亚麻(Linum usitatissimum)脂肪酸去饱和酶基因3(fatty acid desaturase 3,FAD3)是一种脂肪酸脱氢酶基因,编码ω-3多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)脱氢酶,能够将ω-6 PUFAs转化成ω-3PUFAs.本研究利用CRISPR/Cas9介导,将无抗性、无标记的人(Homo sapiens)源化hFAD3基因表达载体C2(5'同源臂-CAG-hFAD3-PolyA-3'同源臂)和M2(5'同源臂-MAR-CAG-hFAD3-PolyA-MAR-3'同源臂)定点敲入牛(Bos taurus)胎儿成纤维细胞NCAPG-LCORL位点.运用气质联用仪对脂肪酸含量进行分析,结果表明hFAD3转基因细胞中ω-6/ω-3 PUFAs比值(0.565)较非转基因细胞(1.549)显著下降(P＜0.01).qRT-PCR结果表明hFAD3基因整合并表达后,脂肪分解相关的过氧化物酶体增殖物激活受体基因(peroxisome proliferator activated receptorl,PPARg)、激素敏感脂肪酶基因(hormone-sensitive lipase,LIPE)和脂蛋白脂酶(lipoprotein lipase,LPL)表达总量上调倍数大于脂肪合成相关的乙酰辅酶A羧化酶基因(acetyl CoA carboxylase,ACC)、硬脂酰辅酶A去饱和酶基因(stearoyl CoA desaturase,SCD)和脂肪酸合酶基因(fatty acid synthase,FASN)表达总量,说明hFAD3基因的表达使得细胞内的脂肪趋于分解.通过流式分选获得59株C2打靶载体单克隆细胞株,PCR鉴定及测序结果表明,其中定点整合阳性单克隆细胞3株,定点整合效率为5.1％.同时检测定点整合阳性单克隆细胞株中脂肪酸的含量以及脂肪相关基因的表达量,结果与转染后细胞一致.比较有无核基质结合区(matrix attachment region,MAR)序列介导两种转基因细胞中hFAD3基因的表达量,结果显示,MAR介导组是无MAR组的5.91倍(P＜0.01).本研究利用CRISPR/Cas9介导可实现hFAD3基因在牛NCAPG-LCORL位点的定点整合以及在细胞水平正常行使其生物学功能,同时MAR的介导可显著提高外源基因的表达量,为安全高效生产转基因动物提供科学依据.

鸟类作为色彩最丰富的陆生脊椎动物,其体表覆盖着颜色多样的羽毛,在伪装、择偶、信号识别等多方面具有重要功能,因此羽毛颜色引起了研究者的极大兴趣.羽毛颜色总体分为由化学物质产生的色素色和由物理结构产生的结构色,其中常见色素有两大类.根据近年来对羽毛色素的研究进展,本文总结了黑色素和类胡萝卜素的类型、合成途径、获取途径以及相关基因,为深入研究羽毛色素合成、代谢的分子调控机制提供科学依据.

