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

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苏瑞波, 陈明, 徐兆师, 李连城, 马庆, 马有志. 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. 作物学报, 2014, 40(1): 63-71
SU Rui-Bo, CHEN Ming, XU Zhao-Shi, LI Lian-Cheng, MA Qing, MA You-Zhi. Improvement of Minimal Gene Cassette Expression Stability by Scaffold Attachment Region (SAR) Sequence in Wheat Transformation. Acta Agronomica Sinica, 2014, 40(1): 63-71 复制到剪切板

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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重大科学工程 / 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

^* 通讯作者(Corresponding authors): 马庆, E-mail:qingma66@163.com; 陈明, E-mail:chenming02@caas.cn

第一作者联系方式: E-mail:suruibo19@163.com, Tel: 010-82108750

收稿日期:2013-09-16

基金: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。

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

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

SU Rui-Bo^1,², CHEN Ming^2,^*, XU Zhao-Shi², LI Lian-Cheng², MA Qing^1,^*, MA You-Zhi²

¹Agriculture College, Inner Mongolia Agricultural University, Hohhot 010018, China

²National 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

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 study, 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 wheat variety Kenong 199. At the 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 withGUS 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 geneGmDREB3 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 showedGmDREB3 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 ofGmDREB3 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.

Keyword: SAR sequence; Transformation with minimal expression cassette; DREB-like gene; Gene gun-mediated transformation

Show Figures

基因枪转化法具有转化效率高、无宿主限制、简单易操作等优点, 在小麦遗传转化中得到广泛应用。传统的小麦基因枪转化法多以环形质粒DNA转化为主, 这种方法可能会导致载体骨架序列伴随目的基因同时整合进植物基因组。转化中所使用的环形质粒载体通常含有一些选择标记基因, 如抗生素抗性基因。这些包含抗生素抗性基因的载体骨架序列的整合, 给转基因植物带来了极大的安全隐患。Barro等^{[ 1]}和Cannell等^{[ 2]}分别对基因枪转化获得的6株转基因阳性植株进行Southern-blot分析, 发现这些阳性植株的子二代中各有3株带有氨苄抗性基因 bla。此外, 载体骨架序列中会存在一些潜在的重组热点序列, 引起外源基因不同程度的重排、断裂, 导致遗传不稳定^{[ 2]}; 质粒的复制原点会促进复制介导的基因重排^{[ 3]}; 载体骨架序列中富含A-T的序列会导致DNA片段的多聚化, 从而引起外源基因的多拷贝插入^{[ 4]}。

鉴于环状质粒的载体骨架序列在遗传转化中存在的问题, Fu等^{[ 5]}用包含启动子、编码框和终止子的DNA最小表达框轰击水稻愈伤组织并获得阳性植株, Southern-blot结果显示转基因植株含1~2个外源基因拷贝, 转基因引起的重排反应频率较低。姚琴等^{[ 6]}利用基因枪法进行小麦最小表达框转化, 虽获得了阳性植株, 但没有进一步验证目标基因表达的稳定性。目前用最小表达框转化法获得转基因小麦的成功报道还很少, 我们也发现最小表达框转化法转化小麦时存在转化基因表达稳定性差的问题。为了推进该转化技术的应用, 提高转化基因的表达效率及稳定性是关键。

核基质结合区SAR (scaffold attachment region)可作为DNA的边界元件与核基质结合, 使两个SAR之间的基因片段被界定成一个独立的染色质环状结构, 从而阻挡邻近染色质区的作用与影响, 提高外源基因的稳定性^{[ 7, 8]}。现已从真核蓝藻、酵母、动物、植物及人类中分离出多种SAR序列, 并对其功能进行了验证。采用来自真核蓝藻SAR序列构建的载体, 能够提高转基因的表达水平, 增强外源基因表达的稳定性^{[ 8]}。从酵母中扩增到SAR序列, 并在最小表达框一端或两端加上SAR序列, 采用基因枪法转化烟草悬浮细胞, 结果加SAR序列能提高目的基因的表达, 而且两端加SAR序列比对照(无SAR序列的最小表达框)目的基因的表达量高20倍以上^{[ 9]}。利用来自烟草中的SAR序列, 同样可以增强目的基因的表达^{[ 10]}。

本研究通过在最小表达框两端加SAR序列, 改进了小麦最小表达框转化技术。通过转化 GUS报告基因比较了不加SAR序列的载体与加SAR载体的转化效率及 GUS基因的表达稳定性。并且采用加SAR序列的载体转化抗逆相关基因 GmDREB3, 通过PCR的方法鉴定转基因小麦中插入片段的完整性, 利用real-time PCR方法估算转化基因的拷贝数。结果证实我们改进的最小表达框转化技术可以提高小麦基因枪转化效率, 提高目的基因在转基因小麦中的表达效率及遗传稳定性, 且片段完整性良好。改进的最小表达框转化技术为获得安全转基因小麦新品种、新种质提供了新思路。

1 材料与方法

1.1 植物材料

烟草( Nicotiana tobacumcv. W38)种植于中国农业科学院作物科学研究所人工气候温室。以科农199为受体的转 GUS基因小麦和以济麦22为受体的转 GmDREB3基因小麦种植于中国农业科学院作物科学研究所可控温室。

