Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.     

甘蓝自交不亲和反应的细胞信号转导

S受体激酶(S—receptor kinase,SRK)和S位点富含半胱氨酸(S-locus cysteine-rich,SCR)分别是甘蓝柱头和花粉中导致自交不亲和反应的决定性蛋白质因子。本文就SRK、SCR的结构和功能加以综述,阐明两者在细胞信号转导中的作用。     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996     

油菜叶绿体16S rRNA基因前导顺序的一级结构

质粒pBN119的3.2kb BamHI片段的PvuⅡ-BglⅡ片段全顺序长为840bp,其中含油菜叶绿体16S rRNA基因5′端的140bp。通过寻找GTTC顺序,发现在395至468位核苷酸之间是tRNA~(Val)基因;在73至118位核苷酸之间是一个蛋白阅读框。和已发表的玉米叶绿体16S rRNA前导顺序进行比较,同样存在三个相应的大肠杆菌RNA聚合酶的结合位点。和大肠杆菌的启动子及相应基因作比较,表明叶绿体基因组具有很明显的原核性,但其tRNA~(Val)基因没有CCA3′顺序。在16S rRNA基因、tRNA~(Val)基因及蛋白阅读框的5′端附近均能找到一个比较稳定的茎环结构。我们推测这些茎环结构可能和位于反问重复顺序上的某些基因的转录调节有关。     

Leaf senescence in Brassica napus: cloning of senescence related genes by subtractive hybridisation

A subtractive hybridisation technique was developed to clone cDNAs representing genes that showed enhanced expression during leaf senescence in Brassica napus. A number of different genes were identified that, when analysed by northern hybridisation, showed different patterns of expression during leaf development but were all expressed at increased levels during senescence. Sequence analysis of these cDNAs showed that several types of genes were found including two different proteases, glutamine synthetase, ATP sulphurylase, catalase, metallothionein, ferritin and an antifungal protein. The possible roles of these gene products in the senescence process are discussed.     

Neither compatible nor self-incompatible pollinations of Brassica napus involve reorganization of the papillar cytoskeleton

Compatible pollination of Brassica napus necessitates pollen hydration, pollen germination and growth of the pollen tube through the loosened walls of stigmatic papillar cells, whereas self-incompatible (SI) pollinations fail at one of these stages. Analyses of the early stages of pollination show that at high (but not low) relative humidities, both compatible and SI pollen hydrates, but SI germination is reduced and the rare pollen tubes generally fail to penetrate the papillar walls, although there is some wall loosening. Inside the papillae, both compatible and SI interactions may induce the formation of callose, but there is no evidence for a major accumulation of cytoplasm or secretory vesicles in the vicinity of the pollen tubes and neither microtubule nor F-actin patterns re-arrange in this zone. These observations indicate that the source of the wall-loosening enzymes is probably the pollen tube or pollen coat, and that the common cellular responses of plants to attempted invasions have become suppressed in the papilla–pollen tube interaction.     

Analysis of self-incompatibility interactions in 30 resynthesized Brassica napus lines. II. Expression of S-locus glycoproteins (SLGs)

Thirty resynthesized Brassica napus lines with defined S-allele constitution and the ancestral B. oleracea and B. campestris lines were used for the analysis of S- locus glycoproteins (SLGs). The aim of this study was to investigate (1) whether the S-specific glycoproteins of the diploid ancestor lines were also expressed in the amphidiploid hybrids and (2) whether the occurrence of SLG bands was correlated with the activity of the respective S-alleles, which had been tested by means of diallele pollination tests in a previous study. Stigma proteins were separated by isoelectric focusing (IEF)-gel electrophoresis, and glycoprotein bands were identified by Western blotting and Con-A/peroxidase reaction. The SLG bands of the B. campestris parent could be detected in all 30 resynthesized B. napus lines. In contrast, B. oleracea SLG bands could only be detected in 12 resynthesized B. napus lines. Only B. napus lines which carried the dominant B. oleracea S-alleles S₈ and S₂₉ showed respective SLG bands in all cases. Nine B. napus lines showed only glycoprotein bands of the B. campestris parent, although the biological functioning of the B. oleracea S-alleles was demonstrated by test-pollinations. New SLG bands different from those of the B. oleracea and B. campestris parents occurred in 16 B. napus lines. The expression level of the SLGs in B. napus was not correlated with the self-incompatibility phenotype, not only in the case of recessive S-alleles (S₂, S₁₅), but also for dominant alleles (e.g. S₁₄, S₃₂, S₄₅). Received: 22 January 1999 / Accepted: 30 January 1999     

