Molecular Techniques for Dicistrovirus Detection without RNA Extraction or Purification

Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.

藻胆体的光谱特性和光能传递

一，螺旋藻藻胆体光谱特性及其光能传递的研究 1，完整藻胆体与解离藻胆体吸收光谱的比较研究 对螺旋藻完整藻胆体和解离藻胆体的吸收光谱中进行了比较研究。随着PBS逐渐解离，其吸收光谱表现出如下变化特点：在紫外区，吸收峰始终位于355nm，尖形峰逐渐变成钝形峰;在红区，完整藻胆体和解离藻胆体都有很强的光吸收，吸收峰呈平顶状，其半带宽逐渐变小，紫外区与红区相对吸收强度比值逐渐变小，四组导数吸收光谱中的小峰数目越来越少。室温荧光发射光谱表明，PBS在低于0.9mol/L的磷酸缓冲液中变得不稳定，并开始逐渐解离，解离的PBS与完整的PBS相比，其荧光发射峰逐渐蓝移。 2，藻胆体在解离过程中荧光发射和光能传递的研究 完整藻胆体的室温荧光发射光谱中只有一个峰，在678nm。说明在完整藻胆体中，光能传递效率高。在77K荧光发射光谱中，完整藻胆体只有一个峰，位于682nm，这是L_(cm)(TE_1)的荧光峰;严重解离的藻胆体的主峰在656nm，是PC的荧光;在679nm有一个小峰，是APC-B的荧光(TE_2)。据此，我们提出螺旋藻藻胆体的光能传递链为：(此处表从略，见全文) 二，螺旋藻藻胆体核心及其与藻蓝蛋白的重组 PC+core混合物，浓缩重组48h后，其室温荧光发射峰位于663nm，与PC的室温荧光发射峰643nm和PC+core混合物（未重组）的室温荧光发射峰648nm相比，说明部分APC与部分PC发生了重组，使部分PC吸收的光能传递给了APC，使荧光发射峰红移;与藻胆体核心室温荧光发射峰664nm相比，则非常接近，说明重组效果较好。PC+core混合物（未重组），其77K荧光发射光谱中有两个峰：654nm,679nm,分别是PC,APC-B的荧光峰，F679/F654的比值为32.0%。我们以F679/F654比值的变化来判断PC与core是否发生了重组。PC+core混合物，经48h浓缩重组后，77K荧光发射光谱中有F657,F679两个峰，F679/F654的比值则为45.9%，比未重组的混合物32.0%升高了，说明部分PC与core发生了重组，部分PC吸收的光能传递给了APC和APC-B，使F679加强，F654减弱。 三，螺旋藻藻胆体一类囊体膜光谱特性与光能传递的研究 藻胆体一类囊体膜的吸收光谱，室温荧光发射光谱和77K荧光发射光谱表明：藻胆蛋白能将捕获的光能传递给叶绿素a，叶绿素a捕获的光能不能逆传给藻胆蛋白。 四，藻胆体一类囊体膜的重组 藻胆体一类囊体膜的吸收光谱说明，一部分被洗下来的PBS能重新结合到类囊体膜上，但并没有达到100%的重组。 五，整体螺旋藻光谱特性及其光能传递的研究 整体螺旋藻光谱特性与PBS-类囊体膜的光谱特性极为相似，表现出同样的规律：PBS的吸收面积与叶绿素a相比，叶绿素a的吸收是主要的。 从PBS-类囊体膜和整体螺旋藻的吸收光谱，室温荧光发射光谱，77K荧光发射光谱的研究中可知，二者表现出极为相似的规律：PBS藻胆蛋白捕获的光能能传递给叶绿素a，叶绿素a捕获的光能不能逆传给PBS藻胆蛋白。主要的捕光物质是叶绿素a。 另外，我们还对Spirulina platensis 6 and Spirulina maxima的藻胆体在解离过程中的荧光发射和光能传递进行了研究，表现规律与Spirulina platensis相同。

