摘要
根据实验室克隆的克氏原螯虾（Procambarus clarkii）HSP70（SCHSP70）的核酸序列，制备内部缺失42bp的核酸片段作为竞争性模板。采用竞争性RT-PCR的方法，定量测定克氏原螯虾组织的SCHSP70mRNA的袁达量。结果显示，SCHSP70有广泛的组织分布，但是在正常情况下表达量均较低。经2h热休克处理，7种组织的SCHSP70 mRNA的表达量均显著上升，表明该SCHSP70可能是一种热诱导型HSP70。心脏组织scHSP70在热激前后的表达量均较高，分别为1．12×10^5和4．80×10^5分子／ug总RNA。而血细胞中SCHSP70的表达在热激前后均处于最低水平，分别为0．191×10^4和1．61×10^4分子μg总RNA。在0～8h长时间的热休克实验中，心脏组织scHSP70的表达随着热休克时间的延长，呈现增强的趋势，提示心脏组织在虾类的应激反应中发挥了重要的作用。
A competitor eDNA fragment was constructed and used as a quantitative template. This fragment was derived from scHSP70 cDNA cloned from Procambarus clarkii. This design created a 42 bp internal deletion of the target scHSP70 eDNA and yielded a 301 bp competitor cDNA template. Competitive RT- PCR was used to quantify the scHSP70 cDNA copies in all tissue samples which were proportional to scHSP70 mRNA levels. The results showed that scHSP70 distributed in all the tissues examination, such as heart, muscle, hemocytes, digestive gland, antennal gland, testis and intestine. However, under a normal condition, the expression levels of scHSP70 mRNA were much lower in all tissues compared to treated ones. Challenge of P. clarkii with 2 h heat shock resulted in dramatic increases in the expression levels of scHSP70 mRNA in all tissues. The upregulated mRNA expression of the scHSP70 following heat shock indicates that scHSP70 gene was apparently inducible by thermal challenge and involves in stress responses. The levels in heart tissues remained the highest under unstressed and stressed conditions, from 1.12× 10^5 to 4.80×10^5 molecules/μg total RNA with heat shock. In heart tissue, the level of scHSP70 mRNA gradually increased compared with untreated crayfish, which showed the heart tissue might play an important physiological role in stress response in the crayfish. The level in hematocytes was still lowest among all tissues, although it was upregulated from 0. 191× 10^4 to 1.61× 10^4 molecules/ μg total RNA.
出处
《海洋科学》
CAS
CSCD
北大核心
2009年第8期21-25,共5页
Marine Sciences
基金
暨南大学人才科研启动基金资助项目(51204019)
