全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

坛紫菜TPS基因的克隆及其原核表达

DOI: 10.7668/hbnxb.2013.05.012, PP. 66-73

Keywords: 坛紫菜,TPS基因,原核表达,重组菌,耐盐性

Full-Text   Cite this paper   Add to My Lib

Abstract:

以叶状体总DNA为模板,用3对DNA引物进行PCR扩增,获得了坛紫菜6-磷酸海藻糖合成酶基因(PhTPS)3个重叠的片段,大小分别为1.3,1.1,0.7kb。用pMD18-T载体克隆这些片段,经测序与序列拼接获得了该基因完整的ORF序列,其长度为2727bp。在已经构建条斑紫菜TPS基因原核表达载体pET22b-PyTPS的基础上,用PhTPS基因替代该表达载体中的PyTPS基因,获得了PhTPS基因的原核表达载体pET22b-PhTPS。重组菌BL21(pET22b-PhTPS)经IPTG诱导,SDS-PAGE显示获得了约100kDa的特异性蛋白条带;目测及OD600的测定结果表明重组菌的耐盐性明显高于对照菌,即PhTPS基因的表达提高了重组菌的耐盐性。为利用PhTPS基因进行作物耐盐等抗逆转基因改良提供了依据。

References

[1]  Adam R P,Kendall E,Kartha K K. Comparison of free sugars in growing and desiccated plants of Selaginella lepidophylla[J]. Biochemical Systematics and Ecology, 1990,18: 107-110.
[2]  Hounsa C G,Brandt E V,Thevelein J, et al. Role of trehalose in survival of Saccharomyces cerevisiae under osmoticstress[J]. Microbiology,1998,144: 671-680.
[3]  Hottiger T,Boller T,Wiemken A. Rapid changes of heat and desiccation tolerance correlated with changes of trehalose content in Saccharomyces cerevisiae cells subjected to temperature shifts[J]. FEBS Lett,1987,220 ( 1) : 113-115.
[4]  Styrvold O B,Storm A R. Synthesis accumulation,and excretion of trehalose in osmotically stressed Escherichia coli K-12 strains: influence of amber suppressors and function of the periplasmic trehalase[J]. J Bacteriol,1991,173: 1187-1192.
[5]  Kaasen I,Falkenberg P,Styvold O B, et al. Molecular cloning and physical mapping of the otsBA genes,which encode the osmoregulatory trehalose pathway of Escherichia coli: evidence that transcription is activated by KatF( AppR)[J]. J Bacteriol,1992,174: 889-898.
[6]  Bell W,Kiaassen P,Ohnacker M, et al. Characterization of the 56 kDa subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIFI,a regulator of carbon catabolite inactivation[J]. Eur J Biochem,1992,209: 951-959.
[7]  Virgilio C D,Burckert N,Bell W,et al. Disruption of TPS2,the gene encoding the 100-kDa subunit of the trehalose- 6-phosphate synthase /phosphatase complex in Saccharomyces cerevisiae,causes accumulation of trehalose- 6-phosphate and loss of trehalose-6-phosphate phosphatase activity[J]. Eur J Biochem,1993,212: 315-323.
[8]  Leyman B,Van Dijck P,Thevelein J M. An unexpected plethora of trehalose synthesizing genes in Arabidopsis thaliana[J]. Trends Plant Sci,2001,6: 5110-5113.
[9]  Ge L F,Chao D Y,Shi M, et al. Overexpression of the trehalose- 6-phosphate phosphatase gene OsTPP1 confers tolerance in rice and results in the activation of stress responsive genes[J]. Planta,2008,228: 191-201.
[10]  Garg A K,Kim J K,Thomas G, et al. Trehalose ac-cumulation in rice plants confers high tolerance levels to different abiotic stresses[J]. Proc Natl Acad Sci,2002,99: 15898-15903.
[11]  Jang I C,Oh S J,Seo J S, et al. Expression of a bifunctional fusion of the Escherichia coli genes for trehalose-6- phosphate synthase and trehalose-6-phosphate phosphatase in transgenic rice plants increases trehalose accumulation and abiotic stress tolerance without stunting growth[J]. Plant Physiol,2003,131: 516-524.
[12]  Dong C L,Zhang M L,Zhang Y Q, et al. Transformation of trehalose synthase gene ( TPS gene) into corn inbred line and identification of drought tolerance[J]. African Journal of Biotechnology,2011,10: 15253-15258.
[13]  Yeo E T,Kwon H B,Han S E, et al. Genetic engineering of drought resistant potato plants by introduction of the tehalose-6-phosphontes synthase( TPS1) gene from Saccharomyces cerevisiae[J]. Mol Cells,2000,10( 3) : 263-268.
[14]  Avonce N,Wuyts J,Verschooten K, et al. The Cytophaga hutchinsonii ChTPSP: first characterized bifunctional TPS-TPP protein as putative ancestor of all eukaryotic trehalose biosynthesis proteins[J]. Mol Biol Evol,2010, 27: 359-369.
[15]  Kondrak M,Marincs F,Antal F, et al. Effects of yeast trehalose synthase gene 1 on gene expression and carbohydrate contents of potato leaves under drought stress conditions[J]. BMC Plant Biology,2012,12: 74.
[16]  Wang G L,Zhao G,Feng Y B, et al. Cloning and comparative studies of seaweed trehalose-6-phosphate sybthase genes[J]. Marine Drugs,2010,8: 2065 -2079.
[17]  郭宝太,毕玉平,李广存,等. 坛紫菜DNA 的提取与纯化[J]. 农业生物技术学报, 1999,7 : 342-352.
[18]  Pan Y T,Carroll J D,Elbein A D. Trehalose-phosphate synthase of Mycobacterium tuberculosis: cloning,expression and properties of the recombinant enzyme[J]. Eur J Biochem,2002,269: 6091-6100.
[19]  Cai Z J,Peng G Y,Cao Y Q, et al. Trehalose-6-phosphate synthase 1 from Metarhizium anisopliae: clone,expression and properties of the recombinant[J]. J Biosci Bioeng,2009,107: 499-505.
[20]  Miranda J A,Avonce N,Suarez R, et al. A bifunctional TPS TPP enzyme from yeast confers tolerance to multiple and extreme abiotic-stress conditions in transgenic Arabidopsis[J]. Planta,2007,226: 1411-1421.
[21]  张艳敏,郭北海,蒋春志,等. 转甜菜碱醛脱氢酶( BADH) 基因小麦的耐盐耐旱性[J]. 华北农学报, 2003,18( 1) : 29-32.

Full-Text

comments powered by Disqus

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133

WeChat 1538708413