Molecular cloning and expression pattern of 14 kDa apolipoprotein in orange-spotted grouper, Epinephelus coioides

A novel fish-specific apolipoprotein (apo-14 kDa) has been recently cloned from eel and pufferfish. However, its expression pattern has not been elucidated. in this study, EcApo-14 has been screened from hypothalamic cDNA library of male orange-spotted grouper, which shows 62.9%, 51%, 46.9%, 43.2%, and 31.9% identities to Apo-14 of European flounder, pufferfish, Japanese eel, gibel carp, and grass carp, respectively. RT-PCR analysis reveals that this gene is first transcribed in neurula embryos and maintains a relatively stable expression level during the following embryogenesis. EcApo-14 transcripts are at a very high level during embryonic and early larval development in the yolk syncytial layer (YSL), and decrease in YSL and form intense staining in liver at 3 days after hatching. In adult tissues, EcApo-14 is predominantly expressed in liver and brain. The data suggested that EcApo-14 might play an important role in liver and brain morphogenesis and growth. (c) 2005 Elsevier Inc. All rights reserved.

Molecular identification and expression analysis of natural resistance associated macrophage protein (Nramp) cDNA from Japanese flounder (Paralichthys olivaceus)

Natural resistance associated macrophage protein (Nramp) controls partially innate resistance to intracellular parasites. Its function is to enhance the ability of macrophages to kill pathogens. However, little is known about the structure and function of Nramp in lower vertebrates such as teleosts. We have recently isolated a cDNA encoding Nramp from Japanese flounder (Paratichthys olivaceus). The full-length cDNA of the Nramp is 3066 bp in length, including 224 bp 5' terminal UTR, 1662 bp encoding region and 1180 bp 3' terminal UTR. The 1662-nt open reading frame was found to code for a protein with 554 amino acid residues. Comparison of amino acid sequence indicated that Japanese flounder Nramp consists of 12 transmembrane (TM) domains. A consensus transport motif (CTM) containing 20 residues was observed between transmembrane domains 8 and 9. The deduced amino acid sequence of Japanese flounder had 77.30%, 82.71%, 82.67%, 79.64%, 80.72%, 90.97%, 91.16%, 60.14%, 71.48%, 61.69%, 72.37% identity with that of rainbow trout Nramp alpha and beta, channel catfish Nramp, fathead minnow Nramp, common carp Nramp, striped sea bass Nramp, red sea bream Nramp, mouse Nramp 1 and 2, human Nramp 1 and 2, respectively. RT-PCR indicated that Nramp transcripts were highly abundant in spleen, head kidney, abundant in intestine, liver and gill, and less abundant in heart. The level of Nramp mRNA in embryos gradually increases during embryogenesis from 4 h (8 cell stage) to 80 h (hatched stage) after fertilization. (c) 2005 Elsevier Ltd. All rights reserved.

Expression pattern and developmental behaviour of cellular nucleic acid-binding protein (CNBP) during folliculogenesis and oogenesis in fish

In vertebrates, folliculogeneis establishes an intricate system for somatic cell-oocyte interaction, and ultimately leads to the acquisition of their respective competences. Although the formation process and corresponding interactions are strikingly similar in diverse organisms, knowledge of genes and signaling pathways involved in follicle formation is very incomplete and the underlying molecular mechanisms remain enigmatic. CNBP has been identified for more than ten years, and the highest level of CNBP transcripts has been observed in adult zebrafish ovary, but little is known about its functional significance during folliculogeneis and oogenesis. In this study, we clone CNBP cDNA from gibel carp (Carassius auratus gibelio), and demonstrate its predominant expression in gibel carp ovary and testis not only by RTPCR but also by Western blot. Its full-length cDNA is 1402 bp, and has an ORF of 489 nt for encoding a peptide of 163 aa. And its complete amino acid sequence shared 68.5%-96.8% identity with CNBPs from other vertebrates. Based on the expression characterization, we further analyze its expression pattern and developmental behaviour during folliculogeneis and oogenesis. Following these studies, we reveal an unexpected discovery that the CagCNBP is associated with follicular cells and oocytes, and significant distribution changes have occurred in degenerating and regenerating follicles. More interestingly, the CagCNBP is more highly expressed in some clusters of interconnected cells within ovarian cysts, no matter whether the cell clusters are formed from the original primordial germ cells or from the newly formed cells from follicular cells that invaded into the atretic oocytes. It is the first time to reveal CNBP relevance to folliculogeneis and oogenesis. Moreover, a similar stage-specific and cell-specific expression pattern has also been observed in the gibel carp testis. Therefore, further studies on CNBP expression pattern and developmental behaviour will be of significance for understanding functional roles of CNBP during gametogenests. (c) 2005 Elsevier B.V. All rights reserved.

