Identification and characterization of genes that control fat deposition in chickens

Background： Fat deposits in chickens contribute significantly to meat quality attributes such as juiciness, flavor, taste and other organoleptic properties. The quantity of fat deposited increases faster and earlier in the fast- growing chickens than in slow-growing chickens. In this study, Affymetrix Genechip~ Chicken Genome Arrays 32773 transcripts were used to compare gene expression profiles in liver and hypothalamus tissues of fast-growing and slow-growing chicken at 8 wk of age. Real-time RT-PCR was used to validate the differential expression of genes selected from the microarray analysis. The mRNA expression of the genes was further examined in fat tissues. The association of single nucleotide polymorphisms of four lipid-related genes with fat traits was examined in a F2 resource population. Results： Four hundred genes in the liver tissues and 220 genes hypothalamus tissues, respectively, were identified to be differentially expressed in fast-growing chickens and slow-growing chickens. Expression levels of genes for lipid metabolism （SULTIB1, ACSBG2, PNPLA3, LPL, AOAH） carbohydrate metabolism （MGAT4B, XYLB, GBE1, PGM1, HKDCl）cholesttrol biosynthesis （FDPS, LSS, HMGCR, NSDHL, DHCR24, IDI1, MEI） HSD17B7 and other reaction or pro- cesses （CYPIA4, CYP1A1, AKR1BI, CYP4V2, DDO） were higher in the fast-growing White Recessive Rock chickens than in the slow-growing Xinghua chickens. On the other hand, expression levels of genes associated with multicellular organism development, immune response, DNA integration, melanin biosynthetic process, muscle organ develop- ment and oxidation-reduction （FRZB, DMD, FUT8, CYP2C45, DHRSX, and CYP2C18） and with glycol-metabolism （GCNT2, ELOVL d, and FASN）, were higher in the XH chickens than in the fast-growing chickens, fiT-PCR validated high expression levels of nine out of 12 genes in fat tissues. The G1257069A and T1247123C of the ACSBG2 gene were significantly associated with abdominal fat weight. The G4928024A of the FASN gene were significantly associ- ated with fat bandwidth, and abdominal fat percentage. The C4930169T of the FASN gene was associated with ab- dominal fat weight while the A59539099G of the ELOVL 6 was significantly associated with subcutaneous fat. The A8378815G of the DDT was associated with fat band width. Conclusion： The differences in fat deposition were reflected with differential gene expressions in fast and slow growing chickens.

Dietary manganese supplementation inhibits abdominal fat deposition possibly by regulating gene expression and enzyme activity involved in lipid metabolism in the abdominal fat of broilers

Excessive abdominal fat deposition seriously restricts the production efficiency of broilers.Several studies found that dietary supplemental manganese(Mn)could effectively reduce the abdominal fat deposition of broilers,but the underlying mechanisms remain unclear.The present study aimed to investigate the effect of dietary supplementation with the inorganic or organic Mn on abdominal fat deposition,and enzyme activity and gene expression involved in lipid metabolism in the abdominal fat of male or female broilers.A total of 4201-d-old AA broilers(half males and half females)were randomly allotted by body weight and gender to 1 of 6 treatments with 10 replicates cages of 7 chicks per cage in a completely randomized design involving a 3(dietary Mn addition)×2(gender)factorial arrangement.Male or female broilers were fed with the Mn-unsupplemented basal diets containing 17.52 mg Mn kg^(-1)(d 1-21)and 15.62 mg Mn kg^(-1)(d 22-42)by analysis or the basal diets supplemented with 110 mg Mn kg^(-1)(d 1-21)and 80 mg Mn kg^(-1)(d 22-42)as either the Mn sulfate or the Mn proteinate with moderate chelation strength(Mn-Prot M)for 42 d.The results showed that the interaction between dietary Mn addition and gender had no impact(P>0.05)on any of the measured parameters;abdominal fat percentage of broilers was decreased(P<0.003)by Mn addition;Mn addition increased(P<0.004)adipose triglyceride lipase(ATGL)activity,while Mn-Prot M decreased(P<0.002)the fatty acid synthase(FAS)activity in the abdominal fat of broilers compared to the control;Mn addition decreased(P<0.009)diacylglycerol acyltransferase 2(DGAT2)mRNA expression level and peroxisome proliferator-activated receptor γ(PPARγ)mRNA and protein expression levels,but up-regulated(P<0.05)the ATGL mRNA and protein expression levels in the abdominal fat of broilers.It was concluded that dietary supplementation with Mn inhibited the abdominal fat deposition of broilers possibly via decreasing the expression of PPARγand DGAT2 as well as increasing the expression and activity of ATGL in the abdominal fat of broilers,and Mn-Prot M was more effective in inhibiting the FAS acitivity.