盐胁迫对螺旋藻光合作用影响的研究

盐胁迫是限制高等植物和藻类生长和产量的主要环境因子之一。PSII对环境胁迫的响应被认为是光合作用适应逆境过程中最重要的一个环节。尽管盐胁迫对PSII的影响已进行了大量的研究，但有关盐胁迫对PSII作用方式和位点的研究仍存在着争议。我们主要研究了盐胁迫对螺旋藻PSII结构和功能的影响，以探讨盐胁迫对PSII的作用方式和位点以及该藻细胞PSII对盐胁迫的适应机理。主要研究结果如下： 1． 用0、0.2、0.4、0.6、0.8M NaCl处理螺旋藻细胞12小时。随盐浓度的增加，螺旋藻细胞的Chla、carotenoid、PC、APC及蛋白含量均呈下降趋势，说明盐胁迫抑制了上述色素及蛋白的合成或加速了它们的降解，从而影响了螺旋藻的光合作用。 2． 随盐浓度的增加，螺旋藻细胞光合放氧活性和PS II电子传递活性显著降低，表明盐胁迫引起藻细胞PS II活性的下降。 3． 通过放氧活性、热致发光（TL）、多相荧光瞬态上升动力学曲线的测定以及Western 杂交，来探讨盐胁迫对螺旋藻细胞PS II供体侧电子传递及OEC33蛋白含量的影响。结果显示：随盐浓度的增加，螺旋藻细胞光合放氧活性和PS II电子传递活性下降;TL B-band和Q-band强度降低，在0-0.6M NaCl下，B-band的周期性振荡清楚，最大值出现在第二次和第六次闪光，而在0.8M NaCl时，S态振荡基本上消失，S 态氧化还原循环受阻;Fm， J、I和P相荧光水平降低。以上结果都表明盐胁迫使PS II的放氧侧受损伤。且随盐浓度的增加，盐分引起螺旋藻细胞外周蛋白OEC33的降解，在蓝藻中首次提出放氧机构的S态循环受阻，放氧活性降低。 4． 通过OJIP曲线的测定以及JIP-test、闪光诱导的可变荧光衰减动力学、热致发光（TL）的分析，我们研究了盐胁迫对螺旋藻细胞PS II受体侧的影响。结果显示： JIP-test的参数Ψo和φEo随盐浓度的增加而下降,显示QA-到QB 电子传递受阻;可变荧光衰减动力学快相组分半衰期延长，所占总可变荧光百分比下降，表明QA-到QB 电子转移变慢，中相组分半衰期延长、所占百分比下降，说明空的QB位点对PQ的结合减慢，有可能PQ分子对QB位点的结合能力下降;TL B-band和Q-band的峰温度出现了位移，可能QA、QB的氧化还原电势发生了改变。以上结果表明，盐胁迫伤害了PSII受体侧的电子传递。 5． 首次运用闪光诱导下的叶绿素荧光上升及其衰减动力学来研究盐胁迫对PS II受体侧的影响。 6． 盐胁迫下，PS II供体侧和受体侧电子传递受抑制，有活性的PSII反应中心数量下降，说明盐胁迫对螺旋藻细胞PSII的伤害也可能是多位点的作用方式。此外，盐胁迫下，藻细胞放氧活性的下降快于受体侧QA 到QB电子传递所占百分比的下降，有可能PS II放氧侧先受损伤，然后是反应中心和受体侧。上述结果表明盐胁迫下PSII活性的降低是由于PSII供体侧和受体侧电子传递的抑制，有活性的PSII反应中心的减少。 7． 借助螺旋藻类囊体膜的Western杂交分析，来研究盐胁迫对螺旋藻类囊体膜PSII相关蛋白的影响。结果表明，上述PSII活性的抑制是由于类囊体膜蛋白的损失。主要与PSII反应中心CP43、CP47和OEC33蛋白含量的下降有关。 8． PS II机构对盐胁迫的适应涉及以下几个方面：降低吸收横截面，（PC/chla，APC/chla比值的降低）;光系统II光化学反应的改变，通过关闭的PS II反应中心比例的增加，使得PS II机构免于过多激发能的伤害而得以保护;提高了剩余的有活性反应中心的耗能效率（DIo/RC增加）;保持有活性反应中心高的激发能转化效率，比如，TRo/RC保持不变;另外，随盐浓度的增加，由藻胆体向光系统I的能量传递增加，避免过量激发能对 PSII的伤害，使螺旋藻细胞适应盐胁迫环境。

