PSMD9对山羊前体脂肪细胞脂质沉积的调控作用研究

doi:10.11843/j.issn.0366-6964.2023.09.007

摘要/Abstract

摘要： 旨在克隆山羊PSMD9基因序列并对其进行生物信息学分析,检测PSMD9在山羊各组织中的表达情况和肌内前体脂肪细胞不同分化时期的表达水平,并进一步揭示过表达和干扰PSMD9基因对山羊肌内前体脂肪细胞脂质沉积的影响。本研究以1周岁健康简州大耳羊公羊(n=12)为试验对象,采用T-A克隆技术获得山羊PSMD9基因序列,进行生物信息学分析,并通过双酶切方法构建PSMD9过表达载体。利用脂质体转染在山羊肌内脂肪细胞中过表达PSMD9,小干扰RNA(small interfering RNA, siRNA)干扰PSMD9表达。通过油红O染色法观察PSMD9对脂滴形成的影响,GPO-Trinder酶学反应检测甘油三酯含量;同时利用实时荧光定量PCR(RT-qPCR)检测PSMD9在山羊不同组织中的表达水平和肌内前体脂肪细胞不同分化时期的表达水平。结果,获得山羊PSMD9基因核苷酸序列763 bp,其中5'UTR 67 bp,CDS区564 bp,3'UTR 132 bp,编码187个氨基酸残基;山羊PSMD9基因在肝脏中的表达量最高,在脾脏中表达量最低,在诱导分化前4 d其表达水平随着山羊前体脂肪细胞分化而逐渐降低,随后上升,并在第8天表达量最高;过表达PSMD9基因,肌内前体脂肪细胞的脂滴明显增加,甘油三酯含量显著提高,同时可显著上调DGAT2、FASN和ACC的相对表达量,DGAT1、PPARγ和SCD1表达量无显著变化;干扰PSMD9基因肌内前体脂肪细胞脂滴减少,甘油三酯含量降低,同时可显著下调DGAT1、PPARγ、FASN和ACC表达量,DGAT2和SCD1无显著变化。本试验成功获得山羊PSMD9基因CDS区序列,其在肝脏组织中表达量最高,山羊PSMD9基因的表达随着肌内前体脂肪细胞的分化而逐渐降低,在第4天表达量最低,之后表达量逐渐升高,PSMD9的表达能够显著促进山羊肌内前体脂肪细胞脂质沉积,这些结果为进一步阐明PSMD9基因对调控山羊肌内脂肪形成的作用机制提供了重要数据支撑。

关键词: 山羊, PSMD9, RNA干扰, 过表达, 肌内前体脂肪细胞

Abstract: The study aimed to clone the PSMD9 gene sequence of goat and conduct the bioinformatics analysis. Then, the expression of PSMD9 in different tissues of goat and the expression level in intramuscular preadipocytes at different stages of differentiation was detected, and further revealed the effect of overexpression and interference of PSMD9 on lipid deposition in goat intramuscular preadipocytes. In this study, the sequence of PSMD9 gene was obtained by T-A cloning technique using 1-week-old healthy Jianzhou big-eared male goats (n=12), bioinformatics analysis was performed, and PSMD9 overexpression vector was constructed by double digestion method. PSMD9 was overexpressed in goat intramuscular adipocytes using liposome transfection, and the small interfering RNA (siRNA) interfered with PSMD9 expression. The effect of PSMD9 on lipid droplet formation was observed by Oil Red O staining and triglyceride content was measured by GPO-Trinder enzymatic reaction. The expression levels of PSMD9 in different tissues of goats and different stages of differentiation in intramuscular preadipocytes were measured by real-time quantitative PCR (RT-qPCR). The results showed that 763 bp nucleotide sequence of goat PSMD9 gene was obtained, including 5'UTR 67 bp, CDS 564 bp, and 3'UTR 132 bp, encoding 187 amino acid residues. The expression of PSMD9 gene in goat was highest in the liver and lowest in the spleen, and its expression level decreased gradually in goat precursor adipocytes in the former 4 days of differentiation induction, subsequently increased, and the expression level was the highest on the 8th day. Overexpression of the PSMD9 gene significantly increased the lipid droplets and triglyceride content in intramuscular preadipocytes and significantly up-regulated the relative expression of DGAT2, FASN and ACC, while the expression of DGAT1, PPARγ and SCD1 did not significantly changed. Interfering with PSMD9 gene decreased the lipid droplets and triglyceride content in intramuscular preadipocytes, while significantly down-regulated the expression of DGAT1, PPARγ, FASN and ACC, the expression of DGAT2 and SCD1 did not significantly changed. In this research, the CDS region sequence of PSMD9 gene was successfully obtained and its expression was highest in liver tissue. The expression of PSMD9 gene decreased gradually during differentiation in intramuscular adipocytes and reached the lowest level at day 4 in goat, after which the expression gradually increased.The expression of PSMD9 can significantly promote lipid deposition in goat's intramuscular preadipocytes. These results provide important data support for further elucidating the mechanism of PSMD9 gene regulating the formation of intramuscular fat in goat.

Key words: goat, PSMD9, RNA interference, overexpression, intramuscular precursor adipocytes

中图分类号:

S827.2

引用本文

邵鹏, 唐崟梅, 林亚秋, 王永, 向华, 黄炼, 朱江江. PSMD9对山羊前体脂肪细胞脂质沉积的调控作用研究[J]. 畜牧兽医学报, 2023, 54(9): 3653-3663.

SHAO Peng, TANG Yinmei, LIN Yaqiu, WANG Yong, XIANG Hua, HUANG Lian, ZHU Jiangjiang. Regulation Effect of PSMD9 on Lipid Deposition in Goat Precursor Adipocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(9): 3653-3663.

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[1]	严晓春, 习海娇, 李金泉, 王志英, 苏蕊. 内蒙古绒山羊绒毛性状基因组育种值估计准确性研究[J]. 畜牧兽医学报, 2024, 55(1): 120-128.
[2]	张晨俭, 李隐侠, 丁强, 刘伟佳, 王慧利, 何南, 吴家顺, 曹少先. CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J]. 畜牧兽医学报, 2024, 55(1): 129-141.
[3]	张德安, 杨若渚, 刘杰, 刘德武, 邓铭, 柳广斌, 孙宝丽, 郭勇庆, 李耀坤. 饲喂青贮黄梁木代替青贮玉米川中黑山羊肝转录组的表达分析[J]. 畜牧兽医学报, 2024, 55(1): 232-244.
[4]	唐崟梅, 李琪, 李海洋, 林亚秋, 王永, 向华, 黄炼, 朱江江. 山羊FATP2基因的克隆及对前体脂肪细胞脂质沉积的影响[J]. 畜牧兽医学报, 2023, 54(9): 3642-3652.
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