Identification of a Spindlin homolog in gibel carp (Carassius auratus gibelio)

Spindlin has been suggested to play an important role during the transition from oocyte maturation to embryo development in mouse, but its homolog similar to the mouse Spindlin in molecular and expression characterization has not been identified up to now in other vertebrates. In this study, a full length of cDNA sequence is cloned and sequenced from the gibel carp (Carassius auratus gibelio). It contains 1240 nucleotides with an open reading frame of 771 nt encoding 257 amino acids. Based on its amino acid sequence alignment and comparison analysis with the known Spin family proteins, the newly cloned Spin is named Carassius auratus gibelio Spindlin (CagSpin). Its product could be detected from mature eggs to blastula embryos, but its content decreased from the two-cell stage, and could not be detected after the gastrula stage. It suggests that the CagSpin should be a maternal protein that is expressed during oocyte maturation, and plays a crucial role in early cleavage of embryogenesis. CagSpin is the first homolog similar to mouse spindlin identified in fish, and also in other vertebrates. GST pull-down assay reveals the first biochemical evidence for the association of CagSpin and p-tubulin, the microtubule component. Therefore, CagSpin may play important functions by interacting with beta-tubulin and other spindle proteins during oocyte maturation and egg fertilization. (c) 2005 Elsevier Inc. All rights reserved.

Identification of three duplicated Spin genes in medaka (Oryzias latipes)

Gene and genomic duplications are very important and frequent events in fish evolution, and the divergence of duplicated genes in sequences and functions is a focus of research on gene evolution. Here, we report the identification and characterization of three duplicated Spindlin (Spin) genes from medaka (Oryzias latipes): OlSpinA, OlSpinB, and OlSpinC. Molecular cloning, genomic DNA Blast analysis and phylogenetic relationship analysis demonstrated that the three duplicated OlSpin genes should belong to gene duplication. Furthermore, Western blot analysis revealed significant expression differences of the three OlSpins among different tissues and during embryogenesis in medaka, and suggested that sequence and functional divergence might have occurred in evolution among them. © 2005 Elsevier B.V. All rights reserved.

Cloning of rainbow trout (Oncorhynchus mykiss) histone H3 promoter and the activity analysis in rare minnow (Gobiocypris rarus)

Rainbow trout historic H3 (RH3) promoter was cloned via high fidelity PCR. The cloned RH3 promoter was inserted into a promoter-lacked vector pEGFP-1, resulting in an expression vector pRH3FGFP-1. The linearized pRH3EGFP-1 was microinjected into fertilized eggs of rare minnows and the sequential embryogenetic processes were monitored under a fluorescent microscope. Strong green fluorescence was ubiquitously observed at as early as the gastrula stage and then in various tissues at the fry stage. The results indicate that RH3 promoter, as a piscine promoter, could serve in producing transgenic Cyprinoid such as rare minnow. Promoter activity of RH3, CMV and common carp beta-actin (CA) were compared in rare minnow by the expression of respective recombinant EGFP vectors. The expression of pCMVEGFP occurred earlier than the following one, pRH3EGFP-1, and then pCAEGFP during the embryogenesis of the transgenics. Their expression activities demonstrated that the CMV promoter is the strongest one, followed by the CA and then the RH3.

Retention of the developmental pluripotency in medaka embryonic stem cells after gene transfer and long-term drug selection for gene targeting in fish

Embryonic stem (ES) cells provide a unique tool for introducing random or targeted genetic alterations, because it is possible that the desired, but extremely rare recombinant genotypes can be screened by drug selection. ES cell-mediated transgenesis has so far been limited to the mouse. In the fish medaka (Oryzias latipes) several ES cell lines have been made available. Here we report the optimized conditions for gene transfer and drug selection in the medaka ES cell line MES1 as a prelude for gene targeting in fish. MES1 cells gave rise to a moderate to high transfection efficiency by the calcium phosphate co-precipitation (5%), commercial reagents Fugene (11%), GeneJuice (21%) and electroporation (>30%). Transient gene transfer and CAT reporter assay revealed that several enhancers/promoters and their combinations including CMV, RSV and ST (the SV40 virus early gene enhancer linked to the thymidine kinase promoter) were suitable regulatory sequences to drive transgene expression in the MES1 cells. We show that neo, hyg or pac conferred resistance to G418, hygromycin or puromycin for positive selection, while the HSV-tk generated sensitivity to ganciclovir for negative selection. The positive-negative selection procedure that is widely used for gene targeting in mouse ES cells was found to be effective also in MES1 cells. Importantly, we demonstrate that MES1 cells after gene transfer and long-term drug selection retained the developmental pluripotency, as they were able to undergo induced differentiation in vitro and to contribute to various tissues and organs during chimeric embryogenesis.

Margulis' theory on division of labour in cells revisited

Division of labour is a marked feature of multicellular organisms. Margulis proposed that the ancestors of metazoans had only one microtubule organizing center (MTOC), so they could not move and divide simultaneously. Selection for simultaneous movement and cell division had driven the division of labour between cells. However, no evidence or explanation for this assumption was provided. Why could the unicellular ancetors not have multiple MTOCs? The gain and loss of three possible strategies are discussed. It was found that the advantage of one or two MTOC per cell is environment-dependent. Unicellular organisms with only one MTOC per cell are favored only in resource-limited environments without strong predatory pressure. If division of labour occurring in a bicellular organism just makes simultaneous movement and cell division possible, the possibility of its fixation by natural selection is very low because a somatic cell performing the function of an MTOC is obviously wasting resources. Evolutionary biologists should search for other selective forces for division of labour in cells.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

