libanotifolia, gingidium), D. maximus and D. muricatus were compared by restriction enzyme analysis. A number of restriction fragment length polymorphisms (RFLPs) were observed. As expected from taxonomic data the degree of plastid DNA homology between D. carota and D. maximus is significantly higher (97%) than between D. carota and D. muricatus (70%). On the basis of RFLPs of plastid DNA the mode of plastid inheritance in interspecific crosses between D. muricatus and D. c. sativus was analysed. The results clearly indicate paternal plastid inheritance. Thus Daucus is the second genus among angiosperms transmitting predominantly male plastids.     

春性甘蓝型油菜BnFY34基因的克隆及序列分析

春性甘蓝型油菜杂交种由于发育期较长,极大地限制了该油菜在高寒地区的推广。为了从春性特早熟甘蓝型油菜中筛选出苗期和蕾期差异表达的基因,该研究以春性特早熟甘蓝型油菜为材料,利用cDNA-AFLP技术筛选出1个春性特早熟甘蓝型油菜苗期特异表达的基因片段,并采用RACE技术成功分离克隆了该基因,命名为BnFY34。通过对该基因的测序和生物信息学分析,发现该基因序列大小为455 bp,包含完整的开放阅读框(ORF),编码一个由71个氨基酸残基组成的蛋白,推测的蛋白质分子量为8.04 kD,理论等电点为10.25,其三级结构中含有3个α螺旋,不含β折叠。同时,将BnFY34基因与甘蓝型油菜基因组序列进行比对,结果显示BnFY34基因位于C4染色体上,由3个外显子和2个内含子组成。另外,通过对该基因与其他植物同源基因的亲缘关系进行了分析,结果表明BnFY34基因与芸薹属植物属于同一亚族,并且与甘蓝型油菜中已报到的未诠释基因XM_013837282.1的相似度达100%,其功能有待进一步研究。该研究结果对春油菜产量和品质的早熟转基因育种具有重要意义。     

Sequence and expression of endogenous S-locus glycoprotein genes in self-compatible Brassica napus

Brassica napus is an amphidiploid plant which is self-compatible even though it is derived from hybridisation of the self-incompatible species B. oleracea and B. campestris. Experiments were undertaken to establish if S-locus glycoprotein (SLG) genes exist in B. napus and whether these are expressed as in self-incompatible Brassica species. Two different stigma-specific cDNA sequences homologous to SLG genes were obtained from the B. napus cultivar Westar. One of these sequences, SLG _WS1, displayed highest homology to class I SLG alleles, whereas the other, SLG _WS2, showed greatest homology to class II SLG genes. Both were expressed at high levels in Westar stigmas following a developmental pattern typical of SLG genes in the self-incompatible diploids. We infer that they represent the endogenous SLG genes at the two homoeologous S-loci. The occurrence of normally expressed SLG genes and its relevance to the self-compatible phenotype of B. napus is discussed.     

The EPSPS gene flow from glyphosate-resistant Brassica napus to untransgene B. napus and wild relative species Orychophragmus violaceus

Gene flow from transgenic plants to compatible wild relatives is one of the major impediments to the development of the culture of genetically engineered crop plants. In this work, the flow of EPSPS (conferring resistance to glyphosate) gene of transgene Brassica napus toward the untransgene B. napus and wild relative species Orychophragmus violaceus in an open field (1 ha) was studied. The data related to only the 2004 and 2005 autumn season on one location of southwest of China. Pollen dispersal and fertilization of the target plants were favored and a detailed analysis of the hybrid offspring was performed. In field, the data studied show that the gene flow frequency was 0.16% between GM and non-GM B. napus at a distance of 1 m from the transgenic donor area. The crosspollination frequency was 0.05% between GM and non-GM B. napus at a distance of 5 m from the transgenic donor area. At a distance of 10 m, no crosspollination was observed. According to the results of this study, B. napus transgene flow was low. However, the wild relative species O. violaceus could not be fertilized by the transgenic pollen of B. napus, no matter what the distance was.     

Molecular characterization and genetic origin of the Brassica napus acetohydroxyacid synthase multigene family

Summary The Brassica napus rapeseed cultivar Topas contains an acetohydroxyacid synthase (AHAS) multigene family consisting of five members (AHAS 1–5). DNA sequence analysis indicate that AHAS1 and AHAS3 share extensive homology. They probably encode the AHAS enzymes essential for plant growth and development. AHAS2 has diverged significantly from AHAS1 and AHAS3 and has unique features in the coding region of the mature polypeptide, transit peptide and upstream non-coding DNA, which raises the possibility that it has a distinct function. AHAS4 and AHAS5 have interrupted coding regions and may be defective. The complexity of the AHAS multigene family in the allotetraploid species B. napus is much greater than reported for Arabidopsis thaliana and Nicotiana tabacum. Analysis of the presumptive progenitor diploid species B. campestris and B. oleracea indicated that AHAS2, AHAS3 and AHAS4 originate from the A genome, whereas AHAS1 and AHAS5 originate from the C genome. Further variation within each of the AHAS genes in these species was found.     

Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype

Summary Genetically transformed repeseed (Brassica napus) roots were obtained by in vitro inoculation of excised stem segments with Agrobacterium rhizogenes. Axenic root organ clones were established and they exhibited a phenotype characteristic of transformed roots: rapid growth, reduced apical dominance and root plagiotropism. Stem regeneration was induced by exposing root fragments to 2,4-dichloroacetic acid (2,4-D) in liquid medium, followed by transfer to solid regeneration medium. The resulting plants exhibited the transformed phenotype observed in other species where similar experiments have been performed. Direct evidence for genetic transformation was obtained from opine assays and molecular hybridization. Sexual transmission of the transformed phenotype was Mendelian, and a probable case of T-DNA insertion into two independent loci within the same plant was detected. The estimated optimal time necessary to obtain transformed oilseed rape plants using this approach is 2 months.     

Paternal inheritance of plastids in Medicago sativa

Summary Plastids are plant cellular organelles that are generally inherited from the maternal parent in the angiosperms. Many species exhibit biparental inheritance of plastids, but usually with a predominantly maternal influence. In contrast to this, we report strong paternal inheritance of plastids in reciprocal crosses of alfalfa, Medicago sativa, by following restriction fragment length polymorphisms for plastid DNA in two normal green plastids. Mitochondrial inheritance remained exclusively maternal.     

甘蓝型油菜BnDGAT1基因表达的特异性分析

该研究从甘蓝型油菜中克隆获得了二酰甘油酰基转移酶基因（DGAT）,命名为BnDGAT1,并对该基因编码的氨基酸序列、蛋白结构域和系统进化树进行分析。结果表明:该基因编码的氨基酸序列包含二酰甘油酰基转移酶等多个功能结构域,并具有8个疏水跨膜结构区。系统进化分析表明,BnDGAT1与芥菜、拟南芥、旱金莲中DGAT1系统进化关系相对较近。利用定量PCR对BnDGAT1基因的RNA转录表达分析表明,在不同组织和角果的不同发育阶段,BnDGAT1基因的表达具有组织特异性,且在角果不同发育阶段,其RNA转录水平随着角果发育的成熟表达明显下调。     

MITE转座子在不同甘蓝型油菜品种中的多态性研究

微型反向重复转座因子(MITE)在真核生物基因组中普遍存在,其活动能够在物种内形成丰富的多态性,在基因组进化和基因调控中均发挥重要作用。该研究利用286对不同MITE家族侧翼序列筛选的特异引物,对101个中国油菜、27个加拿大油菜和29个合成油菜品系(共157个品系)的基因组DNA进行多态性研究,以明确MITE家族的插入/缺失多态性以及在不同油菜品种之间插入的遗传多样性,进而探讨他们之间的亲缘关系。结果显示：(1)286对引物中筛选出60对条带清晰重复性高的多态性引物,多态性比例21.0%;其中Stowaway like家族和Tourist like家族特异引物的多态性比例分别为24.6%和20.0%。(2)PCR扩增结果显示,60对多态性引物对中国、加拿大、合成油菜品系的基因组DNA分别扩增出4 029、1 044、1 087条清晰可辨的条带。(3)UPGMA聚类显示,中国油菜品种间遗传相似系数在0.59~0.95,加拿大油菜品种间遗传相似系数在0.73~0.95;合成油菜品种间遗传相似系数值在0.64~0.93。研究表明,MITE在基因组中大量随机插入,形成种内丰富的多态性,利用MITE家族引物检测不同地区的油菜种质资源的多态性,可为油菜的育种找到许多尚未被开发的遗传变异。     

Paternal inheritance of mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids

Organelle inheritance in intergeneric hybrids of Festuca pratensis and Lolium perenne was investigated by restriction enzyme and Southern blot analyses of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA). All F₁ hybrids exhibited maternal inheritance of both cpDNA and mtDNA. However, examination of backcross hybrids, obtained by backcrossing the intergeneric F₁ hybrids to L. Perenne, indicated that both uniparental maternal organelle inheritance and uniparental paternal organelle inheritance can occur in different backcross hybrids.     

