The integrative expression of GUS gene driven by FCP promoter in the seaweed Laminaria japonica (Phaeophyta)

In this study, the background activity of beta-glucuronidase (GUS) was analyzed histochemically and fluorometrically in the negative control of Laminaria japonica (Phaeophyta) thalli, showing low level of activity. GUS gene transformation without selectable gene in L. japonica was performed using four different promoters, i.e., Cauliflower mosaic virus 35S promoter (CaMV35S) from cauliflower mosaic virus, ubiquitin promoter (UBI) from maize, adenine-methyl transfer enzyme gene promoter (AMT) from virus in green alga Chlorella, and fucoxanthin chlorophyll a/c-binding protein gene promoter (FCP) from diatom Phaeodactylum tricornutum. The GUS transient activity was determined fluorometrically after bombarding sliced parthenogenetic sporophytes explants, and it was found that the activity resulting from CaMV35S and FCP promoters (in 114.3 and 80.6 pmol MU min(-1) (mg protein)(-1), respectively) was higher than for the other two promoters. The female gametophytes were bombarded and regenerated parthenogenetic sporophytes. FCP was the only promoter that resulted in detectable GUS chimeric expression activity during histochemical staining and polymerase chain reaction. Results of Southern blot showed that GUS gene was integrated with the L. japonica genome.

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.

Suppression of zebrafish VEGF gene by cytomegalovirus promoter-driven short hairpin constructs induces vascular development defects and down regulation NRP1 expression

The usage of RNA interference for gene knockdown in zebrafish through expression of the small interfering RNA mediators from DNA vectors has created a lot of excitement in the research community. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against vascular endothelial growth factor (VEGF) gene in zebrafish was tested, and its effects on VEGF-mediated vasculogenesis and angiogenesis were evaluated. Altogether four vectors targeting various locations of VEGF gene were constructed, and pSI-V4 was proven to be the most effective one. Microinjection of pSI-V4 into the zebrafish embryos resulted in defective vascular formation and down regulation of VEGF expression. In situ hybridization analysis indicated that silencing VEGF gene expression by pSI-V4 resulted in down regulation of neuropilin-1 (NRP1), a potent VEGF receptor. Knockdown of VEGF expression by morpholino gave the same result. This provided evidence that the VEGF-mediated angiogenesis in zebrafish was in part dependent on NRP1 expression. The results contributed to a better understanding of molecular mechanisms of cardiovascular development and provided a potential promoter for making inducible knockdown in zebrafish.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Cloning and characterization of beta-carotene ketolase gene promoter in Haematococcus pluvialis

The unicellular green alga Haematococcus pluvialis accumulates a highly valuable ketocarotenoid, astaxanthin, under various environmental stresses. beta-carotene ketolase (BKT) plays a key role in astaxanthin biosynthesis in H. pluvialis. In this paper, an approximate 700 bp 5'-flanking region of the bkt gene containing a putative promoter was cloned through walking upstream. The results of the sequence analysis showed that this bkt 5'-flanking region might have cis-acting elements such as sterol regulatory element (SRE-1)-like motifs, the C-repeat/dehydration responsive element (DRE) and al-3 proximal element (APE)-like motifs, except for typical TATA and CCAAT boxes. The results of the P-galactosidase assay and the transient expression of lacZ driven by a series of sequential deletions revealed that a minimal promoter-like region might exist from -630 to -408 bp, and the highest promoter activity was observed to span the positions from -630 to -308 bp. The results of the site-directed mutagenesis of a C-repeat/DRE and two APE-like motifs in a promoter-like region (-630 to -308 bp) suggested that two APE-like motifs might be essential for transcriptional control of the bkt gene.

Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos. (c) 2007 Elsevier Inc. All rights reserved.

