Cloning and expression of NK4 gene in Raji cells

AIM: To clone NK4 gene and to construct recombinant eukaryotic expression vector for observing its expression in transfected Raji cells. METHODS: Total RNA was extracted from human hepatic tissue. NK4 gene cDNA was amplified by RT-PCR, and then cloned into vector pVITRO2-mcs to construct the recombinant eukaryotic expression vector pVITRO2-mcs-NK4. Raji cells were transfected by recombinant vector pVITRO2-mcs-NK4 and screened by homomycin B. The stable strain of NK4 gene expression was screened by real-time fluorescent quantitative PCR, ELISA, immunocytohistochemistry and semisolid culture. RESULTS: The specific DNA fragment was detected by RT-PCR in Raji cells transfected with NK4 gene. The transfected Raji cells expressed NK4 mRNA and protein stably, which inhibited Raji cell proliferation, metastasis and invasion. CONCLUSION: NK4 gene is cloned and recombined to construct recombinant eukaryotic expression vector pVITRO2-mcs-NK4 successfully. NK4 gene in Raji cells expresses stably.     

Molecular cloning of ING4 gene and ING4-induced apoptosis in HeLa cells

AIM: To isolate a gene encoding mouse ING4, construct pcDNA3.0-ING4 recombinant eukaryotic expression plasmid and investigate its effects on HeLa cells in vitro. METHODS: The mouse ING4cDNA was amplified by RT-PCR from mouse liver. The eukaryotic expression vector pcDNA3.0-ING4 was constructed by DNA recombination technique. The recombinant plasmid pcDNA3.0-ING4 was identified by PCR, restriction enzyme digestion and DNA sequence analysis, then was transfected into HeLa cells by lipofectamine. The expression was determined by RT-PCR. Apoptosis was detected by fluorescence microscope with Hoechst33258 staining and laser scanning confocal microscope. Cell cycle distribution was measured with flow cytometry. RESULTS: RT-PCR product was about 750 bp specific fragment. Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed that results were about 750 bp, DNA sequencing revealed that ING4 cloning were successful. With Hoechst fluorescence staining, we found that the percentage of apoptotic rate in HeLa cells transfected with pcDNA3.0- ING4 (21.25%) was higher than that in HeLa cells transfected with pcDNA3.0 (8.91%,P<0.01). Apoptosis was also detected by laser scanning confocal microscope. Cell cycle analysis reavealed the cell number in S phase of HeLa cells transfected with pcDNA3.0- ING4 increased. CONCLUSION: The gene encoding mouse ING4 and construction of pcDNA3.0- ING4 eukaryotic expression vector were successfully obtained, ING4 could enhance apoptosis in HeLa cells.     

Construction of an eukaryotic expression vector encoding human granzyme B and it's expression in Hep2 cells

AIM: To construct pVAX1-GrB. METHODS: Lymphocytes from human laryngeal carcinoma tissue were separated from tumor tissue. The fragment of granzyme B (GrB) was amplified by RT-PCR and was recombined to the downstream of T7 promoter in the vector pVAX1. The construction was transfected into Hep2 cells with lipofectamine 2000. The expression of protein was identified by indirect immunofluorescent antibody assay. RESULTS: It has been proved that the sequence of the RT-PCR product was totally consistent with the data of GenBank by DNA sequencing analysis. The GrB cDNA fragment was cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB was maintained. The target protein was detected in the transfected Hep2 cells. CONCLUSION: The pVAX1-GrB plasmid was successfully constructed and expressed.     

