同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

同源四倍体水稻雌配子体发育特性及变异胚囊研究

通过秋水仙素诱导获得同源四倍体水稻10个株系，包括6个恢复系、3个保持系和1个不育系，这些株系具有加倍的染色体组。田间观察表明10个株系具有特殊的农艺性状：茎杆变粗壮、植株颜色加深、叶片变厚、叶宽适度增加、分蘖数减少、有效分蘖的比率下降等。根尖有丝分裂鉴定表明，同源四倍体水稻10个株系具有正常的有丝分裂，观察细胞的染色体数目皆为2n=48。花粉母细胞减数分裂鉴定表明10个株系具有比较理想的减数分裂行为，后期I染色体滞后、末期I微核生成和末期II异常小孢子比率较低，能较好的完成减数分裂过程，其中后期I染色体滞后比率约为10%-20%，末期I微核生成比率约为1%-6%，末期II异常小孢子比率约为1%-8%。这提示，染色体联合和分离不规则导致三价体、单价体 和落后染色体等产生，并进一步导致在后期和末期不均横分离产生异常小孢子，这可能是同源四倍体株系结实率不高的原因之一。 同源四倍体水稻正常胚囊为蓼型，变异胚囊具有多种类型，其比率显著高于二倍体对照，变化范围为39.62%－69.85%。按变异胚囊的结构特点和形成方式，分为四种类型：退化型，结构变异型，无融合生殖型和反足细胞增殖型。退化型胚囊的平均比率为29.17%，包括小胚囊（15.04%）和完全退化胚囊（14.13%），前者仍有较小胚囊腔而后者胚囊腔缺失。结构变异胚囊包括结构缺失、结构重复和位置异常，反映了蓼型胚囊八核七细胞结构的变异，其在各株系的平均比率为18.96%。无融合生殖胚囊发生比率极低，平均比率为1.77%，类型包括反足胚和卵细胞胚。反足细胞增殖胚囊是反足细胞团频繁增殖形成，伴随上述三种变异发生使异常胚囊的多样性进一步增加，其在各株系的平均比率为10.62%。相关分析表明，同源四倍体水稻结实可能主要来自三部分：正常胚囊、正常型小胚囊和反足细胞增殖型胚囊。这三种胚囊具有相对完整的蓼型结构，可能具有较好的育性，其对结实率的贡献程度估计值分别为72.44%、15.12%、12.44%。此外，完全退化型胚囊和位置异常型胚囊对结实率分别表现出显著（-0.66）和极显著（-0.92）的负相关，这表明二者可能是结实性的抑制因素。 Ten autotetraploid strains, which include six restoring lines, three maintaining lines and a sterile line, are derived from artificial induction by colchicine treatments. Variations of agronomical traits are observed which include large organs, sturdy plants, long panicle length and deep leaf color and so on. It has been confirmed that autotetraploid strains exhibit normal chromosome behaviors in mitosis and the chromosome numbers are all 48. Moreover, abnormal chromosome behaviors are investigated in meiosis including univalent, trivalent, quatrivalent, chromosome lagging and microkernel and so on. It evaluates that the percentage of chromosome lagging in anaphase I is about 10%-20%, the percentage of microkernel in telophase I is about 1%-6% and the percentage of abnormal microspore in telophase II is about 1%-8%. In all, abnormal behaviors of chromosomes could induce univalent, trivalent and et al. and subsequently induce infertile microspore. That may be one of the causes of low seed sets in autotetraploid strains. Embryo sacs of autotetraploid strains are formed according to the Polygonum type. However, these strains exhibit variations of abnormal embryo sacs at high frequencies (39.62% - 69.85%). The variations are frequently involved in the spikelets of the main panicles and the first tillers, leading to obvious decreases of the percentages of normal embryo sacs among the strains. Four types of abnormal embryo sacs are classified basing on their different structures and origins: degenerated embryo sac (DES), structure variation (SV), apomixis (Apo) and antipodal cell proliferation (ACP). Embryo sacs of DES (29.17%) exhibit small embryo sacs (15.04%) or no embryo sac (14.13%), the former showing embryo sacs with decreased size and the latter showing no sac. Embryo sacs of AS (18.96%) include three subtypes: structure deletion, structure duplication and location variation, which suggests abnormalities of the eight nuclei, seven celled pattern of the Polygonum type. Embryo sacs of Apo (only 1.77%) include two origins of apomictic embryos: antipodal embryo and egg embryo. Embryo sacs of ACP are observed frequently (10.62%) in autotetraploid strains which subsequently increase the variations of abnormal embryo sacs. It evaluates by the Pearson’s correlation analysis that seed set is probably contributed by three origins of embryo sacs: normal embryo sacs, small embryo sacs (normal pattern) and embryo sacs of ACP. These three origins exhibit comparatively good structure of the Polygonum type and could account for seed set at a percentage of 72.44%, 15.12%, 12.44%, respectively. Moreover, the subtype of no embryo sac (NES) negatively related to seed set at the P>0.01 level (-0.92) and the subtype of location variation (LV) negatively related to seed set at the P>0.05 level (-0.66). Which suggest the two subtypes may have strong stress on seed set.

