生物克隆论文克隆技术的论文.pdf

基因组学与应用生物学， 2010 年， 第 29 卷， 第 6 期， 第 1019- 1025 页 Genomics and Applied Biology, 2010, Vol.29, No.6, 1019- 1025 An Article 研究论文 CloningandBioinformaticAnalysisofPotato - AmylaseGene amyA1 Wang YuliWang YonggangMa Jianzhong * Wang QianTao PengfeiSu Yi School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou, 730050 * Corresponding author, majz AbstractIn this research, we amplified a cDNA for potato - amylase by RT-PCR and cloned it. Sequence anal- ysis showed that the cDNA had a 1 224 bp open reading frame and was referred to as amyA1, which encodes for an - amylase with 407 amino acid residues (GenBank accession number: GQ406048.1) with the MW 46.40 kD. After that we used semi-quantitative RT-PCR assay to detective the expression of the amyA1 gene in potato leaves and stems. The result showed that the expression in stems is a little stronger than in leaves. Then we analyzed the amino acid sequence bioinformatically, including its codon usage bias, physical and chemical properties, subcellu- lar localization, and conserved structures. 29 - amylase genes from same or different species were taken from the GenBank for constructing a phylogenetic tree. The bioinformatical analyses showed that the putative protein shared 98% identity with a published potato - amylase (GenBank accession number: M79328.1) at the amino acid level. The deducted - amylase also contains a catalytic domain (PF00128、 SM00624) between 20 to 348 and a C-terminal beta-sheet domain (PF07821)between 349407, which are similar to ones of the amylase family 13. The postulated eight-stranded alpha/beta barrel was also found in the enzyme, which was thought as an active site of - amylase. According to the phylogenetic tree, the two genes from potato presents more close homology to those from cassava and apple than from barley, rice and maize. KeywordsPotato, - amylase, Clone, Bioinformatic analysis 马铃薯 - 淀粉酶基因的克隆及生物信息学分析 王玉丽王永刚马建忠 * 王倩陶鹏飞苏移 兰州理工大学生命科学与工程学院, 兰州 , 730050 * 通讯作者, majz DOI: 10.3969/gab.029.001019 摘要本研究利用 RT-PCR 从马铃薯(Solanum tuberosum)茎段总 RNA 中扩增、 克隆了一 cDNA 分子。该 cDNA 分子含有一长为 1 224 bp 的开放读框， 可编码一含 407 个氨基酸残基的多肽、 理论分子量为 46.40 kD、 可能为亲水性的胞外酶。因其氨基酸序列同源于 - 淀粉酶， 故将该基因命名为 amyA1 (NCBI 收录号: GQ406 048.1)。采用半定量 RT-PCR 方法检测了 amyA1 基因在马铃薯茎、 叶等不同组织中的表达强度， 表明在茎 组织中的表达丰度略高。利用生物信息学软件分析了 amyA1 密码子的偏好性， 以期为选择适宜的表达系 统提供依据； 同时对 amyA1 的理化性质、 细胞内定位、 保守结构及高级结构进行了预测。基于 NCBI 数据 库中有物种代表性的 29 种 - 淀粉酶基因序列构建了基因进化树。与 NCBI 收录的马铃薯 - 淀粉酶基 因(NCBI 收录号: M79328.1)的核苷酸及氨基酸序列同源性达 98%。第 20 至第 348 范围内的氨基酸残基 含有与淀粉酶 13 家族及亚家族相似的催化活性域(PF00128、 SM00624)， 第 349 至第 407 范围内的氨基酸 残基含有 - 淀粉酶 C- 末端 折叠区域(PF07821)。蛋白质结构预测表明氨基酸残基序列有维持淀粉酶 活性的(/)8桶状结构以及其它几个功能域结构。所构建的基因进化树表明， 2 个马铃薯 - 淀粉酶基因 Program fund: This research was supported by grants from the Technical Bureau, Gansu (No. 07- 02- 44) and the Degree Courses on Building Lan Zhou University of Technology (No. 2010- 11) 基因组学与应用生物学 Genomics and Applied Biology 与木薯、 苹果的序列同源性较高， 与菜豆的次之， 与水稻、 大麦和玉米等单子叶植物的序列同源性较低。 关键词马铃薯, - 淀粉酶基因, 克隆, 生物信息学 - amylase family is the largest glycoside hydrol- yse family, which contains more than 30 members, in- cluding hydrolase, transferase and isomerase, and most of which belong to glycoside hydrolase family 13 (GH13) that exists in the nature. Currently, varies of amylase genes cDNA sequence have been cloned from animals, plants, fungi and bacteria by genetic engineer- ing technology. Based on different origins, it can be generally divided into bacteria amylase, fungi amylase, plant amylase and animal amylase, etc. At the same time, people have cloned many extremely resistant - amylase gene by techniques such as mutation, which has extended the application in the field of industry (Fitter, 2005). With the consumption of nonrenewable resources such as petrol and the increasing demand for energy in the development of humanity, finding new renewable resources as the substitute in the post-petrol time is im- minent. As the third highest yield economical crop of the world, potato is potentially advantageous as the ma- terial to produce ethanol (Ghang et al., 2007). From cloning potato self amylase-encoding gene, a transgenic amylase engineering yeast is constructed, using potato as the raw material. Producing fuel ethanol by biologi- cal fermentation can increase the added value of potato and solve the problem of energy crisis. In this research, by cloning - amylase gene in different tissues of pota- to, we studied the distribution in tissues in the course of bourgeon and growth. According to bioinformation analysis, the structure and function of - amylase gene was better understood (MacGregor et al., 2001). This provided theoretical accordance for the expression of potato - amylase gene in different expression systems such as yeast. 