盐藻CDPK基因的克隆与生物信息学分析

为了研究CDPK基因的结构和功能,应用RT-PCR和RACE方法从盐藻Dunaliella salina中克隆得到CDPK基因(GenBank登录号为JQ964113),并对其进行生物信息学分析。结果表明:盐藻CDPK基因的cDNA全长3 052bp。5'-UTR长70bp;开放阅读框长1650bp,编码549个氨基酸;3'-UTR长1332bp。蛋白表现为弱酸性,且稳定、亲水,二级结构中以无规则卷曲和α-螺旋为主,三级结构同源建模成功。亚细胞定位于叶绿体和细胞核中,无跨膜结构域,无信号肽。蛋白序列中存在4个丝氨酸磷酸化位点、4个苏氨酸磷酸化位点和3个酪氨酸磷酸化位点,蛋白激酶结构域在53～331位氨基酸处,蛋白激酶ATP结合域在59～82位氨基酸处,丝氨酸/苏氨酸蛋白激酶活性域在173～185位氨基酸处,4个EF-hand钙结合域分别在357～385、393～421、429～457、465～493位氨基酸处。进化分析结果表明,克隆的CDPK基因与团藻、衣藻、盐藻Dunaliella tertiolecta的CDPK亲缘关系最近。为进一步研究CDPK基因的功能及探究盐藻耐盐机制的信号转导途径奠定了分子基础。

盐生杜氏藻MAPK基因的克隆及表达分析

克隆盐藻MAPK基因(命名为DsMAPK),为研究盐藻耐盐分子机制奠定基础。采用RT-PCR和RACE技术克隆DsMAPKcDNA全长,对其进行生物信息学分析,并通过QRT-PCR方法检测盐胁迫下该基因的表达情况。DsMAPK的cDNA全长序列为1861bp(GenBank AccessionJQ782412),包含一个开放阅读框,长度为1407bp,编码468个氨基酸;DsMAPK蛋白为亲水性蛋白,α-螺旋和自由卷曲是其蛋白质二级结构的主要结构元件,伸展链散布其中。据氨基酸序列同源性分析表明,DsMAPK和衣藻、团藻的MAPK蛋白具有较近的亲缘关系。盐藻在高盐胁迫下,DsMAPK基因表达显著上调,1h后达到最大值,为正常对照组的5倍,差异极显著(P<0.01)。DsMAPK基因可能在盐藻对盐胁迫的反应中起重要作用,是一种参与盐藻耐盐反应的早期快速反应基因。

基于16S和COⅠ基因探讨岩扇贝在扇贝科中的分类地位

采用PCR扩增和序列测定等技术,对岩扇贝(Crassadoma gigantea)线粒体DNA 16S rRNA和COⅠ基因片段进行初步研究,得到16S rRNA基因片段的大小在613.0～634.0 bp之间,A、T(U)、G、C的平均含量分别为26.5%、29.7%、24.5%、19.3%;COⅠ基因片段的大小为660 bp,碱基A、T、G、C的平均含量分别为17.2%、40.3%、27.2%、15.3%。16S基因在岩扇贝中偏好使用GUU、UUG、AUU、ACG、UAU、CAA、GAG、UUU、UCU、GAU、GGU、GAG、AGG和GGA等25个密码子。COⅠ系统进化树表明,与岩扇贝亲缘关系最近的是柿孔扇贝(Azumapecten farreri),其次是冰岛扇贝(Chlamys islandica)。16S系统进化树亲缘关系由近到远依次是北美扇贝(Patinopecten caurinus)、虾夷扇贝(Mizuhopecten yessoensis)、Chlamys islandica。

盐藻丝氨酸/苏氨酸蛋白激酶基因DsSTPK的克隆、原核表达及纯化

前期研究表明,盐藻丝氨酸/苏氨酸蛋白激酶DsSTPK在转录水平上的表达受盐胁迫诱导。为了进一步研究该蛋白激酶的生理功能,通过RT-PCR扩增DsSTPK的开放阅读框,并将其克隆至pET32a(+)载体,得到重组表达载体pET32a(+)-DsSTPK。将重组表达载体转化E.coli.BL21(DE3),IPTG诱导表达,然后对融合蛋白进行表达形式分析,利用His60 Ni离子重力柱进行纯化,所得产物进行SDS-PAGE和Western-blot鉴定。本研究成功构建了重组表达载体pET32a(+)-DsSTPK,经IPTG诱导后表达出的融合蛋白的分子量与预期相符;表达形式分析显示该融合蛋白在上清和包涵体中均存在;将上清蛋白纯化后获得了纯度较高的融合蛋白;Western-blot检测显示该融合蛋白能被抗组氨酸抗体特异性识别,具有良好的免疫学活性。DsSTPK的成功表达、纯化,为下一步制备抗体,然后进一步在蛋白质水平上研究其在盐藻耐盐机制中的作用奠定了基础。

