莱芜猪心脏组织cDNA文库的构建及部分表达序列标签的分析
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摘要
莱芜猪是我国地方名猪,具有五千多年的悠久历史,具有繁殖性能高、抗逆性强、肉质优良、耐粗食、杂交优势明显等特点。目前,以莱芜猪为原料,开发冷鲜肉、烤肉、香肠三大系列20多个类型的肉制品,已在省内外形成良好的品牌效应,产品销往全国各地,备受消费者青睐。可以预计莱芜猪的市场空间大,发展前景广阔。因而,合理地开发利用和有效的保护这一珍稀的遗传资源具有重要的理论和应用研究价值。
本研究以莱芜猪为供试材料,用改进的异硫氰酸胍法提取心脏组织总RNA,通过SMART技术反转录成单链cDNA,以长距离PCR法合成双链cDNA:PCR产物经蛋白酶K消化、纯化后,用Sfi I酶切；将酶切产物分级分离,回收大于0.5 kb的cDNA片段,并与λTriplEx2载体连接；连接产物经体外包装,形成莱芜猪心脏组织原始文库,其文库滴度为2.0×105pfu/mL,重组率为94.4%。原始文库扩增后获得扩增文库,检测滴度为3.0×108pfu/mL。随机挑取重组克隆感染大肠杆菌BM25.8,使λ噬菌体cDNA文库质粒化,形成pTriplEx2。再从转化的平板上随机挑取克隆菌落,依载体两端序列设计引物,进行PCR扩增,结果显示大部分克隆的插入片段长度在0.75 kb以上。
从扩增文库中随机挑取重组克隆进行5′和3′测序,获得8条有效的EST,长度在244～711 bp之间,分析8个克隆的ESTs序列所包含的ORF,并进行BLASTp比对,推断其相关的功能。结果表明,LW13、LW15、LW16、LW17克隆的最长ORF均不完整,数据库中无与其匹配的蛋白质序列,推测可能为新基因的一部分；LW19克隆的最长ORF为609 bp,编码202个氨基酸,分子量为22.4 kD,该蛋白质序列含有硫氧还蛋白保守结构域,推测与维持体内稳定的氧化还原状态有关；LW20克隆的最长ORF为198 bp,编码65个氨基酸；LW22克隆的最长ORF为105 bp,仅编码34个氨基酸；LW24克隆的最长ORF不完整。在数据库中,均未发现与LW20、LW22和LW24三个克隆相匹配的蛋白质序列,推测可能为新基因的一部分。
经BLASTx和BLASTn两个方面的综合比对分析,说明LW19和LW22两个克隆的EST编码的氨基酸序列可能在细胞分化、细胞周期调控、细胞衰老和死亡以及机体免疫等基础研究中具有一定的理论意义,对研究莱芜猪某些优良性状的表现机理也起到非常重要的作用,为后续工作的开展也做了重要的铺垫。
通过SMART技术构建莱芜猪心脏组织cDNA文库,不仅保存了稀有的地方种猪遗传资源,而且为莱芜猪有明确功能的特异基因的克隆、表达以及新基因的筛选与功能研究提供依据。
Laiwu pig is one of our local pig varieties for five thousand years. It has the characters of high reproductive capacity, disease-resistance, good meat quality, resistance to coarse grain, heterosisr. A serial of the porcine products have been developed including popular cold fresh meat, roast meat, sausage.Laiwu pig has formed a good brand effect in our country. It is hopeful of its having a big market and a good future. Therefore, it is a very important value in theory and practice to develop and protect this porcine rare genetic resource.
Total RNA was extracted form the heart tissue of Laiwu pig using improved isothiocyanate method. The total RNA was reverse-transcribed into single-strand cDNA by SMART (the Switch Mechanism At the 5'end of RNA Templates) technique. Long distance PCR (LD-PCR) was then carried out to synthesize the double-strand cDNA that were digested by Sfi I, and the digested fragments were fractionated by CHROMA SPIN-400. Then cDNA fragments longer than 0.5 kb were collected and ligated toλTriplEx2 vector. Ligation products were packed in vitro intoλphage particulates in order to gain unamplified cDNA library. The titer of primary cDNA library was 2.0×105 pfu/ml checked strictly by conventional titer determination and the rate of recombinant was about 94.4%. The titer of the amplified library was 3.0×108 pfu/ml. The recombinantλTriplEx2 clones picked from the amplified library infected E.coli BM25.8 to convert into pTriplEx2 plasmids. Plaques picked randomly from transformed pTriplEx2 plasmids were tested using PCR with universal primers derived from the sequence flanking the vector. The length of the most inserted fragments was above 0.75 kb.