肌肉生长抑制素(myostatin,MSTN)属于转化生长因子β(transforming growth factor β,TGF-β)超家族成员,在骨骼肌中高表达;敲除MSTN后,肌肉质量增加、脂肪含量变少,但抑制MSTN表达后,如何影响脂肪沉积,尚未研究清楚.为了探究抑制MSTN表达后对脂肪相关基因表达的影响,本研究利用反义RNA技术,成功构建了双向表达载体pMSTN-CMV-CAG-antiMSTN,并通过Western blot筛选出抑制效果最佳的MSTN的反义载体.利用最佳反义MSTN载体在牛(Bos taurus)肌肉卫星细胞中有效干扰MSTN表达后,检测了脂肪合成相关基因(脂肪酸合酶(fatty acid synthase,FASN),乙酰CoA羧化酶(acetyl-CoA carboxylase,ACC)和硬脂酰辅酶A去饱和酶1(stearoyl-CoA desaturase-1,SCD-1))、脂肪酸氧化分解相关基因(肉毒碱棕榈酰转移酶ⅠB(carnitine palmitoyltransferase 1b,CPT-ⅠB),乙酰辅酶A氧化酶(acyl-CoAoxidase,ACOX1)和烯酰辅酶A水合酶(enoyl-CoA hydratase 1,ECHDC1))以及脂肪转运蛋白(fatty acidtransport proteins,FATP)表达的变化.结果显示,在MSTN基因被干扰后,肌肉卫星细胞中脂肪酸合成相关基因表达量上调,脂肪氧化分解相关基因中ACOX1和CPT-1B表达量上调,而ECHDC1基因表达量下调,另外,FATP表达基本没有变化.MSTN功能缺失后,脂肪合成和脂肪氧化分解过程都得到了加强,所以MSTN敲除所导致的脂肪减少并不是由于抑制了脂肪合成,而是由于脂肪分解加强,为肌肉提供更多的能量.MSTN下调后,CPT-1B的表达上调最为显著,而CPT-1B是脂肪酸β氧化的关键基因,所以研究结果提示MSTN对于脂肪的影响主要是通过影响β氧化过程实现的.研究结果为MSTN下调所导致的脂肪含量下降提供了理论依据,为MSTN作用于脂肪沉积的机制做出了初步探讨.

In the present study, follistatin (FST) gene expression vectors with either a bicistronic gene transfer cassette alone, or a bicistron gene cassette carrying a matrix attachment region (MAR) were constructed and transfected to bovine fetal fibroblasts. Evaluations of both the integration and expression of exogenous FST indicated that the pMAR-CAG-FST-IRES-AcGFP1-polyA-MAR (pMAR-FST) vector had higher capacity to form monoclonal transgenic cells than the vector without MAR, though transient transfection and integration efficiency were similar with either construct. Remarkably, protein expression in transgenic cells with the pMAR-FST vector was significantly higher than that from the bicistronic vector. Exogenous FST was expressed in all of the pMAR-FST transgenic mice at F-0, F-1 and F-2. Total muscle growth in F-0 mice was significantly greater than in wild-type mice, with larger muscles in fore and hind limbs of transgenic mice. pMAR-FST transgenic mice were also found with more evenly distributed muscle bundles and thinner spaces between sarcolemma, which suggests a correlation between transgene expression-associated muscle development and the trend of muscle growth. In conclusion, a pMAR-FST vector, which excluded the resistant genes and frame structure, enhances and stabilizes FST gene expressions in both transfected cells and transgenic mice.

肌肉生长抑制素(myostatin,MSTN),也称为生长分化因子8(GDF8),是转化生长因子β(TGF-β)超家族成员,主要在骨骼肌中表达,在负向调控肌细胞的生长发育中起关键作用.本文对MSTN研究的最新进展进行了综述,讨论了MSTN信号通路与其他通路之间的相互关系,重点阐述了试验条件下MSTN对肌肉发育相关基因的作用,介绍了通过基因操作或使用抗体的方法抑制MSTN的表达对于提高动物产肉量、治疗肌肉萎缩等疾病以及延缓衰老的重要意义.

基质结合区（matrix attachment regions, MAR）是一段能与核基质特异性结合的DNA序列，在进化上较为保守，对于真核生物基因组中的染色体折叠有着非常重要的作用。将其构建到外源基因两侧时可以增加转基因的表达水平，并在一定程度上降低个体之间的差异。目前MAR序列已成为动物和植物基因工程领域的研究热点之一。本文主要综述MAR序列的特征、分布及功能、对转基因动物的影响及作用机制等。