Loss of p15/Ink4b accompanies tumorigenesis triggered by complex DNA double-strand breaks

DNA double-strand breaks (DSBs) are the most deleterious lesion inflicted by ionizing radiation. Although DSBs are potentially carcinogenic, it is not clear whether complex DSBs that are refractory to repair are more potently tumorigenic compared with simple breaks that can be rapidly repaired, correctly or incorrectly, by mammalian cells. We previously demonstrated that complex DSBs induced by high-linear energy transfer (LET) Fe ions are repaired slowly and incompletely, whereas those induced by low-LET gamma rays are repaired efficiently by mammalian cells. To determine whether Fe-induced DSBs are more potently tumorigenic than gamma ray-induced breaks, we irradiated 'sensitized' murine astrocytes that were deficient in Ink4a and Arf tumor suppressors and injected the surviving cells subcutaneously into nude mice. Using this model system, we find that Fe ions are potently tumorigenic, generating tumors with significantly higher frequency and shorter latency compared with tumors generated by gamma rays. Tumor formation by Fe-irradiated cells is accompanied by rampant genomic instability and multiple genomic changes, the most interesting of which is loss of the p15/Ink4b tumor suppressor due to deletion of a chromosomal region harboring the CDKN2A and CDKN2B loci. The additional loss of p15/Ink4b in tumors derived from cells that are already deficient in p16/Ink4a bolsters the hypothesis that p15 plays an important role in tumor suppression, especially in the absence of p16. Indeed, we find that reexpression of p15 in tumor-derived cells significantly attenuates the tumorigenic potential of these cells, indicating that p15 loss may be a critical event in tumorigenesis triggered by complex DSBs.

Rapid molecular weight determination of recombinant hirudin by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The molecular weight of recombinant hirudin ( rHV-2) was determined rapidly by matrix-assisted laser desorption/ionization time of fight mass spectrometry (MALDI-TOF-MS). The effects of the three types of matrixes were compared and discussed, alpha-cynao-4-hydroxycinnamic acid was proved to be the best matrix. It showed that MALDI-TOF-MS was superior to the traditional method of molecular weight determination of the biological macromolecules. The mass spectrum data proved that the primary structure of rHV-2 was correct and there was no amino acid deletion, mutation and modification in its expression, refolding and purification.

Chromosomal localization and molecular marker development of the lipopolysaccharide and beta-1,3-glucan binding protein gene in the Zhikong scallop Chlamys farreri (Jones et Preston) (Pectinoida, Pectinidae)

Zhikong scallop Chlamys farreri(Jones et Preston) is an economically important species in China. Understanding its immune system would be of great help in controlling diseases. In the present study, an important immunity-related gene, the Lipopolysaccharide and Beta-1,3-glucan Binding Protein (LGBP) gene, was located on C. farreri chromosomes by mapping several lgbp-containing BAC clones through fluorescence in situ hybridization (FISH). Through the localization of various BAC clones, it was shown that only one locus of this gene existed in the genome of C. farreri, and that this was located on the long arm of a pair of homologous chromosomes. Molecular markers, consisting of eight single nucleotide polymorphism (SNPs) markers and one insertion-deletion (indel), were developed from the LGBP gene. Indel marker testing in an F1 family revealed slightly distorted segregation (p = 0.0472). These markers can be used to map the LGBP gene to the linkage map and assign the linkage group to the corresponding chromosome. Segregation distortion of the indel marker indicated genes with deleterious alleles might exist in the surrounding region of the LGBP gene.

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

Interaction between Thymidylate Synthase and Its Cognate mRNA in Zebrafish Embryos

Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 mu M 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 mu M ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5'-UTR of TS mRNA, which corresponded to nt 13-32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.