猕猴体细胞核移植影响因素的研究：着床前胚胎表观遗传重编程和供体细胞周期同步化

体细胞核移植（somatic cell nuclear transfer）克隆技术的成功，特别 是运用终末分化的淋巴细胞和嗅觉神经元细胞成功克隆出小鼠，证实了分化的体 细胞核潜在的发育全能性。该技术已经在多个物种上成功地得到克隆后代，在转 基因动物、基因敲除动物和疾病模型动物生产中也得到成功应用，在结合干细胞 技术的治疗性克隆和再生医学方面也取得了初步成果，展现出了具有深远意义的 应用前景。但是，目前该领域仍然存在着很多急待解决的重要问题：克隆成功率 低，克隆胚和克隆动物经常呈现发育异常，妊娠和出生前后的高死亡率。对哺乳 动物早期胚胎发育过程中DNA 甲基化、组蛋白修饰等表观遗传重编程 （epigenetic reprogramming）机制的深入了解，有助于研究体细胞核在去核卵 母细胞中的表观遗传重编程事件，进而改善克隆胚重编程效率和发育能力。 猕猴是一种重要的实验动物，在人类疾病模型和生物医药研究中有重要的意 义。本研究主要围绕猕猴体细胞克隆胚胎早期发育过程中的表观遗传重编程事件 和核移植前体细胞同步化处理这两方面展开。1），首次详细地了描绘了猕猴着床 前胚胎发育过程中整体水平的DNA 甲基化表观遗传重编程事件，研究发现在受精 卵中父本基因组形成原核后迅速地发生了去甲基化，在2 细胞期后的卵裂过程 中，母本基因组才开始逐渐地去甲基化，到桑葚胚达到最低水平，然后开始重新 （de novo）甲基化,到囊胚期时形成不对称的甲基化模式，滋养外胚层（TE）呈 现高甲基化状态，而内细胞团（ICM）呈现低甲基化状态,这一不对称模式可能是 灵长类动物特有的，其他哺乳动物呈现正好相反的不对称模式。2），研究发现， 大多数猕猴克隆胚胎的DNA 甲基化重编程存在异常,效率低。很多2 细胞期克隆 胚（67%）和8 细胞期克隆胚（50%）的核DNA 甲基化水平显著高于对应的体 外受精胚，8 细胞克隆胚之间呈现多种不同的表观遗传特征。大多数克隆囊胚的 ICM 细胞核的甲基化水平显著高于IVF 囊胚，这些异常可能是导致克隆胚胎移 植到代孕母体后发育时间不长就失败的原因。3），在核移植前对猕猴成纤维细 胞同步化处理的研究中发现，血清饥饿，细胞周期阻断剂DMSO（二甲基亚砜）、 roscovitine、aphidicolin 和indirubin 的处理都有显著的同步化效果，提高了G0+G1 期细胞的比例。经过BrdU 标记法证实了这几种处理方法抑制细胞增殖的效果，并且证实了这种周期阻滞作用是可逆的。用原位末端标记法（TUNEL）分析证 实，血清饥饿1 到4 天后细胞凋亡比例显著上升，在贴壁的细胞中约有6%发生 凋亡，而正常对照只有1%左右，而周期阻断剂处理没有增加细胞凋亡率，这提 示这些周期阻断剂可能是一种相对安全且有效的猕猴成纤维细胞处理方法。核移 植前对猕猴成纤维细胞进行处理，有助于优化体细胞核移植技术，也是改善体细 胞核在克隆胚中重编程效率的重要途径。

哺乳动物体细胞核移植影响因素的研究

题。 在已有的基础上，建立了基于单个植入前胚胎的逆转录-聚合酶链反应（RT-PCR）技术，成功实现了针对单个猕猴植入前胚胎进行可重复的，多个特定基因的表达检测。在此基础上，对一些重要的早期胚胎发育相关基因在猕猴体细胞核移植胚胎和体外受精胚胎中的表达进行了比较研究。这其中包括核仁相关蛋白基因nucleolin，nucleophosmin，fibrillarin，PAF53，UBF和mRNA早期装配相关蛋白snrpn，这些基因被认为与植入前胚胎的再程序化有着非常密切的关系。我们的研究结果发现，相对于体外受精胚胎，被检测基因在核移植胚胎中的转录出现了不同程度的异常，表现为表达丰度相对较低，这意味着体细胞核移植植入前胚胎的再程序化，或者说供体核的再程序化可能出现了异常。通过进一步对fibrillarin（胚胎合子基因组启动的标志基因）的表达研究，比较了合子基因组启动这一再程序化分子事件在核移植胚胎和体外受精胚胎中的发生时序。研究结果证实，体细胞核移植胚胎的合子基因组启动出现了明显的滞后。我们还发现这与核移植胚胎的异常卵裂速度有着一定的关联，同时说明某些源自核移植操作技术和方法的未知因素可能是造成核移植胚胎再程序化异常的主要原因。为了验证合子基因组滞后对核移植胚胎后续发育的影响，本论文研究分析了着床相关基因Mamu-AG在猕猴核移植胚胎中的表达。研究首次发现Mamu-AG mRNA只在囊胚期开始出现，这与在人类中的情况有所不同（始于8细胞时期）。在正常体外受精囊胚中，不论发育时间如何，第6和第7天的囊胚均正常表达Mamu-AG mRNA。而在核移植囊胚中，仅有第7天的囊胚检测到该mRNA，第6天的囊胚中则没有，这进一步说明了胚胎合子基因组启动的延迟是影响核移植胚胎发育的重要环节。 本研究从分子水平上对猕猴植入前胚胎基因转录和表达进行了分析研究，首次发现了在猕猴体细胞核移植胚胎中发育相关基因表达异常，以及合子基因组启动滞后的分子证据，这为我们改进现有的猕猴核移植操作程序提供了新的思路，同时也为进一步研究猕猴植入前胚胎发育的再程序化奠定了基础。