Identifying the complex genetic architecture of growth and fatness traits in a Duroc pig population

In modern pig breeding programs,growth and fatness are vital economic traits that significantly influence porcine production.To identify underlying variants and candidate genes associated with growth and fatness traits,a total of 1067 genotyped Duroc pigs with de-regressed estimated breeding values(DEBV)records were analyzed in a genome wide association study(GWAS)by using a single marker regression model.In total,28 potential single nucleotide polymorphisms(SNPs)were associated with these traits of interest.Moreover,VPS4 B,PHLPP1,and some other genes were highlighted as functionally plausible candidate genes that compose the underlying genetic architecture of porcine growth and fatness traits.Our findings contribute to a better understanding of the genetic architectures underlying swine growth and fatness traits that can be potentially used in pig breeding programs.

Negative effects of long-term feeding of high-grain diets to lactating goats on milk fat production and composition by regulating gene expression and DNA methylation in the mammary gland

Background： It is well known that feeding a high concentrate（HC） diet to lactating ruminants likely induces subacute ruminal acidosis（SARA） and leads to a decrease in milk fat production. However, the effects of feeding a HC diet for long periods on milk fatty acids composition and the mechanism behind the decline of milk fat still remains poorly understood. The aim of this study was to investigate the impact of feeding a HC diet to lactating dairy goats on milk fat yield and fatty acids composition with an emphasis on the mechanisms underlying the milk fat depression. Seventeen mid-lactating dairy goats were randomly allocated to three groups. The control treatment was fed a low-concentrate diet（35% concentrate, n = 5, LC） and there were two high-concentrate treatments（65% concentrate, HC）, one fed a high concentrate diet for a long period（19 wks, n = 7, HL）; one fed a high concentrate diet for a short period of time（4 wk, n = 5, HS）. Milk fat production and fatty acids profiles were measured. In order to investigate the mechanisms underlying the changes in milk fat production and composition,the gene expression involved in lipid metabolism and DNA methylation in the mammary gland were also analyzed.Results： Milk production was increased by feeding the HC diet in the HS and HL groups compared with the LC diet（P 〈 0.01）, while the percentage of milk fat was lower in the HL（P 〈 0.05） but not in the HS group. The total amount of saturated fatty acids（SFA） in the milk was not changed by feeding the HC diet, whereas the levels of unsaturated fatty acids（UFA） and monounsaturated fatty acids（MUFA） were markedly decreased in the HL group compared with the LC group（P 〈 0.05）. Among these fatty acids, the concentrations of C15：0（P 〈 0.01）, C17：0（P 〈 0.01）, C17：1（P 〈 0.01）, C18：1 n-9 c（P 〈 0.05）, C18：3 n-3 r（P 〈 0.01） and C20：0（P 〈 0.01） were markedly lower in the HL group, and the concentrations of C20：0（P 〈 0.05） and C18：3 n-3 r（P 〈 0.01） were lower in the HS group compared with the LC group. However, the concentrations of C18：2 n-6 c（P 〈 0.05） and C20：4 n-6（P 〈 0.05） in the milk fat were higher in the HS group. Real-time PCR results showed that the m RNA expression of the genes involved in milk fat production in the mammary gland was generally decreased in the HL and HS groups compared with the LC group. Among these genes, ACSL1, ACSS1 ＆ 2, ACACA, FAS, SCD, FADS2, and SREBP1 were downregulated in the mammary gland of the HL group（P 〈 0.05）, and the expressions of ACSS2, ACACA, and FADS2 m RNA were markedly decreased in the HS goats compared with the LC group（P 〈 0.05）. In contrast to the gene expression, the level of DNA methylation in the promoter regions of the ACACA and SCD genes was increased in the HL group compared with the LC group（P 〈 0.05）. The levels of ACSL1 protein expression and FAS enzyme activity were also decreased in the mammary gland of the HL compared with the LC group（P 〈 0.05）.Conclusions： Long-term feeding of a HC diet to lactating goats induced milk fat depression and FAs profile shift with lower MUFAs but higher SFAs. A general down-regulation of the gene expression involved in the milk fat production and a higher DNA methylation in the mammary gland may contribute to the decrease in milk fat production in goats fed a HC diet for long time periods.