甘蓝型油菜PGK基因的克隆与表达分析

为研究甘蓝型油菜磷酸甘油酸激酶(PGK)基因表达特性,在对拟南芥PGK基因家族生物信息学分析的基础上,通过电子克隆方法获得3个甘蓝型油菜PGK基因(BnPGK1、BnPGK2、BnPGK3)。分别设计特异引物,以甘蓝型油菜雄性不育系09A和保持系09B的cDNA为模板克隆BnPGK基因全长序列。根据获得的cDNA序列设计实时荧光定量特异引物,采用实时荧光定量PCR技术,研究油菜雄性不育系与保持系PGK基因表达差异。结果显示:BnPGK基因在甘蓝型油菜雄性不育系09A和保持系09B的根、茎、叶、花蕾中均有表达,属组成性表达。除茎中的BnPGK3外,BnPGK其它基因在根、茎、叶中的表达均表现为09A高于09B,而在花蕾中均为09B高于09A,BnPGK1和BnPGK3在09B中的表达量是09A中的2倍以上。     

油菜花粉发育相关基因RALFbn的克隆与表达分析

根据美国NCBI数据库中快速碱化因子(RALF)类基因序列的已知信息,克隆了油菜的快速碱化因子基因RALFbn,对其核酸序列及预测蛋白进行了生物信息学分析,并在油菜多种组织内观测其表达情况.结果表明:(1)经克隆获得油菜RALFbn基因的cDNA序列全长为510 bp,无内含子,编码79个氨基酸.(2)生物信息学分析发现,油菜RALFbn蛋白具有RALF类蛋白保守的“YIXY”区和4个保守的半胱氨酸残基,并且含有N豆蔻酰化位点、酪氨酸激酶Ⅱ磷酸化位点、蛋白激酶C磷酸化位点、和酪氨酸激酶磷酸化位点等多个生物活性位点,说明该蛋白在油菜中潜在的生理调节能力较为活跃.(3) RT-PCR检测RALFbn基因在油菜生殖器官中的表达结果发现,RALFbn主要在油菜雄蕊中表达,而在雌蕊、花瓣和萼片中没有表达.提示RALFbn基因极可能与油菜雄蕊中花粉的发育相关.     

甘蓝型油菜苗期生长阶段对NaCl胁迫的生理响应

以甘蓝型油菜自交系2205(强耐盐型)、487(中耐盐型)和1423(敏盐型)为材料,采用土培+水培方法于五叶期研究了0(无盐胁迫,CK)、60 mmol·L-1(低盐)、120 mmol·L-1(中低盐)、180 mmol·L-1(中盐)、240mmol·L-1(高盐)NaCl胁迫后的叶片MDA、可溶性糖、甜菜碱和叶绿素含量的变化特征,为油菜耐盐性评价提供理论依据。结果显示:(1)叶片MDA含量在低盐和中低盐胁迫下降低且显著低于CK,在中盐和高盐胁迫下显著升高,并以品系2205含量最低且升幅最小,品系1423最高且升幅最大。(2)叶片可溶性糖含量在低盐和中低盐胁迫下减少(但2205在中低盐胁迫下显著高于CK),在中盐和高盐胁迫下升高且显著高于CK,并以品系2205含量最高增幅最大且显著高于品系487和1423,品系1423含量最低且增幅最小。(3)叶片甜菜碱含量随NaCl浓度升高增加,品系2205和487在中低以上盐浓度胁迫下显著升高,品系1423仅在高盐胁迫下显著升高,并以品系2205含量最高且增幅最大,1423含量最低且增幅最小。(4)叶片叶绿素含量在低盐和中低盐胁迫下显著增加,且以2205叶片叶绿素含量最高且增幅最大,但在中盐和高盐胁迫下显著减少,并以品系1423含量最低且降幅最高。研究表明,120mmol·L-1以下NaCl胁迫对油菜苗期生长可能有促进作用,180mmol·L-1以上NaCl胁迫则有明显抑制作用,且NaCl浓度越高油菜受伤害越重;油菜苗期生长阶段,NaCl胁迫浓度在120~180mmol·L-1时各生理指标发生显著变化,可能是鉴定耐盐性强弱的适宜浓度;综合分析认为,品系2205具有强的耐盐性,品系1423耐盐性最差,这与之前萌芽期和幼苗期鉴定结果一致。     

Stability of cloned Brassica napus chloroplast DNA fragments in the cyanobacterium Anacystis nidulans R2

Summary Brassica napus (cv. Triton) chloroplast (cp) DNA BamHI gragments were inserted into a bacteria-cyanobacteria shuttle vector pCB4. The chloroplast genomic library was screened in Escherichia coli and 28 individual clones, which represent 94% of the total chloroplast genome, were isolated. Cyanobacterium Anacystis nidulans R2 was transformed with each member of the clone bank by selection for ampicillin resistance. A study of transformation efficiency showed dramatic variation (up to 200-fold) among recombinant clones. Furthermore, plasmid DNA reisolated from some cyanobacterial transformants exhibited instability. Variations in transformation efficiency and plasmid instability were shown to be DNA sequence specific. B. napus cpDNA clones were thus classified into three types according to their stability in the cyanobacterial host.     

