家蚕BmICE-912基因的克隆与功能研究
摘要
家蚕发育过程的独特性、品系的多样性、遗传变异的多态性,使其具备成为模式生物的良好潜质,是进行细胞凋亡等研究的理想材料。细胞凋亡是家蚕完成变态发育的重要手段,对凋亡的研究是解析家蚕变态发育机制的重要途径。Caspase家族在细胞凋亡中起着重要的作用,但是家蚕中Caspase基因的相关研究仍处于初级阶段。本实验以NCBI中提交的Bmlce设计特异引物,获得了该基因的一个新的剪接体BmICE-912,并对该基因进行了信息分析和功能研究。同时,对ICE基因4个剪接体间关系也进行了初步探讨。结果如下：
1. BmICE-912基因的克隆与序列特征
本实验克隆了BmICE-912基因,序列全长912 bp,包含完整的开放阅读框(ORF)912 bp,共编码303个氨基酸,预测分子量为35.1 kD。将克隆的BmICE-912与家蚕基因组序列进行比对,结果表明该基因具有8个外显子和7个内含子。编码蛋白具有Caspase家族成员特有的酶活性中心位点QACRG序列和经典的大小亚基。家蚕ICE基因的4个剪接体很可能是由两个同源基因通过两种剪接方式产生的。
2. BmICE-912蛋白具有Caspase-1酶活性
我们采用了caspase-1、caspase-3、caspase-9活性检测试剂盒对原核表达产生的BmICE-912目的蛋白进行酶活检测,利用紫外分光光度计测定每一份样品,对得到的405 nm的A值数据分析,结果表明,目的蛋白的活性在加入了caspase-1作用底物后催化产生的pNA量最高。即通过原核表达产生的BmICE-912蛋白具有caspase-1酶活性。
3. BmICE-912有抑制细胞凋亡的功能且影响家蚕翅的发育
将滞后质粒1180hrs 1000A4-BmICE-912-sv40以一定的浓度梯度转染BmE细胞,UVB处理BmE-SWU1细胞,正常培养24h, TUNEL染色后,经流式细胞仪检查凋亡情况,结果显示：转染组的细胞凋亡率为3.16%,远低于对照组35.74%的凋亡率。这些变化提示,在我们所研究的UVB诱导转染组BmE-SWU1细胞凋亡过程中,BmICE-912基因起到了明显的抑制细胞凋亡的功能。除此之外,利用RANi技术,通过蚕体注射siRNA(BmICE-912),我们发现降低BmE-912基因的表达,影响了干涉组蛾子翅的发育,导致翅明显变小。
Silkworm not only has the potential to became a good model organism,but also has been the ideal material to study apoptosis, it is dependent on the uniqueness of the silkworm, strain diversity, genetic variation of the polymorphism. Apoptosis is a necessary process for silkworm to complete metamorphosis. Analysis of apoptosis in silkworm is an important way to understand mechanism of metamorphosis. Caspase family plays an important role in apoptosis, but the research is still in its infancy. In this study, according to the Bmlce gene (GenBank accession number:AY885228), the special primers were designed, a new novel isoform of ICE named BmICE-912 is obtained from silkworm, analysis the function and information of the new novel isoform. At the same time, we analysis the relationship between the four novel isoforms. The results are as follows:
1 Gene cloning and sequence analysis of BmICE-912 gene
The open reading frame(ORF) of the new novel isoform contain 912 base pairs, encoding 303 amino acids. Sequence analysis of BmICE-912 showed that it contains 8 exons and 7 introns. Protein of BmICE-912 gene contains the specific active site sequences QACRG and classic large and small subunits which is unique in caspase family member. The two homologous genes produce four novel isoforms of ICE in silkworm by two splicing ways.
2 BmICE-912 protein has enzyme activity of caspase-1
The activity of BmICE-912 protein of prokaryotic expression was studied by caspase-1, caspase-3, caspase-9 activity Assay Kit. Determination of each sample using UV spectrophotometric, Analysis the A-value data in 405 nm, the results may indicate that the activity of the target protein cleave the colorimetric substrate Ac-YYAD-pNA under saturated substrate concentrations in caspase-1. That is means that BmICE-912 protein has enzyme activity of caspase-1.