Papaya improves non-alcoholic fatty liver disease in obese rats by attenuating oxidative stress,inflammation and lipogenic gene expression

BACKGROUND Non-alcoholic fatty liver disease(NAFLD)is a global health issue that is correlated with obesity and oxidative stress.AIM To evaluate the anti-NAFLD effect of papaya in high fat diet induced obesity in rats.METHODS Four-week-old male Sprague-Dawley rats were divided into four groups after 1 wk of acclimatization:Group 1 was the rats fed a normal diet(C);group 2 was the rats fed a high fat diet(HFD);group 3 was the rats fed a HFD with 0.5 mL of papaya juice/100 g body weight(HFL),and group 4 was the rats fed a HFD with 1 mL of papaya juice/100 g body weight(HFH)for 12 wk.At the end of the treatment,blood and tissue samples were collected for biochemical analyses and histological assessment.RESULTS The results of the HFH group showed significantly reduced body weight(HFH vs HFD,P<0.01),decreased NAFLD score(HFH vs HFD,P<0.05),and reduced hepatic total cholesterol(HFL vs HFD,P<0.01;HFH vs HFD,P<0.001),hepatic triglyceride(HFH vs HFD,P<0.05),malondialdehyde(HFL,HFH vs HFD,P<0.001),tumour necrosis factor-α(HFH vs HFD,P<0.05)and interleukin-6(HFH vs HFD,P<0.05)when compared to the HFD group.However,the liver weight showed no significant difference among the groups.The activities of catalase and superoxide dismutase significantly increased in HFH when compared with the HFD group(P<0.05 and P<0.001,respectively).The suppression of transcriptional factors of hepatic lipogenesis,including sterol regulatory elementbinding protein 1c and fatty acid synthase,were observed in the papaya treated group(HFH vs HFD,P<0.05).These beneficial effects of papaya against HFDinduced NAFLD are through lowering hepatic lipid accumulation,suppressing the lipogenic pathway,improving the balance of antioxidant status,and lowering systemic inflammation.CONCLUSION These current results provide experimental-based evidence suggesting papaya is an efficacious medicinal fruit for use in the prevention or treatment of NAFLD.

Transcriptome analysis reveals steroid hormones biosynthesis pathway involved in abdominal fat deposition in broilers

Excessive abdominal fat deposition reduces the feed efficiency and increase the cost of production in broilers.Therefore,it is an important task for poultry breeders to breed broilers with low abdominal fat.Abdominal fat deposition is a highly complex biological process,and its molecular basis remains elusive.In this study,we performed transcriptome analysis to compare gene expression profiles at different stages of abdominal fat deposition to identify the key genes and pathways involved in abdominal fat accumulation.We found that abdominal fat weight(AFW)increased gradually from day 35(D35)to 91(D91),and then decreased at day 119(D119).Accordingly,after detecting differentially expressed genes(DEGs)by comparing gene expression profiles at D35 vs.D63 and D35 vs.D91,and identifying gene modules associated with fat deposition by weighted gene co-expression network analysis(WGCNA),we performed intersection analysis of the detected DEGs and WGCNA gene modules and identified 394 and 435 intersecting genes,respectively.The results of the Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses showed that the steroid hormone biosynthesis and insulin signaling pathways were co-enriched in all intersecting genes,steroid hormones have been shown that regulated insulin signaling pathway,indicating the importance of the steroid hormone biosynthesis pathway in the development of broiler abdominal fat.We then identified 6 hub genes(ACTB,SOX9,RHOBTB2,PDLIM3,NEDD9,and DOCK4)related to abdominal fat deposition.Further analysis also revealed that there were direct interactions between 6 hub genes.SOX9 has been shown to bind to proteins required for steroid hormone receptor binding,and RHOBTB2 indirectly regulates the steroid hormones biosynthesis through cyclin factor,and ultimately affect fat deposition.Our results suggest that the genes RHOBTB2 and SOX9 play an important role in fat deposition in broilers,by regulating steroid hormone synthesis.These findings provide new targets and directions for further studies on the mechanisms of fat deposition in chicken.