油菜分子标记图谱构建及抗菌核病性状的QTL定位

以油菜品种０８５（抗菌核病）与湘油１３号杂交得到的Ｆ２代作为作图群体,以ＲＡＰＤ标记来构建油菜连锁图谱。通过对３００个１０－ｍｅｒ随机引物的筛选,共获得２００个多态性ＲＡＰＤ位点。经Ｍａｐｍａｋｅｒ／ＥＸＰ３．０软件处理,构建了１张含１９３个标记位点、１９个连锁群、覆盖长度为１３２４ｃＭ的连锁图谱。在此基础上利用Ｍａｐｍａｋｅｒ／ＱＴＬ １．１将抗菌核病基因定位在第四、八和十四３个连锁群上,即：Ｓ     

甘蓝型油菜温敏不育系417S育性遗传及RAPD分析

通过对新选育的甘蓝型油菜高温敏感生态型不育系417S与30份中外品种(系)进行测交、杂交和回交,研究其温敏不育性的遗传特点.结果表明:(1)30份品种(系)可不同程度的恢复417S的育性,其中24个品种(系)可完全恢复417S育性,占所配组合的80.0%;5个品种(系)可高度恢复417S的不育性,占所配组合的17.2%.(2)自然条件下同一组合正反交F2群体育性分离结果不同,人工控温条件下不同组合F2分离群体中可育与不育呈现15∶1分离,回交群体中出现3∶1分离,表明417S温敏不育性受细胞质和2对隐性重叠核基因共同控制.(3)以417S与1521C构成的回交一代群体为试材,采用分离群体分组分析法(BSA法)对回交一代群体构成的不育集团和可育集团筛选育性基因的RAPD标记,从247个供试引物中筛选出2个引物BA392(5′-AGTCACTCCC-3′)、S113(5′-GACGCCACAC-3′)在不育群体和可育群体间扩增出多态性产物.经进一步对F2分离群体的单株检测,引物BA392扩增出了得到重复验证和可靠的多态性扩增产物,其产生的特异条带BA392-400bp与417S温敏雄性不育的恢复基因连锁,遗传图距为6.0cM,可作为417S温敏不育恢复基因的连锁标记.     

Detection of 5S and 25S rRNA genes in Sinapis alba, Raphanus sativus and Brassica napus by double fluorescence in situ hybridization

Different ribosomal RNA (5S and 25S) genes were investigated simultaneously by fluorescence in situ hybridization (FISH) in Sinapis alba, Raphanus sativus and Brassica napus. The chromosomes of S. alba carried four 5S and six 25S gene sites, and those of R. sativus four sites of each gene, respectively. These two species have one chromosome pair with both rDNA genes; the two are closely located on a short arm of S. alba, while in R. sativus one is distal on the short arm (5S) and the other more proximal on the long arm (25S). In B. napus we have confirmed 12sites of 25S rDNA. The detection of 5S rDNA genes revealed 14 signals on 12 chromosomes. Of these, six chromosomes had signals for both rDNA genes. The FISH with 5S rDNA probes detected two sites closely adjacent in four chromosomes of B napus. These results are discussed in relation to a probable homoeologous chromosome pair in B. oleracea. Received: 20 July 1999 / Accepted: 8 October 1999     

Expression levels of meristem identity and homeotic genes are modified by nuclear-mitochondrial interactions in alloplasmic male-sterile lines of Brassica napus

Homeotic conversions of anthers were found in cytoplasmic male sterile (CMS) plants of Brassica napus derived from somatic hybrids of B. napus and Arabidopsis thaliana. CMS line flowers displayed petals reduced in size and width and stamens replaced by carpelloid structures. In order to investigate when these developmental aberrations appeared, flower development was analysed histologically, ultrastructurally and molecularly. Disorganized cell divisions were detected in the floral meristems of the CMS lines at stage 4. As CMS is associated with mitochondrial aberrations, ultrastructural analysis of the mitochondria in the floral meristems was performed. Two mitochondrial populations were found in the CMS lines. One type had disrupted cristae, while the other resembled mitochondria typical of B. napus. Furthermore, expression patterns of genes expressed in particular floral whorls were determined. In spite of the aberrant development of the third whorl organs, BnAP3 was expressed as in B. napus during the first six stages of development. However, the levels of BnPI were reduced. At later developmental stages, the expression of both BnAP3 and BnPI was strongly reduced. Interestingly the expression levels of genes responsible for AP3 and PI activation such as LFY, UFO and ASK1 were higher in the CMS lines, which indicates that activation of B-genes in the CMS lines does not occur as in B. napus. Disrupted and dysfunctional mitochondria seem to be one of the first aberrations manifested in CMS which result in a retrograde influence of the expression levels of genes responsible for the second and third whorl organ differentiation.     