高温胁迫对螺旋藻光系统II影响的研究

高温胁迫是限制高等植物和藻类生长和产量的主要环境因子之一。光系统II（PSII）对环境胁迫的响应被认为是光合作用适应逆境过程中最重要的一个环节。高温胁迫对螺旋藻PSII的研究相对较少，对PSII受体侧的研究更加少了。我们借助热致发光及QA-再氧化动力学，这两种检测完整光合生物PSII供体侧和受体侧电子传递的有效、简单、无损伤的方法，为高温胁迫如何影响供体侧和受体侧的电子传递提供更直接的依据，获得高温胁迫对PSII功能影响的更精确的消息。另外，有关螺旋藻在高温胁迫下的能量传递过程研究较少，希望在荧光光谱研究的结果上探求其对高温胁迫的适应机理。主要研究结果如下： 1.高温胁迫抑制螺旋藻PS II的活性， PSII原初光能转化效率Fv/Fm随处理温度的提高而下降。高温去除后Fv/Fm可以得到部分恢复（5-15%）。 2.高温胁迫对闪光诱导的可变荧光衰减动力学有显著影响，分别代表QA-到QB 的直接电子传递和PQ分子扩散到空的QB位点后QA-到QB电子传递的快相(半衰期160 ms)和中相(半衰期2 ms)占整个可变荧光的比例，随处理温度的升高显著降低，而代表S2QA- 电荷重组的慢相（半衰期约4s）显著增加。显示高温导致QA到QB的电子传递以及PQ与QB位点的结合受阻，从而促进了QA-与放氧复合体S2态的重组过程。同时我们发现，经过5分钟的恢复，这些光系统II还原侧电子传递的功能抑制能够大部分得到恢复，显示高温胁迫对受体侧电子传递的影响具有可逆性。 3. 通过采用77K低温荧光光谱等手段，我们研究了高温胁迫对螺旋藻细胞光合能量传递的影响。研究显示，高温胁迫对580nm和436nm激发的低温荧光光谱都有显著影响。高温胁迫对PS I的发射峰F725和F751没有显著影响，显示高温没有影响藻胆体到光系统I的激发能传递。而高温胁迫引起了PBS对PS II荧光发射比值的上升，说明高温抑制了藻胆体到光系统II之间的激发能传递。但藻蓝蛋白的发射峰643nm在高温处理后基本没有变化，显示高温抑制PBS到PS II的能量传递不是由于藻蓝蛋白到别藻蓝蛋白之间的能量传递受阻造成的。结果还显示，高温胁迫对藻胆体到光系统II能量传递的抑制也不是由于藻胆体与光系统II发生分离，而是抑制了别藻蓝蛋白到CP43和CP47的能量传递，原因可能是由于藻胆体内部结构的改变引起的。 ４.热致发光（TL）和荧光衰减动力学的测定和分析结果显示，高温胁迫改变了S2QA-和S2QB-重组体的稳定性，其中S2QA-的稳定性降低，S2QB-的稳定性升高。根据具有异质性的TL信号，我们推测有活性PSII中有可能存在对高温胁迫敏感度不同的两种亚基，它们具有不同的QB/QB-氧化还原势能。当高温胁迫造成相当数量的反应中心失活，QA到QB正常的电子传递受阻时，光系统有可能通过保存更多的具有较高能量的反应中心亚基，达到促进QA到QB的电子传递的目的。 5. OJIP荧光瞬态上升曲线在高温胁迫后出现标志性K峰，说明螺旋藻的放氧复合物受到伤害，放氧S态引起的多次闪光下的TL振荡，显示这个高温处理对S1→S2的转变没有影响，却抑制了S2→S3的转变，这同OJIP荧光瞬态上升曲线的结果相一致，说明高温对螺旋藻放氧复合体造成了伤害。