猪FATP1基因5'调控区多态性及其与脂肪性状的相关分析

脂肪酸转运蛋白1(Fatty acid transport protein 1,FATP1)是脂肪酸转运蛋白家族(FATPs)的一员,具有促进脂肪细胞吸收利用长链和极长链脂肪酸的功能,在脂肪沉积过程中发挥着重要的作用。采用直接测序法寻找FATP1基因5'调控区+64～-723 bp片段的单核苷酸多态性,在-586 bp处发现了一个T→C突变SNP(单核苷酸多态性)座位,利用PCR-RFLP的方法检测它在3个试验猪群体中的基因型,发现该座位在皮特兰和金皮F2代猪群中存在多态性,而在金华猪中仅表现为FATP1g-586TFATP1g-586T基因型。建立混合模型方程组分析该座位对188头金皮F2代猪脂肪性状的效应,结果显示,猪FATP1基因第-586座位的不同基因型与肩背膘厚、6～7肋背膘厚、最后一根肋骨处背膘厚、平均背膘厚和板油重显著相关(P<0.05),表明FATP1基因5'调控区的第-586位上的T→C突变对猪脂肪沉积有一定的影响。

fat-1基因对结肠癌HT-29细胞增殖的抑制作用

目的探讨fat-1基因在结肠癌HT-29细胞凋亡、增殖以及细胞周期中所起的作用。方法构建真核表达载体(pEGFP-fat-1),用脂质体介导的方法转染到结肠癌HT-29细胞,通过荧光显微镜观察及RT-PCR检测fat-1基因的表达,气相色谱分析检测fat-1基因对HT-29细胞n-6/n-3多聚不饱和脂肪酸(PUFAs)比例的影响,MTT法分析fat-1基因对HT-29细胞增殖的影响,流式细胞术检测fat-1基因对HT-29细胞凋亡和细胞周期的影响。结果成功构建了真核表达载体pEGFP-fat-1,并在HT-29细胞内有效表达。fat-1基因通过降低HT-29细胞内n-6/n-3PUFAs的比例抑制HT-29细胞的增殖,促进其凋亡,细胞增殖主要被阻滞在S期。结论fat-1基因改变HT-29细胞n-6/n-3PUFAs比例后,通过一定的信号转导途径促进大部分HT-29细胞在S期凋亡,抑制了其增殖。

线虫fat-1基因密码子优化设计

本研究旨在优化线虫fat-1基因,使其在牛肌肉组织中高效表达,为进一步生产fat-1转基因肉牛提供目的基因。试验在不改变fat-1基因编码氨基酸序列的基础上,参考了11个牛肌肉蛋白相关基因的密码子使用频率,对fat-1基因进行了密码子优化。结果发现,共改变了65个碱基,涉及65个密码子、7个氨基酸,约占fat-1基因总碱基数的5.38%,G+C含量从45.08%上升到50.04%,且分布均匀利于基因的表达。结构预测结果显示,该优化后的基因能够在牛肌肉中高效表达。

fat-1转基因小鼠的构建与鉴定

目的构建和鉴定携带有外源fat-1基因的转基因小鼠。方法将fat-1基因的cDNA与动物表达载体pEF-neo连接,构建pEF-fat-1重组质粒,酶切、测序鉴定正确后,以显微注射法把线性化重组质粒注射到小鼠受精卵的雄原核中,并将受精卵移植到受体鼠的输卵管中产出转基因小鼠,通过PCR、Southern blot杂交等方法确立阳性整合有目的基因的G0代小鼠。结果成功构建了pEF-fat-1重组质粒,将其显微注射到小鼠受精卵中,得到G0代小鼠,PCR、Southern blot杂交确立了4只整合有fat-1基因的首建鼠。结论fat-1基因可整合到小鼠体内,得到的转基因小鼠为研究fat-1基因的生物学功能提供了动物模型。

