克隆植物对异质性生境的适应对策研究

植物的生境在时间和空间上都是异质性的，即使在很小的尺度上这种异质性也是存在的。克隆生长使得克隆植物在理论上更适应利用异质性环境，本文以几种克隆植物为对象，采用实验生态学方法，着重从生理生态特性、信号物质传导方面探讨克隆植物对异质性环境的适应对策。 以匍匐茎克隆植物野草莓(Fragaria vesca)为对象，研究了不同海拔梯度种群(1800m和3900m)对光照和养分资源斑块性分布生境的响应。研究结果显示：与资源的空间同质性处理(I) 和(II) 相比, 资源的空间异质性处理(III) 和(IV) 两个种群野草莓的近端、远端和整个克隆片段的生物量和分株数均获得显著增加。经历低光高养近端分株与经历高光低养的远段分株相连时，相比与低光高养的同质生境，来自两个海拔的种群分配更多的生物量到根；经历高光低养近端分株与经历低光高养远端分株相连时，相比于高光低养的同质生境，来自两个海拔的种群分配更少的生物量到根，类似的生物量分配格局在远端分株也被观察到。相比于高光低养同质性生境，当与低光高养远端分株相连时经历高光低养近端分株有更大的叶面积；相比于高光低养同质性生境，当与低光高养近端分株相连时经历高光低养远端分株有更大叶面积。实验结果表明, 资源交互斑块性生境中野草莓发生了克隆内分工。通过克隆内分工, 克隆植物能有效的利用异质性分布的资源, 缓解资源交互斑块性分布对克隆植物生长的不利影响。 以匍匐茎克隆植物蛇莓(Duchesnea indica)为对象, 研究其在高光照低水分斑块和低光照高水分斑块组成的资源交互斑块性生境中的克隆内分工。结果显示，当生长于高光照低水分(HL)条件下近端分株(basal ramets)与生长于低光照高水分(LH)条件下的远端分株(apical ramets)之间的匍匐茎连接时，近端分株根冠比显著下降，而远端分株根冠比显著增加，近端分株叶面积和远端分株总根长显著增加；当与低光照高水分条件下的远端分株相连时，近端分株叶片光合速率和叶绿素含量也相应增加。此外，克隆分株间资源交互传输显著提高蛇莓的生长表现（生物量和分株数）。因此，在光、水资源交互斑块性环境中克隆植物蛇莓分株在生物量分配、资源获取器官形态和生理特性方面发生了环境诱导的功能特化。这种对局部丰富资源的趋富特化在一定的程度上增强了克隆分株对资源的吸收利用能力，克隆内资源共享有助于缓解资源交互性斑块生境对克隆植物生长的不利影响，有效地提高克隆植物在其生境中存活与定居能力。 一个盆栽实验被采用以便调查克隆整合对经受局部沙埋的根状茎克隆植物沙生苔草(Carex praeclara)的影响，结果显示随着沙埋深度的增加，切断分株间的根状茎连接将显著降低经受沙埋处理分株的存活。当克隆植物经历局部沙埋时，切断分株间根状茎连接对其克隆生长（生物量、分株数和叶片数量）有显著负影响。耗-益(cost-benefit)分析显示，当与经历沙埋处理的远端分株相连时，近端分株的生长表现没有遭受任何负面影响。与经历沙埋处理远端分株相连时，近端分株的光合能力随沙埋深度的增加而增加。分株间的源-汇反馈调节机制所导致的补偿性反应减缓了局部沙埋对克隆植物生长的负效应。因此，克隆整合有助于提高经历局部沙埋克隆植物的存活，克隆植物在沙化地区植被恢复与重建方面具有重要意义。 克隆植物分株间的匍匐茎或根状茎连接不仅可以传输水分、矿质养分、光合产物，而且还可以传输信号物质。以根状茎克隆植物黑褐苔草(Carex alrofusca)为对象，采用盆栽实验研究外源茉莉酸诱导克隆片段相连分株间信号物质传导。结果显示，相比中龄和老龄分株，幼年分株对1mM茉莉酸诱导有显著反应。茉莉酸引起幼年分株叶片浓缩单宁含量显著增加，同时其叶片可溶性碳水化合物和氮含量降低。茉莉酸诱导后，幼年分株被昆虫咬食叶面积比率显著下降。因此匍匐茎或根状茎传也是克隆植物分株间信号物质传导重要通道，克隆植物通过分株间的风险扩散策略增强了对幼嫩植物组织器官的保护，这对克隆植物的存活或生长具有重要意义。