Construction of eukaryotic expression vector and expression of outer membrane lipoprotein LipL41 gene of Leptospiria lai

AIM:To construct a eukaryotic expression vector expressing outer membrane lipoprotein LipL41 of Leptospira lai and express it in mammalian cell. METHODS:LipL41 gene was amplified by PCR from genome of Leptospira lai 017 strain, and was subcloned into vector pGEX-4T-1. After sequencing, LipL41 gene digested by restriction endonuclease and cloned into vector pcDNA3. After confirming the correctness of the eukaryotic recombinant vector by restrication enzyme digestion, it was transfected into COS7 cells by liposome. Its expression was analyzed by RT-PCR. RESULTS:A fragment of 1 011 bp was amplified, and sequence analysis showed it had a 98% homology with Leptospira kirschneri. The analysis of restriction enzyme indicated that the eukaryotic recombinant vector was correctly constructed. A specific amplified fragment was showed in the cells transfected with recombinant plasmid by RT-PCR, but the cell transfected with blank plasmid did not show this band. CONCLUSIONS:The LipL41 gene of Leptospira lai was successfully inserted into eukaryotic expression plasmid and the recombinant plasmid expressed the LipL41 mRNA.     

番茄S-腺苷蛋氨酸脱羧酶基因SlSAMDC1的克隆与序列分析

 以蔓陀罗S-腺苷蛋氨酸脱羧酶全长cDNA序列为信息探针, 筛选NCBI番茄EST数据库, 依据同源EST信息,经人工拼接、RT-PCR及RACE技术验证,获得了1个新的SAMDC基因家族成员,命名为SlSAMDC1(GenBank登录号：EF550528),并利用染色体步移技术克隆了879 bp的上游调控区域。SlSAMDC1 cDNA序列全长1 847 bp, 5′-UTR和3′-UTR分别长523 bp、241 bp;存在3个ORF(微型uORF、小型uORF和主ORF),主ORF编码360个氨基酸的SAMDC酶原;SlSAMDC1基因组序列全长3 648 bp,有3个内含子,均位于5′-UTR,所有内含子的剪切位点均符合真核生物“GT-AG”规则。SlSAMDC1基因与其它植物来源SAMDC基因同源性较高,与人、大肠杆菌以及酵母的同源性较低。表达谱分析发现,SlSAMDC1基因在番茄根、茎、叶、花蕾、果实等器官中均表达,果实中的表达量相对较高。生物信息学分析表明,SlSMDC1基因的上游调控序列存在多个顺式作用元件,如W-box、TATA-box、CAAT-box等。     

青花菜快速碱化因子RAL F 的克隆与序列分析

 以一个与甘蓝显性核不育相关的差异表达片段序列为信息探针, 在NCBI与TAIR网站数据库中进行同源EST序列搜索, 经人工拼接、RT - PCR克隆与序列分析验证, 获得了青花菜快速碱化因子RALF(Rapid Alkalinization Factors) 基因的cDNA全长序列, 命名为BoRALFL1 (GenBank序列登录号DQ059310)。该cDNA全长240 bp, 编码79个氨基酸, 与电子克隆获得的序列完全相同。序列分析表明, 编码蛋白存在前导信号肽与多个磷酸化位点, 与同源基因RALFL8核酸序列在88 bp上有82%的一致性, 推导的氨基酸序列在74个氨基酸上存在56%的一致性, 不同植物间氨基酸序列N - 端差异大, C - 端具有较高的保守性。     

大白菜温度敏感雄性不育育性转化相关XET基因的电子克隆

以从大白菜温敏雄性育性转化相关基因差异表达的分析中获得的EST-58为标签,采用电子克隆的方法对其进行延伸,并对电子克隆的结果进行RT-PCR验证,结果表明:电子克隆到1个由1197bp核苷酸组成的序列,该序列具有完整的开放阅读框架(ORF),编码蛋白为292个氨基酸。经RT-PCR扩增,连续样品间EST-58表达的带型变化趋势与cDNA-AFLP的差异显示情况一致,且RT-PCR获得的序列与电子克隆的序列一致性达98%。用该序列在GenBank数据库中进行blastX同源性分析,确定该序列为大白菜的XET基因(GenBank登陆号为EU579461)。     