甘蓝型油菜游离小孢子培养的胚胎发生

以生长在非控温控光下的４个冬性甘蓝型油菜（ＢｒａｓｓｉｃａｎａｐｕｓＬ．）品种为供体,进行游离小孢子培养。研究发现,多数品种在开花３－７天取材最为适宜。在甘蓝型油菜小孢子培养中只有单核晚期的小孢子才可能发育成胚状体,而花药培养时处于单核早期的小孢子易于发育成胚状体。在适当花期选取发育比较一致的单核晚期小孢子培养,经数小时后,部分小孢子便开始膨大,这是小孢子发育成胚的最早标志,膨大的小孢子中,有部分形成多细胞球并进一步发育成胚。用春性甘蓝型油菜为材料进行蔗糖浓度的实验结果表明：培养３天后,在１６％蔗糖培养基中存活的小孢子最多,达１６．１３％;培养３０天后,胚状体诱导频率则以１３％蔗糖浓度为最高,每花蕾可达１４４个胚状体。如果在１６％蔗糖培养基中培养３天后,添加等体积的１３％蔗糖培养基,能够大大提高胚状体的诱导频率,为仅用１３％蔗糖培养基培养的３．７倍。这一实验体系正在用于抗菌核病的诱变与筛选,并作为外源基因导入的实验体系。     

STS markers linked to Phoma resistance genes of the Brassica B-genome revealed sequence homology between Brassica nigra and Brassica napus

The RFLP and AFLP techniques are laborious and expensive and therefore of limited use for marker-assisted selection, demanding a high throughput of samples in a short time. But marker-assisted selection is most useful for traits which are hard to score on single plants and influenced by environmental factors. Four RFLP and three AFLP markers have been found to be linked to genes of the B-genome of Brassica mediating resistance against Phoma lingam in oilseed rape. One RFLP and one AFLP marker were converted into three PCR-based STS markers: one of dominant, as well as one of codominant inheritance separated in a standard agarose gel and a third one of codominant inheritance to be separated in a polyacrylamide gel on an automated sequencer. As expected, the STS markers mapped at the same position as the original RFLP and AFLP markers. The STS markers are efficient in marker-assisted backcross programs of the resistant B-genome/Brassica napus recombinant lines with most of the tested oilseed rape varieties and breeding lines. More than 90% of the tested oilseed rape varieties and breeding lines exhibited no resistance marker alleles. The mapping results obtained with the markers, as well as comparative sequencing of the marker alleles, indicate synteny and homology between the B-genome resistance gene donors and B. napus in the region of the resistance genes. The location of the resistance genes in the B-genome/B. napus recombinant lines is most likely on the A genome. Thus the transfer of the B-genome resistance genes into Brassica campestris is also possible. Received: 9 December 1999 / Accepted: 21 June 2000     

甘蓝型油菜中一类AP2/ERF转录因子的克隆和生物信息学分析

AP2/ERF转录因子家族是植物中广泛存在的一类转录因子,AP2/ERF这类转录因子主要参与植物的细胞周期、生长发育以及生物和非生物胁迫相关基因的表达调控。由于拟南芥和油菜同属于芸薹属,具有相似的基因信息,利用油菜UniGene数据库,以拟南芥ERF转录因子保守序列为探针,通过电子克隆从一个UniGene Cluster中分离得到三个同类的AP2/ERF转录因子,从cDNA序列、氨基酸序列的相似性、组成成分、理化性质、疏水性/亲水性分析、序列比对、进化树、功能域、二级结构、三级结构、无序化特性进行了预测和较为全面的分析。结果显示油菜来源的BnaERF1、BnaERF2和BnaERF3属于AP2/ERF转录因子的B-2亚族,是亲水性蛋白,在蛋白质的三级结构上与AtERF1相似。蛋白质无序化分析发现,油菜BnaERF1、BnaERF2和BnaERF3无序化程度小于拟南芥AtERF1。设计引物通过PCR和RT-PCR方法分别从甘蓝型双低油菜沪油15幼苗的DNA和cDNA中扩增了上述基因,初步分析BnaERF2没有内含子,BnaERF1和BnaERF3有内含子。另外,通过EST丰度分析显示,该类转录因子的表达最高峰在种子中,其次为花,在芽、茎和分生组织中没有检测出表达的存在。     

