|
|
Effects of IAG Recombinant Plasmid Injection on the Expression of IAG, mhc, gasp and Secondary Sexual Characteristics of Procambarus clarkii |
CHEN Yi-Jun, XIE Ying-Lin, ZHAO Tong, YING Ke, LI Yan-He* |
Key Laboratory of Freshwater Biological Breeding, Ministry of Agriculture and Rural Affairs/National Experimental Teaching Demonstration Center for Aquaculture, Huazhong Agricultural University, Wuhan 430070, China |
|
|
Abstract The insulin-like androgenic gland hormone (IAG) gene plays an important role in sexual differentiation and growth in decapod crustaceans. To investigate the effects of recombinant plasmids containing IAG on the expression of endogenous genes (IAG) and growth-related genes myosin heavy chain(mhc) and growth arrest-specific protein (gasp) as well as secondary sexual characteristics in Procambarus clarkii. This study involved injecting different doses (0.5, 1.0 and 3.0 μg/g) of IAG recombinant plasmids into the muscle of healthy minor female P. clarkii weighing between 6.5~16.5 g. The weight, abdominal development, and expression of genes IAG, mhc, and gasp in the tissues of the experimental shrimp were monitored at 1, 7, 14, and 28 d, respectively. The results showed that compared with the control group, the copy number of plasmids in the experimental group was positively correlated with the concentration of injection. The copy number in each tissue showed a distribution pattern of muscle>gonads>appendages>hepatopancreas. The degree of plasmid digestion was the highest between 3 and 7 d, and at 28 d, the plasmid was almost completely digested. After 1 d of injection, the expression of mhc in muscles significantly increased at injection concentrations of 1.0 and 3.0 μg/g (P<0.05), and body weight gain was also significantly higher than that of the control group (P<0.05). Within 28 d after injection, female crayfish with an injection concentration of 3.0 μg/g had the fastest ovarian development, while the control group had the slowest development. No masculinization was observed in the abdomen of all female shrimps. Before the mass digestion of plasmids, the expression levels of IAG in the gonads and gasp in the muscles of the experimental group were lower than those in the control group, and their expression was inhibited. The results showed that the injection of IAG recombinant plasmid had certain effects on ovarian development and expression of IAG, mhc, gasp. This study provides reference for research on IAG function and gender control.
|
Received: 04 August 2024
|
|
Corresponding Authors:
*liyanhe@mail.hzau.edu.cn
|
|
|
|
[1] 曹志涛, 张卫林. 2011. 质粒浓度对氯化钙转化法转化效率的影响[J]. 生物学通报, 46(4): 41-42. (Cao Z T, Zhang W L.2011. The effect of plasmid concentration on the conversion efficiency of calcium chloride conversion method[J]. Bulletin of Biology, 46(4): 41-42.) [2] 韩晓磊, 马强, 李小蕊, 等. 2011. 不同地区克氏原螯虾群体的形态差异分析[J]. 水生态学杂志, 32(03): 82-87. (Han X L, Ma Qiang, Li X R, et al.2011. Morphological variations analysis of different geographic populations of Procambarus clarki[J]. Journal of Hydroecology, 32(03): 82-87.) [3] 胡春生, 张庆林, 张通. 2011. 基因治疗用质粒DNA生产面临的问题及研究进展[J]. 中国生物工程杂志, 31(04): 119-123. (Hu C S, Zhang Q L, Zhang T.2011. Research progress in large-scale production of plasmid DNA for gene therapy[J]. China Biotechnology, 31(04): 119-123.) [4] 胡倩, 王齐帅, 刘小龙, 等. 2023. 雌雄分养对克氏原螯虾养殖效果及性腺发育的影响[J]. 湖北农业科学, 62(11): 113-117. (Hu Q, Wang Q S, Liu X L, et al.2023. Effect of isolating female from male culture on the culture performance and gonad development of Procambarus clarkii[J]. Hubei Agricultural Sciences, 62(11): 113-117.) [5] 吉红, 曹福余, 王毅, 等. 2008. 克氏原螯虾在安康地区三种养殖条件下生长情况的比较[J]. 