桃成花基因PpLFY的克隆与表达及多克隆抗体制备

 以‘八月脆’桃成花诱导期腋芽为试材,克隆得到一个FLORICAULA/LEAFY同源基因,命名为PpLEAFY（PpLFY）,GenBank登录号为EF175869,该基因cDNA全长1 314 bp,ORF为1 248 bp,编码一个含416个氨基酸残基的蛋白,与其他物种中的LEAFY蛋白的同源性较高,为（74.22%~84.69%）。时期及组织特异性表达分析表明,PpLFY主要在叶片、花瓣及成花诱导期及花芽形态分化初期中表达。构建了pGEX-4T-1/PpLFY原核表达系统,获得了可溶性融合蛋白rPpLFY,分子量约为36 kD。利用该蛋白免疫新西兰大白兔,获得了抗rPpLFY的多克隆抗体,ELISA效价为1:10 000。     

柿花发育相关的MADS2box基因克隆与表达

以雌花型柿(Diospyros kaki) ‘阳丰’花为试材, 克隆得到一个与其花发育相关的MADS2box基因, 命名为DkMADS1, GenBank登录号为DQ412058。该基因cDNA全长1 193 bp, ORF为747 bp, 编码一个含有249个氨基酸的蛋白。DkMADS1 具保守的MADS区及半保守的K区, 与其它植物中的MADS2box蛋白有很高同源性。RT-PCR表达分析表明, 该基因在‘阳丰’花的萼片、花瓣、子房和‘禅寺丸’的雄蕊中均有表达。     

苹果铁结合蛋白基因的克隆及表达特性

 根据已报道的植物铁结合蛋白基因( ferritin) 的保守区域设计引物, 用RT2PCR 的方法从‘嘎拉’苹果叶片中获得铁结合蛋白基因的全长序列。该基因cDNA共有1 043个碱基, 其中编码区834bp, 编码277个氨基酸残基的蛋白质, 终止密码子后有209 bp左右的3’端尾随序列区。Southern杂交结果显示ferritin基因在‘嘎拉’苹果基因组中至少有7个拷贝存在。0.5 mmol·L ^{- 1}的柠檬酸铁处理‘嘎拉’苹果试管苗不同时间后的定量RT-PCR分析表明, ‘嘎拉’苹果的铁结合蛋白基因随着诱导时间的延长表达量增加, 说明该基因可能在转录水平上受铁诱导表达。     

荔枝APETALA1（AP1）同源基因cDNA全长克隆及其表达研究

 应用RT-PCR方法克隆得到荔枝AP1同源基因cDNA全长,命名为LcAP1（基因登录号：JN214349）。LcAP1基因开放阅读框738 bp,编码245个氨基酸,推测蛋白质分子量为28.39 kD,等电点为9.69。序列分析显示,LcAP1基因编码的蛋白在1 ~ 61氨基酸含有1个MADS盒结构域,在89 ~ 179氨基酸有1个K盒结构域。蛋白质二级结构预测表明,LcAP1基因编码的蛋白有3个α螺旋,2个β折叠区,8个β转角。同源分析表明,LcAP1基因在不同植物中的一致性为72% ~ 82%。半定量RT-PCR分析表明,在‘三月红’荔枝花芽分化期,LcAP1基因在成熟叶、幼叶、老茎、嫩茎、花芽和花梗中均表达,在花芽中表达最多。     

柿果ACC合成酶基因的克隆与植物表达载体的构建

以柿果组织中分离的总RNA为模板,经RT-PCR扩增到约1.1 kb的富有柿果ACC合成酶(Fuyu-ACS)的 基因片段,将此片段克隆到pGEMR-T easy vector上,经测序分析与Genbank中平核无柿果的DK-ACS1基因核苷酸 序列的同源性为99％。设计了2对带限制性内切酶位点的特异性引物,以测序质粒为模板,PCR扩增到2个ACS- Fuyu片段。2个片段经双酶切消化后,分别以正反2个方向插入到植物表达载体pBI121的35 S启动子和NOS终止 子之间,构建成Fuyu-ACS基因的植物表达载体。     