The polymorphism of lysozyme gene in Zhikong scallop (Chlamys farreri) and its association with susceptibility/resistance to Listonella anguillarum

Lysozyme functions as a crucial biodefence effector against the infection of bacterial pathogens in innate immunity. The nucleotide sequence polymorphisms in promoter region of a nuclear goose type lysozyme gene from Zhikong scallop Chlamys farreri (designated as CFLysG) were investigated to explore their association with susceptibility/resistance to Listonella anguillarum infection. Eight sites of single nucleotide polymorphisms (SNPs) and two sites of insert-deletion (ins-del) polymorphisms were identified in the promoter region of CFLysG. Two of them, -753 TATCTCGATCAGG ins-del polymorphism and -391 A-G SNP were selected to analyze their distribution in the susceptible and resistant stocks, which were identified according to the survival time after L. anguillarum challenge. Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), two genotypes were found at each site, which were ins/del and ins/ins at locus -753, and A/A and A/G at locus -391, respectively. The -753 ins/del genotype was more prevalent in the resistant stock than that in the susceptible stock, 30% vs 16.67% in frequency, but there was no significant difference in the frequency distribution between these two stocks (P=0.15). In contrast, the frequency of -391A/G genotype in the resistant stock was significantly higher (30%) than that in the susceptible stock (7.14%) (P=0.007), indicating a significant association with the resistance of Zhikong scallop to L anguillarum. To confirm the presumption, another independent challenge experiment was performed, in which the cumulative mortality of scallops with -391 A/A genotype (96.8%) was significantly higher than those with -391 A/G genotype (64.5%) (P=0.001), which further validate the association between -391 A/G genotype and the resistance of Zhikong scallop to L anguillarum. These results suggested that the -391 A/G could be a potential marker applied in future selection of Zhikong scallop with enhanced resistance to L anguillarum. (C) 2008 Elsevier Ltd. All rights reserved.

Domain analysis of the Edwardsiella tarda ferric uptake regulator

Recent studies have shown that the ferric uptake regulator (Fur) of Edwardsiella tarda (Fur(Et)) shares high sequence identity with the Escherichia coli Fur (Fur(Ec)) at the N-terminal DNA-binding region. In the present study, the functional importance of the C-terminal region of Fur(Et) was investigated. It was found that Fur(Et) bearing deletion of the C-terminal 12 residues still possesses most of the repressor activity, whereas Fur(Et) bearing deletions of the C-terminal 16 and more than 16 residues are severely affected in activity. Domain swapping analyses indicated that the chimeric Fur proteins (Et75Ec73 and Et75Vh74) consisting of the N-terminal 1-75 region of Fur(Et) fused to the C-terminal 76-148 region of Fur(Ec) and the C-terminal 76-149 region of the Vibrio harveyi Fur (Fur(Vh)), respectively, are fully active. C92 of Fur(Ec) and C137 of Fur(Vh), which are functionally essential in Fur(Ec) and Fur(Vh), respectively, are also essential in Et75Ec73 and Et75074, respectively. Further study identified an artificial Fur protein, EtMF54, which is composed of the N-terminal 49 residues of Fur(Et) and five artificial residues. Compared to Fur(Et), EtMF54 possesses partial Fur activity that is iron-dependent. These results (I) indicate that there exist certain functional/structural compatibilities among Fur(Et), Fur(Ec), and Fur(Vh) at the C-terminal region; (ii) provide insights to the potential location of the regulatory ion-binding site of Fur(Et).

The genomic structure, alternative splicing and immune response of Chlamys farreri thioester-containing protein

MEP is a member of thioester-containing protein (TEP) family found in Zhikong scallop Chlamys farreri and is involved in innate immunity against invading microbes. In the present study, the genomic DNA of CfTEP was cloned and characterized. The genomic DNA sequence of CfTEP consisted of 40 exons and 39 introns spanning 35 kb with all exon-intron junction sequences agreeing with the GT/AG consensus. The genomic organization of CfTEP was similar to human and mouse 0 rather than ciona C3-1 and Drosophila dTEP2. By RT-PCR technique, seven different cDNA variants of CfTEP (designated as CfTEP-A-CfTEP-G) were cloned from scallop gonad. CfTEP-A-CfTEP-F were produced by alternative splicing of six mutually exclusive exons (exons 19-24), respectively, which encoded the highly variable central region. While in CfTEP-G, the deletion of all the six exons introduced a new translation stop site and might trigger nonsense mediated decay (NMD). The mRNA expression and the proportion of the seven CfTEP variant transcripts were examined in the gonad of scallops after bacterial challenge. The fragments containing the highly variable central region of UTEP were amplified by RT-PCR and a 100 positive clones were sequenced randomly. The expression profiles of the seven MEP variants were different and displayed the sex and bacteria dependent manner. In the blank, sea water and Listonella anguillarum challenged subgroups of male scallops, all the transcripts detected were CfTEP-G isoform. In the Micrococcus luteus challenged subgroup, the isoforms expressed and their proportions were CfTEP-F (54%), CfTEP-B (23%), CfTEP-A (10%), CfTEP-C (7%) and CfTEP-E (6%). However, in the gonad of female scallops, only CfTEP-A were found in blank and sea water challenged subgroups. After L anguillarum or M. luteus challenge, four and five isoforms were detected, respectively, with CfTEP-F isoform being the most one in the both subgroups. These results suggested that the evolution of TEP genes was very complex, and that the diverse CfTEP transcripts generated by alternative splicing played an important role as pattern recognition receptors in the innate immune defense of scallops. (C) 2009 Elsevier Ltd. All rights reserved.