体细胞核移植影响因素的研究

尽管体细胞核移植(somatic cell nuclear transfer, SCNT)技术仍然处于 起步发展阶段，但是随着体细胞核移植技术在近些年来的飞速发展，人们已经得 到了多种哺乳动物体细胞核移植的存活后代。体细胞核移植技术在生物医药、农 业及其它领域的应用显示了这项技术的巨大发展前景。另一方面，人们发现体细 胞核移植效率低下并且体细胞克隆动物存在许多缺陷，这些主要是由于核移植技 术，供体细胞选择，体外培养系统以及卵母细胞的状态等的差异导致的。本研究 主要围绕着这些因素在体细胞核移植过程中对于克隆胚胎植入前后发育的影响 而开展，目的在于提高体细胞核移植的效率。 实验一，研究了几个因素对克隆胚胎发育和克隆效率的影响，为提高体细胞 核移植效率提供一些依据。这部分研究主要包括四倍体半克隆小鼠研究和蛋白酶 体抑制剂在克隆胚胎和孤雌激活胚胎发育中的影响。四倍体体细胞半克隆 （tetraploid semi-cloned , TSC）胚胎的体外发育结果表明，虽然TSC 胚胎可 能避免二倍体半克隆胚胎发育过程中的非整倍体现象，但是仍然不能形成胎儿。 另外，利用蛋白酶体抑制剂MG132 处理克隆胚胎的结果表明，虽然MG132 通过抑 制成熟促进因子（maturation promoting factors，MPF）活性的降低可以提高 克隆胚胎的体外发育率，但是没有改变克隆胚胎的质量。 实验二，研究了克隆技术在克隆胚胎构建和发育上的影响。首先，我们研究 了完整颗粒细胞注射到卵母细胞中所引起的变化。我们发现完整的颗粒细胞注入 到卵母细胞中之后很快引起细胞膜裂解和细胞核碎裂，从而导致激活后重构胚胎 的碎裂。这一实验表明完整供体细胞核注入的方法不适用于小鼠体细胞克隆。接 下来我们研究了不同的核移植技术――利用piezo 的直接注射法（piezoelectric microinjection，PEM）和电融合法（Electrofusion，EF）――对植入前后的克隆胚 胎以及出生后的克隆动物的影响。研究的结果发现，在PEM 法中，提高piezo 脉 冲的强度会导致供体细胞核DNA 断裂增加，使植入前克隆胚胎的细胞数目减少， 凋亡增加，从而降低克隆胚胎的质量。相反，在用EF 法产生的胚胎中细胞核DNA 的断裂很少，克隆胚胎质量相对较好。实验的结果表明，由于两种克隆技术利用 的原理不同，会对克隆胚胎的发育产生一定的影响，从而改变克隆的效率。 实验三，研究了猕猴体细胞克隆胚胎早期发育过程中细胞核内有丝分裂器蛋 白[Nuclear Mitotic Apparatus Protein，NuMA]的表达和分布。尽管在有些猕猴体 细胞核移植胚胎中没有检测到NuMA 的正确表达和分布并且有些胚胎出现了微 管组装异常，但是大多数猕猴体细胞核移植胚胎具有正常的NuMA 蛋白表达和 正常的核型。通过改进操作技术我们得到了可以发育到正常囊胚的猕猴体细胞核 移植胚胎。这些结果提示猕猴SCNT 胚胎发育失败的原因不是NuMA 等重要蛋 白的缺失。

