THE FUNCTION AND MECHANISM OF BURSICON GENE IN CARIDINA
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摘要: 为探讨米虾鞣化激素在其蜕皮周期及表皮角质层形成过程中的作用, 采用PCR技术克隆得到了米虾鞣化激素两个亚基基因的开放阅读框(ORF)序列。bursicon-α ORF全长441 bp, 共编码146个氨基酸; bursicon-β ORF全长411 bp, 共编码136个氨基酸。利用实时荧光定量PCR分析米虾整个蜕皮周期中鞣化激素2个亚基基因的表达特征, 结果发现, 鞣化激素bursicon-α和bursicon-β在米虾蜕皮周期的各个阶段的相对表达量存在差异, 在蜕皮前期(D期)相对表达量开始上升, 到D3期时相对表达量最高, 蜕皮期E期相对表达量最低。RNA干扰(RNA interference, RNAi)介导bursicon-α和bursicon-β基因沉默后, 发现米虾的蜕皮周期延长, 表皮角质层明显变薄。结果提示, 鞣化激素(Bursicon)与新形成的外骨骼中角质层的加厚与硬化密切相关, 进而影响蜕皮时间。Abstract: To study the function of bursicon gene in Caridina during molting and cuticle formation, its open reading frame (ORF) containing two subunits genes were amplified by PCR respectively. The ORF of bursicon-α is 441 bp in length, encoding a protein with 146 amino acid residues. The ORF of bursicon-β is 411 bp in length, encoding a protein with 136 amino acid residues. The dynamic changes in the expression of both bursicon subunit genes during the molt cycle in Caridina have been analyzed by the real-time quantitative PCR. Relative expression of both bursicon-α and bursicon-β had different levels in different stages of the molt cycle, which increased through premolt stage (D stage) and reached their peak level at the D3 stage. The mRNA accumulation decreased to its lowest level at the molt stage (E stage). RNA interference-mediated knockdown of bursicon-α and bursicon-β retard the molting process and ecdysis behavior of Caridina. The cuticle of dsRNA-treated Caridina was thinner than that in the control group. These findings demonstrate that bursicon is involved in the thickening and hardening of cuticle in newly formed exoskeleton. Moreover, bursicon plays an important role in molt cycle.
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Keywords:
- Caridina /
- Bursicon /
- Cuticular tanning /
- Molt cycle /
- Real-time quantitative PCR /
- RNA interference
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图 1 米虾和其他物种Bursicon-α亚基系统进化树
物种名称及其Bursicon-α亚基的GenBank登录号Species and GenBank accession numbers of their Bursicon-α subunits: 蓝蟹Callinectes sapidus (ACG50067.1); 普通滨蟹Carcinus maenas (ABX55995.1); 克氏原螯虾Procambarus clarkii (ADY80040.1); 斑节对虾Penaeus monodon (AKJ74864.1); 米虾Caridina (MG766223); 水蚤Daphnia arenata (ABX55998.1); 大型溞Daphnia magna (KZS13469.1); 烟草天蛾Manduca sexta (Q4FCM6.1); 家蚕Bombyx mori (CAH89262.2); 西方蜜蜂Apis mellifera (NP_001091704.1); 德国小蠊Blattella germanica (CUTO8823.1); 温带臭虫Cimex lectularius (XP_014256820.1); 黑腹果蝇Drosophila melanogaster (NP_650983.1); 家蝇Musca domestica (ABO20870.1)。标尺示遗传距离Scale bar indicates the genetic distance
Figure 1. Phylogenic tree of bursicon-α subunit in Caridina and other species
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图 2 米虾和其他物种Bursicon-β亚基系统进化树
物种名称及其Bursicon-β亚基的GenBank登录号Species and GenBank accession numbers of their Bursicon-β subunits: 斑节对虾Penaeus monodon (ALO17552.1); 欧洲龙虾Homarus gammarus (ADI86243.1); 米虾Caridina (MG766224); 普通滨蟹Carcinus maenas (ABX55996.1); 蓝蟹Callinectes sapidus (ACG50066.1); 水蚤Daphnia arenata (ABX55997.1); 大型溞Daphnia magna (KZS13470.1); 烟草天蛾Manduca sexta (ABB92831.1); 家蚕Bombyx mori (NP_001037289.1); 西方蜜蜂Apis mellifera (CAM06632.1); 中欧山松大小蠹Dendroctonus ponderosae (XP_019755392.1); 赤拟谷盗Tribolium castaneum (NP_001107780.1); 黑腹果蝇Drosophila melanogaster (NP_609712.1); 家蝇Musca domestica (ABO20869.1)。标尺示遗传距离Scale bar indicates the genetic distance