The recombinant pTriplEx2 plasmids carried the inserts more than 500 bp were picked from the transformed plates with E.coli BM25.8 and sequenced from 5' end and 3'end. Eight pieces of effective ESTs were obtained totally between 244 and 711 bp in length. The putative open reading frames (ORFs) of these 8 ESTs were analyzed through Open Reading Frame Finder in NCBI and by using the BLASTp program, and the functions of amino acid sequences translated from these ORF of the eight ESTs were predicted. The results indicated that the longest ORF of LW13、LW15、LW16 and LW17 clones were not complete, and their amino acid sequences translated from their ESTs had unmatched with the protein sequences in database, they might be one part of some new genes;the longest ORF of LW19 clone was was 609 bp in length and encoded a 202 amino acid residues,22.4 kD in molecular weight. The amino acid sequences had a matching protein sequence in database, its containing thioredoxin-like superfamily conserved domain which was related with oxidation-reduction levels in vivo. The longest ORF of LW20 clone was 198 bp in length and encoded 65 amino acid residues. The longest ORF of LW22 clone was 105 bp in length and encoded only 34 amino acid residues. The longest ORF of LW24 clone was not complete. Three clones of LW20, LW22 and LW24 had unmatched protein sequences in database, they might be representative of one part of a unknown gene.
By combined alignment of BLASTx and BLASTn programs in NCBI, it was shown that the amino acid sequences encoded by two ESTs of the clones LW19 and LW22 functioned in foundmental researches of cell differentiation, cell cycle control, cell aging and death, and body immunity, and might play a very important role in the expression mechanism of some favourite characters in Laiwu pig. These also laid some foundations for further research.
The cDNA library from the heart tissue of Laiwu pig constructed by SMART technique might be helpful to preservation of local rare porcine resources, but also provided a valuable clues for cloning and expression of functional genes and for screening of unknown genes.
本研究以莱芜猪为供试材料,用改进的异硫氰酸胍法提取心脏组织总RNA,通过SMART技术反转录成单链cDNA,以长距离PCR法合成双链cDNA:PCR产物经蛋白酶K消化、纯化后,用Sfi I酶切；将酶切产物分级分离,回收大于0.5 kb的cDNA片段,并与λTriplEx2载体连接；连接产物经体外包装,形成莱芜猪心脏组织原始文库,其文库滴度为2.0×105pfu/mL,重组率为94.4%。原始文库扩增后获得扩增文库,检测滴度为3.0×108pfu/mL。随机挑取重组克隆感染大肠杆菌BM25.8,使λ噬菌体cDNA文库质粒化,形成pTriplEx2。再从转化的平板上随机挑取克隆菌落,依载体两端序列设计引物,进行PCR扩增,结果显示大部分克隆的插入片段长度在0.75 kb以上。
从扩增文库中随机挑取重组克隆进行5′和3′测序,获得8条有效的EST,长度在244～711 bp之间,分析8个克隆的ESTs序列所包含的ORF,并进行BLASTp比对,推断其相关的功能。结果表明,LW13、LW15、LW16、LW17克隆的最长ORF均不完整,数据库中无与其匹配的蛋白质序列,推测可能为新基因的一部分；LW19克隆的最长ORF为609 bp,编码202个氨基酸,分子量为22.4 kD,该蛋白质序列含有硫氧还蛋白保守结构域,推测与维持体内稳定的氧化还原状态有关；LW20克隆的最长ORF为198 bp,编码65个氨基酸；LW22克隆的最长ORF为105 bp,仅编码34个氨基酸；LW24克隆的最长ORF不完整。在数据库中,均未发现与LW20、LW22和LW24三个克隆相匹配的蛋白质序列,推测可能为新基因的一部分。
经BLASTx和BLASTn两个方面的综合比对分析,说明LW19和LW22两个克隆的EST编码的氨基酸序列可能在细胞分化、细胞周期调控、细胞衰老和死亡以及机体免疫等基础研究中具有一定的理论意义,对研究莱芜猪某些优良性状的表现机理也起到非常重要的作用,为后续工作的开展也做了重要的铺垫。
通过SMART技术构建莱芜猪心脏组织cDNA文库,不仅保存了稀有的地方种猪遗传资源,而且为莱芜猪有明确功能的特异基因的克隆、表达以及新基因的筛选与功能研究提供依据。
Laiwu pig is one of our local pig varieties for five thousand years. It has the characters of high reproductive capacity, disease-resistance, good meat quality, resistance to coarse grain, heterosisr. A serial of the porcine products have been developed including popular cold fresh meat, roast meat, sausage.Laiwu pig has formed a good brand effect in our country. It is hopeful of its having a big market and a good future. Therefore, it is a very important value in theory and practice to develop and protect this porcine rare genetic resource.