Check list of plankton of the northern Red Sea

Qualitative estimation of phytoplankton and zooplankton of the northern Red Sea and Gulf of Aqaba were carried out from four sites: Sharm El-Sheikh, Taba, Hurghada and Safaga. A total of 106 species and varieties of phytoplankton were identified including 41 diatoms, 53 dinoflagellates, 10 cyanophytes and 2 chlorophytes. The highest number of species was recorded at Sharm El-Sheikh (46 spp), followed by Safaga (40 spp), Taba (30 spp), and Hurghada (23 spp). About 95 of the recorded species were previously mentioned by different authors in the Red Sea and Gulf of Suez. Eleven species are considered new to the Red Sea. About 115 species of zooplankton were recorded from the different sites. They were dominated by four main phyla namely: Arthropoda, Protozoa, Mollusca, and Urochordata. Sharm El-Sheikh contributed the highest number of species (91) followed by Safaga (47) and Taba (34). Hurghada contributed the least (26). Copepoda dominated the other groups at the four sites. The appearances of Spirulina platensis, Pediastrum simplex, and Oscillatoria spp. of phytoplankton in addition to the rotifer species and the protozoan Difflugia oblongata of zooplankton impart a characteristic feature of inland freshwater discharge due to wastewater dumping at sea in these regions resulting from the expansion of cities and hotels along the coast.

Solar UV radiation drives CO2 fixation in marine phytoplankton: A double-edged sword

Photosynthesis by phytoplankton cells in aquatic environments contributes to more than 40% of the global primary production (Behrenfeld et al., 2006). Within the euphotic zone (down to 1% of surface photosynthetically active radiation [PAR]), cells are exposed not only to PAR (400-700 nm) but also to UV radiation (UVR; 280-400 nm) that can penetrate to considerable depths (Hargreaves, 2003). In contrast to PAR, which is energizing to photosynthesis, UVR is usually regarded as a stressor (Hader, 2003) and suggested to affect CO2-concentrating mechanisms in phytoplankton (Beardall et al., 2002). Solar UVR is known to reduce photosynthetic rates (Steemann Nielsen, 1964; Helbling et al., 2003), and damage cellular components such as D1 proteins (Sass et al., 1997) and DNA molecules (Buma et al., 2003). It can also decrease the growth (Villafane et al., 2003) and alter the rate of nutrient uptake (Fauchot et al., 2000) and the fatty acid composition (Goes et al., 1994) of phytoplankton. Recently, it has been found that natural levels of UVR can alter the morphology of the cyanobacterium Arthrospira (Spirulina) platensis (Wu et al., 2005b). On the other hand, positive effects of UVR, especially of UV- A (315-400 nm), have also been reported. UV- A enhances carbon fixation of phytoplankton under reduced (Nilawati et al., 1997; Barbieri et al., 2002) or fast-fluctuating (Helbling et al., 2003) solar irradiance and allows photorepair of UV- B-induced DNA damage (Buma et al., 2003). Furthermore, the presence of UV-A resulted in higher biomass production of A. platensis as compared to that under PAR alone (Wu et al., 2005a). Energy of UVR absorbed by the diatom Pseudo-nitzschia multiseries was found to cause fluorescence (Orellana et al., 2004). In addition, fluorescent pigments in corals and their algal symbiont are known to absorb UVR and play positive roles for the symbiotic photosynthesis and photoprotection (Schlichter et al., 1986; Salih et al., 2000). However, despite the positive effects that solar UVR may have on aquatic photosynthetic organisms, there is no direct evidence to what extent and howUVR per se is utilized by phytoplankton. In addition, estimations of aquatic biological production have been carried out in incubations considering only PAR (i. e. using UV-opaque vials made of glass or polycarbonate; Donk et al., 2001) without UVR being considered (Hein and Sand-Jensen, 1997; Schippers and Lurling, 2004). Here, we have found that UVR can act as an additional source of energy for photosynthesis in tropical marine phytoplankton, though it occasionally causes photoinhibition at high PAR levels. While UVR is usually thought of as damaging, our results indicate that UVR can enhance primary production of phytoplankton. Therefore, oceanic carbon fixation estimates may be underestimated by a large percentage if UVR is not taken into account.

