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作物学报 ›› 2014, Vol. 40 ›› Issue (01): 63-71.doi: 10.3724/SP.J.1006.2014.00063

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

用核基质结合区(SAR)序列提高小麦最小表达框转基因表达的稳定性

苏瑞波1,2,陈明2,*,徐兆师2,李连城2,马庆1,*,马有志2   

  1. 1内蒙古农业大学农学院, 内蒙古呼和浩特 010018; 2 农作物基因资源与基因改良国家大科学工程 / 农业部麦类生物学与遗传育种重点实验室 / 中国农业科学院作物科学研究所, 北京100081
  • 收稿日期:2013-06-17 修回日期:2013-09-16 出版日期:2014-01-12 网络出版日期:2013-10-22
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)项目(2012AA10A309)和国家转基因生物新品种培育科技重大专项(2013ZX08002-002)资助。

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 Published:2014-01-12 Published online:2013-10-22

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摘要：

采用最小表达框技术转化植物可以规避由骨架序列引起的安全风险。核基质结合区序列SAR (scaffold attachment region)可作为边界元件与核基质结合阻挡转基因片段邻近染色质区的作用与影响, 提高外源基因稳定性。本研究在最小表达框序列两端添加SAR序列, 提高小麦最小表达框转基因表达的稳定性, 提高转化基因的表达效率。首先, 以GUS为目的基因构建带有SAR序列的最小表达框, 以科农199为受体进行基因枪转化, 同时以不加SAR序列的最小表达框片段为对照。带有SAR序列的最小表达框片段共轰击857个幼胚, T0代获得40株再生植株, PCR检测到16株阳性植株, 转化效率为1.87%; 对这16个阳性单株进行GUS染色, 15株显色; 从来自4个T0阳性植株的18个T1代植株中随机选取18株进行PCR和GUS染色检测, 有15株表现为阳性。不带SAR序列的对照片段轰击1012个幼胚, 获得31株再生植株, 其中5株PCR阳性, 转化效率0.49%, 这5个阳性植株中仅2株为GUS染色阳性; 来自于5个T0代PCR阳性株系的10个T1代单株中没有发现PCR和GUS染色阳性株。表明SAR序列可以提高基因枪转基因效率和目的基因表达稳定性。为了创制抗旱转基因小麦, 以来自大豆的抗旱相关转录因子基因GmDREB3为目的基因, Bar基因为筛选标记基因, 转化受体小麦济麦22, 共轰击6045个幼胚, 获得再生植株130株, PCR检测阳性植株30株, 转化效率为0.50%; 随机选取6株PCR阳性植株进行RT-PCR分析, 其中5株可检测到外源基因的转录。进一步对这5株RT-PCR阳性植株插入片段完整性进行分析, 其中4株插入片段基本完整。通过real-time PCR分析, 发现T0代6个RT-PCR阳性植株的外源GmDREB3的拷贝数为1~3个。以上结果证明, 在最小表达框两端加上SAR序列后可以提高小麦最小表达框转基因表达的稳定性。

关键词: SAR序列, 最小表达框转化法, DREB类基因, 基因枪转化法

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

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