1 Results 1.1 Extraction of total-RNA and amplification of gene by RT-PCR Total-RNA of leaf and stem from potato culture seedling was extracted according to Trizol RNA extrac- tion manufacturers instruction. Absorbance of RNA so- lution originated from leaf stem was examined by Cary 50 under 280 nm and 260 nm UV spectrophotometer, separately. A260/A280(ratio, R) were 1.92 and 1.97 sepa- rately. R value ranged from 1.802.0. The quality of RNA met the requirements. Meanwhile, the integrity of RNA was examined by agarose gel electrophoresis. Bands 28S and 18S was bright and clear, and the illu- mination of 28S band was twice as much as that of 18S (Figure 1). The quality of RNA was very fine. The first chain of cDNA was synthesized by re- verse transcription fron total-RNA, and according to which - amylase gene was amplified by PCR. PCR productwasexaminedby1.0%agarose gel electrophore- sis. The product could be amplified from cDNA both o- riginated from leaf and stem. It was 1.2 kb and was consistent as expected and was named - amylase, ab- breviated amyA (Figure 2). 1.2 Analysis of - amylase gene expression in differ- ent tissues During the germination of potato tuber, the activity of - amylase gene was much relevant to its growth. It could facilitate the germination of the tuber and the growth of early seedlings. Also, expression - amylase gene was found in other tissues and cells during the Figure 2 RT-PCR results ofTotal-RNA from potato leaf (1,2) and from potato stem (3,4) Note: M: DL2000 DNA Marker Figure 1 Total-RNA frompotato leaf(1)and frompotato stem(2) 1020 growth. Analysis by semi-quantitative RT-PCR showed that (Figure 2) expression of - amylase gene was found in both leaf and stem, and the abundance of which was higher in stem tissue. Further clone and sequence anal- ysis showed that amylase was found to express in all different tissues during the growth of potato. 1.3 Identification of the recombinant Positive clone was attained by screening. And re- combined plasmid pET-amy A was attained by bacteria shaking cultivation and plasmid extraction. The correct- ness of the recombined plasmid was examined by the Not site designed as a part of the primer. As Figure 3 showed, the recombined plasmid was digested by Not and produced a band of 1 230 bp which was the same size as expected. It was sent to Shanghai Sangon for se- quencing. 1.4 Nucleotide sequence and codon analysis of amyA1 It was known from the sequence result that the at- tained - amylase gene was 1 224 bp as the whole length, and it coded 407 amino acids. After analysis, integritating and BLAST, it was named amyA1. It was handedtoNCBIandgotanaccessionnumber GQ406048.1. By analyzing with CLC Protein Work- bench5.0 Software, it was found to contain one ORF (open reading frame). Homological analysis of nu- cleotide sequence by NCBI/Blast showed that the ho- mology of this sequence and the nucleotide sequence of - amylase gene from potato as M79328.1 registered in GenBank was as high as 98%. And the homology of both amino acid sequences was 98%. And the one with AK323104.1 tomato - amylase gene nucleotide se- quence was as high as 97%. It might be due to the dif- Figure 3 Results of Notdigesting assay of positive clone Note: M: DL2000 Marker; 1,2: Positive clone ferences between genius and species. It was meaningful to the research of the diversity of this gene sequence (P- ark et al., 2000). The amyA1 gene statistics was carried out accord- ing to the attained - amylase gene sequence, using CHIPS and Codon W Software and we got effective codon. It showed that except CCC that coded Pro and CGA that coded Arg (R), codons that coded all the oth- er amino acids appeared in the gene sequence. Its Enc was 53.57, close to 60, which showed that during the synthesis of potato - amylase, the degeneration and bias of the codon was not obvious. Besides, the amount ofGC was 44.1%, and the amount of GC3s was 39.95%, which showed that most of the G and C bases in this gene were at the third of the codon. From the ratio of the usage frequencyof the effective codon in potato - amy- lase gene sequence and the yeast bias codon it was found that the difference between the codon bias of potato and yeast was little. Among all the codon “/1000” of potato - amylase gene sequence and yeast, 17 were not less than 2.0 or not more than 0.5. According to this, we could primarilypredict that this gene could express well. Meanwhile, this provided the theoretic basis for the se- lection of proper expression system and the improve- ment ofgene expression amount ofcodon. 