甘蓝型油菜小孢子培养技术的几项改进

本研究在NLN-16和NLN-13的培养基中分别加入0.1mg/L 6-BA和0.05%的活性碳,结果表明6-BA对小孢子再生胚有明显地促进作用,再生胚的频率比对照增加26枚/皿,经分析达到显著水平;而0.05%的活性碳对小孢子再生胚促进作用不显著。对甘蓝型油菜小孢子培养再生植株的染色体加倍及移栽的研究结果表明,在小孢子培养初期加50mg/L秋水仙碱加倍效率最佳,加倍率达到67.6%。小孢子培养的再生苗移栽至大田后,采用遮阳网覆盖小苗,移栽成活率达到87.6%。 Abstract:The application of microspore culture technique was restricted because of its low frequency of embryogenesis and chromosome doubling.Two methods of enhancing the frequency of embryogenesis were employed in the study,namely,activated charcoal treatment in NLN-13 media and 6-BA treatment in NLN-16 media.The treatment with 0.05% activated charcoal produced 24 embryos per plate,which increased 1.7 embryos per plate,as compared with the treatment without activated charcoal.However,the analysis of T-test showed that it was not significant.After adding 0.1mg/L 6-BA in NLN-16 media,the frequency of embryogeny was 38.3 embryos per plate,and it was 26 embryos more per plate than that of CK.Analysis of T-test is significant.This indicates that 6-BA promotes embryogeny in microspore culture.Adding 50mg/L colchicines in NLN-16 media,the doubling frequency was 67.6%.The plantlets transplanted into field with two methods of light-covered net and plastic films were investigated.A survival rate of 87.6% was obtained using light-covered method whereas 57.7% survived using plastic film method.     

中国甘蓝型油菜遗传多样性的RAPD分子标记

本文利用ＲＡＰＤ方法和统计学分析,对我国７省市和国外引进的总计４０份甘蓝型油菜品种的遗传多样性进行了研究。结果表明,４０个品种的甘蓝型油菜存在着广泛的遗传变异,根据ＲＡＰＤ指纹图谱,通过在ＤＮＡ分子水平上的聚类分析可以将它们分为３大类群,反映出这些品种之间的亲缘关系,并对如何引进甘蓝型油菜资源进行了初浅的讨论。     

Characterization of S tester lines in Brassica oleracea: polymorphism of restriction fragment length of SLG homologues and isoelectric points of S-locus glycoproteins

 Forty three S tester lines of Brassica oleracea were characterized using DNA and protein gel-blotting analyses. DNA gel-blot analysis of HindIII-digested genomic DNA with class-I and class-II SLG probes revealed that 40 lines could be classified as class-I S haplotypes while three lines could be classified as class-II S haplotypes. The band patterns in the S tester lines were highly polymorphic. Although the S tester lines typically showed two bands corresponding to SLG and SRK in the analysis with the class-I SLG probe, only one band was observed in the S ²⁴ homozygote. This band was identified as SRK, suggesting that this haplotype has no class-I SLG band. In the analysis using the class-II SLG probe, one plant yielded a different band pattern from the known class-II haplotypes, S ² , S ⁵ and S ¹⁵ . Unexpectedly, this plant was reciprocally cross-incompatible with the S ² haplotype. Therefore, it was designated as S ^2-b . We found an S ¹³ haplotype having a restriction fragment length polymorphism different from that of the S ¹³ homozygotes of the S tester line. These findings indicate that S homozygous lines with the same S specificity do not necessarily show the same band pattern in the DNA gel-blot analysis. Soluble stigma proteins of 32 S homozygotes were separated by isoelectric focusing and detected using anti-S ²² SLG antiserum. S haplotype-specific bands were detected in 27 S homozygotes but not in five S homozygotes, including the S ²⁴ homozygote. This is consistent with the observation that the S ²⁴ haplotype had no SLG band. Received: 13 July 1998 / Accepted: 29 September 1998     

应用基因芯片分析甘蓝型油菜柱头特异表达基因

以甘蓝型油菜(Brassica napus)野生型(宁油10号)及其柱头授粉功能缺失突变体FS-M1为材料,使用油菜基因表达谱芯片筛选甘蓝型油菜柱头特异表达基因。在含有16 540个基因的油菜基因表达谱芯片中(43 803探针),获得了4 410条差异表达探针,选择部分差异表达基因进行实时定量PCR,所得结果与芯片检测结果相吻合。其中,野生型较FS-M1显著上调且获得209个功能注释的探针,对应198个基因,这些特异表达的基因主要富集在水解酶、转移酶、氧化还原酶和转录因子中;涉及较大的基因家族包括:细胞色素P450基因、GDSL脂肪酶/水解酶基因、ABC转运蛋白基因、myb转录因子基因、bHLH转录因子基因、过氧化物酶家族和受体激酶基因等。推测这些基因与甘蓝型油菜柱头发育及授粉功能有关。