家畜生态学报, 29(06): 76-81. (Ji H, Cao F Y, Wang Y, et al. 2008. Growth pattern comparison of Procambarus clarkii under three rearing conditions[J]. Acta Ecologiae Animalis Domastici, 29(06): 76-81.) [6] 李浪平, 吕建林, 龚世园, 等. 2006. 克氏原螯虾形态参数关系的初步研究[J]. 水利渔业, (03): 40-42. (Li L P, Lv J L, Gong S Y, et al. 2006. A preliminary study on the relationship between morphological parameters of Procambarus clarkii[J]. Reservoir Fisheries, (03): 40-42.) [7] 彭波, 谭云飞, 蓬国辉, 等. 2021. 克氏原螯虾体征性状与肌肉质量的相关及通径分析[J]. 水产科学, 40(5): 718-725. (Peng B, Tan Y F, Peng G H, et al.2021. Path analysis of effects of phenotypic traits attributes on abdomen meat weight of red swamp crayfish Procambarus clarkii[J]. Fisheries Science, 40(5): 718-725.) [8] 唐建清, 宋胜磊, 吕佳, 等. 2003. 克氏原螯虾种群生长模型及生态参数的研究[J]. 南京师大学报(自然科学版), (01): 96-100. (Tang J Q, Song S L, Lv J, et al.2003. A Study on growth model and ecological parameters of Cambarus clakii[J]. Journal of Nanjing Normal University (Natural Science Edition), (01): 96-100.) [9] 王佳佳, 李健, 葛倩倩, 等. 2019. 脊尾白虾肌球蛋白重链和肌球蛋白轻链基因的克隆与表达分析[J]. 中国海洋大学学报(自然科学版), 49(01): 16-27. (Wang J J, Li J, Ge Q Q, et al.2019. Isolation and expression analyses of myosin heavy and light chain encoding genes of Exopalaemon carinicauda[J]. Journal of Ocean University of China, 49(01): 16-27.) [10] 徐滨, 李忠, 魏开金, 等. 2019. 6个克氏原螯虾群体形态学分析[J]. 淡水渔业, 49(06): 27-32. (Xu B, Li Z, Wei K J, et al.2019. Morphological variations among six populations of Procambarus clarkii[J]. Freshwater Fisheries, 49(06): 27-32.) [11] 张龙, 石林林, 李艳和. 2019. 克氏原螯虾形态与体重的通径分析[J]. 中国农学通报, 35(18): 148-153. (Zhang L, Shi L L, Li Y H.2019. Path analysis of the morphology and body weight of Procambarus clarkii[J]. Chinese Agricultural Science Bulletin, 35(18): 148-153.) [12] 郑宏坤, 谢熙, 郑亮, 等. 2020. 甲壳动物胰岛素样促雄腺激素功能及作用机制的研究进展[J]. 动物学杂志, 55(05): 670-680. (Zheng H K, Xie X, Zheng L, et al.2020. Research advance in functions and related mechanisms of insulin-like androgenic gland hormone in crustaceans[J]. Chinese Journal of Zoology, 55(05): 670-680.) [13] Chandler J C, Aizen J, Elizur A, et al.2015. Discovery of a novel insulin-like peptide and insulin binding proteins in the Eastern rock lobster Sagmariasus verreauxi[J]. General and Comparative Endocrinology, 215: 76-87. [14] Chung S J, Manor R, Sagi A.2011. Cloning of an insulin-like androgenic gland factor (IAG) from the blue crab, Callinectes sapidus: Implications for eyestalk regulation of IAG expression[J]. General and Comparative Endocrinology, 173(1): 4-10. [15] Collins C, Lester K, Del-Pozo J, et al.2021. Non-Lethal sequential individual monitoring of viremia in relation to dna vaccination in fish-example using a salmon alphavirus DNA vaccine in Atlantic salmon salmo salar[J]. Vaccines (Basel), 9(2): 163. [16] Evdokiou A, Cowled P A.1998. Growth-regulatory activity of the growth arrest-specific gene, GAS1, in NIH3T3 fibroblasts[J]. Experimental Cell Research, 240(2): 359-67. [17] Ge H L, Tan K, Shi L L, et al.2020. Comparison of effects of dsRNA and siRNA RNA interference on insulin-like androgenic gland gene (IAG) in red swamp crayfish Procambarus clarkii[J]. Gene, 752: 144783. [18] Kanellos T, Sylvester I D, Colin R H, et al.1999. DNA is as effective as protein at inducing antibody in fish[J]. Vaccine, 17: 965-972. [19] Halagowder D, Ayithan N.2006. Molecular mechanisms regulating molting in a crustacean[J]. The FEBS Journal, 273(4): 839-846. [20] Helinski D R.1979. Bacterial plasmids: Autonomous replication and vehicles for gene cloning[J]. CRC Critical Reviews in Biochemistry, 7: 83-101. [21] Hughes T S, Langer S J, Johnson K W, et al.2009. Intrathecal injection of naked plasmid DNA provides long-term expression of secreted proteins[J]. Molecular Therapy: The Journal of the American Society of Gene