全长cDNA克隆的三种方法的比较研究

为了得到一段EST所在基因的全长,采用了快速扩增cDNA末端（RACE）、cDNA文库构建、λphage测序引物与特异性引物结合,非放射性探针进行噬菌体原位杂交等方法分别对该基因进行了克隆,结果得到了一致的全长cDNA,三种不同实验方法的应用,为更方便、有选择性地克隆全长cDNA基因奠定了基础。     

SMART技术构建栀子cDNA文库

目的:构建栀子叶片cDNA文库。方法:提取栀子叶片总RNA。利用SMART技术合成双链cDNA。双链cDNA经限制酶Sfil酶切后与pDNR-LIB质粒连接。利用电刺激转化法将重组质粒导入E.coli DH5α而获得文库。利用PCR法检测文库的重组率。结果:原始文库滴度为2.63×105cfu/ml。随机检测文库中的15个克隆,表明重组率约为86.7%。选择14个插入片段的长度在400bp以上的克隆进行测序和生物信息学分析,结果预测的全长基因占所检测序列的64.3%。结论:成功构建了栀子叶片的cDNA文库,为栀子基因的结构和功能的研究提供了基础。     

大乳头水螅RACE cDNA文库的构建

目的:为筛选和克隆大乳头水螅发育调控相关基因的全长cDNA,构建大乳头水螅RACE cDNA文库.方法:提取大乳头水螅总RNA后从其中分离mRNA,运用SMART技术构建RACE cDNA文库.为鉴定所构建文库的质量,根据GenBank中大乳头水螅actin基因cDNA序列设计5'RACE和3'RACE的引物及用于扩增actin基因编码区全长序列的引物.结果:琼脂糖凝胶电泳结果表明,RACE cDNA文库中全长cDNA的长度集中在500-2 000bp之间.5'RACE、3'RACE PCR及扩增actin基因编码区全长序列时均以本文构建的大乳头水螅RACE cDNA文库为模板,这3个PCR反应均能扩增出产物,产物大小与目标片段预计大小相似.PCR产物分别经T/A克隆及测序后证明为大乳头水螅actin基因cDNA的相应序列.结论:RACE cDNA文库的成功构建为通过RACE方法获得大乳头水螅功能基因cDNA全长序列奠定了基础.     

水杨酸诱导湖北海棠全长cDNA文库的构建及应用

以'湖北海棠'为材料,经水杨酸处理后,用改良CTAB法提取总RNA,纯化后构建全长cDNA文库,并进行PGIP基因的克隆.结果表明:(1)提取的总RNA无降解,无污染;mRNA弥散带主要集中在500～2 000 bp左右,没有rRNA 残留.(2)ds cDNA弥散带主要分布于300～2 000 bp之间,PCR验证后片段大小分布于200～2 000 bp之间,说明合成ds cDNA质量较好,成功地构建了全长cDNA文库.(3)通过PCR从该cDNA文库中克隆了PGIP基因,命名为MhPGIP,GenBank登录号为FJ449708;其核苷酸序列及推导氨基酸序列与苹果的一致性分别为98%和97%,该序列含有两个串联的亮氨酸重复序列.综上所述,构建的全长cDNA文库质量很好,该文库的建成可以用于今后抗病新基因的挖掘、克隆和利用,为苹果抗病机理的研究奠定基础.     

灰绿藜RNA提取方法及在cDNA文库构建中的应用

目的:为分离灰绿藜耐盐相关新基因,构建叶片cDNA文库,探索从盐生植物中提取高质量RNA的方法.方法:以灰绿藜叶片为材料,应用RNA Plant Reagent法(Tiangen)、Plant RNA purification Reagent法(Invitrogen)、UNIQ柱式总RNA试剂盒提取法(Sangon)、SDS-LiCl法、TRIZOL试剂法(Invitrogen)等进行总RNA的提取,并构建其全长cDNA文库和抑制消减文库.结果:RNAPlant-Reagent法、Plant RNA purification Reagent法和UNIQ柱式总RNA试剂盒提取法获得的RNA完整性均不好;SDS-LiCl法提取的RNA完整性好(28S条带亮度是18S的2倍、条带锐利清晰)、纯度高(A280/A260为1.89±0.03,A260/A230为2.23±0.02),适合用于直接反转录及构建全长cDNA文库,并获得成功;TRIZOL试剂法简单快捷,产率高(2724±82μg/g),完整性较好,可经mRNA纯化后用于抑制消减文库的构建并获得成功.结论:建立了用于构建高质量cDNA文库的灰绿藜RNA的制备方法.     