牙鲆和大黄鱼经济性状候选基因的DNA分子标记研究

本文利用不同的分子标记方法，分别对牙鲆及大黄鱼不同养殖群体的生长、抗病等经济性状的候选基因进行了序列多态性研究，检测到了几个SNP位点和微卫星的多态性位点，并分析了它们与经济性状之间的相关性；同时，利用微卫星的多态性位点对牙鲆2个养殖群体的遗传变异进行了分析，这些均为海水鱼类遗传育种及标记辅助选育工作提供了基础数据。 在牙鲆胶南养殖群体中，以100个个体为实验材料，根据其生长激素（GH）基因的6个外显子序列设计引物，通过SSCP分析技术显示该群体GH基因的第4外显子存在多态性，检测到2种基因型，AA型和AB型。DNA测序结果表明，AB型在第1763位发生碱基突变，c→t，与AA型同源性达到99%。连锁分析结果表明：这2种基因型的个体在体重和头长上表现出显著的差异，AB型个体的体重和头长都明显大于AA型个体(P<0.05)，由此推测等位基因B是一个对牙鲆体重和头长都有利的等位基因；这2种基因型个体之间在其体型性状上也存在显著差异（P<0.05）；同时，该多态位点的Hardy-Weinberg平衡性检验结果表明，该群体处于Hardy-Weinberg平衡状态。在牙鲆GH基因第1外显子区域还发现了一个微卫星位点，对该位点进行多态性分析，检测到5种基因型、3种等位基因，one-way ANOVA统计结果显示，基因型AC个体的体重、头长和体高明显大于其它基因型个体（P<0.05），C是一个对体重、头长和体高有利的等位基因。 对2个大黄鱼养殖群体的GH基因进行SSCP分析后发现，浙江群体大黄鱼GH基因在第196位存在1个SNP（g→a）位点，检测到2种基因型，AA和AB。t检验结果表明，AA型个体的体高比AB型个体的高(P≤0.05)，但AB型个体在体长/体高上占优势(P≤0.05)，提示该突变位点可以作为大黄鱼体型性状的候选标记。福建群体大黄鱼GH基因在第692位有1个SNP位点(t→c)，共检测到2种基因型，CC型和CD型，其中，CD基因型个体的体重和全长显著大于CC基因型个体(P≤0.01)，提示该位点可以作为大黄鱼体重和头长性状的候选标记。 在牙鲆胶南和日照2个养殖群体中，采用牙鲆GHR基因5’端Promoter区的一个微卫星标记，进行了群体遗传变异的研究，并探索了该基因多态性位点与牙鲆生长性状之间的相关性。结果表明，2个群体在该位点检测到的等位基因数为12和9个，有效等位基因数为6.26和5.04个。两个群体该位点的Hardy-Weinberg遗传偏离指数均为正值，并没有显示出杂合子缺失，但各基因型分布频率都在一定程度上偏离Hardy-Weinberg平衡(P<0.01)。连锁分析发现，在胶南群体中，IM基因型对应的个体在全重、全长、体长、头长、体高和眼径形态学数据中均是最大的，但仅在体重上极其显著的大于全部其它基因型个体；在日照群体中，BC基因型对应的个体在全重、全长、体高、尾柄高、尾柄长和眼径数据中均是最大的；而CJ基因型对应的个体在体长和头长这两组数据中是最大的。由此认为，该位点IM基因型可以作为牙鲆体重性状的潜在标记。 在进行牙鲆抗病性状标记的筛选时，利用迟缓爱德华氏菌（Edwardsiella tarda）LSE40对牙鲆鱼进行攻毒感染实验，得到死亡群体和未死亡群体。选择Toll样受体基因中的TLR2、TLR3和TLR9基因作为候选基因，分别对这3个基因中的部分序列共设计7对引物进行扩增，同时对扩增产物进行RFLP多态性分析，目前只在TLR3基因内检测到一个EcoRI的酶切多态性位点，测序后发现，这是由于在TLR3基因第3806位的EcoRI酶切位点在某些个体中缺失所致。酶切产物共呈现出3种基因型，分别定义为AA，AB和BB。χ2检验证明该多态性位点与牙鲆抗迟缓爱德华氏菌LSE40的能力有一定关系。利用多因素非条件Logistic回归分析对死亡组和存活组牙鲆的各种形态学数据以及不同基因型之间进行了分析，发现体长、头长和体高均具有显著的相关性（P<0.05），而这几个因素与体重的相关性不显著（P>0.05）。多因素非条件Logistic分析后发现：AA基因型对死亡率具有显著的影响（P<0.05），是主要的危险因素，而AB基因型的作用不显著（P>0.05）；头长是主要的保护因素（P<0.05），体重对死亡率的影响很小。χ2检验证明，等位基因A是对死亡的主要危险等位基因，B是对存活有利的主要等位基因。推测该位点可以作为牙鲆抗迟缓爱德华氏菌的潜在标记。

长牡蛎SNP标记的开发及多态性检测

长牡蛎是重要的经济养殖贝类，良种化、抗逆性状及快速生长个体的培育是长牡蛎养殖业得以持续发展的基础。目前飞速发展的分子标记辅助育种技术为优良品种的快速培育提供了理论基础和实践经验。本研究以长牡蛎为主要研究材料，探讨了长牡蛎SNP标记的筛选和多态性评价。 本研究利用已有长牡蛎EST库中的序列进行单核苷酸多态（SNP）标记开发。通过对长牡蛎（Crassostrea gigas）已有的EST序列数据库检索，经过序列聚类和拼接得到EST簇4548个，含有不少于4条EST序列的簇共1079个，经过进一步设置筛选条件，整理出可供利用的EST簇313个，得到候选SNP位点共计1140个。目前根据候选SNP位点共设计引物82组，通过片段长度差异等位基因特异性PCR（fragment length discrepant allele specific PCR，FLDAS-PCR）的分型方法，在一野生群体中进行检测和验证，结果共有17个SNP候选位点显示多态性，期望杂合度分布区间为0.088至0.506，观测杂合度分布区间为0.091至0.667；通过哈代-温伯格(HW) 平衡、连锁不平衡检验，结果显示除3个SNP位点的差异显著（P值<0.05）,不符合HW平衡之外，其他14个位点没有明显的连锁不平衡。对含有17个SNP的EST的共同序列进行BlastX分析，推测其功能并确定开放阅读框，从而预测17个SNP的性质。 本研究表明对于目前基因组学研究尚处在初级阶段的海洋生物物种，通过基于EST数据库的SNP开发是一条重要途径，可以有效弥补海洋生物基因组学滞后影响SNP标记开发的现状。