猕猴胚胎干细胞自我更新机理及WNT 信号通路在猕猴胚胎早期发育中作用的研究

尽管大部分动物实验是在啮齿类动物上开展的，我们仍然相信涉及人类的许多问题，如胚胎干细胞的体内功能、调亡和肿瘤形成等只有在非人灵长类模型上才能得到最好的回答。猕猴(标准的非人灵长类动物模型)在解剖、生理和代谢方面都和人类非常相似。人类很多神经疾病，如阿尔茨海默氏病、帕金森病，只能在非人灵长类模型上才能精确建模。所以研究猕猴胚胎干细胞自我更新的原理及猕猴胚胎早期发育，对研究免疫排斥，检测基于胚胎干细胞的治疗的可行性，安全性和有效性具有重要意义。本文一方面对胚胎干细胞维持自我更新和多潜能性的机理研究进行了综述，另一方面对以下两个方面的内容进行了研究： 1）运用寡核苷酸芯片和定量PCR 验证的方法来分析五株猕猴饲养层细胞的表达模式，期望发现在支持性和非支持性的饲养层细胞中差异性表达的基因。我们着重定位于饲养层胞外空间和细胞膜上的细胞因子，因为这些因子可以通过直接接触或通过膜结合受体激活下游信号通路，并最终促进猕猴胚胎干细胞的自我更新。我们发现在支持性的饲养层中有八个基因是高表达的，他们是GREM2， bFGF，KITLG，DKK3，GREM1，AREG，SERPINF1 和LTBP1； 经定量PCR 验证的SCF，bFGF 和GREM2 的表达情况都和芯片数据吻合。 2）为了描述在IVF (in vitro fertilized, 体外受精)，ICSI (intracytoplasmic sperm injection, 单精注射)，SCNT (somatic cell nuclear transfer, 体细胞核移植)和孤雌生殖猕猴囊胚中WNT 信号通路的表达情况，我们运用了信号通路特异性PCR Array 系统及免疫细胞化学来检测mRNA 和蛋白表达水平。其中，ICSI 作为IVF 胚胎的参照组，以排除显微操作对胚胎质量的影响。结果，我们发现非经典WNT/JNK 信号，而不是经典WNT 信号通路，在IVF 正常胚胎发育中起作用。而体细胞核移植和孤雌生殖的胚胎的WNT 信号通路基因表达明显高于正常胚胎。WNT 信号通路基因的表达模式可以作为胚胎质量的一个指示标准，有助于回答为什么猕猴 SCNT 和孤雌生殖胚胎发育异常。

