蓝藻Synechococcus elongatus PCC7 942荧光实时定量RT-PCR实验体系的优化

A study on an optimized real time fluorescent quantitative PCR method for detecting gene expression in Synechococcus elongatus PCC 7942

蓝藻(Synechococcu elongatus PCC 7942)是最简单的原核生物钟模式生物,研究其生物钟基因表达模式具有重要的科学意义.然而由于该蓝藻高质量RNA提取困难,目前应用荧光实时定量RT-PCR(qPCR)实验方案对其特定基因表达模式进行研究的报道较少.研究尝试通过对影响RNA提取质量的溶菌酶作用时间、起始细胞数量等因素,以及对影响qPCR反应特异性和重现性的c DNA模板浓度、引物浓度、引物退火温度等条件进行优化,初步建立了一套适用于S.elongatus PCC 7942的qPCR实验体系.结果表明震荡培养至OD750≈0.4时,取10 m L蓝藻细胞起始提取实验,使用3 mg/m L溶菌酶处理10 min后,再经试剂盒法提取总RNA的策略,RNA得率最高且质量最好.蓝藻的qPCR优化结果显示c DNA终质量浓度0.1μg/μL,引物终浓度4 pmol/L,并根据引物对Tm值调整退火温度是最佳反应条件.

The circadian rhythm in cyanobacterium Synechococcus elongatus PCC 7942,which is driven by a central oscillator consisted of Kai A,Kai B and Kai C protein,is considered to be the simplest model to explain the endogenous time keeping mechanism in prokaryotic cells.To make a better understanding of how the clock genes are arranged together in this cyanobacterium,it＇s important to study the expression patterns of these clock genes under entrained conditions.Due to the complex cell wall structure of S.elongatus PCC 7942,which makes getting high quality and high quantity RNA quite difficult,there are quite few reports on the studies of the expression patterns of the clock genes via qPCR method. In the present study,an optimized qPCR system to S. elongatus PCC7942 cells was established by optimizing the key factors which affected the efficiency and accuracy of qPCR,such as the initial cells amount for RNA isolation,the lysozyme digestion time of the cell walls,the removal method of genomic DNA,the purification method of c DNA templates,as well as the concentration of the primers and annealing temperature of PCR,etc.The results indicated that 10 m L cyanobacterial cells with OD750≈0.4（ about 1.1 mg in dry weight） treated by 3 mg / m L lysozyme for 10 mins,were the best for total RNA isolation by a RNA extraction Kit; The process of removing the residual genomic DNA from the total RNA by DNase1 before being applied as templates for c DNA synthesis,and the purification of c DNA by a universal DNA recovery kit after reverse transcription reaction would significantly promote the quantity and repeatability of following qPCR.It is conclude that adjusting c DNA template concentration to 0. 1 μg / μL and primers concentration to 4 pmol / L,while performing the real-time qPCR under the optimized annealing temperatures on the basis of semi-quantity RT-PCR play a pivotal role in analyzing the clock genes expression profiles in cyanobacterium S.elongatus PCC 7942 by qPCR.