悦目金蛛丝腺SMART RACE cDNA文库的构建与鉴定

运用SMART 技术构建了悦目金蛛丝腺SMART RACE cDNA文库.经检测,文库所含全长cDNA的长度主要集中在500 bp～2000 bp 之间;把双链cDNA通过T/A克隆、随机挑选阳性克隆并测序后,得到1条752 bp的全长cDNA序列.以该文库为模板,用依据这条全长cDNA序列设计的基因特异性引物与接头引物进行RACE, 3'RACE 和5'RACE的产物拼接后的全长序列与上述全长cDNA序列一致.结果表明,该文库适于用RACE方法从中分离在悦目金蛛丝腺中表达基因的全长cDNA.本文还对SMART 技术的特点和局限性进行了讨论.     

毛冠鹿大脑组织全长cDNA文库构建

运用SMART技术构建了毛冠鹿(Elaphodus cephalophus)大脑组织全长cDNA文库。提取大脑组织总RNA,Oligotex mRNA Kit纯化、获得poly(A) RNA,以CDSⅢ/3′PCR引物进行逆转录,LD-PCR扩增获得全长双链cDNA,经SfiⅠ酶切及柱层析分离后,500 bp以上的片段与载体λTripIEx2连接,体外包装得到cDNA文库。经鉴定原始文库滴度为5.1×105pfu/ml,扩增后文库滴度为1.5×109pfu/ml,重组率达到85%以上,插入片断平均长度约为1.0 kb,说明构建文库质量符合要求,可用于大脑特异表达基因的筛选。从该文库中克隆到了rig基因全长,包含5′和3′非编码区,从第43至477个核苷酸为一完整阅读框(ORF),此阅读框可编码一个145氨基酸的rig蛋白。     

烤烟品种南江3号均一化全长cDNA文库构建

目的:为了获得功能基因的信息,构建烤烟品种南江3号的均一化cDNA文库.方法:用RNeasy Plant Mini Kit提取烤烟南江3号叶片和花的RNA,用反转录酶逆转录合成第一链eDNA.以LD-PCR扩增获得的双链cDNA为模板,采用基于双链特异性核酸酶(Duplex-Specific Nuclease,DSN)的均一化cDNA文库技术,构建南江3号盛花期的均一化cDNA文库.结果:构建了南江3号盛花期的均一化cDNA文库,文库重组率大为97.47%,库容量约为1.26×10<'6>,插入片段平均长度大于1.2kb.从文库中随机48个克隆进行PCR检测,挑取20个克隆进行测序,序列通过BLAST比对结果显示文库可能包含大量基因和ESTs序列.结论:该文库为研究南江3号的基因功能和资源提供材料来源.     

中华大蟾蜍精巢组织全长cDNA文库的构建及泛素延伸蛋白基因Bg-ubi52的获得

运用SMART技术构建了中华大蟾蜍(Bufo bufo gargarizans)精巢全长cDNA文库。提取中华大蟾蜍精巢总RNA,用Clontech公司SMARTTM cDNA文库构建试剂盒反转录合成第一链cDNA,LD-PCR扩增获得全长cDNA双链;经SfiⅠ酶切、层析柱分离后,500bp以上的片段与λTriplEx2载体连接并包装,建成原始文库。经鉴定,原始文库滴度为2.21×106pfu/ml,重组率为91%;文库扩增后的滴度为2.94×109pfu/ml,重组率为93.7%。插入片段大小分布于0.4~2.0kb之间,平均长度约为1.0kb,说明已构建文库质量较高,为进一步筛选、克隆精巢特异表达基因奠定了基础。从该文库中克隆到了泛素延伸蛋白基因,全长561bp,包含完整的5′和3′非编码区,编码128个氨基酸,即泛素的76个氨基酸后融合了52个氨基酸的核糖体L40蛋白。     

鞘蕊苏贝壳杉烯酸氧化酶基因的克隆及表达载体的构建

为提高鞘蕊苏有效成分的含量,开展Coleus Ent-kaurenoic acid oxidase(KAO)基因的克隆以及表达载体构建方面的研究。提取鞘蕊苏叶片的总RNA,利用套式PCR技术克隆获得KAO基因全长,将该基因连接到克隆载体p MD-18T,经测序鉴定正确后,再将该基因连接到p ET-28a表达载体中。结果显示,成功克隆出的鞘蕊苏KAO基因大小为1 500 bp,对克隆基因进行测序及酶切鉴定,均得到正确大小的DNA片段,说明表达载体构建成功。本研究成功克隆得到鞘蕊苏贝壳杉烯酸氧化酶基因并构建其表达载体,为提高鞘蕊苏有效成分含量提供了基础。     

A method to construct cDNA library of the entomopathogenic fungus, <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis>, in the hemolymph of the infected locust