Fat-1基因多态性与脊柱结核易感关联性研究

目的寻找和分析与脊柱结核易感性相关的新的候选基因。方法收集2013年6月—2015年12月杭州市红十字会医院收治的脊柱结核患者193例为病例组,同时选取同时期本院体检健康者207例为对照组,采用全基因组外显子技术捕获6例脊柱结核患者外显子区DNA后,进行高通量测序,并结合现代生物信息学方法进行分析,结果初步筛选到Fat-1基因多态性(rs1280098,c.8798A>C,p.Q2933P)与脊柱结核的发生相关;随后,采用Sanger测序法对对照组和病例组患者做上述突变位点的验证。检测Fat-1基因型分布,采用实时荧光反转录聚合酶链式反应(RT-q PCR)法检测Fat-1基因相对表达水平,酶联免疫吸附试验(ELISA)法检测血清白介素(IL)-12和γ-干扰素(IFN-γ)含量,同时检测生化指标。结果病例组AA基因型129例(66.8%)、AC基因型33例(17.1%)、CC基因型31例(16.1%);对照组分别为157例(75.8%)、35例(16.9%)、15例(7.3%)。两组Fat-1基因型分布比较,差异有统计学意义(χ~2=7.885,P=0.019)。不同遗传模型的Logistic回归分析结果显示:在相加模型中,CC纯合突变基因型与脊柱结核是否发病相关,AC杂合突变基因型与脊柱结核是否发病不相关(P>0.05);在显性模型中,AC+CC突变基因型与脊柱结核是否发病相关(P<0.05);在隐性模型中,CC纯合突变基因型与脊柱结核是否发病相关(P<0.05);且该多态性位点最小等位基因C与脊柱结核是否发病相关(P<0.05)。病例组患者外周血Fat-1基因相对表达水平低于对照组,血清IL-12和IFN-γ含量高于对照组,脂蛋白A、C反应蛋白、红细胞沉降率和嗜酸粒细胞计数高于对照组,清蛋白水平低于对照组(P<0.05)。结论 Fat-1基因rs1280098位点多态性与脊柱结核的发病相关。此外,Fat-1基因该位点的突变导致Fat-1基因转录水平下调,以及血清炎性因子IL-12和IFN-γ水平上调,并最终导致脊柱结核易感性增加。这些检测结果为脊柱结核的早期诊断提供了分子依据。

fat-1转基因动物研究进展

转染秀丽隐杆线虫fat-1基因后的哺乳动物具备了将n-6多不饱和脂肪酸(PUFAs)转化为n-3 PUFAs的能力,可降低动物机体的n-6/n-3 PUFAs比例。n-3 PUFAs有益于人类健康,可减少多种相关疾病发生的风险,但人体内不能合成n-3 PUFAs,其必须依赖于富含n-3 PUFAs的食品,fat-1转基因动物将成为人类必需的n-3 PUFAs的重要来源。对fat-1及其转基因动物的研究现状进行综述。

转Fat-1基因猪外源基因遗传稳定性分析

测定转Fat-1基因猪外源基因遗传稳定性,为转基因猪育种选择优质新材料提供基础数据。采用PCR和Southern印迹杂交方法对转Fat-1基因传代生产仔猪进行检测,了解Fat-1基因在猪基因组中的整合情况,统计传代猪转基因阳性比率,与自然基因单等位基因正常遗传比率进行差异显著性分析。结果显示,F2代传代仔猪Fat-1基因阳性比率为57.14%,F3代传代仔猪阳性比率为46.67%。F1代公猪外源Fat-1基因是1拷贝基因,传代配种母猪使用非转基因母猪,仔猪阳性比率与自然基因单等位基因正常遗传比率(50%)差异不显著。Fat-1基因在猪育种传代过程中遗传力没有下降,转Fat-1基因公猪能够稳定遗传生产后代猪。