The FAD3B was cloned from the seed of flax(Linum usitatissimum Linn)using RT-PCR.The gene was constructed as the recombinant eukaryotic expression vectors pIRES-AcGFP-CMV-FAD3B and pIRES-AcGFP-CAG-FAD3B with different promoters.The expression level of FAD3B was detected by qRT-PCR in transfected cells.Different over-expression levels of transfected cells were used as the experimental subjects.Nine candidate reference genes were evaluated,including ACTB,GAPDH,18S rRNA,UXT,PPP1R11,RPS15A,SF3A1,EEF1A2 and HMBS.Three statistical algorithms(geNorm,NormFinder and BestKeeper)were used to rank genes by their stability values.The overall rank ordering of candidate internal control genes was PPP1R11>EEF1A2>18S rRNA>RPS15A>GAPDH>HMBS>UXT>ACTB>SF3A1,PPP1R11 and EEF1A2 can be considered as the most stable reference genes.Selection of stable control genes can normalize the expression level of gene,laid a solid foundation so as to elaborate the function of genes.

DREB transcription factors play an important role in tolerance to abiotic stress in high plants. In this work, two new DRE-binding protein genes MfDREB1 and MfDREB1s cDNA that encoded an AP2/EREBP type transcription factor were isolated by RT-PCR from Medicago falcate seedlings. Sequence analysis showed MfDREB1 and MfDREB1s were almost identical except that there was a 202bp fragment at the 3' end of the MfDREB1s cDNA that is absent in MfDREB1 cDNA. The MfDREB1 has a open reading frame of 651bp, which encodes 216 amino acid residues. The putative protein is deduced a predicted molecular mass of 24.6kDa and a pI of 5.95. The MfDREB1s has a open reading frame of 555bp, the putative protein is 184 amino acid long with a predicted molecular weight of 20.8kDa, pI 9.11. The Protein Blast data revealed that the two proteins can be classified as a typical member of the AP2/EREBP family of DNA-binding proteins. The comparison of the MfDREB1 cDNA and MfDREB1s cDNA with their corresponding genes in genomic DNA showed that the size and nucleotide sequence of the cDNA was identical to that of the genomic DNA. This suggested that the genomic MfDREB1 gene and MfDREB1s gene had no introns. Southern blot analysis indicated that MfDREB1 and MfDREB1s are multi-copy genes in Medicago falcate genome. Northern blot analysis indicated that the MfDREB1 and MfDREB1s genes were induced by low temperature stress.

Objective:Construction of Multidrug resistance gene-1 shRNA vector and research of its function for human resistance leukemia cells.Methods: Artificial synthesized DNA oligonucleotide encoding the small hairpin RNA of mdr1 gene was recombined into pSilencer4.1-CMV.The expression vector obtained eukaryotic RNAi was introduced into K562/A cells with LipofectamineTM2000,and positive colonies were screened by Hygromycin B;The expression of mdr1 in K562/A cells before and after transfection were detected by RT-PCR,Western Blotting,and MTT method.Results: The mRNA expression of mdr1 in K562/A group was decreased by 43.55% with control group by the above small hairpin RNA,and the protein expression was decreased by 69.46%.The multidrug reversable resistance was 23 times by MTT method.Conclusions: The specific small hairpin RNA could inhibit the expression of mdr1 gene.This study of multidrug reversible resistance and treatment of leukemia has theoretical and practical significance.

Objective:To investigate the effect of over expression of fhit(fragile histidine triad gene,fhit) gene on the growth and apoptosis of HeLa cells,we constructed the bicistronic expression vector and co-expression vector of fhit and egfp gene.Method:According to the mRNA sequence of fhit gene,the cDNA was cloned by RT-PCR from the liver tissue total RNA,which is 493bp.Then it was inserted into the cloning vector pMD18-T.After selecting the recombined plasmids which the fhit gene inserted into correctly,We digested the plasmids with restriction endonuclease in order to recover the target fragments.These fragments were ligated into the multiple cloning site(MCS) of the bicistronic expression vector pIRES2-EGFP and co-expression vector pEGFP-C1.Then the vectors were transfected into HeLa cells with lipofectamineTM Regent respectively.G418 was used to select the stable transfected cells.Then we detected the expression level of fhit gene in genome,transcript and protein by PCR,RT-PCR,immunocytochemistry and TUNEL test.Results:In this study,we achieved the 493bp frgment of fhit gene through PCR from the genome of transgenic cells,it proved that the target gene had been integrated into cells genome stably.From the result of RT-PCR we concluded that the gene was transcripted into RNA constantly.Furthermore,immunocytochemistry and TUNEL test indicated that fhit gene had been steadily inherited and expressed in HeLa cells.Conclusions:The results in this study confirmed that fhit gene over expression can efficiently accelerate apoptosis of HeLa cells.

