Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level ofcyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and c...Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level ofcyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption. With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction （PCR） and real-time quantitative PCR （qPCR） techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macao Storage Reservoir （MSR）. The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4-12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional micro- scopic enumeration for the ecosystems monitoring program.展开更多
通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3(serine/arginine-rich specific kinase 3,SRPK3)的全长基因CDS区...通过对猪SRPK3基因初步的研究,为猪分子遗传育种提供基础分子生物学信息,为猪的遗传育种提供分子标记。以大白猪为实验材料,采用RT-PCR方法克隆了精氨酸-丝氨酸蛋白激酶3(serine/arginine-rich specific kinase 3,SRPK3)的全长基因CDS区;采用生物信息学方法分析了SRPK3基因核酸序列并对其所编码的的蛋白序列进行了预测与分析编码蛋白序列的结构特点;采用PCR-SSCP方法对大白猪,野猪,民猪及野家杂交猪的SRPK3基因的多态性进行了检验;采用实时荧光定量PCR(Real-time)方法检测了SRPK3在1日龄和30日龄大白猪及杜洛克的心脏、肌肉、脾脏、肝脏、肾脏、肺脏、胃、小肠、大肠、脑的表达情况;采用皮下注射的方式构建猪骨骼肌损伤模型用于研究在骨骼肌修复过程中SRPK3基因表达特性。经拼接所得到的1 708bp核苷酸片段,涵盖了SRPK3基因的全长CDS(1 701bp),该基因编码含567个氨基酸片段;蛋白存在两个S_TKc结构域,猪SRPK3蛋白序列与人和牛的相似性较高。PCR-SSCP检测发现第6外显子上A629→G629,T653→T653的突变,氨基酸变化为Pro→His,Ile→Thr;第9外显子处的G1059→A1059,氨基酸无突变。利用荧光定量PCR研究发现,表达结果显示该基因表达具有组织和种间特异性。SRPK3基因的表达在整个骨骼肌细胞损伤修复过程中逐渐升高。SRPK3基因主要在肌肉和心肌内表达,骨骼肌损伤修复过程中伴随骨骼肌细胞分化SRPK3的表达持续升高,推测其可能与骨骼肌细胞发育相关。展开更多
文摘Freshwater algal blooms have become a growing concern world-wide. They are caused by a high level ofcyanobacteria, predominantly Microcystis spp. and Cylindrospermopsis raciborskii, which can produce microcystin and cylindrospermopsin, respectively. Longtime exposure to these cyanotoxins may affect public health, thus reliable detection, quantification, and enumeration of these harmful algae species has become a priority in water quality management. Traditional manual enumeration of algal bloom cells primarily involves microscopic identification which limited by inaccuracy and time-consumption. With the development of molecular techniques and an increasing number of microbial sequences available in the Genbank database, the use of molecular methods can be used for more rapid, reliable, and accurate detection and quantification. In this study, multiplex polymerase chain reaction （PCR） and real-time quantitative PCR （qPCR） techniques were developed and applied for monitoring cyanobacteria Microcystis spp. and C. raciborskii in the Macao Storage Reservoir （MSR）. The results showed that the techniques were successful for identifying and quantifying the species in pure cultures and mixed cultures, and proved to be a potential application for water sampling in MSR. When the target species were above 1 million cells/L, similar cell numbers estimated by microscopic enumeration and qPCR were obtained. Further quantification in water samples indicated that the ratio of the estimated number of cell by microscopy and qPCR was 0.4-12.9 for cyanobacteria and 0.2-3.9 for C. raciborskii. However, Microcystis spp. was not observed by manual enumeration, while it was detected at low levels by qPCR, suggesting that qPCR is more sensitive and accurate. Thus the molecular approaches provide an additional reliable monitoring option to traditional micro- scopic enumeration for the ecosystems monitoring program.
基金Financial supported by China High Technology (863) Project (2001AA241191)Fund of Nation Natural Science(30370901)fund of doctoral of university from ministry of education P.R.C(20040389009)
文摘在甘蓝型油菜基因组中鉴定出18个BnGPDH基因,参考拟南芥AtGPDH基因家族分类信息将它们分为BnGPDHp、BnGPDHm和BnGPDHc 3个亚家族。基因结构、保守模块分析发现亚家族内的基因成员具有较高的保守性;染色体位置定位显示BnGPDH分布在10条染色体上;qRT-PCR分析发现BnGPDHc家族成员在所检测的组织部位中具有不同的表达模式。此外,在花后21 d(day after flower,DAF)和30DAF的种子中BnGPDHc1-2、BnGPDHc2-1、BnGPDHc2-2的相对表达量在高油材料中要显著高于低油材料,表明这些基因可能与油菜种子中三脂酰甘油的形成相关。