Cytological Mechanism on the Integration of Heterologous Genome or Chromosomes in the Unique Gynogenetic Carassius auratus gibelio

银鲫（ＣａｒａｓｉｕｓａｕｒａｔｕｓｇｉｂｅｌｉｏＢｌｏｃｈ）是行天然雌核发育生殖的两性型三倍体鱼类，与普通两性融合生殖鱼类相比，具有独特的育种优势。八十年代以来，异育银鲫、复合四倍体异育银鲫的发现表明，雌核发育卵子不但具有保持自身全部染色体的能力，还能整合异源精子的部分遗传物质或整个基因组，影响雌核发育后代的性状。因此，搞清楚异源基因组的整合机制对于进一步弄清其发育模式以及诱导复合多倍体银鲫均具有十分重要的作用。两性融合发育鱼类的精子入卵后，精核在促精核活化因子的诱导下，可以逐渐解凝并形成雄性原核；而在

Clonal diversity and genealogical relationships of gibel carp in four hatcheries

To conserve and utilize the genetic pool of gynogenetic gibel carp (Carassius auratus gibelio), the Fangzheng and Qihe stock hatcheries have been established in China. However, little information is available on the amount of genetic variation within and between these populations. In this study, clonal diversity in 101 fish from these two stock hatcheries and 35 fish from two other hatcheries in Wuhan and Pengze respectively was analysed for variation in serum transferrin. Thirteen clones were found in Fangzheng and Qihe, of which 12 were novel. Six clones were specific to Fangzheng and three specific to Qihe, whereas four were shared among the Fangzheng and Qihe fish. To obtain more knowledge on genetic diversity and genealogical relationships within gibel carp, the complete mitochondrial DNA (mtDNA) control region (similar to 920 bp) was sequenced in 64 individuals representing all 14 clones identified in the four hatcheries. Differences in the mtDNA sequences varied remarkably among hatcheries, with the Fangzheng and Qihe lines demonstrating high diversity and Wuhan and Pengze showing no variation. The Fangzheng and Qihe lines might represent two distinct matrilineal sources. One of the Qihe samples carried the haplotype shared by a most widely cultivated Fangzheng clone, indicating that a Fangzheng clone escaped from cultivated ponds and moved into the Qihe hatchery. Four Fangzheng samples clustered within the lineage formed mainly by Qihe samples, most likely reflecting historical gene flow from Qihe to Fangzheng. It is suggested that clones in Wuhan originated from Fangzheng, consistent with their introduction history, supporting the hypothesis that gibel carp in Pengze were domesticated from individuals in the Fangzheng hatchery.

Site-directed gene integration in transgenic zebrafish mediated by cre recombinase using a combination of mutant Lox sites

With current gene-transfer techniques in fish, insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences, and multiple gene interactions make gene expression experiments difficult to interpret and fish phenotype less predictable. To meet different fish engineering needs, we describe here a gene targeting model in zebrafish. At first, four target zebrafish lines, each harboring a single genomic lox71 target site, were generated by zebrafish transgenesis. The zygotes of transgenic zebrafish lines were coinjected with capped Cre mRNA and a knockin vector pZklox66RFP. Site-specific integration event happened from one target zebrafish line. In this line two integrant zebrafish were obtained from more than 80,000 targeted embryos (integrating efficiency about 10(-4) to 10(-5)) and confirmed to have a sole copy of the integrating DNA at the target genome site. Genomic polymerase chain reaction analysis and DNA sequencing verified the correct gene target events where lox71 and lox66 have accurately recombined into double mutant lox72 and wild-type loxP. Each integrant zebrafish chosen for analysis harbored the transgene rfp at the designated egfp concatenates. Although the Cre-mediated recombination is site specific, it is dependent on a randomly placed target site. That is, a genomic target cannot be preselected for integration based solely on its sequence. Conclusively, an rfp reporter gene was successfully inserted into the egfp target locus of zebrafish genome by Cre-lox-mediated recombination. This site-directed knockin system using the lox71/lox66 combination should be a promising gene-targeting platform serving various purposes in fish genetic engineering.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Characterization of transgene integration pattern in F4 hGH-transgenic common carp (Cyprinus carpio L.)