Therapy, 17(1): 88-94. [22] Kianann T, Miao Z, Huigong J, et al.2020. siRNA-Mediated MrIAG silencing induces sex reversal in Macrobrachium rosenbergii[J]. Marine Biotechnology, 22(3): 456-466. [23] Lezer Y, Aflalo E D, Manor R, et al.2015. On the safety of RNAi usage in aquaculture: The case of all-male prawn stocks generated through manipulation of the insulin-like androgenic gland hormone[J]. Aquaculture, 435: 157-166. [24] Li F J, Bai H K, Zhang W Y, et al.2015. Cloning of genomic sequences of three crustacean hyperglycemic hormone superfamily genes and elucidation of their roles of regulating insulin-like androgenic gland hormone gene[J]. Gene, 561(1): 68-75. [25] Maeda S, Ohmori K, Kurata K, et al.2004. Expression of LacZ gene in canine muscle by intramuscular inoculation of a plasmid DNA[J]. Journal of Veterinary Medical Science, 66(3): 337-339. [26] Martinelli D C, Fan C M.2007. The role of Gas1 in embryonic development and its implications for human disease[J]. Cell Cycle, 6: 2650-2655. [27] Sebé-Pedrós A, Grau-Bové X, Richards T A, et al.2014. Evolution and classification of myosins, a paneukaryotic whole-genome approach[J]. Genome Biology and Evolution, 6(2): 290-305. [28] Sharabi O, Manor R, Weil S, et al.2016. Identification and characterization of an insulin-like receptor involved in crustacean reproduction[J]. Endocrinology, 157(2): 928-41. [29] Shi L L, Han S X, Fei J M, et al.2019. Molecular characterization and functional study of insulin-like androgenic gland hormone gene in the red swamp crayfish, Procambarus clarkii[J]. Genes, 10(9): 645. [30] Sroyraya M, Chotwiwatthanakun C, Stewart J M, et al.2010. Bilateral eyestalk ablation of the blue swimmer crab, Portunus pelagicus, produces hypertrophy of the androgenic gland and an increase of cells producing insulin-like androgenic gland hormone[J]. Tissue and Cell, 42(5): 293-300. [31] Teige L H, Aksnes I, Røsæg M V, et al.2020. Detection of specific Atlantic salmon antibodies against salmonid alphavirus using a bead-based immunoassay[J]. Fish Shellfish Immunology, 106: 374-383. [32] Ventura T, Fitzgibbon Q, Battaglene S, et al.2015. Identification and characterization of androgenic gland specific insulin-like peptide-encoding transcripts in two spiny lobster species: Sagmariasus verreauxi and Jasus edwardsii[J]. General and Comparative Endocrinology, 214:126-133. [33] Wang Q, Yang J X, Zhou G Q, et al.2011. Length-weight and chelae length-width relationships of the crayfish Procambarus clarkii under culture conditions[J]. Journal of Freshwater Ecology, 26(2): 287-294. [34] Wolff J A, Malone R W, Williams P, et al.1990. Direct gene transfer into mouse muscle in vivo[J]. Science, 247(4949 Pt 1): 1465-1468. [35] Yaara L, Eliahu D A, Rivka M, et al.2015. On the safety of RNAi usage in aquaculture: The case of all-male prawn stocks generated through manipulation of the insulin-like androgenic gland hormone[J]. Aquaculture, 435: 157-166. [36] Yan J, Zheng B Y, Tan K, et al.2021. Effects of two exogenous proteins on the insulin-like androgenic gland hormone gene expression in Procambarus clarkii[J]. Aquaculture Research, 52(12): 6602-6611. [37] Zhang L, Li Y H, Wu M L, et al.2021. The SNP polymorphisms associated with WSSV-resistance of prophenoloxidase in red swamp crayfish (Procambarus clarkii) and its immune response against white spot syndrome virus (WSSV)[J]. Aquaculture, 530(2021): 735787. |
[1] |
SHEN Heng-Rui, ZHANG Ai-Fang, WANG Qi-Shuai, ZHOU Zhi-Yong, LIU Yun-Long, XU Xian-Dong, WANG Jing-Yu, ZHONG Ke-Er, LI Yan-He, HUANG Jiang-Feng. Genetic Diversity and Structure Analysis of Seven F1 Hybrid Population of Procambarus clarkii[J]. 农业生物技术学报, 2025, 33(7): 1580-1588. |
|
|
|
|