Endostatin cDNA was obtained by RT-PCR and then inserted into pEF1-4SX.Then the fragment of endostatin linking with IRES was cut and ligated into a eukaryotic expression vector to construct the tricistronic eukaryotic expression vector pES-IRES-FHIT-IRES-EGFP.This recombinant vector was transfected into Hela cells with LipofectamineTM 2000.Total RNA was extracted from those stably transfected Hela cells,and then was checked by RT-PCR Southern blotting.The results indicated that the moncistrons of endostatin,FHIT and EGFP as well as the tricistron of these three genes all had been transcribed successfully in the transfected Hela cells.Further more,the results of immunocytochemistry showed that FHIT and endostatin had been translated in the transfected Hela cells.These results provided some basic information for further research on multigene therapy strategies in tumor treatment.

In order to establish a simple,practical technique with high transformation frequency in alfalfa(Medicago sativa L.),the pollen-tube pathway method was used for alfalfa transformation.The alfalfa cultivar 'Algonquin' was used as a recipient and the plant binary vector pPZP221 containing gus gene was used as donor DNA.The plasmid DNA solution was applied to the stigma at 6h,9h,12h,20h,24h,28h,32h and 48h after hand-pollination,respectively.PCR results showed that the transformation rate of T0 seedlings from 20h to 48h was relatively high and the highest rate was up to 16.7% at 32h after pollination.This results suggested that foreign DNA was inserted into the genome of transgenic T0 seedlings.

PTEN,the first-discovered tumor suppressor with dual-specificity phosphatase activity,inhibits cell migration through dephosphorylating focaladhension kinase.In our previous research,the prokaryotic recombinant His-PTEN protein was obtained and proved to have a significant tumor suppression activity.The phosphatase activity of recombinant His-PTEN,as well as the effect on cell migration and FAK phosphorylation,was further analyzed in the present research.Apparent lipid phosphatase activity of recombinant His-PTEN was showed by in vitro dephosphorylation of [D3-32P]PIP3.After the transfection of His-PTEN into prostate cancer cell DU-145,FAK phosphorylation level was significantly reduced,indicating an obvious protein phosphatase activity of the recombinant protein.His-PTEN also inhibited the migration of cancer cell DU-145,leading to a conclusion that the recombinant PTEN might be a potential drug to suppress tumor invasion.

In order to use Cucumis melo L.cv Hetao as a bioreactor for production of human augmenter of liver regeneration(hALR) protein,471 bp cDNA hALR was amplified from the human fetal liver mRNA by RT-PCR with two artificially synthesized primers,then ligated into vector pMD18-T to construct pMD-ALR.The sequence analysis of pMD-ALR showed that the sequence was completely identical with that of hALR cDNA in GenBank.The fruit-specific expression vector pPZP-CAN was constructed by inserting hALR which was obtained from pMD-ALR by HindⅢ and SacⅠrestrictive digesting,into plant expression vector pPZP-CGN which contains Cucumis melo L.cv cucumisin gene promoter.63 hand-pollinating flowers were obtained via pollen-tube pathway transformation method in testing-farm,by which 27 fruits were achieved with 42.9 percentage of fruit-set.The DNA from T0 seedings were analyzed by PCR,PCR-southern and Dot boltting analysis and 13 positive were obtained.