The integration pattern and adjacent host sequences of the inserted pMThGH-transgene in the F4 hGH-transgenic common carp were extensively studied. Here we show that each F4 transgenic fish contained about 200 copies of the pMThGH-transgene and the transgenes were integrated into the host genome generally with concatemers in a head-to-tail arrangement at 4-5 insertion sites. By using a method of plasmid rescue, four hundred copies of transgenes from two individuals of F4 transgenic fish, A and B, were recovered and clarified into 6 classes. All classes of recovered transgenes contained either complete or partial pMThGH sequences. The class I, which comprised 83% and 84.5% respectively of the recovered transgene copies from fish A and B, had maintained the original configuration, indicating that most transgenes were faithfully inherited during the four generations of reproduction. The other five classes were different from the original configuration in both molecular weight and restriction map, indicating that a few transgenes had undergone mutation, rearrangement or deletion during integration and germline transmission. In the five types of aberrant transgenes, three flanking sequences of the host genome were analyzed. These sequences were common carp beta-actin gene, common carp DNA sequences homologous to mouse phosphoglycerate kinase-1 and human epidermal keratin 14, respectively.

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.

Monolithic integration of sampled grating DBR with electroabsorption modulator by combining selective-area-growth MOCVD and quantum-well intermixing

We present the monolithic integration of a sampled-grating distributed Bragg reflector (SC-DBR) laser with a quantum-well electroabsorption modulator (QW-EAM) by combining ultra-low-pressure (55 mbar) selective-area-growth (SAG) metal-organic chemical vapour deposition (MOCVD) and quantum-well intermixing (QWI) for the first time. The QW-EAM and the gain section can be grown simultaneously by using SAG MOCVD technology. Meanwhile, the QWI technology offers an abrupt band-gap change between two functional sections, which reduces internal absorption loss. The experimental results show that the threshold current I-th = 62 mA, and output power reaches 3.6 mW. The wavelength tuning range covers 30 nm, and all the corresponding side mode suppression ratios are over 30 dB. The extinction ratios at available wavelength channels can reach more than 14 dB with bias of -5 V.

Design of novel three port optical gates scheme for the integration of large optical cavity electroabsorption modulators and evanescently-coupled photodiodes

This paper presents a novel scheme to monolithically integrate an evanescently-coupled uni-travelling carrier photodiode with a planar short multimode waveguide structure and a large optical cavity electroabsorption modulator based on a multimode waveguide structure. By simulation, both electroabsorption modulator and photodiode show excellent optical performances. The device can be fabricated with conventional photolithography, reactive ion etching, and chemical wet etching.

Optical response of high-level bandgap in one-dimensional photonic crystal applying in-plane integration

A new broadband filter, based on the high level bandgap in 1-D photonic crystals (PCs) of the form Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si is designed by the plane wave expansion method (PWEM) and the transfer matrix method (TMM) and fabricated by lithography. The optical response of this filter to normal-incident and oblique-incident light proves that utilizing the high-level bandgaps of PCs is an efficient method to lower the difficulties of fabricating PCs, increase the etching depth of semiconductor materials, and reduce the coupling loss at the interface between optical fibers and the PC device. (c) 2007 Society of Photo-Optical Instrumentation Engineers.

Optical response of high-order band gap photonic crystal applying in-plane in one-dimensional integration

A new broadband filter, based on the high-order band gap in one-dimensional photonic crystal (PCs) of the form Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si vertical bar air vertical bar Si, has been designed by the plane wave expansion method (PWEM) and transfer matrix method (TMM) and fabricated by lithography. The optical response of this filter to normal-incident and oblique-incident light proves that utilizing the high-order band gaps of PCs is an efficient method to lower the difficulties of fabricating PCs, increase the etching depth of semiconductor materials, and reduce the coupling loss at the interface between optical fibers and PC device. (c) 2007 Elsevier B.V. All rights reserved.