A method was developed to construct cDNA library of pathogenic fungus in the blood of the infected insect for cloning the fungal genes expressed in the host. This method is designed to take advantage of the obvious difference between the cell structures and components of the pathogen cells and that of the host cells. The host blood cells only have cell membrane, which can be disrupted by using SDS/proteinase K (PK). The fungal cells grown in the animal blood have cell wall, which can protect the fungal cell from the disruption of SDS/proteinase K (PK). By this method, the blood cells were disrupted by SDS/proteinase K (PK) and then the released animal RNA and DNA were digested completely with RNase and DNase. Therefore, the fungi grown in the blood were harvested without any contamination of host RNA and DNA. The pure fungi harvested from the infected blood can be used for mRNA extraction and cDNA library construction. The purity of the fungal mRNA was confirmed by PCR and RT-PCR with specific primer pairs for the host and specific primer pairs for the fungus, respectively, and the clones of cDNA library constructed by using the fungal mRNA was also analyzed. The results showed that there was no detectable contaminated insect DNA or RNA existing in the fungal mRNA. Randomly selected cDNA clones from cDNA library were sequenced and analyzed against GenBank using Blastx; no selected sequences had significant similarity with insects’ genes in comparison with the data of GenBank. The results further confirmed that the method to purify the pathogenic fungus from the host animal is reliable and the mRNA extracted from the fungus is eligible for cDNA library construction, and other molecular analysis including RT-PCR. This method may be applied to other pathogenic fungi and their host animals.     

人肺腺癌细胞系cDNA文库的建立

本文以Uni-ZAP XR为载体,成功地建立了人肺腺癌高、低转移细胞系的cDNA文库。文中同时也探讨了总RNA的提取和mRNA的分离,cDNA的第一条链及第二条链的合成及克隆到载体等在建立cDNA文库过程中,几个关键性的问题。该项工作的完成,为下一步在这两株细胞系cDNA之间进行消减式杂交,筛选转移相关基因奠定了坚实基础。     

家蝇cDNA文库的构建

自Ｂｏｍａｎ小组 (Ｓｔｅｉｎｅｒ ,1981)从惜古比天蚕(Ｈｙａｌｏｐｈｏｒａｃｅｃｒｏｐｉａ)中分离、纯化出第一种抗菌肽天蚕素以来 ,人们已在昆虫和其他无脊椎动物中发现了 170多种抗菌肽 (Ｌｏｗｅｎｂｅｒｇｅｒｅｔａｌ ,1999)。目前 ,人们不仅搞清楚了多数抗菌肽的氨基酸序列、结构和功能特点 ,而且还对一些抗菌肽的基因进行了克隆 (陈留存和王金星 ,1999)。我们以家蝇为材料 ,分离、纯化出了抗菌肽 ,在此基础上 ,构建了经过诱导的家蝇ｃＤＮＡ文库 ,以克隆其基因。1　材料和方法1 1　实验材料家蝇 (Ｍｕｓｃａｄｏｍｅｓｔｉｃａ)…     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress （Thellungiella halophila）, a close relative of the model plant Arabidopsis thaliana L., is an extremophile that is adapted to harsh saline environments. To mine salt-tolerance genes from this species, we constructed an entry cDNA library from the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombinationassisted cDNA library construction method that is completely free of manipulations involving restriction enzymes and DNA ligase. This cDNA library construction procedure is significantly simplified and the quality of the cDNA library is improved. This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for plant transformation and expression via recombination-assisted cloning. The library is plant transformation ready and is used to transform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

Construction of a Plant Transformation-ready Expression cDNA Library for Thellungiella halophila Using Recombination Cloning

Salt cress(Thellungiella halophila),a close relative of the model plant Arabidopsis thaliana L.,is an extremophile that isadapted to harsh saline environments.To mine salt-tolerance genes from this species,we constructed an entry cDNA libraryfrom the salt cress plant treated with salt-stress by using a modified cDNA synthesis and an improved recombination-assisted cDNA library construction method that is completely free of manipulations involving restriction enzymes andDNA ligase.This cDNA library construction procedure is significantly simplified and the quality of the cDNA library isimproved.This entry cDNA library was subsequently shuttled into the destination binary vector pCB406 designed for planttransformation and expression via recombination-assisted cloning.The library is plant transformation ready and is used totransform Arabidopsis on a large scale in order to create a large collection of transgenic lines for functional gene mining.     