1.5 Protein sequence analysis of amyA1 1.5.1 Prediction ofphysics and chemistryproperties The amyA1 had 407 amino acids, MW 46395.1, molecular formula C2087H3141N577O606S12, theoretic pI was 6.95. Among them, the number of residues with negative charge (Asp+Glu) was 46, 11.3% of the total amino acid amount; the number of the residues with positive charge (Arg+Lys) was 45, 11.06% of the total amino acids amount. The parameter of instability was 45.7, which showed that it belonged to instable protein. The half life was 30 hours. The hydrophobic nature of the protein was predicted by Kyte and it showed that amino acids 68, 103, 108, 187, 212 and 294 had active function, which related to the binding to calcium and zinc ions. 1.5.3 Advanced structural prediction Prediction according to Swiss-Model showed that prediction of spatial structure of this sequence was based on PDB: 1rp8 ChainA as template and the se- quence consistency was 44.853%. Spatial structure was showed in Figure 4 that it contained typical (/)8barrel structure at active Catalytic region. Along with predic- tion of secondary structure, it showed that C-end had typical - fold structure, generally identical to the spa- tial structure of the amylase known. Most amylases had eight centers of (/)8barrel structures and domain B, C and D. Domain B had three - folds and three - helix- es, whose length and structure varied with the different origins; domain C was backward of catalytic domain, which was composed by - folds and was known to protect the stability of hydrophobic amino acids in catalytic active center. 1.6 Phylogenetic tree analysis based on - amylase gene Figure 5 showed the evolutional distance between different species. Different nodes stood for different classification units. It showed the evolutional distance between species as well as diversity of the same species from different sources or between varieties. Besides, comparison of homological sequences originated from different - amylase gene revealed that homology of - amylase gene sequences from microbes, animals and plants was less than 10%. But regions A, B, C and D a- mong those - amylases were found to have high con- servation and it suggested that those conservative re- gions related to binding and catalytic centers. 2 Discussion With the consumption of nonrenewable resources such as petrol and the increasing command for energy in the development of humanity, finding new renewable resources as the substitute in the post-petrol time is im- minent. As the third highest yield economical crop of the world, potato is potentially advantageous as the ma- Figure 4 AmyA1 protein spatial structure predicted by Swiss- Model 1022 terial to produce ethanol (Ghang et al., 2007). From cloning potato self amylase gene, a transgenic amylase engineering yeast is constructed, using potato as the raw material. Producing fuel ethanol by biological fer- mentation can increase the added value of potato and solve the problem of energy crisis. Starch was the main storing material in potato whose digestion was the key step of tuber germination and growth. Therefore the expression and regulation of amylase played an important role in germination of potato tuber. Considering the specificity, and traits such as high rates of hydrolysis and utility of potato starch by self amylase, this article reported cloning of potato self - amylase gene, analysis of physics and chemistry properties as well as sequence structure, etc. of its nu- cleotide and peptide chain, alignment with yeast bias codon, and prediction of its expression in yeast. Analy- sis showed that potato - amylase gene belonged glyco- side hydrolyse family 13. It had integrate functional ac- tive domain. Meanwhile, at active position, it had com- Figure 5 Phylogenetic tree of different species based on - amylase gene monly catalytic amino acids: Asp, Glu and Asp (Sun, 2006). It provides theoretic foundation for the transfor- mation of - amylase gene in bioengineering strains, such as in yeast expression system, using potato raw starch to express amylase, fuel alcohol was produced. Also, the kinetic parameters of this enzyme was re- searched and the activity, thermo stability and the resis- tance of acid and alkali. According to available results of bioinformation analysis, the gene coded this enzyme was reconstructed (Juge et al., 2006). On the one hand, the utility rate was improved. On the other hand, the stability and resistance was strengthened so that its ap- plication in industry was extended. 