Welcome to Acta Agronomica Sinica，

Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (01): 63-71.doi: 10.3724/SP.J.1006.2014.00063

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

Improvement of Minimal Gene Cassette Expression Stability by Scaffold Attachment Region (SAR) Sequence in Wheat Transformation

SU Rui-Bo1,2,CHEN Ming2,*,XU Zhao-Shi,LI Lian-Cheng,MA Qing1,*,MA You-Zhi2   

  1. 1Agriculture College, Inner Mongolia Agricultural University, Hohhot 010018, China; 2 National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture / Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2013-06-17 Revised:2013-09-16 Online:2014-01-12 Published:2013-10-22

RichHTML

PDF

905

Cited

2

Abstract:

The minimal expression cassette only containing the promoter, coding sequence and terminator sequence is transformed into plants genome, which will reduce the security risks that may be caused by the vector skeleton sequence. Scaffold attachment region (SAR) combining with the nuclear matrix to separate transformed DNA fragment with adjacent genome sequence, which block the influence of the neighboring chromatins and improve the stability of exogenous gene. In this research, a new vector was constructed with the minimal expression cassette flanked with SAR sequences. This vector was used in wheat transformation by micro-particle bombardment aiming at improving stability of exogenous gene expression. GUS as a reporter gene was constructed in the minimal expression cassette flanked with SAR sequences, and this DNA fragment was transformed into what variety Kenong 199. At same time, the fragment with the minimal expression cassette but without the SAR was used as a control. A total of 857 immature embryos were bombarded using the minimal expression cassette with GUS and SAR sequences, and 40 T0 plants were obtained, of which 16 plants were positive by PCR testing and 15 plants were positive by GUS staining. The transformation efficiency was 1.87%. In the 18 individuals randomly selected from the T1 generation derived from four positive T0 plants, 15 plants showed positive reactions in PCR testing and GUS staining. In contrast, transformation efficiency of the control was only 0.49% (five PCR positive plants/1012 immature embryos), and only two PCR positive plants were confirmed by GUS staining. In 10 T1 plants of the control derived from five T0 PCR positive lines, no positive plant was identified by either PCR assay or GUS staining. Using this improved method, a new drought-related transcription factor gene GmDREB3 from soybean was transformed into wheat receptor Jimai 22. A total of 130 T0 plants were obtained from 6045 immature embryos bombarded, of which 30 plants were positive by PCR testing with transformation efficiency of 0.50%. Six positive plants were randomly selected for expression analysis, and five showed GmDREB3 expression by RT-PCR assay. Integrality of inserted fragment in transgenic wheat genome was further identified in the five plants by PCR assay, and four transgenic plants had amplified products similar to the fragment transformed. Real-time PCR results showed 1–3 copies of GmDREB3 genes in the six positive T0 plants. These results indicate that the transformation stability is significantly improved by adding SAR sequences flanked with the minimal expression cassette.

Key words: SAR sequence, transformation with minimal expression cassette, DREB-like gene, Gene gun-mediated transformation

[1]Barro F, Cannell M, Lazzeri P, Barcelo P. The influence of auxins on transformation of wheat and Tritordeum and analysis of transgene integration patterns in transformants. Theor Appl Genet, 1998, 97: 684–695



[2]Cannell M, Doherty A, Lazzeri P, Barcelo P. A population of wheat and Tritordeum transformants showing a high degree of marker gene stability and heritability. Theor Appl Genet, 1999, 99: 772–784



[3]Linden R M, Winocour E, Berns K I. The recombination signals for adeno-associated virus site-specific integration. Proc Natl Acad Sci USA, 1996, 93: 7966–7972



[4]Müller A E, Kamisugi Y, Grüneberg R, Niedenhof I, Hörold R J, Meyer P. Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum. J Mol Biol, 1999, 291: 29–46



[5]Fu X, Duc L T, Fontana S, Bong B B, Tinjuangjun P, Sudhakar D, Twyman R M, Christou P, Kohli A. Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res, 2000, 9: 11–19



[6]姚琴, 丛玲, 汪越胜, 陈明洁, 杨广笑, 何光源. 无载体框架序列转基因小麦中外源基因表达框的遗传分析. 遗传, 2006, 28: 695–698



Yao Q, Cong L, Wang Y S, Chen M J, Yang G X, He G Y. Inheritance of the foreign gene 1Ax 1 in transgenic wheat (Triticum aestivum L.) with gene cassettes lacking vector backbone sequences. Hereditas (Beijing), 2006, 28: 695–698 (in Chinese with English abstract)



[7]王天云, 袁保梅, 薛乐勋. 核基质结合区与转基因沉默. 生物学通报,2004, 39(12): 15–17 (in Chinese)



Wang T Y, Yuan B M, Xue L X. Scaffold attachment region and Transgene Silence. Bull Biol, 2004, 39(12): 15–17 (in Chinese)



[8]Wang T, Xue L, Hou W, Yang B, Chai Y, Ji X, Wang Y. Increased expression of transgene in stably transformed cells of Dunaliella salina by matrix attachment regions. Appl Microbiol Biotechnol, 2007, 76: 651–657



[9]Allen G C, Hall G E, Childs L C, Weissinger A K, Spiker S, Thompson W F. Scaffold attachment regions increase reporter gene expression in stably transformed plant cells. Plant Cell, 1993, 5: 603–613



[10]Breyne P, Van Montagu M, Depicker N, Gheysen G. Characterization of a plant scaffold attachment region in a DNA fragment that normalizes transgene expression in tobacco. Plant Cell, 1992, 4: 463–471



[11]Weng H, Pan A, Yang L, Zhang C, Liu Z, Zhang D. Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol Biol Rep, 2004, 22: 289–300



[12]Thi Loc N, Tinjuangjun P, Gatehouse A M, Christou P, Gatehouse J A. Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance. Mol Breed, 2002, 9: 231–244



[13]Vain P, Worland B, Kohli A, Snape J W, Christou P, Allen G C, Thompson W F. Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny. Plant J, 1999, 18: 233–242



[14]Spiker S, Thompson W F. Nuclear matrix attachment regions and transgene expression in plants. Plant Physiol, 1996, 110: 15–21



[15]Holmes-Davis R, Comai L. Nuclear matrix attachment regions and plant gene expression. Trends Plant Sci, 1998, 3: 91–97



[16]van der Geest A H M, Hall G E Jr, Spiker S, Hall T C. The β-phaseolin gene is flanked by matrix attachment regions. Plant J, 1994, 6: 413–423



[17]Allen G C, Hall G, Michalowski S, Newman W, Spiker S, Weissinger A K, Thompson W F. High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell, 1996, 8: 899–913
No related articles found!
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
Add: No.12 Zhongguancun South Street, Beijing 100081,China Email:xbzw@chinajournal.net.cn
Supported by Beijing Magtech Co.Ltd