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Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (10): 1801-1808.doi: 10.3724/SP.J.1006.2011.01801

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Molecular Cloning and Expression Analysis of C4 Phosphoenolpyruvate Carboxylase Gene from A. hypochondriacus L.

FENG Rui-Yun1,2,BAI Yun-Feng2,**,LI Ping3,ZHANG Wei-Feng2,*,WANG Yuan-Yuan1,YANG Wu-De1,*   

  1. 1 College of Agronomy, Shanxi Agricultural University, Taigu 030801, China; 2 Institute of Crop Sciences, Shanxi Academy of Agricultural Sciences, Taiyuan 030032, China; 3 Bio-engineering School of Shanxi University, Taiyuan 030006, China
  • Received:2011-03-23 Revised:2011-06-25 Online:2011-10-12 Published:2011-07-28
  • Contact: 张维锋, E-mail: zhwfen@sina.com; 杨武德, E-mail: sxauywd@126.com E-mail:byfok@126.com

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Abstract: Phosphoenolpyruvate carboxylase (PEPC) plays a crucial role in primarily fixing atmospheric CO2 in C4 and CAM plants. This paper aimed at cloning both cDNA and genomic DNA of PEPC gene from Amarnthus hypochondriacus in order to provide candidate genes for C4 genetic engineering. The cDNA was cloned by RT-PCR, and the corresponding gDNA was cloned by LA-PCR. The sequence characteristics were analyzed using bioinformatics softwares including Blast, ClustalX (1.8), DNAMAN, PlantCARE. The gene expression pattern was analyzed by using semi-quantitative RT-PCR. The results indicated that the cDNA with the complete coding region was 2 895 bp in length which encoded 964 putative amino acids. The amino acid at site 774 was the serine that is an invariant residue in all C4 phosphoenolpyruvate carboxylases regardless of their taxonomic. The corresponding gDNA was 6 785 bp in length which harbored ten exoned and nine introns. The longest intron was the first intron with 1 662 bp in length. A computer scan disclosed that the first intron harbored 15 light-induced elements, nine hormone-induced elements, 29 TAAT motifs and 72 TATA motifs.In comparisons of PEPC genes between different species the exons are usually conserved but the introns have often diverged in both nucleotide sequences and lengths. The GC ratios of exons are positively correlated with those of introns in PEPC genes from different species. And the GC ratios of PEPC gene in dicotyledon A. hypochondriacus, potato, A. thaliana and ice plant are lower than those in monocotyledon maize, rice and wheat. The semi-quantitative RT-PCR analysis revealed that the cloned PEPC gene was light-inducible leaf-specifically expressed and classified as C4-type sub-family groups.

Key words: Amarnthus hypochondriacus, C4 photosynthetic type, PEPC, gDNA, Leaf-specific, Light-inducible expression