3 Materials and Methods 3.1 Materials High starch potato tissue culture seedling was (cv. Shepody) from our laboratory. E. coli DH5 and plasmid pET- 32a (Ampr) were preserved in our laboratory. Cloning and Bioinformatic Analysis of Potato - Amylase Gene amyA1 马铃薯 - 淀粉酶基因的克隆及生物信息学分析 1023 基因组学与应用生物学 Genomics and Applied Biology Trizol, T4 DNA Lingase, pfu DNA Polymerase, dNTP, PrimeScriptTMReverse Transcriptase, Calf In- testinal Alkaline Phosphatase (CIAP), Gel Extraction Kit (Zymo), DEPC and restriction endonuclease were purchased from TaKaRa (Dalian, China). All other che- mical reagents were of analytical purity from Shanghai Sangon. 3.2 Extraction of total RNA and synthesis of the first chain of cDNA Total RNA of potato leaf and stem was extracted following the manufacturers instruction for Trizol (Sangon), with slight changes. The first chain of cDNA was synthesized with downstream primer referring to the manufacturers instruction for reverse transcriptase (TaKaRa), and was preserved in - 20. 3.3 Amplification of - amylase gene by RT-PCR The primers of - amylase gene in leaf and stem of potato (cv. Shepody) tissue culture seedlings are design- edaccording to - amylase gene(GenBank accession number: M79328.1) sequence of potato. Upstream primer: 5- GCGGCCGCTGTATAATCT CAACTACAATCCATT- 3. Downstream primer: 5- GCGGCCGCTTTAAGTA ACGGAATAGCTGTAAGA- 3. A Not site was designed in both upstream and downstream primer each. - amylase gene was ampli- fied by PCR from reverse transcript production as tem- plate. Condition for amplification: 95 1 min; 95 45 s, 65 45 s, 72 1 min, 30 cycles; 72 10 min. After purification with Purification Kit, the PCR prod- uct was phosphated according to the manufacturers in- struction and preserved. 3.4 Construction of vector pET-amy A Plasmid pET- 32a was prepared by alkaline lysis method, and digested using EcoR , dephosphorylated with CIAP, extracted and preserved. PCR product proceeded with T4 Polynucleotide Kinase was cloned to blunt ended plasmid pET- 32a to construct recombined plasmid pET-amy A. The recom- bined plasmid was transformed to E. coli DH5, and screened through Amprfor positive clone. 3.5Sequencingandbioinformationanalysisof - amylase gene The positive clone was proved by digesting with endonuclease and sent to be sequenced in Sangon. The result sequence was analyzed and jointed by CLC Pro- tein Workbench 5.0 Software and the correct - amy- lase gene sequence and amino acid sequence was at- tained. Each was submitted to GenBank and aligned with the gene sequence and protein sequence recorded in NCBI separately by BLAST. Meanwhile, the se- quence result was analyzed and predicted the nucleic and protein properties and structures. 3.5.1 Analysis of nucleotide properties The primary structure of nucleotide depends on the reference frequency and order of the four bases. DNA regions with different orders of bases in a DNA molecule constructs particular functional units and de- termines the types and order of amino acids and the functions of advanced structure of protein. Statistic count of effective number of codons (ENc) of - amy- lase gene was made by CHIPS Software. Enc as well as GC amount in CDS region, GC amount as the third base in a codon and usage frequency of codons were made by Codon W and CUSP. Finally we compared the codon usage bias frequency/1 000 of - amylase gene with that of yeast genome, and predicted its expression in yeast (Zhao et al., 2008). 3.5.2 Analysis of protein properties An alysis of protein physics chemistry: Physics chemistry properties of the sequence such as MW, the- oretic pI, amino acid ingredient were analyzed by Prot- Param tool (/tools/protparam. html); prediction and analysis of hydrophobic property: The hydrophobic property of amino acid sequence was analyzedbyProgram ProtScale on ExPASy(http:/www. expasy.ch/tools/protscale.html); prediction of secondary structure: The secondarystructure ofthe protein sequen- ce was analyzed by 3D-PSSM (http:/www.sbg.bio.ic.ac