中矮1号梨砧木(S_2)PGIP基因的克隆及序列分析

利用RT-PCR技术,根据GenBank中梨属PGIP基因序列设计1对特异引物,以梨矮化砧木S2叶片总RNA为模板,克隆到1条约1100bp的cDNA片段,将其与pMD18-Tvector连接后转化Escherichia coli JM109,对筛选到的阳性克隆进行序列测定并使用生物信息学方法对所得结果进行综合分析。结果表明,克隆片段为梨PGIP基因,该cDNA编码330个氨基酸,预测分子量为36388ku,第1～24个氨基酸残基是信号肽。与梨属其它种中已获得的PGIP核苷酸序列开放阅读框(Open reading frame,ORF)的同源性为97.5%～99.6%,氨基酸序列的同源性为97.6%～99.1%。     

百合肌动蛋白基因lilyActin 的克隆与表达分析

 为了在百合功能基因表达研究中选择一个理想内参基因,依据岷江百合cDNA 文库所获得的百合肌动蛋白（Actin）基因的EST 序列,采用RACE 技术进行该基因cDNA 全长克隆,并利用实时荧光定量PCR 分析其在不同组织中的表达模式,获得百合肌动蛋白基因cDNA 全长序列（GenBank 登录号：JX826390）,命名为lilyActin。该基因cDNA 全长1 367 bp,其中,5′非编码区91 bp,3′非编码区233 bp,开放读码框1 134 bp,编码377 个氨基酸。序列比对发现,该基因与其它15 种植物肌动蛋白核苷酸序列的相似性均在80%以上,氨基酸序列的相似性达98%。进化分析显示,百合肌动蛋白与郁金香肌动蛋白的亲缘关系最近。实时荧光定量PCR 结果显示,该基因在百合的花蕾、叶片和鳞片组织中恒定表达,表明相对于其他物种的内参基因,lilyActin 更适宜作为百合属植物的内参基因。     

Effect of estrogen receptor 2 on the cell proliferation in prostate cancer cell line PC-3M

AIM: To construct the recombination plasmid pcDNA3.1-hERβ with the human estrogen receptor 2 (ESR2) full length cDNA and transfect it into hormone-independent prostate cancer PC-3M cell line, and to study the effects of ESR2 on proliferation in transfected cells. METHODS: The complete cDNA of ESR2 was obtained from human ovary tissue by RT-PCR technique and cloned into eukaryotic expression vector pcDNA3.1 by using gene recombination technique to construct the pcDNA3.1-hERβ recombination plasmid. The plasmid was detected by endonuclease digestion and DNA sequencing and was transfected into PC-3M cells. MTT and FCAS assay were used to test the effects of ESR2 on the ability of proliferation in PC-3M cells. RT-PCR and Western blotting were used to detect the expressions of cyclinD1 and P21Cip1. RESULTS: The results of sequencing and endonuclease digestion demonstrated that the construction of pcDNA3.1-hERβ recombination plasmid was successful. The sequence analysis suggested that the ESR2 sequence detected by PCR was identical to that published in GenBank, and the product of endonuclease was as long as the complete human ESR2 gene. 48 h after transfected the pcDNA3.1-hERβ into PC-3M cells, the expression of ESR2 mRNA and protein levels increased significantly detected by RT-PCR and Western blotting. Compared to the cells transfected with vector as control, the PC-3M cells transfected with pcDNA3.1-hERβ showed that cell population decreased and proliferation activity degraded. FCAS showed that the cells in G0/G1 stage increased and in S stage or G2/M stage decreased. RT-PCR and Western blotting showed that the expression of cyclinD1 gene reduced and expression of P21Cip1 increased. CONCLUSION: The recombination of plasmid pcDNA3.1-hERβ is constructed and transfected into the PC-3M cells successfully. The activity of cell proliferation is inhibited after pcDNA3 transfection.1-hERβ. It is possible that ESR2 inhibits cell proliferation by the expression of proliferation related genes cyclinD1 and P21Cip1.     