线虫Fat-1基因密码子的优化及其真核表达载体构建

目的:为使线虫Fat-1基因能够在牛中高效表达,通过密码子优化及全基因合成方法制备Fat-1基因,并构建其转基因表达载体。方法:通过生物信息学手段,将来源于线虫的Fat-1基因以牛为表达宿主进行密码子优化,将优化后的Fat-1基因命名为bFat-1;通过全基因合成方法获得bFat-1基因,构建pcDNA3.1-bFat-1真核表达载体。结果:通过密码子优化,影响Fat-1基因在牛中表达的密码子适应指数、优势密码子频率及GC含量等各项指标均有显著改善;全基因合成、克隆及测序结果显示已获得bFat-1基因;构建并鉴定了pcDNA3.1-bFat-1载体。结论:获得了适合牛体表达的Fat-1基因并构建了其转基因载体,为获得高效表达Fat-1基因的转基因牛细胞系奠定了基础。

转Fat-1基因体细胞克隆草原红牛研究

Fat-1基因的转基因动物克隆,为研究ω-3多不饱和脂肪酸的功能提供了高效、准确的医学、营养学动物模型。试验成功建立了草原红牛耳部成纤维细胞系,将Fat-1基因转染到草原红牛成纤维细胞,获得转基因阳性细胞克隆。以转基因细胞为核供体,体外成熟牛卵母细胞为核受体构建转基因克隆囊胚,比较未转基因与转基因克隆胚胎的体外发育情况。结果表明,体细胞重构胚体外囊胚率分别为26.04%和26.04%,两者之间无显差异著(P>0.05)。转Fat-1基因的草原红牛重构桑葚胚或早期胚胎移植到延边黄牛子宫内,有7头妊娠,但没能够获得妊娠足月个体。

转fat-1基因动物模型和n-3 PUFAs功能作用机理研究进展

研究fat-1基因功能的作用机理对充分合理利用多不饱和脂肪酸(PUFAs)资源具有重要意义,建立转fat-1基因动物模型是了解fat-1基因功能、作用机理的重要方法。文章详细阐述了fat-1基因通过n-3 PUFAs而发挥的生理功能、转fat-1基因动物神经系统疾病模型、转fat-1基因动物肿瘤疾病模型、转fat-1基因炎症动物疾病模型及作用机理研究进展,并对转fat-1基因动物抗肥胖作用机理、转fat-1基因动物在优质家畜育种改良中的作用、利用转fat-1基因动物模型研究骨骼肌发育机理和糖尿病的治疗方法等方面的进展进行了总结,以期为合理利用PUFAs资源和优质家畜改良育种提供理论依据。

Fat-1基因过表达抑制宫颈癌Hela细胞生长机制研究

目的:探讨fat-1基因对宫颈癌Hela细胞生长的影响,并研究PI3K/Akt/mTOR通路是否参与fat-1对Hela细胞生长的调控作用。方法:构建真核表达载体(p-EGFP-fat-1),并转染人宫颈癌Hela细胞,使其过表达fat-1基因,为fat-1组,以转染空载质粒的Hela细胞为阴性对照组,以未经任何处理的Hela细胞为空白对照组。采用气相色谱法检测fat-1基因对ω-3多不饱和脂肪酸(ω-3PUFA)和ω-6多不饱和脂肪酸(ω-6PUFA)含量的影响;采用MTT和Hoechst实验检测fat-1基因对细胞增殖和凋亡的影响;采用Transwell和细胞划痕实验检测fat-1基因对细胞侵袭和迁移的影响;采用Western blot检测fat-1基因对PI3K/Akt/mTOR通路相关蛋白表达情况的影响。结果:Fat-1组细胞中fat-1基因表达水平高于空白对照组和阴性对照组(P<0.01)。Fat-1组细胞中ω-3PUFA含量高于空白对照组和阴性对照组,ω-6PUFA含量和ω-6PUFA/ω-3PUFA比例均低于空白对照组和阴性对照组(P均<0.01)。Fat-1组细胞的增殖、侵袭和迁移能力均低于空白对照组和阴性对照组,Fat-1组细胞凋亡水平高于空白对照组和阴性对照组(P均<0.01)。Fat-1组细胞中p-PI3K、p-Akt和p-mTOR表达水平均低于空白对照组和阴性对照组(P<0.01)。结论:Fat-1基因可通过增加细胞内ω-3PUFA含量,靶向作用于PI3K/Akt/mTOR通路,抑制宫颈癌Hela细胞的增殖、侵袭和迁移,促进细胞凋亡。