A potent anti-oxidant property: fluorescent recombinant alpha-phycocyanin of Spirulina

To express and product a fluorescent antioxidant holo-alpha-phycocyanin (PC) of Spirulina platensis (Sp) with His-tag (rHHPC; recombinant holo-alpha-phycocyaninof Spirulina platensis with His-tag) in 5-l bench scale. A vector harbouring two cassettes was constructed: cpcA along with cpcE-cpcF in one cassette; ho1-pcyA in the other cassette. Lyases CpcE/F of Synechocystis sp. PCC6803 (S6) could catalyse the 82 site Cys in apo-alpha-PC of Sp linking with bilin chromophores, and rHHPC was biosynthesized in Escherichia coli BL21. The constant feeding mode was adopted, and transformant reached the biomass of rHHPC up to 0.55 g l(-1) broth in 5-litre bench scale. rHHPC was purified by Ni2+ affinity column conveniently. The absorbance and the fluorescence emission spectra of rHHPC had lambda(max) at 621 and 650 nm, respectively. The IC50 values of rHHPC were 277.5 +/- 25.8 mu g ml(-1) against hydroxyl radicals and 20.8 +/- 2.2 mu g ml(-1) against peroxyl radicals. Combinational biosynthesis of rHHPC was feasible, and the constant feeding mode was adopted to produce good yields of rHHPC. Fluorescent rHHPC with several unique qualitative and quantitative features was effective on scavenging hydroxyl and peroxyl radicals. A potent antioxidant rHHPC was co-expressed, produced and characterized for nutritional and pharmacological values, which would help to develop phycobiliproteins' applications in their fluorescent and biological activities.

Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria

Cyanobacteria are an ancient group of gram-negative bacteria with strong genome size variation ranging from 1.6 to 9.1 Mb. Here, we first retrieved all the putative restriction-modification (RM) genes in the draft genome of Spirulina and then performed a range of comparative and bioinformatic analyses on RM genes from unicellular and filamentous cyanobacterial genomes. We have identified 6 gene clusters containing putative Type I RMs and 11 putative Type II RMs or the solitary methyltransferases (MTases). RT-PCR analysis reveals that 6 of 18 MTases are not expressed in Spirulina, whereas one hsdM gene, with a mutated cognate hsdS, was detected to be expressed. Our results indicate that the number of RM genes in filamentous cyanobacteria is significantly higher than in unicellular species, and this expansion of RM systems in filamentous cyanobacteria may be related to their wide range of ecological tolerance. Furthermore, a coevolutionary pattern is found between hsdM and hsdR, with a large number of site pairs positively or negatively correlated, indicating the functional importance of these pairing interactions between their tertiary structures. No evidence for positive selection is found for the majority of RMs, e. g., hsdM, hsdS, hsdR, and Type II restriction endonuclease gene families, while a group of MTases exhibit a remarkable signature of adaptive evolution. Sites and genes identified here to have been under positive selection would provide targets for further research on their structural and functional evaluations.

Comparative studies on the efficiency of energy transfer of two R-phycoerythrin-C-phycocyanin conjugates constructed from sodium periodate and glutaraldehyde respectively

The C-phycocyanin and the R-phycoerythrin were purified from the blue-green alga Spirulina platensis and red alga Polysiphonia urceolata respectively. Both sodium periodate and glutaraldehyde are effective coupling agents being capable of constructing the R-phycoerythrin-C-phycocyanin conjugate, which was also called phycobiliproteins energy transfer model. The two artificial conjugates constructed with different methods were purified by Sephadex G-200 chromatography respectively. Spectra analysis indicated that energy transfer occurred in the two conjugates. The conjugate with sodium periodate had the higher efficiency of energy transfer than that with glutaraldehyde conjugate.

The stabilization effect of glutaraldehyde on the Spirulina plateusis phycobilisomes

The spectral properties of the glutaraldehyde-treated phycobilisomes were studied. The results showed that glutaraldehyde was effective in preventing phycobilisomes from dilution-induced dissociation and preserving the intra-phycobilisomes energy transfer.