Random peptide libraries displayed on the ribosome are becoming a new tool for the in vitro selection of biologically relevant macromolecules, including epitopes, antagonists, enzymes, and cell-surface receptors. Ribosome display is a cell-free system of coupling individual nascent proteins (phenotypes) to their corresponding mRNA (genotypes) by the formation of stable protein-ribosome-mRNA complexes and permitting the selection of a functional nascent protein by iterative cycles of panning and reverse transcription-polymerase chain reaction (RT-PCR) amplification in vitro. The complexity of the random peptide library is critical for the success of a panning experiment; greater the diversity of sequences within the library, the more likely it is that the library comprises sequences that can bind a given target with specific affinity. Here, we have used the cell-free system Escherichia coli S30 lysate to construct high-complexity random peptide libraries (>10(14) independent members) by introducing strategies that are different from the methods described by Mattheakis et al. and Lamla et al. The key step in our method is to produce nanomole (nmol) amounts of DNA elements that are necessary for in vitro transcription/translation by using PCR but not plasmid DNA. Library design strategies and protocols that facilitate rapid identification are also presented.

A new DRE-binding protein gene MsDREB1 cDNA that encoded an EREBP/AP2 type transcription factor was isolated by RT-PCR from alfalfa (Medicago sativa L.) seedlings.The MsDREB1 had an open reading frame of 651 bp,which encoded 216 amino acid residues.The putative protein was deduced to have a predicted molecular mass of 24.9 kDa and a p1 of 6.11.The Protein Blast data revealed that this protein could be classified as a typical member of the EREBP/AP2 family of DNA-binding proteins.The comparison of the MsDREB1 cDNA with the MsDREB1 gene in genomic DNA showed that the size and nucleotide sequence of the cDNA were the same as that of the genomic DNA.This indicates that the genomic MsDREB1 gene has no introns. Southern blot analysis indicates that it is a double-copy gene.

The human LKB1 tumor suppressor has been implicated as an important regulator of many cellular processes and signaling pathways, indicating that it could be a good candidate for anticancer drugs. The failure of its obtain high-level expression has been a major obstacle to study its protein structure and function in vitro. Here, we describe the high-level expression of human LKB1 in Escherichia coli and show its kinase activity and anticancer effects on a tumor cell line. The gene encoding LKB1 was optimized by replacing rare codons with codons frequently used in E. coli and synthesized with overlapping primers. The recombinant His-LKB1 was expressed in hosts BL21(DE3) (BL) and Rosetta-gami(DE3)pLysS (RG). His-LKB1 from BL was present mainly as inclusion body. The soluble His-LKB1 from RG accounted for 34.1% of total proteins and the yield of purified His-LKB1 was approximately 92 microg/ml. Purified His-LKB1 protein from both hosts was functionally active, as shown by reversible autophosphorylation and kinase activity in the absence of any other associated kinase. The growth inhibitory ratio of the purified BL-derived and RG-derived His-LKB1 on hepatic carcinoma SMMC-7721 cells was 24.97% and 45.68%, respectively, and both could produce significant cell-cycle arrest.

Ovine cumulus-oocyte complex was matured for 24 hours and fertilised for 17 hours in the vitro culture.Blastocysts rate was compared between ultracentrifugation and the control groups.By applying the method of ultracentrifugation could move the opaque lipid droplets to the edge of the zygotes so that their pronuclears were clear visualization.The breast specific expression of ALR and the non-specific expression of EGFP gene vector DNA were injected into the pronuclear of the fertilized eggs.Blastocysts rate of control were higher than ultracentrifugation group,but no significant difference(P>0.05).After microinjection for 2 days the transgenic zygotes expressed green fluorescence.The five positive zygotes were tested by PCR.GFP can be used as a genetic marker to screen transgenic embryos.The establishment of transgenic embryo screening methods using GFP as genetic marker will greatly increase efficiency of gene transfection and the production of transgenic sheep.