莱芜猪肝脏组织全长cDNA文库的构建和鉴定

目的:构建莱芜猪肝脏组织全长cDNA文库,以便研究与莱芜猪优良性状相关的基因。方法:采用改良的异硫氰酸酸胍一步法制备总RNA;利用SMART技术,以PrimeScript反转录酶逆转录合成第一链cDNA,通过LD-PCR扩增获得cDNA双链;经蛋白酶K消化和CHROMA SPIN-400柱分级分离后,收集500 bp以上的cDNA片段,并与pMD18-T载体连接,转化大肠杆菌DH5α感受态细胞,建成原始文库;随机挑取单菌落,用HindⅢ和EcoRⅠ进行双酶切鉴定重组子插入片段大小。结果:经鉴定,原始文库的滴度为2.8×105 cfu/mL,重组率约为98%,插入片段大小为0.5～2 kb,平均插入片段长度大于1 kb。结论:建立的cDNA文库质量良好,可以用于目的基因的筛选。     

三丁基锡暴露条件下杂色鲍肝胰腺均一化cDNA文库的构建

用RDP试剂提取三丁基锡暴露诱导的杂色鲍肝胰腺总RNA,经Oligotex纯化得到 mRNA;应用SMART技术合成双链cDNA,双链特异核酸酶(DSN)进行双链cDNA的均一化,构建了杂色鲍三丁基锡暴露诱导下的均一化cDNA文库.原始文库的库容为4.3× 106 CFU/ml,重组率为 97.9%.从文库中随机挑选了3 288个克隆进行测序,得到3 048个高质量EST序列,其中有 370条 Contigs,2 103条 Singlets,Unigenes共 2 473条,冗余率为18.86%.以上结果说明该文库质量较好,为进一步筛选相关功能基因打下基础;较低的冗余率说明该文库值得继续使用大规模ESTs测序的方法寻找相关功能基因,并为进一步使用基因芯片技术研究相关功能基因的表达谱提供便利.     

人尿道(阴茎)鳞癌上皮细胞(HUS-98)cDNA文库的构建及鉴定

为了进一步分离人尿道(阴茎)鳞癌组织特异性表达基因和鳞癌特异性相关基因,采用SMART技术,构建了人尿道 (阴茎)鳞癌上皮细胞cDNA文库,从人尿道(阴茎)鳞癌上皮细胞中分离总RNA并纯化mRNA,利用经修饰的oligo(dT)引物 合成cDNA第一链,利用SMART核苷酸作为cDNA第一链在mRNA5′端延伸出去的模板,采用LD-PCR合成双链cDNA,双链 cDNA经酶切和过柱分级分离后,克隆入λTriplEx2载体后经体外包装而成cDNA文库。结果表明原始人尿道(阴茎)鳞癌上 皮cDNA文库获得1.57×107个重组子,重组率达到98%。文库扩增后,滴度达到4.0×109pfu/ml,插入cDNA平均长度为2.5kb。 构建的人尿道(阴茎)鳞癌上皮cDNA文库具有良好的质量,该cDNA文库为进一步筛选鳞癌抑癌基因及鳞癌特异性表达基因 奠定了基础。     

正常人体淋巴细胞cDNA文库的构建

应用GIBCOBRL建库试剂盒建立了正常人体淋巴细胞cDNA文库。取新鲜的正常人外周血,分离出淋巴细胞,进行体外培养,提取总RNA,纯化mRNA,并将其反转录成cDNA,与SalI和NotI接头连接后插入λZipLox载体,体外包装后转染到Y1090宿主菌中,进行滴度测试及文库扩增。构建的正常人淋巴细胞cDNA文库含2-6×106重组子,克隆效率为5×1012重组子/g cDNA,插入片段长度约为1~5kb。扩增后的文库浓度为3×107重组子/μl,将文库稀释到10-6时所产生的噬菌斑密度最为适宜。试验结果表明,该库符合标准,所构建的正常人淋巴细胞cDNA文库为进一步筛选目的基因、制作基因芯片等提供了有效的工具。 Abstract:A lymphocyte cDNA library of normal human was constructed in order to obtain specific gene and prepare lymphocyte gene chips to detect the relative genes between psychiatric diseases and immunity.The lymphocyte was abstracted from fresh normal human blood and cultured in vitro.Total RNA of lymphocyte was extracted from the cultured cells and then mRNA was extracted further.Moreover,single-strand cDNA and double-strand cDNA were synthesized in turn.The double-strand cDNAs were ligated to SalI and NotI adaptor,which were later ligated to arms of λZipLox.Ligated-cDNAs were packed in vitro,and then infected E.coli Y1090.Titering the phage and amplifying the library.The lymphocyte cDNA library consisted of 2-6×106 recombinants with the length of 1~5kb and the cloning efficiency was 5×1012 recombinants/g cDNA.The amplified library was 3×107recombinants/μl in concentration and the number of bacteriophage plagues was the most suitable in density after it was diluted to 10-6 in concentration.The constructed cDNA library of normal human lymphocyte would be helpful to further detecting target genes and preparing gene chips etc.