不同抗旱性青稞LEA2/LEA3蛋白基因的克隆与表达

作物的抗旱性是一个多基因控制的、极为复杂的数量性状，植物对干旱在分子水平上的差异反应通过植物组织生理和细胞生物学水平，最终表现为植物抗旱性的不同。在我国，旱地农业超过耕地面积的50%，但水资源短缺，因此培育和选育抗旱高产作物是发展节水型农业最有效的途径。 青藏高原气候恶劣、年均降雨量少，也是世界大麦初生起源中心，因而蕴藏了十分丰富的与抗逆相关的种质资源材料，从这些特殊的资源材料克隆抗旱基因，不仅对培育抗旱、优质、高产大麦新品种具有重要理论意义和经济价值，而且对整个作物抗旱基础和育种应用研究都具重大促进作用。 为了筛选青稞（裸大麦，Hordeum vulgare ssp. vulgare）抗旱性材料，本研究选用来自青藏高原不同地区的84份青稞为材料，在叶片失水率(water loss rate, WLR)检测分析的基础上，选择失水率值差异显著的12个品种，通过相对含水量（relative water content, RWC）和反复干旱法评价其抗旱性，并通过植株对干旱胁迫下的丙二醛（MDA）含量和游离脯氨酸（free-proline）含量变化，了解不同抗旱性材料的生理反应特性。选择抗旱性强弱不同的品种各两份进行LEA2蛋白基因(Dhn6基因)、LEA3蛋白基因(HVA1基因)的克隆，比较LEA蛋白结构差异与作物抗旱性之间的关系。同时，对抗旱性不同的青稞品种受到干旱时间不同的失水变化率（dynamics water loss rate, DWLR）进行了检测；对抗旱性不同的青稞对照材料进行2 h、4 h、8 h和12 h的快速干旱处理，通过SYBR Green实时荧光定量RT-PCR技术对Dhn6基因、Dhn11基因、Dhn13基因和HVA1基因在不同抗旱性材料受到不同干旱时间处理后的相对表达水平进行了检测。本研究对LEA蛋白基因在抗旱性不同的青稞材料中的干旱胁迫分子水平上的差异反应进行了研究，也对植物的抗旱机理进行了初步探讨。主要研究结果如下： 1. 青稞苗期进行离体叶片失水率测定结果表明，来自青藏高原的84份青稞材料的WLR在0.086~0.205gh-1g-1DW之间。选择WLR低于0.1gh-1g-1DW和WLR高于0.18gh-1g-1DW的品种各6份，并对苗期分别进行未干旱及干旱12小时的处理。相对含水量检测结果表明，低失水率青稞材料干旱后的具有更高的相对含水量，盆栽缺水试验也显示叶片失水率低的材料耐旱能力强于失水率高的材料。通过水合茚三酮法测定离体叶片游离脯氨酸的含量，结果表明，所有品种未干旱处理时，游离脯氨酸含量差异不大（17.10~25.74 µgg-1FW）；干旱12小时后，低失水率的品种游离脯氨酸含量明显增高（32.99~53.45µgg-1FW），高失水率品种的游离脯氨酸含量与干旱前变化不明显(P<0.05)。硫代巴比妥酸法测定离体叶片丙二醛(MDA)含量，结果显示，12份所选对照品种中，丙二醛的含量在0.97~2.74nmolg-1FW，干旱12小时后丙二醛的含量显著上升（1.46~4.74nmolg-1FW），高失水率的6个品种的丙二醛含量在未干旱和干旱处理时都明显高于低WLR品种。本研究结果表明青稞的低失水率、低丙二醛含量、高相对含水量和高脯氨酸含量具相关性（P<0.05）。综上研究，我们认为作物失水率的测定可以作为快速检测作物抗旱性的指标之一，因此，强抗旱品种喜玛拉10号(TR1)、品比14号(TR2)和弱抗旱品种冬青8号(TS1)、QB24 (TS2)被选作抗旱基因克隆和表达分析的研究材料。 2. 高等植物胚胎发育晚期丰富蛋白(late embryogenesis abundant proteins, LEA proteins)与植物耐脱水性密切相关，为了探讨青稞LEA蛋白结构差异性与植物抗旱性的关系，本研究以强抗旱品种（喜玛拉10号、品比14号）和弱抗旱品种（冬青8号、QB24）为材料，利用同源克隆法，通过RT-PCR，分别克隆了与抗旱性密切相关的Dhn6基因和HVA1基因。Dhn6基因序列分析结果表明，强抗旱品种品比14号和弱抗旱品种冬青8号Dhn6基因所克隆到的序列为1026bp，它们之间只有5个碱基的差异；喜玛拉10号和QB24克隆到的序列长963bp。在强弱不同的抗旱品种中有22个核苷酸易突变位点，相应的脱水素氨基酸序列推导结果表明，22个核苷酸突变位点中，仅有8个位点导致相应的氨基酸残基的改变，其余的位点系同义突变，另外，21个富含甘氨酸序列的缺失并没有联系作物抗旱性特征。推测这些同义突变位点的氨基酸残基对维持青稞DHN6蛋白的正常结构和功能起着非常重要的作用，也可能DHN6蛋白对青稞长期适应逆境胁迫和遗传进化的结果。对HVA1基因的序列分析结果表明，冬青8号、QB24、品比14号和喜玛拉10号的目的基因核苷酸序列全长分别为661bp、697bp、694bp和691bp，它们都包含1个完整的开放阅读框。相应的LEA3蛋白氨基酸序列结果表明，11个高度保守的氨基酸残基组成基元重复序列的拷贝数与青稞抗旱性之间没有必然关系，在强抗旱品种（喜玛拉10号、品比14号）中三个共同的氨基酸突变位点Gln32、Arg33和Ala195可能对抗旱蛋白的结构和功能有影响；另外，强抗旱青稞品种LEA3蛋白质中11-氨基酸保守基元序列拷贝数和极性氨基酸占蛋白的比例更高，推测LEA3蛋白中基元序列拷贝数和极性氨基酸占蛋白的比例对该蛋白的结构和功能影响更大。 3. LEA蛋白基因的表达水平的上调与植物的耐脱水性密切相关，我们对强抗旱性材料（喜玛拉10号、品比14号）和弱抗旱材料（冬青8号、QB24）进行干旱处理2 h、4 h、6 h、8 h和10 h的失水变化率进行测定，结果表明弱抗旱品种在2~4小时之间失水率变化最明显，而四个对照品种的失水率在8小时后和24小时的失水率值变化不大。进一步提取青稞苗期进行2 h、4 h、8 h和12 h的干旱处理后的总RNA，通过SYBR Green实时荧光定量RT-PCR技术对青稞脱水素基因(Dhn6、Dhn11和Dhn13)和LEA3蛋白基因(HVA1)的相对表达水平受干旱时间和作物抗旱性的影响进行了检测。研究发现，抗旱性不同的青稞品种随干旱处理的时间延长，Dhn6、Dhn11、Dhn13和HVA1基因的相对表达水平不同。 Dhn6基因的相对表达水平在强抗旱青稞品种干旱8小时后快速上升，但在弱抗旱青稞品种干旱处理12小时后检测到更高表达量；Dhn11基因在对照青稞抗旱品种的表达累积水平随干旱时间的延长持续下降；整个干旱过程中，Dhn13基因的相对表达水平在弱抗旱品种持续上升，在强抗旱品种中干旱处理8小时快速上升并达到最高，干旱12小时后降低。与脱水素基因相比较，强抗旱青稞品种在干旱2小时后HVA1基因的相对表达水平显著升高，相对表达量随干旱处理的时间持续上升，在干旱12小时后达到最高；与之相比较，在整个干旱过程中，弱抗旱品种的相对表达水平显著低于强抗旱品种，在干旱8小时之前弱抗旱品种的相对表达水平变化不明显；在干旱8~12小时后却显著上升。上述结果表明，不同的LEA蛋白在植物耐脱水过程中的干旱表达累积水平不同；干旱不是诱导高等植物Dhn11基因表达的主要因素；植物的抗旱性不同，不同LEA蛋白基因对干旱的反应有差异。推测某些LEA蛋白基因的干旱胁迫早期表达累积程度与植物的抗旱性直接相关；其中，Dhn11基因和Dhn12基因不同的表达模式可能与干旱调控表达顺式作用成分(dehydration responsive element, DRE)的有无或结构上的差异有关。 本研究结果认为，(1)失水率和相对含水量可作为植物抗旱性检测的指标之一；(2) DHN6同义突变位点的氨基酸残基对维持该蛋白的正常结构和功能起着重要作用；(3) 11-氨基酸保守基元序列拷贝数和极性氨基酸的比例对LEA3蛋白结构和功能有重要影响；(4)LEA蛋白表达随着干旱胁迫程度而增加，但Dhn11基因并不受干旱诱导表达；(5)作物的抗旱性不同，LEA蛋白对干旱的累积反应并不相同，干旱早期LEA蛋白的累积程度可能会影响植物的抗旱性。 Drought resistance was a complex trait which involved multiple physiological and biochemical mechanisms and regulation of numerous genes. Because its complex traits, it is difficult to understand the mechanisms of drought resistance in plants. Plants respond to water stress through multiple physiological mechanisms at the cellular, tissue, and whole-plant levels. Tibetan hulless barley, a pure line, is a selfing annual plant that has predominantly penetrated into the Qinghai-Tibetan Plateau and remains stable populations there. The wide ecological range of Tibetan hulless barley differs in water availability, temperature, soil type and vegetation, which makes it possess a high potential of adaptive diversity to abiotic stresses. This adaptive genetic diversity indicates that the potential of Tibetan hulless barley serves as a good source for drought resistance alleles for breeding purposes. 12 contrasting drought-tolerant genotypes were selected to measure relative water content (RWC), maldondialdehyde (MDA) and proline content, based on values of water loss rate (WLR) and repeated drought methods from Tibetan populations of cultivated hulless barley. As a result of the screening, sensitive and tolerant genotypes were identified to clarify relationships between characteristics of LEA2/LEA3 genes sequences and expression and drought-tolerant genotypes, associated with resistance to water deficit. In addition, dynamics water loss rate (DWLR) was measured to observe the changes on diffrential drought-tolerant genotypes. Real-time quantitative RT-PCR was applied to detect relative expression levels of Dhn6, Dhn11, Dhn13 and HVA1 genes in sensitive and tolerant genotypes with 2 h, 4 h, 8h and 12 h of dehydration. In the present study, differential sequences and expression of LEA2/LEA3 genes were explored in Tibetan hulless barley, associated with phenotypically diverse drought-tolerant genotypes. 