Sequence analysis of Arthrospira maxima based on fosmid library

Arthrospira (Spirulina) (Setchell& Gardner) is an important cyanobacterium not only in its nutritional potential but in its special biological characteristics. An unbiased fosmid library of Arthrospira maxima FACHB438 that contains 4300 clones was constructed. The size distribution of insert fragments is from 15.5 to 48.9 kb and the average size is 37.6 kb. The recombination frequency is 100%. Therefore the library is 29.9 equivalents to the Arthrospira genome size of 5.4 Mb. A total of 719 sample clones were randomly chosen from the library and 602 available sequences, which consisted of 307,547 bases, covering 5.70% of the whole genome. The codon usage of A. maxima was not strongly biased. GC content at the first position of codons (46.9%) was higher than the second (39.8%) and the third (45.5%) positions. GC content of the genome was 43.6%. Of these sequences, 287 (47.7%) showed high similarities to known genes, 63 (10.5%) to hypothetical genes and the remaining 252 (41.8%) had no significant similarities. The assigned genes were classified into 22 categories with respect to different biological roles. Remarkably, the high presence of 25 sequences (4.2%) encoding reverse transcriptase indicates the RT gene may have multiple copies in the A. maxima genome and might play an important role in the evolutionary history and metabolic regulation. In addition, the sequences encoding the ATP-binding cassette transport system and the two-component signal transduction system were the second and third most frequent genes, respectively. These genomic features provide some clues as to the mechanisms by which this organism adapts to the high concentration of bicarbonate and to the high pH environment.

Spectral changes of C-phycocyanin with different molar ratios of SPDP

Pure C-phycocyanin was prepared from Spirulina platensis using one-step anion-exchange chromatography. The C-PC obtained was with an absorption maximum at 620 nm and a fluorescence emission maximum at 640 nm when excited by 580 nm. SPDP is an excellent heterobifunctional crosslinker for thiolating amines. Different molar ratios of SPDP have remarkable influence on the absorption and fluorescence spectra of C-phycocyanin. The absorption maximum and fluorescence emission maximum both decreased and blue-shifted from 640 run to 630 nm as the molar ratios of SPDP increased. It was found that the molar ratios of SPDP to C-phycocyanin was not more than 100 was appropriate to being conjugated with other biomolecules from the absorption and fluorescence spectra of C-phycocyanin.

海洋藻类功能性蛋白质的纯化、鉴定及其应用的研究

使用膨化柱和离子交换或羟基磷灰石柱层析相结合的方法，分别从多管藻、坛紫菜及钝顶螺旋藻中分离纯化了R-藻红蛋白溶液和C-藻蓝蛋白。光谱检测及电泳分析结果证明完全符合经典的藻胆蛋白纯度标准。彭化床最突出的优点是克服了常规分离方法堵塞色谱柱的难题，纯化速度快、产量高、不需要常规色谱方法所要求的填料的平衡及粗提液的预处理，仅需一步操作就可以得到满足一般食品添加剂纯度要求的藻胆蛋白，极大地简化了后续的纯化程序，减少了分离纯化的步骤和时间，而其产率及纯度均高于常规的藻胆蛋白分离方法。这同时也降低了藻胆蛋白分离纯化的成本。 本文通过戊二醛或环氧氯丙烷交联的方法，合成了四种壳聚糖-氨基酸共聚小球。并选取吸附性好的戊二醛交联孔球和戊二醛交联微球系统测定了其对R-藻红蛋白和C-藻蓝蛋白的吸附和缓释性能。 纯化了藓羽藻中与其细胞器团聚密切相关的一种凝集素并进行了部分性质的鉴定。N端前15个氨基酸序列及LC-ESI-MS质谱分析结果证明此凝集素属于一种新的蛋白质族。实验证明，凝血活性与细胞器团聚活性并不完全依赖于此凝集素分子相同的结构域。 通过异双功能试剂SPDP处理藓羽藻凝集素使之衍生化，DTT处理R-PE在其分子内引入外源巯基，然后将活化的R-藻红蛋白与凝集素进行交联反应。交联产物经凝胶过滤纯化并检测，但电泳及荧光显微镜检测结果并不能证明交联探针的成功制备。