Figure 2. Phylogenic tree of bursicon-β subunit in Caridina and other species
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图 3 米虾bursicon-α和bursicon-β在不同蜕皮阶段的相对表达量
D0-4. 蜕皮前期, E. 蜕皮期, A-B. 蜕皮后期, C. 蜕皮间期; 利用SPSS Statistics 17.0进行单因素ANOVA分析, Duncan氏多重比较法对组间基因的相对转录水平进行检验(P<0.05), 柱上不同的小写字母表示差异显著, 相同的小写字母表示差异不显著; 下同
Figure 3. Relative expression levels of bursicon-α and bursicon-β in Caridina under different molt stages
D0-4. premolt stage, E. molt stage, A-B. postmolt stage, C. intermolt stage. The data were analyzed by SPSS Statistics 17.0 through one-way ANOVA analysis of variance. The relative expresstion levels of genes among groups were tested by Duncan’s test (P<0.05). Different lowercase letters above columns indicate statistically significant differences and the same lowercase letters are not significantly different. The same applies below
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图 4 RNA干扰后bursicon-α和bursicon-β相对表达量随时间的变化
dsGFP. 注射dsGFP的对照组; dsburs α. 注射dsburs α的实验组; dsburs β. 注射dsburs β的实验组; 小写字母表示组内显著性差异; 大写字母表示组间显著性差异
Figure 4. The relative expressions of bursicon-α and bursicon-β change over time after interfering the dsRNA
dsGFP. control group interfered with dsGFP; dsburs α. experimental group interfered with dsburs α; dsburs β. experimental group interfered with dsburs β; Lowercase letters indicate statistically significant differences within the group; Capital letters indicate statistically significant differences among groups
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图 5 dsRNA干扰对米虾蜕皮过程的影响
dsGFP. 注射dsGFP的对照组, 处于蜕皮前期D3期; dsburs α. 注射dsburs α的实验组, 处于蜕皮前期D2期; dsburs β. 注射dsburs β的实验组, 处于蜕皮前期D2期; dsburs α+dsburs β. 按质量浓度1﹕1注射dsburs α和dsburs β的实验组, 处于蜕皮周期D2期; 下同; RZ. 上皮回缩所形成的透明区; TS. 管形套; NS. 新刚毛
Figure 5. Effect of dsRNA interference during molting process of Caridina
dsGFP. control group interfered with dsGFP, at stage D3; dsburs α. experimental group interfered with dsburs α, at stage D2; dsburs β. experimental group interfered with dsburs β, at stage D2; dsburs α +dsburs β. experimental group interfered with an equal concentration of dsburs α and dsburs β, at stage D2. The same applies below. RZ. retracted zone; TS. tubular sheath; NS. new setae
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图 6 dsRNA干扰对米虾蜕皮时间的影响
Figure 6. Effect of dsRNA interference on ecdysis time of Caridina
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图 7 由dsRNA介导的bursicon-α和bursicon-β敲降对米虾角质层形成和鞣化的影响
Figure 7. The bursicon-α and bursicon-β knockdown mediated by dsRNA impairs the complete formation and tanning of cuticle of Caridina
表 1 本研究所用的引物序列
Table 1 Primers used in the study
引物
Primers片段大小
Product size
(bp)引物序列
Primer sequences
(5′—3′)β-actin F 21 TGTGACGATGAAGTAGCAGCA β-actin R 22 AATCTTTCTGACCCATTCCAAC bursicon-α F 26 ATGCACTTAAAGAAGGTTAATACAAG bursicon-α R 22 TTAAATGAAGGGTACGTTTCCC bursicon-β F 24 ATGTGGTCACGGTGGGTATGGATT bursicon-β R 26 TTATCGTGTGGAATCACCACATTTGG bursicon-α QPCR F 20 ATTCCAAGCCCATTCCATCC bursicon-α QPCR R 20 TCCCTCTTCAACATCGGTGC bursicon-β QPCR F 23 TCCATCAACCATACACATTTCCA bursicon-β QPCR R 20 ATCCAGTCAAGCGATTTCCG dsbursα F 27 GCTCTAGAATTCCAAGCCCATTCCATC dsbursα R 27 GGAATTCGTTCCCTCTTCAACATCGGT dsbursβ F 28 GCTCTAGATCACGGTGGGTATGGATTGT dsbursβ R 27 GGAATTCGTGTATTGACGGAGGGTTGG 
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