中国农业科学 ›› 2019, Vol. 52 ›› Issue (15): 2706-2715.doi: 10.3864/j.issn.0578-1752.2019.15.014

• 畜牧·兽医·资源昆虫 • 上一篇    下一篇

慢病毒介导shRNA干扰MAT2A与MAT2B基因抑制猪肌内脂肪细胞分化

赵存真,易本驰,陈培荣,李建柱,赵云焕,朱忠珂()   

  1. 信阳农林学院牧医工程学院,河南信阳 464000
  • 收稿日期:2018-06-14 接受日期:2019-07-03 出版日期:2019-08-01 发布日期:2019-08-06
  • 通讯作者: 朱忠珂
  • 基金资助:
    国家自然科学基金(31702096);河南省教育厅项目(18B230010);信阳农林学院校级科技创新团队(CXTD-201701)

Lentivirus Mediated Interference Silencing MAT2A and MAT2B Inhibited Differentiation of Porcine Intramuscular Preadipocytes

ZHAO CunZhen,YI BenChi,CHEN PeiRong,LI JianZhu,ZHAO YunHuan,ZHU ZhongKe()   

  1. College of Animal Science and Veterinary Medicine, Xinyang College of Agriculture and Forestry, Xinyang 464000, Henan
  • Received:2018-06-14 Accepted:2019-07-03 Online:2019-08-01 Published:2019-08-06
  • Contact: ZhongKe ZHU

摘要:

【目的】腺苷甲硫氨酸转移酶(MAT)在ATP的作用下,催化生成体内重要的甲基供体S-腺苷甲硫氨酸(SAMe)。通过构建慢病毒介导的pLenti-H1干扰载体,探究腺苷甲硫氨酸转移酶MAT2A和MAT2B对猪肌内脂肪细胞分化的影响。【方法】无菌条件下采集3—7日龄小猪背最长肌,采用差速贴壁法分离猪肌内脂肪细胞。根据GenBank中猪MAT2A基因序列(Accession No.NM_001167650.1)和MAT2B基因序列(Accession No. NM_001142832.1),获得其CDS序列。利用Invitrogen 公司在线软件BLOCK-iTTM RNAi Designer分别设计shRNA靶序列, 将合成的单链寡核苷酸退火形成双链,与经过Bam H I和Xho I (TaKaRa) 双酶切后的pLenti-Hl载体连接,转化,并提取质粒进行酶切和测序鉴定。采用X-tremeGENE-HP DNA转染试剂与测序成功的重组质粒以及包装质粒(CMV-Δ8.9和CMV-VSVG)共转染293T细胞,48 h后观察绿色荧光蛋白(GFP)的表达,并进行滴度测定。在猪原代脂肪细胞密度达到70%—80%时,侵染病毒;细胞密度融合时诱导分化。提取分化第8天的猪肌内脂肪细胞的RNA,按照反转录试剂盒操作说明进行反转录,合成cDNA第一链。采用primer primer 5软件设计MAT2A、MAT2B、PPARγ、aP2、CEBP/α、β-actin基因的定量引物,实时定量和Western blot试验检测MAT2A和MAT2B基因的干扰效率。油红O染色和实时定量鉴定MAT2A和MAT2B基因对猪肌内脂肪脂质积累的影响。【结果】酶切及测序证明重组慢病毒载体pLenti-Hl-MAT2A/MAT2B构建成功;包装的慢病毒sh-MAT2A和sh-MAT2B病毒滴度分别为6.7×10 7和7×10 7 pfu/mL,侵染肌内脂肪细胞72 h后,可出现90%的绿色荧光蛋白(GFP),表明所包装的病毒可满足侵染猪前体肌内脂肪细胞需要。实时定量结果显示其显著抑制了MAT2A和MAT2B的mRNA水平表达,其干扰效率分别在70%和60%以上;进一步采用Western blot试验及蛋白分析表明,干扰MAT2A基因后,MAT2A蛋白表达水平降低40%左右,差异达到极显著水平(P<0.01);干扰MAT2B基因后,MAT2B蛋白表达水平降低25%左右,差异达到显著水平(P<0.05)。油红O染色和吸光度定量结果显示,与sh-scramble对照组相比,干扰MAT2A和MAT2B基因后,脂滴聚积能力显著抑制猪肌内脂肪细胞脂质积累。实时定量结果显示,干扰MAT2A和MAT2B基因抑制成脂标志基因PPARγ,aP 2及CEBP/α的表达。 【结论】成功构建了猪MAT2A和MAT2B基因的慢病毒sh-MAT2A和sh-MAT2B干扰载体。获得的重组慢病毒感染细胞后,能有效降低猪肌内前体脂肪细胞内MAT2A和MAT2B基因的mRNA和蛋白水平表达;进一步试验证明,干扰MAT2A及MAT2B基因后,显著抑制猪肌内脂肪细胞的脂质积累以及成脂关键基因表达。