甘蓝型油菜BnFLA基因的克隆及表达分析

利用同源克隆法从甘蓝型油菜中获得了1个类成束阿拉伯半乳聚糖蛋白基因(FLA),命名为BnFLA。BnFLA基因开放阅读框长为1 200bp,编码399个氨基酸,分子量为42 885.9Da,等电点为6.37。预测的BnFLA蛋白包含N-端信号肽、2个AGP-like结构域、2个fasciclin-like结构域和C-端GPI-anchor序列。系统进化分析表明BnFLA氨基酸序列与BrFLA17和AtFLA2进化关系较近,一致性分别为98%和87%。qRT-PCR分析表明,BnFLA基因在油菜各组织均有表达,并以下胚轴中表达量最高,其次为子叶,茎秆中表达最少;BnFLA基因的表达受到GA3、BR、IAA、ABA和NaCl的诱导,但受6-BA、蔗糖、低温和PEG抑制。研究认为,油菜中BnFLA基因可能参与激素信号转导途径和非生物胁迫应答。     

Paternal inheritance of chloroplast DNA and maternal inheritance of mitochondrial DNA in loblolly pine

Summary The inheritance of organelle DNAs in loblolly pine was studied by using restriction fragment length polymorphisms. Chloroplast DNA from loblolly pine is paternally inherited in pitch pine x loblolly pine hybrids. Mitochondrial DNA is maternally inherited in loblolly pine crosses. The uniparental inheritance of organelle genomes from opposite sexes within the same plant appears to be unique among those higher plants that have been tested and indicates that loblolly pine, and possibly other conifers, must have special mechanisms for organelle exclusion or degradation or both. This genetic system creates an exceptional opportunity for the study of maternal and paternal genetic lineages within a single species.     

甘蓝型油菜BnTCP7基因的克隆和表达分析

该研究克隆获得了甘蓝型油菜预测转录因子TCP7-like同源编码基因,命名为BnTCP7,与甘蓝型油菜TCP7-like NC_027757基因的核苷酸序列相似性为95.68%。BnTCP7基因开放阅读框全长为648bp,编码215个氨基酸。BnTCP7氨基酸序列与十字花科(Brassicaceae)中其他19条TCP7或者TCP7-like氨基酸序列比对发现,BnTCP7与芸薹属和萝卜属等TCP7同源蛋白具有很强的相似性和保守性,尤其在靠近N端的TCP保守结构域,具有典型的螺旋-环-螺旋结构,且BnTCP7属于TCP家族Ⅰ类,为亲水性不稳定蛋白。通过BnTCP7和拟南芥(Arabidopsis thaliana)AtTCP7(NC_003076)蛋白的二级和三级结构在线预测比对分析,进一步证实BnTCP7具有TCP家族典型结构。进化树分析表明,BnTCP7蛋白与甘蓝型油菜TCP7-like NC_027757蛋白聚在同一分支,两者亲缘关系最近。利用转录组数据对不同时期不同器官BnTCP7基因表达模式分析发现,其表达量呈现一定差异性,其中营养器官中的表达量高于生殖器官。非生物胁迫及激素处理对甘蓝型油菜幼苗植株中BnTCP7基因的表达影响分析发现,BnTCP7基因不仅响应了冷、热、损伤等非生物胁迫,而且参与了ABA和GA3激素的信号转导,表明BnTCP7基因在植物维持正常生长发育和逆境胁迫中可能发挥重要作用。     

甘蓝型油菜热激转录因子的克隆及表达分析

以甘蓝型纯系油菜为试材,用RT-PCR和RACE方法首次从油菜种子中获得油菜热激转录因子的cDNA全长序列,命名为BnHSFA4a,GenBank登录号为HQ435241。结果表明:该cDNA长1 667bp,编码1个具有389aa的蛋白质,该基因编码的氨基酸序列与拟南芥热激转录因子AtHSFA4a编码的氨基酸序列间相似度为82%。对34条编码不同热激转录因子的氨基酸序列进行多重比对,结果在100～190aa处序列间氨基酸总相似性超过60%,显示出较高的保守性。构建原核表达载体并成功表达出预期蛋白。半定量RT-PCR结果显示,该基因的表达先于油菜肌醇半乳糖苷合成酶(BnGOLS1)基因,符合该基因调控BnGOLS1的假设。推测AtHSFA4a基因可能通过调控BnGOLS1的表达量进而在油菜种子脱水耐性获得过程中发挥一定的作用。