龙眼胚胎F3H基因的cDNA克隆及序列分析

采用IEF-SDS-PAGE双向电泳技术,对‘立冬本'龙眼合子胚发育不同时期的蛋白质进行分离,通过MALDI-TOF-TOF鉴定到其中一个上调差异表达的蛋白为黄烷酮-3-羟化酶 (flavanone 3-hydroxylase,F3H)。以‘立冬本’龙眼胚胎cDNA为模板,采用RT-PCR和RACE技术,获得F3H基因cDNA 1 404 bp全长序列。序列分析表明,F3H基因共编码365个氨基酸,其cDNA序列与柑橘、苹果、棉花等F3H基因的同源性均高达80%以上,该基因在GenBank中登录号为EF468104。     

杨梅酸性转化酶基因cDNA分离及表达分析

杨梅果实富含蔗糖,酸性转化酶是蔗糖代谢关键酶,根据植物酸性转化酶基因保守区序列设计引物,提取杨梅叶片RNA,逆转录获得cDNA,以此为模板通过PCR技术扩增到长度为516bp的基因片段,克隆入pMD18-T载体中,命名为MrIVR1(GenBank:DQ339699)。测序及同源性检索表明,该基因推导氨基酸序列与君子兰、葡萄、草莓、胡萝卜等酸性转化酶基因氨基酸序列同源性为60%～69%。运用ClustalX软件对植物转化酶基因进行了系统树分析,结果显示,MrIVR1编码的蛋白质属于细胞壁酸性转化酶。半定量RT-PCR表达分析显示,MrIVR1基因在杨梅果实发育早期表达量最高,随着果实的发育表达量下降,在成熟果实中表达水平较低。     

茄子GA响应因子SmGAI的克隆与分析

采用RT-PCR和RACE技术从茄子地方品种杭州红茄中扩增克隆了GA响应因子相关基因片段,命名为SmGAI,其cDNA序列为1 176 bp,含有972 bp的阅读框,编码323个氨基酸;基因编码的氨基酸序列与番茄单性结实调控基因SlDELLA同源性为76.8 %,高度相似。     

Cloning and sequencing of encoding region of heme oxygenase-1 gene from Tibetan antelope

AIM: To clone and analyze the encoding region of heme oxygenase-1(HO-1) gene from Tibetan antelope(Pantholops hodgsonii). METHODS:The total RNA was isolated from the liver of Tibetan antelope and the HO-1 gene was amplified by RT-PCR. The PCR product was cloned into pGEM-T vector and sequenced. The nucleotide sequences were compared with the data from GenBank by BLAST method. RESULTS:The encoding region of HO-1 gene from Tibetan antelope was obtained and deposited in GenBank as accession number JQ809687. The encoding sequence was 897 bp in full length, which encoded 298 amino acids. Sequence similarity analysis showed that the HO-1 gene cloned from Tibetan antelope shared 98% and 96% in the nucleotides, and shared 92% and 97% in the amino acids with those of Capra hircusand Bos taurus, respectively. The sequence similarity of nucleotides and amino acids also shared 86% and 87% with other vertebrates, which were highly conservative. The molecular phylogenetic tree based on the amino sequence of HO-1 showed that Tibetan antelope and Capra hircus was classified to one cluster, which was basically concerted to the evolutionary relationship of traditional species. CONCLUSION:The encoding region of Tibetan antelope HO-1 gene is successfully cloned, which provides a foundation for exploring the molecular and biological mechanisms of high altitude adaptation and cell protection in Qinghai-Tibet Plateau species under hypoxic condition.