线虫fat-1基因的原核表达、纯化及其抗体的制备

通过RT-RCR得到线虫f a t-1基因全长,将其N端亲水区克隆至原核表达载体pGEX-4T-2中,构建重组质粒pGEX-f a t1-N,将其转化大肠杆菌,并进行诱导表达。对表达产物G ST-f a t1-N融合蛋白进行纯化,制备其抗体,并对抗体效价进行了检测。结果表明,线虫f a t-1基因能在大肠杆菌中表达,制备的抗体能识别在原核表达系统内表达的G ST-f a t1-N融合蛋白,且效价很高,达到1∶107。

fat-1基因密码子优化及在家兔胎儿成纤维细胞中的初步表达

根据家兔基因密码子使用偏好性,对引自GenBank的秀丽隐杆线虫fat-1基因编码序列进行密码子优化,通过全基因合成的方法获得优化后的fat-1基因（命名为opfat-1）;从秀丽隐杆线虫中RT-PCR扩增获得fat-1。将扩增的fat-1和合成的opfat-1基因分别构建到绿色荧光蛋白真核表达载体pEGFP-C1中。采用脂质体Lipo-fectamineTM转染法将fat-1和opfat-1基因转入家兔胎儿成纤维细胞（Rabbit fetal fibroblast cells,rFFCs）中。对比不同DNA和转染试剂用量以及细胞暴露于DNA、转染试剂中作用时间探讨影响rFFCs转染效率的参数,经过优化,最优的rFFCs转染条件为：LipofectamineTM用量2~3μl,DNA量0.8~1.0μg,LipofectamineTM与DNA、细胞共培养时间为8 h。通过绿色荧光标记和RT-PCR检测转染细胞中融合蛋白和目的基因的表达。密码子优化的opfat-1与fat-1相比,体外瞬时表达效率有极为显著的提高;G418抗性筛选获得转基因单克隆细胞,RT-PCR初步验证fat-1和opfat-1基因均在家兔胎儿成纤维细胞中稳定转录表达。

C. elegans fat-1基因对SGC7901细胞的促凋亡作用

目的通过在人胃癌SGC7901细胞系上转入外源C.elegans fat-1基因观其对细胞增殖或对调亡的影响。方法构建携带有能够编码n-3多聚饱和脂肪酸脱氢酶的小秀丽隐杆线虫fat-1基因的重组质粒,转染人胃癌细胞SGC7901,检测n-6/n-3脂肪酸的比例,观察胃癌细胞的增殖和凋亡情况。结果转染成功后携带有fat-1基因的胃癌细胞,显示了对于n-3脂肪酸饱和酶的高表达。脂类分析表明,n-6PUFAs的比例大幅度降低,n-3PUFAs水平明显提高,n-6/n-3脂肪酸比例,从10.45下降到了0.79,尤其是花生四烯酸和二十碳戊烯酸的比率。相应地,在表达有fat-1基因的胃癌细胞中,来源于n-6PUFAs的类花生酸含量有显著减少。同时,fat-1基因的转移导致大量胃癌细胞的凋亡,抑制胃癌细胞的增殖。结论n-3脂肪酸去饱和酶的基因转移,能够有效调整人肿瘤细胞n-6/n-3脂肪酸的比例,起到抗癌作用,证明在癌症的预防和治疗中,n-6与n-3脂肪酸的比例扮演着一个重要的角色。