关键词: 腺苷甲硫氨酸转移酶2A, 腺苷甲硫氨酸转移酶2B, 慢病毒, 肌内脂肪细胞,

Abstract:

【Objective】 Methionine adenosyltransferase (MAT), an essential cellular enzyme responsible for S-adenosylmethionine biosynthesis. The purpose of this study was to construct the lentivirus-mediated pLenti-H1 vector and investigated the effect of adenosine methionine transferase MAT2A and MAT2B on the differentiation of porcine intramuscular adipocytes.【Method】Porcine longissimus dorsi tissue was collected from 3 to 7 days piglets under sterile conditions and isolated the porcine intramuscular preadipocytes by differential adhesion method. According to MAT2A gene (Accession No.NM_001167650.1) and MAT2B gene sequence (Accession No.NM_001142832.1), Invitrogen online software BLOCK-iTTM RNAi Designer was used to respectively design MAT2A and MAT2B shRNA target sequence. The synthesized single-chain oligonucleotide was annealed to form double strands DNA, and was ligated with pLenti-Hl vector after BamH I and Xho I (TaKaRa) double digestion, then the plasmid DNA was extracted and further identified by restriction enzyme digestion and sequencing. X-tremeGENE-HP DNA transfection reagent was used to co-transfect 293T cells with the correct recombinant plasmid and packaging plasmid (CMV-Δ8.9 and CMV-VSVG). The expression of green fluorescent protein (GFP) was observed after 48 h and then the virus titer was tested. The primary porcine intramuscular preadipocytes were infected with viruses at 70% to 80% cell density and induced adipogenic differentiation when cells at full confluence. Total RNA was extracted from adipocytes and the mRNA reverse transcription was performed by using a Revert Aid First-strand cDNA Synthesis Kit. The gene primer sequences of MAT2A, MAT2B, PPARγ, aP2, CEBP/α and β-actin were designed by Primer primer 5.The interference efficiency of MAT2A and MAT2B gene on the porcine intramuscular preadipocytes was detected by Real-time quantitative PCR and Western blot. The effects of MAT2A and MAT2B on porcine intramuscular adipogenesis were detected by Oil O stating and RT-qPCR. 【Result】Recombinant lentivirus vector pLenti-Hl-MAT2A/MAT2B was successfully constructed. The virus titer of lentivirus sh-MAT2A and sh-MAT2B were respectively 6.7×10 7 pfu/mL and 7×10 7pfu/mL. After preadipocytes infected with adenovirus for 72 h, Microscopic fluorescence imaging showed 90% of green fluorescent protein (GFP) appeared, indicated that porcine intramuscular preadipocytes were successfully infected by lentivirus sh-MAT2A and sh-MAT2B. Real-time qPCR results showed that the expression of MAT2A and MAT2B was separately decreased 70% and 60% compared with sh-scramble. The Western blot experiment and protein analysis showed that interference with MAT2A, the expression of MAT2A had an approximately 40% decrease and the difference reached high significant level (P<0.01). The expression of MAT2B had a 25% drop when inhibited with MAT2B and the different tendency towards statistical significance (P<0.05). Oil O staining and quantitative analysis showed that, the lipid accumulation of porcine intramuscular adipocytes markedly reduced upon silencing of MAT2A and MAT2B. Real-time qPCR results showed that knockdown of MAT2A and MAT2B inhibited the mRNA levels of PPARγ,aP2 and CEBP/α.【Conclusion】 Lentivirus-mediate sh-MAT2A and sh-MAT2B interference system was successfully esablished. The recombinant lentivirus can effectively reduce the mRNA and protein expression of MAT2A and MAT2B in porcine intramuscular adipocytes. Further experiments showed that interfering with MAT2A and MAT2B genes, the lipid accumulation and adipogenic key genes expression in porcine intramuscular adipocytes were significantly inhibited.

Key words: adenosine methionine transferase MAT2A, MAT2B, lentivirus, intramuscular preadipocyte, pig