富齿岩虫南移养殖与营养成分分析

该研究通过南移养殖的方法,测定了富齿岩虫(Marphysa maxidenticulata)在广西北海地区的生长状况,并比较了南北方虫体营养成分。结果显示,经240 d的养殖,虫体平均体长达(12.66±1.52)cm,平均体质量达(2.30±0.86)g。试验前期,海泥底质组虫体的特定生长率显著高于粗砂底质组;而粗砂底质组的虫体在养殖后期表现出较高的特定生长率。营养组成分析结果显示,广西北海养殖和山东乳山野生虫体均含有18种氨基酸,但其基本营养组成存在显著差异(P<0.05)。北海养殖虫体氨基酸总量(TAA)、呈味氨基酸(DAA)、非必需氨基酸(NEAA)、半必需氨基酸(SEAA)、必需氨基酸(EAA)含量均高于乳山野生虫体。在脂肪酸组成方面,2个地区的富齿岩虫均含有27种脂肪酸,且以棕榈酸和油酸为主要脂肪酸;乳山野生虫体饱和脂肪酸含量和多不饱和脂肪酸含量与广西北海养殖虫体无显著差异;北海养殖虫体单不饱和脂肪酸总含量显著高于乳山野生虫体(P<0.05)。

Effects of temperature on keeping-alive without water and antioxidant enzyme of the rock scallop Crassadoma gigantea

Temperature was one of the key factors for the living environment of aquatic organisms, especially for shellfish. The effects of temperature was studied on the survival rate and the median lethal time （LT50） of the rock scallop Crassadoma gigantea, and the effects of temperature stress on the antioxidant enzymes of rock scallop Crassadoma gigantea were studied in order to explore the effects of temperature on keeping-alive without water and antioxidant system of the rock scallop Crassadoma gigantea. The rock scallop Crassadoma gigantea （shell length （39.7± 5） mm） at different temperatures （5℃ and 15℃）, humidity （moisture treatment, not moisturizing treatment） and oxygen （aerobic treatment, not aerobic treatment） in laboratory was studied by the survival analysis and the analysis of factors affecting the effects of the median lethal time （LTso） in order to investigate the effects of temperature on the survival rate and the median lethal time of the rock scallop Crassadoma gigantea. Results showed that low temperature, moisture and aerobic treatment can significantly improve the survival rate and the median lethal time of the rock scallop Crassadoma gigantea （P〈0.05）. The effect of temperature on the survival rate and the median lethal time of the rock scallop Crassadoma gigantea was most significant, followed by the effect of oxygen, and that of humidity was the most weakest. Interaction between temperature and humidity, temperature and oxygen significant interaction（P〉0.05）, and interaction between humidity and oxygen interaction was not significant （P〈0.05） in the interaction of three factors. It provided an important basis for the determination of the rock scallop Crassadoma gigantea in the seedling transportation time. In the article, the rock scallop Crassadoma gigantea larvae antioxidant enzymes activity was detected, including SOD activity,CAT activity ,T-A℃ activity （superoxide dismutase activity, catalase activity, total antioxidant capacity） changes through raising the temperature from the low temperature （5℃, 10℃, 15℃, 20℃, 25℃）, to explore the change of the antioxidant enzymes activity under the influence of temperature on the rock scallop Crassadoma gigantea larvae. The SODactivity was significantly lower than that of the other groups （P〈0.05） at 5℃, and the CAT activity had no significant difference between groups （p=0.05）, and the T - AOC level was the lowest significantly below 15℃, 25℃ （P〈0.05）.The SOD and CAT activity had no significant difference in the same experimental time （P〉0.05）, and the T - AOC activity temperature was significantly higher than the rest of the group （P=〈0.05） when temperature at 10℃ and 20℃. The SOD activity had no significant difference （P〉0.05）between groups at 25℃, and the CAT activity was significantly higher than 5℃ 10℃, 15℃ group （P〈0.05）. The T-AOC activity was only lower than 15℃ group （P〉0.05）. It indicated that the high temperature had a clear inducing effect on the CAT activity in the rock scallop Crassadoma gigantea larvae, and had a significant inhibitory effect on the T-AOC activity. The low temperature had a significant inhibitory effect on the activity of T-AOC in the tissue fluid of the scallop. The above results showed that the high and low temperature had a significant effect on the antioxidant enzymes activity of the rock scallop Crassadoma gigantea larvae.

Effect of temperature on the growth of juvenile rock scallop (Crassadoma gigantea)

In order to study the effect of temperature on the growth of juvenile rock scallop(Crassadoma gigantea),learn more about its growth with temperature changes under the conditions of indoor circulating water temperature-controlled culture.Four temperature gradients of10℃,12.5℃,15℃and17.5℃were set,observed the growth of juvenile rock scallop at different test temperatures during two month.The results show:With the increase of test temperature,the growth rate first increased and then decreased.One-way analysis of variance showed that temperature had a significant effect on the growth rate(P<0.05),the optimum temperature was15℃,under this condition,the shell height average daily growth was significantly higher than the other three groups,the average daily growth of shell length and wet weight had no significant difference with17.5℃experimental group but significantly higher than the previous two groups.Within the experimental range,the change of temperature had no significant effect on the survival rate of juvenile rock scallop(P>0.05).