昆虫分子生物学研究
(
网络版
), 2012
年
,
第
1
卷
,
第
1
篇
,
第
1
-
7
页
Kunchong Fenzi Shengwuxue Yanjiu (Online), 2012, Vol.1, No.1, 1
-
7
7
3.8
Bm3HAD
基因的组织表达分析
利用
Primer script
TM
RT reagent
试剂盒，按照说
明书步骤反转录家蚕各组织
cDNA
，
以反转录获得
的各组织
cDNA
为模板进行
RT-PCR
分析。按照
SYBR premix Ex Taq
TM
试剂盒说明书进行荧光定量
PCR
。
荧光定量
PCR
反应体系
(20
μL)
：
10.0
μmol/L
正、反向引物各
0.4
μL
，
cDNA
模板
1
μL
，
2
×SYBR
Premix Ex Taq
TM
10
μL
，
ddH
2
O 8.2 μL
。
反应程序为：
95
℃
10
s
；
95
℃
7
s
，
60
℃
20
s,
共
40
个循环。每个
定量反应重复
3
次。用相对定量法，以
β-
actin
为内标
基因，以
Ct
值衡量
Bm3HAD
基因和
β-
actin
基因在每
个样品的表达水平，
ΔCt
作为计算
Bm3HAD
基因相
对于
β-
actin
基因的差异表达水平，用
2
－
⊿⊿
Ct
公式计
算
Bm3HAD
基因的相对表达量，分析
Bm3HAD
基因
在病毒感染蚕和和正常蚕各组织中的相对转录水
平
(
Livak and Schmittgen, 2001)
。
作者贡献
本论文署名作者中王秀负责该论文实验的设计、操作
及文章的撰写；吴萍负责前期的基因芯片检测、提供本文所
研究基因的
EST
序列并指导实验的设计；高坤、覃光星和
刘挺负责试验中的病毒保存、接种及家蚕饲养；郭锡杰为本
论文的通讯作者，全权负责本论文实验设计、实验过程及文
章撰写的指导。
致谢
本研究由国家自然科学基金项目
(
No.30972143)
和江苏
省自然科学基金项目
(
BK2010353)
资助。感谢两位匿名评审
人对本文提出的评审意见。
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