1. The assessments of WLR and RWC were considered as an alternative measure of plant water statues reflecting the metabolic activity in plants, and the parameters of MDA and proline contents were usually consistent with the resistance to water stress. The values of detached leaf WLR of the tested genotypes were highly variable among 84 genotypes, ranging from 0.086 to 0.205 g/h.g DW. The 12 most contrasting genotypes (6 genotypes with the lowest values of WLR and 6 genotypes with the highest values of WLR) were further validated by measuring RWC, MDA and free-proline contents, which were well watered and dehydrated for 12 h. Results of RWC indicated that the values of 12 contrasting genotypes RWC ranged from 89.94% to 93.38% under condition of well water, without significant differences, but 6 genotypes with lower WLR had higher RWC suffered from 12 h dehydration. The results indicated that lower MDA contents, lower scores of WLR and higher proline contents were associated with drought-tolerant genotypes in hulless barley. Remarkably, proline amounts were increased more notable in 6 tolerant genotypes than 6 sensitive genotypes after excised leaves were dehydrated for 12 h, with control to slight changes under condition of well water. Results of MDA contents showed that six 6 tolerant genotypes had lower MDA contents than the 6 sensitive genotypes under both stressed and non-stressed conditions. As a result of that screening, drought- resistant genotypes (Ximala 10 and Pinbi 14) and drought-sensitive genotypes (Dongqing 8 and QB 24) were chosen for comparing the differential characteristics of LEA2/LEA3 genes and their expression analysis. It was conclusion that measurements of WLR could be considered an alternative index as screening of drought-tolerant genotypes in crops. 2. Late embryogenesis abundant (LEA) proteins were thought to protect against water stress in plants. To explore the relationships between configuration of LEA proteins and phenotypically diverse drought-tolerant genotypes, sequences of LEA genes and their deduced proteins were compared in Tibetan hulless barley. Results of comparing Dhn6 gene in Ximala 10 and QB24 indicated that absence of 63bp was found, except that only 5 mutant nucleotides were found. While 22 mutant sites were taken place in Dhn6 gene between sensitive and tolerant lines, 14 synonymous mutation sites appeared in the contrasting genotypes. The additional/absent polypeptide of 21 polar amino acid residues was not consistent with phenotypically drought-tolerant genotypes in hulless barley. It was deduced that synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein. The sequencing analysis results indicated that each cloned HVA1 gene from four selected genotypes contained an entire open reading frame. The whole sequence of HVA1 gene from Dongqing 8, QB24, Pinbi 14 and Ximala 10 was respectively 661bp, 697bp, 694bp and 691bp. Results of DNA sequence analyses showed that the differences in nucleotides of HVA1 gene in sensitive genotypes were not consistent with that of tolerant genotypes, except for absence of 33 nucleotides from +154 to +186 (numbering from ATG) in QB24. Database searches using deduced amino acid sequences showed a high homology in LEA3 proteins in the selected genotypes. Multiple sequence alignments revealed that LEA3 protein from Dongqing 8 was composed of 8 repeats of an 11 amino acid motif, less the fourth motif than Pinbi 14, Ximala 10 and QB24. Consistent mutant amino acid residues appeared in contrasting genotypes by aligning and comparing the coding sequence region, including Gln32, Arg33 and Ala195 in tolerant genotypes as compared to Asp32, Glu33 and Thr195 (Thr184 in Dongqing 8) in sensitive lines. It was concluded that consistent appearance of Gln32, Arg33 and Ala195 would contributed to functions of LEA3 protein in crops, as well as higher proportion of 11-amino-repeating motifs and polar amino acid residues. 