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Mechanism of Faba Bean Upregulating Type Ⅰ Collagen Expression of Grass Carp Based on the TGF-β/Smads Signal Pathway

Author: LvChiBo
Tutor: XieJun
School: Shanghai Ocean University,
Course: Aquaculture
Keywords: grass carp crisp grass carp TGF-β/Smads signaling pathway type Ⅰcollagen antisense
CLC: S965.112
Type: Master's thesis
Year: 2013
Downloads: 20
Quote: 0
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Abstract


The grass carp is one of china’s main freshwater aquaculture fish, feeding broadbeans in particular stage as a single source of bait for120days, its meat would be hardand crisp, the embrittlement grass carp is known as “crisp grass carp”. The fleshyhardness, springiness, chewiness and collagen content of crisp grass carp aresignificantly enhanced, studies suggest that these changes in meat quality areconnected with muscle collagen content. Collagen is the major component of theextracellular matrix, in the muscle collagen present in connective tissue in space of themuscle fibers and the myofibrils. Type I collagen is the main type of collagen, mayplay an important role in the process of grass carp embrittlement as a result of changesin type I collagen. TGF-β/Smads signaling pathway is the main way to regulatethe expression of collagen type I has been proved in mammals. In view of Smad4is inthe central part of TGF-β signal transduction, using antisense Smad4vector to blockthe signal transduction is a commonly used means in the study ofTGF-β/Smads signaling pathways. In order to explore whether the crisp grass carpprocess after feeding broad beans contacted with the increase of type I collagen content,and whether type I collagen expression regulated by TGF-β/Smads signalingpathway. This experiment cloned Smad4gene cDNA sequence of grass carp andbuild antisense Smad4eukaryotic expression vector pcDNA3.1(+)-AntiSmad4,detectedtype Icollagen α1, type Icollagen α2, TGF-β1, Smad2and Smad4genemRNA and protein expression leveles of broad beans feeding grass carp in120-day,the antisense Smad4eukaryotic expression vector is injected in the grass carp backmuscles, and to detect the protein expression levels of Smad4, type I collagen α1and typeI collagen α2, and the mRNA expression levels of of type I collagen α1and type I collagen α2. The experiments is as follows:(1) In order to study the role of TGF-β/Smads signaling pathway played intype I collagen expression of grass carp (Ctenopharyngodon idellus), and the role ofSmad4in TGF-β/Smads signaling pathway, a full length of2974bp of Smad4cDNAsequence from grass carp was obtained, a1644bp CDS (coding sequence) encoding apolypeptide of547aa was determined in the Smad4of grass carp. Bioinformaticsanalysis showed that the relative molecular mass of grass carp Smad4protein is59690.3, the molecular formula is C2632H4073N753O791S24, isoelectric point is6.5, is ahydrophobic protein. The Smad4protein basic structure divided into MH1and MH2,two highly conserved functional domains and the intermediate link area, the secondarystructure containing5α helix domain and12β-folded, the other is coil. BLASThomology analysis showed higher homology of grass carp Smad4protein withcommon carp and zebrafish,94.23%and92.97%respectively. We also reversely inserted the open reading frame of grass carp Smad4gene into the expression vectorpcDNA3.1(+), and constructed the antisense Smad4gene eukaryotic expression vectorpcDNA3.1(+)-AntiSmad4, which is expected to lay a foundation for follow-up studyof molecular mechanism of Smad4gene.(2) In order to explore the relationship between changes of expression levels oftype I collagen and TGF-β/Smads signaling pathway in the process of broad beansfeeding grass carp, we feeding soaked broad beans to grass carp4months, and takesamples of grass carp back muscles at the0,30,60,90,120days, detected the proteinand mRNA expression levels of type I collagen α1and α2and signaling pathwaysassociated protein TGF-β1, Smad2, Smad4. the experimental results show that inThroughout process, type I collagen α1, α2, TGF-β1, Smad2, and Smad4proteinlevels were increasing trend over time; type I collagen α1, α2mRNA expression levelof the first two months change no significantly (P>0.05), expressed in90and120days increased significantly(P <0.05); the mRNA expression levels ofTGF-β1and Smad2overall increased first and then decreased, and the differencesbetween two adjacent months was significantly (P <0.05), the expression level of thehighest value in60days; the mRNA expression levels of Smad4was significantlyincreased after1month (P <0.05), and no significant change in other times. We inducedthat TGF-β/Smads signaling pathway played an important role in the increase oftype I collagen in the process of broad beans feeding grass carp, and the signalingpathway may also exist other mechanism to control the TGF-β signal transduction.(3)To confirm that the broad beans regulation of expression of grass carpcollagen is based on TGF-β/Smads signalling pathway, the experimental is to studyexpression of type I collagen after blocking-up of TGF-β/Smads signaling pathway byantisense Smad4gene. The antisense Smad4gene eukaryotic expressionvector pcDNA3.1the (+)-AntiSmad4builded before was injected to the back musclesof crisp grass carp, the injection volume is1ml at a concentration of200ng/μl, thentake back muscle samples after injected for0,1,2,5,10,15and20days, detectedprotein expression levels of Smad4, type I collagen α1and α2, and themRNA expression levels type I collagen α1and α2. The results showed that afterintramuscular injection, Smad4protein after two days, and reached the minimumon the10th day, after15days the expression level of recovery to the level beforeinjection; protein expression of type I collagen α1and α2same trend,1day afterinjection start to reduce, reached the minimum on the5th day,15days after theprotein level recovery even more than before the injection; mRNA expression oftype I collagen α1and α2slight increase after the injection, but the difference was notsignificant(P>0.05), the expression of type I collagen α1and α2increased15dayslater.To sum up, Faba bean upregulates the expression of type I Collagen of grasscarp muscle through a mechanism involving the TGF-β/Smads signal pathway.

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CLC: > Agricultural Sciences > Aquaculture, fisheries > Aquaculture technology > A variety of fish farming > Freshwater fish > Grass carp ( grass carp )
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