Construction and characterization of a live, attenuated esrB mutant of Edwardsiella tarda and its potential as a vaccine against the haemorrhagic septicaemia in turbot, Scophthamus maximus (L.)

The esrB gene of Edwardsiella tarda, which encodes a regulator protein of the type III secretion system, was mutated by the unmarked deletion method and reintroduced by allelic exchange into the chromosome of E. tarda LSE40 by means of the suicide vector pRE 112. The LSE40 esrB mutant was highly attenuated when inoculated intraperitoneally into turbot Scophthamus maximus L., showing a 50% lethal dose of 10(8.1) cfu/fish. The esrB mutants were not recoverable from the internal organs at 14 days post-inoculation. Vaccination with a single dose of 10(5)-10(7) cfu/fish of the esrB mutant elicited significant protection against the wildtype strain of E. tarda LSE40 (relative percentage survival > 50%). The protection correlated well with the antibody titres in the serum of vaccinated fish. (c) 2006 Elsevier Ltd. All rights reserved.

Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur(vh)) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur(Ec)) and could complement a mutant of Fur(Ec). Like Fur(Ec), Fur(Vh), possesses two cysteine residues at positions 92 and 95, yet unlike Fur(Ec), in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur(Vh) proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur(vh) are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur(Vh) are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur(Vh) is possibly different from that of Fur(Ec); and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur(Vh); and (iii) provided insights into the potential function of the local structure involving C137 and K138.

EscC is a chaperone for the Edwardsiella tarda type III secretion system putative translocon components EseB and EseD

Edwardsiella tarda is a Gram-negative enteric pathogen that causes disease in both humans and animals. Recently, a type III secretion system (T3SS) has been found to contribute to Ed. tarda pathogenesis. EseB, EseC and EseD were shown to be secreted by the T3SS and to be the major components of the extracellular proteins (ECPs). Based on sequence similarity, they have been proposed to function as the 'translocon' of the T3SS needle structure. In this study, it was shown that EseB, EseC and EseD formed a protein complex after secretion, which is consistent with their possible roles as translocon components. The secretion of EseB and EseD was dependent on EscC (previously named Orf2). EscC has the characteristics of a chaperone; it is a small protein (13 kDa), located next to the translocators in the T3SS gene cluster, and has a coiled-coil structure at the N-terminal region as predicted by COILS. An in-frame deletion of escC abolished the secretion of EseB and EseD, and complementation of Delta escC restored the export of EseB and EseD into the culture supernatant. Further studies showed that EscC is not a secreted protein and is located on the membrane and in the cytoplasm. Mutation of escC did not affect the transcription of eseB but reduced the amount of EseB as measured by using an EseB-LacZ fusion protein in Ed. tarda. Co-purification studies demonstrated that EscC formed complexes with EseB and EseD. The results suggest that EscC functions as a T3SS chaperone for the putative translocon components EseB and EseD in Ed. tarda.

There are two 5 '-flanking regions of bkt encoding beta-carotene ketolase in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a commercially valuable astaxanthin, with levels reaching up to 4% dry weight under environmental stress. In recent years, much effort has been devoted to understanding the molecular mechanisms regulating astaxanthin biosynthetic pathways. Beta-carotene ketolase (bkt), with control being exhibited at the transcription level, plays an important role in astaxanthin biosynthesis by H. pluvialis. Here we demonstrate the presence of two separate 5'-flanking regions [1.5 kilobase (kb) and 2 kb] of bkt (bkt1 and bkt2) that possess regulatory elements similar to those of known stress-responsive genes in plants. Results of 5'-deletion constructs and transient beta-galactosidase expression assays demonstrate that there may be positive regulatory elements governing expression in the shorter promoter at -1060/-900 from the 1.5 kb 5' region, and in the longer promoter at -1838/-1219 and at -1046/ -734 from the 2 kb 5' region relative to each homologous ATG start codon. Furthermore, our present studies reveal that the first intron (+371/+497) downstream from the 1.5 kb 5' untranslated region of bkt1 may function as a negative regulatory element to regulate its own promoter.