3. Most of the LEA genes are up-regulated by dehydration, salinity, or low temperature, are also induced by application of exogenous ABA, which increases in concentration in plants under various stress conditions and acts as a mobile stress signal. Higher levels of proteins of LEA group 3 accumulated was correlated well with high level of desiccation tolerance in severely dehydrated plant seedlings. Dehydrins (DHNs), members of LEA2 protein, are an immunologically distinct protein family, and Dhn genes expression is associated with plant response to dehydration. Dynamic water loss rate was measured between sensitive genotypes and tolerant genotypes after they were dehydrated for 2 h, 4 h, 6h and 8 h. Detailed measurements of WLR at the early stage of dehydration (2, 4, 6, and 8 h) showed that WLR was stabilizing after 8 h, and there were no significant changes between these values and WLR after 24 h. Drought stress was applied to 10-day-old seedlings by draining the solution from the container for defined dehydration periods. Leaf tissues of the selected genotypes were harvested from control plants (time 0); and after 2, 4, 8, and 12 h of dehydration. Differential expression trends of Dhn6, Dhn11, Dhn13 and HVA1 genes were detected in phenotypically diverse drought-tolerant hulless barleys, related to different time of dehydration. Results of quantitative real-time PCR indicated that relative level of HVA1 expression was always higher in tolerant genotypes, rapidly increasing at the earlier stages (after 2-4 h of dehydration). However, HVA1 expressions of sensitive genotypes had a fast increase from 8 h to 12 h of stress. Significant differences in expression trends of dehydrin genes between tolerant genotypes and sensitive lines were detected, mainly in Dhn6 and Dhn13 gene, depending on the duration of the dehydration stress. The relative expression levels of Dhn6 gene were significantly higher in tolerant genotypes after 8 h dehydration, by control with notable higher expression levels after 12 h water stress in sensitive ones. The relative expression levels of Dhn13 gene tended to ascend during exposure to dehydration in drought-sensitive genotypes. However, fluctuate trends of Dhn13 expression level were detected in drought-resistant lines, including in lower expression levels of 12 h dehydration as compared to 8 h water stress. It was conclusion that (1) diverse LEA proteins would play variable roles in resisting water stress in plants; (2) expression of Dhn11 gene was not induced by dehydrated signals because of the trends of expression descended in contrasting genotypes suffered from water deficit and (3) variable accumulations on LEA proteins would be appear in diverse drought-tolerant genotypes during dehydrations. It is deduced that higher accumulations of Dhn6 and Dhn13 expression in 8 h dehydration are related to diverse drought-tolerant lines in crops. The present results indicated that different dehydrin genes would play variable functional roles in resisting water stress when plants were suffered from water deficit. The authors suggest physiologically different reactions between resistant and sensitive genotypes may be the results of differential expression of drought-resistant genes and related signal genes in plants. In addition, contrarily induced expression of Dhn11 and Dhn12 was related to dehydration responsive element (DRE) in barleys. The present study indicated that (1) measurements of WLR and RWC could be considered as one index of drought-tolerant screenings; (2) synonymous mutation sites would play important roles in holding out right configurations and functions on DHN6 protein, (3) higher proportion of 11-amino-repeating motifs and polar amino acid residues would contribute to functions on LEA3 protein, (4) the longer drought, the more accumulation on LEA proteins, except for Dhn11 gene in crops and (5) differential responses on expression of LEA protein genes would result in physiological traits of drought tolerance in plants.

Induction of triploidy in large yellow crocker Pseudosciaena crocea (Richardson, 1846): effects of pressure shocks and growth performance in the first rearing year

The precociously sexual maturation in large yellow crocker Pseudosciaena crocea has become a serious problem. In an attempt to solve this problem, the production of sterile triploids could be an effective strategy. In this study, triploid P. crocea was obtained by subjecting fertilized eggs to pressure shock. Flow-cytometry analysis was used to assess ploidy level. In terms of triploid rate and hatching rate, the optimal conditions of pressure shock for triploidy induction in P. crocea were 7500 psi for 3 min shock at 3 min after fertilization at 20 degrees C. With the application of these parameters, 100% triploid fish were produced. During the first rearing year, triploid P. crocea had a similar growth performance compared with its diploid counterpart before the age of 8 months and showed a significant advantage at the age of 10 and 12 months in body weight and body length (P < 0.05). At the age of 12 months, the carcass weight of triploids was markedly higher than that of diploid control, and gonadal somatic index was significantly lower than that of their diploid control. During the first rearing year, survival in triploid group was 76.44%, inferior to its diploid control (83.21%).