[1]Caemmerer S, Furbank R T. The C4 pathway: an efficient CO2 pump. Photosynthesis Res, 2003, 77: 191–207
[2]Schaeffer A R, Sheen J. Maize C4 photosynthesis involves differential regulation of phosphoenolpyruvate carboxylase gene. Plant J, 1992, 2: 221–232
[3]Engelmann S, Blaesing O E, Svensson P, Westhoff P. Molecular evolution of C4 phosphoenolpyruvate carboxylase in the genus Flaveria: a gradual increase from C3 to C4 characteristics. Planta, 2003, 217: 717–725
[4]Blaesing O E, Ernst K, Streubel M, Westhoff P, Svensson P. The non-photosynthetic phosphoenolpyruvate carboxylases of the C4 dicot Flaveria trinervia-implications for the evolution of C4 photosynthesisl. Planta, 2000, 215: 448–456
[5]Izui K, Matsumura H, Furumoto T, Kai Y. Phosphoenolpyruvate carboxylase: a new era of structural biology. Annu Rev Plant Biol, 2004, 55: 69–84
[6]Ogren W L, Photorespiration: pathways, regulation and modification. Annu Rev Plant Physiol, 1984, 35: 415–442
[7]Haesler R E, Hirsch H J, Kreuzaler F, Peter haensel C. Overexpression of C4-cycle enzymes in transgenic C3 plants: a biotechnological approach to improve C3-photosynthesis. J Exp Bot, 2002, 53: 591–607
[8]Zhou B-Y(周宝元), Ding Z-S(丁在松), Zhao M(赵明). Alleviation of drought stress inhibition on photosynthesis by over expression of PEPC gene in rice. Acta Agron Sin (作物学报), 2011, 37(1): 112–118 (in Chinese with English abstract)
[9]Zhang F, Chi W, Wang Q, Zhang Q D, Wu N F. Molecular cloning of C4-specific Ppc gene of sorghum and its high level expression in transgenic rice. Chin Sci Bull, 2003, 48: 1835–1840
[10]Yang R-Z(杨荣仲), Tan Y-M(谭裕模), Zhang M-Q(张木清), Chen R-K(陈如凯), Li Y-R(李杨瑞). Primary study of genetic transformation of tobacco with sugarcane C4 phosphoenolpyruvate carboxylase gene. Chin J Trop Crops (热带作物学报), 2004, 25(2): 61–65 (in Chinese with English abstract)
[11]Shukla S, Bhargava A,Chatterjee A, Srivastava A, Singh S P. Genotypic variability in vegetable amaranth (Amaranthus tricolor L.) for foliage yield and its contributing traits over successive cuttings and years. Euphytica, 2006, 151: 103–110
[12]Bai Y-F(白云凤), Guo Z-H(郭志华), Bai D-M(白冬梅), Wang X-Q(王小琦), Zhang W-F(张维锋). An improved method for extration and identification of potato RNAs. Acta Hortic Sin (园艺学报), 2007, 34(4): 1059–1062 (in Chinese with English abstract)
[13]Engelmann S, Blaesing O E, Westhoff P, Svensson P. Serine 774 and animo acids 296 to 437 comprise the major C4 determinants of the C4 phosphoenolpyruvate carboxylase of Flaveria trinervia. FEBS Lett, 2002, 524: 11–14
[14]Blaesing O E, Westhoff P, Svensson P. Evolution of C4 phosphoenolpyruvate carboxylase in Flaveria, a conserved serine resudue in the carboxyl-terminal part of the enzyme is a major determinant for C4-specific characteristics. J Biol Chem, 2000, 275: 27917–27923
[15]Levisky V G. RECON: a program for prediction of nucleosome formation potential. Nucl Acids Res, 2004, 32: 346–349
[16]Chen X Q, Zhang X D, Liang R Q, Zhang L Q, Yang F P, Cao M Q. Expression of the intact C4 type PEPC gene cloned from maize in transgenic winter wheat. Chin Sci Bull, 2004, 49: 2137–2143
[17]Xiang X-C(向珣朝), He L-B(何立斌), Sun J-M(孙建明), Li J-H(李季航), Yao Y-P(姚嫣萍), Li P(李平). Effect of maize PEPC gene in different genetic backgrounds of CMS maintainers and tolerance to photooxidation in the PEPC transgenic line. Chin J Rice Sci (中国水稻科学), 2009, 23(3): 257–262 (in Chinese with English abstract)
[18]Ku M S B, Agarie S, Nomura M, Fukayama H, Tsuchida H, Ono K, Hirose S, Toki S, Miyao M, Matsuka M. High-level expression of maize phosphoenolpyruvate carboxylase in transgenic rice plants. Nat Biotechnol, 1999, 17: 76–81
[19]Hudaspeth R L, Grula J W, Dai Z Y, Edwards G E, Ku M S B. Expression on maize phosphoenolpyruvate carboxylase in transgenic tobacco. Plant Physiol, 1982, 98: 458–464
[20]Kawabe A, Miyashita N T. Patterns of codon usage bias in three dicot and four monocot plant species. Genes Genet Systems, 2003, 78: 343–352
[21]Li Ping(李平), Bai Y-F(白云凤), Feng R-Y(冯瑞云), Wang Y-Y(王原媛), Zhang W-F(张维锋). Analhysis of codon bias of NAD-ME gene in Amaranthus hypochondriacus. Chin J Appl Environ Biol (应用和环境生物学报), 2011, 17(1): 12–17 (in Chinese with English abstract)
[22]Furger A, O’Sullivan J M, Binnie A, Lee B A, Proudfoot N J. Promoter proximal splice sites enhance transcription. Gene Dev, 2002, 16: 2792–2799
[23]Jia Q, Wu H T, Zhou X J, Gao J, Zhao W, Ar Q S, Wei J S, Hou L H, Wu S Y, Zhang Y. A “GC-rich” method for mammalian gene expression: a dominant role of non-coding DNA GC content in regulation of mammalian gene expression. Sci China: Life Sci, 2010, 53: 94–100
[24]Wang Y B, Lang Z H, Zhang J, He K L, Song F P, Huang D F. Ubi1 intron-mediated enhancement of the Bt cry1AH gene in transgenic maize (Zea mays L.). Chin Sci Bull, 2008, 53(20): 3185–3190
[25]Jeong Y M, Mun J H, Kim H. An upstream region in the first intron of petunia action depolymerizing factor 1 affects tissue-   specific expression in transgenic Arabidopsis. Plant J, 2007, 50:
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