Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (15): 2706-2715.doi: 10.3864/j.issn.0578-1752.2019.15.014

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

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 E-mail:zhzhongke@163.com

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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

Table 1

Design of shRNA targeting on porcine MAT2A and MAT2B gene"

RNAi 序列 Primer sequence (5′ to 3′)
sh-MAT2A-1

sh-MAT2A-2

sh-MAT2A-3

sh-MAT2B-1

sh-MAT2B-2

sh-MAT2B-3

sh-scramble		 5′-GATCCGCAGCAGTCACCAGATATTGCctcgagGCAATATCTGGTGACTGCTGCTTTTTC-3′
5′-TCGAGAAAAAGCAGCAGTCACCAGATATTGCctcgagGCAATATCTGGTGACTGCTGCG-3′
5′-GATCCGCACACAAGCTCAATGCCAAActcgagTTTGGCATTGAGCTTGTGTGCTTTTTC-3′
5′-TCGAGAAAAAGCACACAAGCTCAATGCCAAActcgagTTTGGCATTGAGCTTGTGTGCG-3′
5′-TCGAGAAAAAGCCAAGTGGCAGATTTGTTATctcgagATAACAAATCTGCCACTTGGCG-3′
5′-GATCCGGAGAGTGCCGTGACTGTTATctcgagATAACAGTCACGGCACTCTCCTTTTTC-3′
5′-GATCCGCACAGCGACGAGTACAAGActcgagTCTTGTACTCGTCGCTGTGCTTTTTC-3′
5′-TCGAGAAAAAGCACAGCGACGAGTACAAGActcgagTCTTGTACTCGTCGCTGTGCG-3′
5′-GATCCGGAGAGTGCCGTGACTGTTATctcgagATAACAGTCACGGCACTCTCCTTTTTC-3′
5′-TCGAGAAAAAGGAGAGTGCCGTGACTGTTATctcgagATAACAGTCACGGCACTCTCCG -3′
5′-GATCCGCGAACACCATTTCGAATTGGctcgagCCAATTCGAAATGGTGTTCGCTTTTTC-3′
5′-TCGAGAAAAAGCGAACACCATTTCGAATTGGctcgagCCAATTCGAAATGGTGTTCGCG-3′
5′-GATCCGACACCTACGCAAAACCCTctcgagGACACCTACGCAAAACCCTTTTTTC-3′
5′-TCGAGAAAAAGACACCTACGCAAAACCCTctcgagGACACCTACGCAAAACCCTG-3′

Table 2

Specific primers for real-time quantitative PCR"

Gene 序列号Accession No. 序列Primer sequence (5′ to 3′)
MAT2A NM_001167650.1 S: GTGGTTCGTGAAACCATTAAG
A: ATCAGTGGCATAACCAAACAT
MAT2B NM_001142832.1 S: TAGGAGCTGCTGTTTTGAGA
A: CACACGCCATTTCATACTTG
PPARγ NM_214379.1 S:AGGACTACCAAAGTGCCATCAAA
A:GAGGCTTTATCCCCACAGACAC
aP2 HM_453202 S:GAGCACCATAACCTTAGATGGA
A:AAATTCTGGTAGCCGTGACA
CEBP/α AF_103944.1 S: GCACTTGCAGTTCCAGATCG
A:ACCCGGTACTCGTTGCTGTT
β-actin NM_007393 S: GGACTTCGAGCAGGAGATGG
A: AGGAAGGAGGGCTGGAAGAG

Fig. 1

Construction and identification of recombinant plasmid LentiH1-shRNA (A): Produce shRNA interference fragments sense and antisense oligo nucleotides were annealed to form complementary double strands DNA. M: DNA marker (DL 5000); 1-3: sh-MAT2A RNA; 4-6: sh-MAT2B RNA. (B): LentiH1 shuttle plasmid was double digested by Xho I and BamH I. 1-2: the product of LentiH1 shuttle plasmid was double digested; all released two fragments (7612 bp and 59 bp). M: DNA marker (DL 5000). (C): recombinant plasmids were identified by digesting. M: DNA marker (DL 5000). 1, 3, 5, 7, 9, 11: digested plasmid (as a control); 2, 4, 6, 8, 10, 12: undigested plasmid (2-6: sh-MAT2A; 8-12: sh-MAT2B)"

Fig. 2

The packaging of pLentiH1-MAT2A/2B shRNA vector A-C: The GFP expression of sh-scramble and sh-MAT2A and sh-MAT2B that separately infected for 48 h"

Fig. 3

The efficiency of cultured preadipocytes that were infected with recombinant lentivirus A-C Porcine preadipocytes were separately infected by sh-scaramble (as a control), sh-MAT2A and sh-MAT2B for 72 h. D-E: Real-time qPCR to analyze the relative expression of MAT2A and MAT2B (Data are presented as means ± SEM, n=3. *P<0.05, **P<0.01)"

Fig. 4

The detection of MAT2A and MAT2B interference efficiency A-B: The Interference efficiency of MAT2A and MAT2B was detected using Western blot. C-D: Quantification to analysis the protein level of MAT2A and MAT2B (Data are presented as means ± SEM, n=3. *P<0.05, **P<0.01)"

Fig. 5

Silencing MAT2A and MAT2B inhibited differentiation of porcine intramuscular adipocytes A-B: Cells were stained with Oil red O and quantification to assess lipid accumulation. C: The relative mRNA expression of MAT2A and MAT2B detected by RT-qPCR. (Data are presented as means ± SEM; n=3; *P<0.05, **P<0.01)"

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