两种蓝藻真核型Ser/Thr激酶基因功能的初步验证

Ser/Thr蛋白激酶（serine/threonine kinases，STK）在真核生物的信号转导通路中具有重要作用，而且已经成为对抗肿瘤、结核等多种人类疾病的药物作用靶点。上世纪九十年代，有研究发现STK在原核生物的信号转导中也发挥重要作用。本论文以聚球藻PCC7942（Synechococcus sp. PCC7942）和钝顶螺旋藻（Spirulina platensis）为材料，对几个真核型的Ser/Thr蛋白激酶基因的功能进行了初步验证。 蓝藻兼具细菌和植物的特点，具有成熟的转化体系，为真核生物基因功能的研究提供了新的模式宿主。聚球藻PCC7942是一种单细胞的淡水蓝藻，具有天然的外源DNA转化系统，是蓝藻分子遗传学研究的模式生物。通过基因敲除及表达差异分析发现聚球藻PCC7942中的Ser/Thr蛋白激酶stk196参与高温胁迫的信号传递。钝顶螺旋藻是原核丝状蓝藻，由于其蕴涵高品质营养成分而成为一类具有重要经济价值的微藻，该研究利用半定量RT-PCR方法，分析四个具有跨膜结构域的Ser/Thr蛋白激酶在正常生长温度下和经低温、高温诱导后表达量的变化情况，发现stk2103在低温诱导后的表达量降低，高温诱导后的表达量升高，提示该基因的表达可能参与了钝顶螺旋藻对温度的适应。 蓝藻中真核型Ser/Thr蛋白激酶功能的研究为我们进一步研究真核生物的Ser/Thr蛋白激酶功能提供了借鉴，并对植物抗逆胁迫的研究提供重要的理论依据。

螺旋藻基因组结构分析和藻胆蛋白的适应性进化

螺旋藻 (Spirulina，或称节旋藻 Arthrospira) 是一类丝状不形成异型胞的蓝藻，常分布于湖泊、池塘和半咸水中。作为目前利用最广泛的经济微藻，螺旋藻的规模培养、生理生化以及分子遗传等都受到了广泛的关注。但由于缺乏足够的基因组信息，影响了螺旋藻的品种改良以及进一步的基因功能产品的开发。 本研究从挑取单藻丝开始，建立了钝顶螺旋藻 (Spirulina platensis) 的纯培养体系，并构建插入片段分别为1-2Kb和4-6Kb的基因组文库，利用鸟枪法进行全基因组测序，共得到具有8倍覆盖度的7.4Mb拼接后的基因组数据。在该草图中，预测有7795个开放读码框 (ORFs)，其中包括长度小于120个氨基酸的ORFs和非全长ORFs。在所有蛋白编码序列中，约有39%的基因是螺旋藻特有的，此外，还有389个基因相对于其他蓝藻，与非蓝藻基因有着更高的相似性。通过Pfam结构域聚类分析发现，螺旋藻含有特有的结构域，如Peptidase_MA、DMT、OB、ATP-grasp、Flavokinase 等。 由于目前尚未有成熟的螺旋藻遗传转化系统，而其体内复杂的限制性内切酶系可能是妨碍外源基因顺利导入和整合的关键因素。在此草图的基础上，我们利用比较基因组学和分子进化的手段，深入分析了螺旋藻及其他蓝藻的限制修饰系统 (Restriction-modification system)，为建立螺旋藻的遗传转化技术，并实现后续的螺旋藻基因功能验证模式奠定了基础。 螺旋藻及其他蓝藻的基因组计划提供了大量基因组序列和结构信息，有助于我们深入了解光合微生物基因的结构、功能和进化，以及环境因子或特定突变是如何塑造其基因组的。本文以藻胆蛋白为例，利用分子进化和群体遗传学理论，描绘出一幅藻胆蛋白进化的动态图景，并分析了环境 - 结构 - 功能之间的相互联系，为藻类捕光色素进化机制理论提供了科学依据。 本研究结果发现低光适应型原绿球藻和海洋聚球藻的藻红蛋白中正选择位点分布有着显著差异，提示二者的藻红蛋白基因有不同的进化模式；正选择作用位点多集中在藻胆蛋白的色基结合区域及XY发卡结构处，这些结构域主要与藻胆蛋白的光能捕获、能量传递和结构组装有关。该研究结果不仅揭示了正选择作用的重要性，而且提示光质、光强和能量传递压等可能是潜在的正选择压力。此外，又深入研究了高光和低光适应型两个生态群体的原绿球藻藻红蛋白基因系统发育、种内多态性和种间变异度，以此揭示环境因子对藻胆蛋白进化的影响。 本研究首次对螺旋藻全基因组进行测序，并以藻胆蛋白为例，从分子进化角度揭示其结构和功能的关系，并首次提出正选择压力可能是促使藻胆蛋白功能分化的主要原因，并认为原绿球藻中藻红蛋白的进化呈现出不同的模式，新的捕光色素的出现以及其所处的海洋环境可能使藻红蛋白获得了新的功能。