3 BmICE-912 can inhibit apoptosis in silkworm cells and affect the growth of wing of silkworm
We use lagged plasmid 1180hrsl000A4-BmICE-912-sv40 to transfection BmE cell in certain concentration, then BmE-SWU1 cells dealled with UVB, after 24 hours, The flow cytometry assay examination show that the apoptosis number of tansfection cells is 3.16%, but the control is 35.74%. These changes suggest that BmICE-912 can inhibit apoptosis in silkworm cells. In addition to, using RANi technology, by injection of siRNA (BmICE-912) into silkworm body, we found that the reduction of BmICE-912 gene expression, affecting the growth of wing of silkworm and resulting smaller wings in RNAi silkworm.
1. BmICE-912基因的克隆与序列特征
本实验克隆了BmICE-912基因,序列全长912 bp,包含完整的开放阅读框(ORF)912 bp,共编码303个氨基酸,预测分子量为35.1 kD。将克隆的BmICE-912与家蚕基因组序列进行比对,结果表明该基因具有8个外显子和7个内含子。编码蛋白具有Caspase家族成员特有的酶活性中心位点QACRG序列和经典的大小亚基。家蚕ICE基因的4个剪接体很可能是由两个同源基因通过两种剪接方式产生的。
2. BmICE-912蛋白具有Caspase-1酶活性
我们采用了caspase-1、caspase-3、caspase-9活性检测试剂盒对原核表达产生的BmICE-912目的蛋白进行酶活检测,利用紫外分光光度计测定每一份样品,对得到的405 nm的A值数据分析,结果表明,目的蛋白的活性在加入了caspase-1作用底物后催化产生的pNA量最高。即通过原核表达产生的BmICE-912蛋白具有caspase-1酶活性。
3. BmICE-912有抑制细胞凋亡的功能且影响家蚕翅的发育
将滞后质粒1180hrs 1000A4-BmICE-912-sv40以一定的浓度梯度转染BmE细胞,UVB处理BmE-SWU1细胞,正常培养24h, TUNEL染色后,经流式细胞仪检查凋亡情况,结果显示：转染组的细胞凋亡率为3.16%,远低于对照组35.74%的凋亡率。这些变化提示,在我们所研究的UVB诱导转染组BmE-SWU1细胞凋亡过程中,BmICE-912基因起到了明显的抑制细胞凋亡的功能。除此之外,利用RANi技术,通过蚕体注射siRNA(BmICE-912),我们发现降低BmE-912基因的表达,影响了干涉组蛾子翅的发育,导致翅明显变小。
Silkworm not only has the potential to became a good model organism,but also has been the ideal material to study apoptosis, it is dependent on the uniqueness of the silkworm, strain diversity, genetic variation of the polymorphism. Apoptosis is a necessary process for silkworm to complete metamorphosis. Analysis of apoptosis in silkworm is an important way to understand mechanism of metamorphosis. Caspase family plays an important role in apoptosis, but the research is still in its infancy. In this study, according to the Bmlce gene (GenBank accession number:AY885228), the special primers were designed, a new novel isoform of ICE named BmICE-912 is obtained from silkworm, analysis the function and information of the new novel isoform. At the same time, we analysis the relationship between the four novel isoforms. The results are as follows:
1 Gene cloning and sequence analysis of BmICE-912 gene
The open reading frame(ORF) of the new novel isoform contain 912 base pairs, encoding 303 amino acids. Sequence analysis of BmICE-912 showed that it contains 8 exons and 7 introns. Protein of BmICE-912 gene contains the specific active site sequences QACRG and classic large and small subunits which is unique in caspase family member. The two homologous genes produce four novel isoforms of ICE in silkworm by two splicing ways.
2 BmICE-912 protein has enzyme activity of caspase-1
The activity of BmICE-912 protein of prokaryotic expression was studied by caspase-1, caspase-3, caspase-9 activity Assay Kit. Determination of each sample using UV spectrophotometric, Analysis the A-value data in 405 nm, the results may indicate that the activity of the target protein cleave the colorimetric substrate Ac-YYAD-pNA under saturated substrate concentrations in caspase-1. That is means that BmICE-912 protein has enzyme activity of caspase-1.
3 BmICE-912 can inhibit apoptosis in silkworm cells and affect the growth of wing of silkworm
We use lagged plasmid 1180hrsl000A4-BmICE-912-sv40 to transfection BmE cell in certain concentration, then BmE-SWU1 cells dealled with UVB, after 24 hours, The flow cytometry assay examination show that the apoptosis number of tansfection cells is 3.16%, but the control is 35.74%. These changes suggest that BmICE-912 can inhibit apoptosis in silkworm cells. In addition to, using RANi technology, by injection of siRNA (BmICE-912) into silkworm body, we found that the reduction of BmICE-912 gene expression, affecting the growth of wing of silkworm and resulting smaller wings in RNAi silkworm.
引文
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