Total RNA was extracted form the heart tissue of Laiwu pig using improved isothiocyanate method. The total RNA was reverse-transcribed into single-strand cDNA by SMART (the Switch Mechanism At the 5'end of RNA Templates) technique. Long distance PCR (LD-PCR) was then carried out to synthesize the double-strand cDNA that were digested by Sfi I, and the digested fragments were fractionated by CHROMA SPIN-400. Then cDNA fragments longer than 0.5 kb were collected and ligated toλTriplEx2 vector. Ligation products were packed in vitro intoλphage particulates in order to gain unamplified cDNA library. The titer of primary cDNA library was 2.0×105 pfu/ml checked strictly by conventional titer determination and the rate of recombinant was about 94.4%. The titer of the amplified library was 3.0×108 pfu/ml. The recombinantλTriplEx2 clones picked from the amplified library infected E.coli BM25.8 to convert into pTriplEx2 plasmids. Plaques picked randomly from transformed pTriplEx2 plasmids were tested using PCR with universal primers derived from the sequence flanking the vector. The length of the most inserted fragments was above 0.75 kb.
The recombinant pTriplEx2 plasmids carried the inserts more than 500 bp were picked from the transformed plates with E.coli BM25.8 and sequenced from 5' end and 3'end. Eight pieces of effective ESTs were obtained totally between 244 and 711 bp in length. The putative open reading frames (ORFs) of these 8 ESTs were analyzed through Open Reading Frame Finder in NCBI and by using the BLASTp program, and the functions of amino acid sequences translated from these ORF of the eight ESTs were predicted. The results indicated that the longest ORF of LW13、LW15、LW16 and LW17 clones were not complete, and their amino acid sequences translated from their ESTs had unmatched with the protein sequences in database, they might be one part of some new genes;the longest ORF of LW19 clone was was 609 bp in length and encoded a 202 amino acid residues,22.4 kD in molecular weight. The amino acid sequences had a matching protein sequence in database, its containing thioredoxin-like superfamily conserved domain which was related with oxidation-reduction levels in vivo. The longest ORF of LW20 clone was 198 bp in length and encoded 65 amino acid residues. The longest ORF of LW22 clone was 105 bp in length and encoded only 34 amino acid residues. The longest ORF of LW24 clone was not complete. Three clones of LW20, LW22 and LW24 had unmatched protein sequences in database, they might be representative of one part of a unknown gene.
By combined alignment of BLASTx and BLASTn programs in NCBI, it was shown that the amino acid sequences encoded by two ESTs of the clones LW19 and LW22 functioned in foundmental researches of cell differentiation, cell cycle control, cell aging and death, and body immunity, and might play a very important role in the expression mechanism of some favourite characters in Laiwu pig. These also laid some foundations for further research.
The cDNA library from the heart tissue of Laiwu pig constructed by SMART technique might be helpful to preservation of local rare porcine resources, but also provided a valuable clues for cloning and expression of functional genes and for screening of unknown genes.
引文
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