1.2 最小表达框表达载体的构建

以烟草W38基因组DNA为模板, 设计引物(表1)扩增SAR序列, 反应条件为94℃ 5 min; 94℃ 30 s, 57℃ 30 s, 72℃ 2 min, 循环35次; 72℃ 10 min。分别将2个SAR序列插入pBluescriptSK载体多克隆位点的5′和3′端, 构建重组载体pSAR12。用 Hind III单酶切pAHC25-GUS回收 GUS基因全长片段, 插入pSAR12构建表达载体pSGUS (图1)。为了转化抗逆相关基因, 设计引物(表1)扩增大豆转录因子基因 GmDREB3, 反应条件为94℃ 5 min; 94℃ 30 s, 58℃ 30 s, 72℃ 50 s, 循环35次; 72℃ 10 min。用 GmDREB3替换pSGUS中的 GUS基因, 构建表达载体pSHGmDREB3。对构建完成的载体均进行酶切验证和测序验证。

表1 目的基因片段及其对应扩增引物序列 Table 1 Target gene fragment and its corresponding amplification primers

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	图1 pSGUS表达载体构建Fig. 1 pSGUS expression vector construction

1.3 基因枪法转化小麦

用 SpeI和 KpnI双酶切pSGUS质粒和pSHGmDREB3质粒, 分别回收6.6 kb包含 SAR序列、启动子、 GUS报告基因、终止子和 SAR序列的线性片段和5.8 kb包含 SAR序列、启动子、 GmDREB3基因、终止子和 SAR序列的线性片段(图2); 用 EcoR I单酶切pAHC25-Bar质粒, 回收2.89 kb包含启动子、 Bar筛选基因和终止子的线性片段; 用 Hind III单酶切pAHC25-GUS, 回收4.3 kb包含启动子、 GUS基因、终止子的线性片段。将上述胶回收的线性片段产物浓缩为1 μg μL^-1, -80℃储存备用。

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	图2 pSGUS (A)和pSHGmDREB3 (B)表达载体的结构Fig. 2 Structure of pSGUS (A) and pSHGmDREB3 (B) vectors

将制备好的含 SAR序列和不含 SAR序列的 GUS线性片段和 GmDREB3线性片段, 分别与不含 SAR序列的 Bar基因线性片段按1∶1 ( V/ V)混合制弹, 进行基因枪共转化。其中 GUS基因转化的受体为科农199, GmDREB3基因转化的受体为济麦22, 金粉用量为每枪45 μg, DNA用量为每枪1 μg。轰击后将愈伤组织在原培养基上培养16~18 h, 转移到SD培养基(MS培养基的无机盐成分中添加VB₁ 1 mg L^-1、天门冬酰胺150 mg L^-1和2,4-D 2.0 mg L^-1)上恢复培养2周。将恢复培养的愈伤组织再转移到筛选分化培养基上培养至愈伤组织分化出幼苗, 将苗转移到生根培养基上继续筛选3次。壮苗后, 待植株生长到15 cm左右时移栽到花盆中, 在可控温室中培养生长, 生长光照设置长日照为 16 h光照/8 h黑暗, 短日照为 10 h光照/14 h黑暗, 室温度设置为18℃。

1.4 转 GUS基因小麦的验证

取转 GUS基因的小麦单株叶片约0.1 g, 采用植物基因组DNA提取试剂盒(TIANGEN, 北京)提取基因组DNA, 并进行 GUS基因的PCR检测。所用检测引物为t-GUS-F/R (表1), 反应条件为94℃ 5 min; 94℃ 30 s, 57℃ 30 s, 72℃ 30 s, 循环35次; 72℃ 10 min。取PCR检测为阳性的转基因植株的根、胚乳、小花置于GUS染色液, 在37℃恒温箱中静置8 h后用75%酒精漂洗5次, 用VHX-600 (Japan)和Nikon8700 (Japan)显微镜观察并照相。

1.5 转 GmDREB3基因小麦的验证及插入片段拷贝数分析

取T₀代转 GmDREB3基因的小麦单株叶片(约0.1 g)的基因组DNA进行 GmDREB3基因PCR检测, 检测引物为GmDREB3-F2/R2 (表1), PCR反应条件为94℃ 5 min; 94℃ 30 s, 56℃ 30 s, 72℃ 25 s, 循环35次; 72℃ 10 min。从PCR检测为阳性的植株中随机选取6株, 按照植物总RNA提取试剂盒(TIANGEN, 北京)的操作说明, 提取转基因阳性小麦单株的总RNA, 取5 μg总RNA反转录cDNA第一链。利用RT-PCR的方法检测目的基因 GmDREB3能否正常转录, 引物为GmDREB3-F1/R1 (表1), 反应条件为94℃ 4 min; 94℃ 30 s, 54℃ 30 s, 72℃ 50 s, 循环35次; 72℃ 10 min。为确认含SAR序列的 GmDREB3基因最小表达框在小麦基因组中的插入完整性, 将全长片段(5.3 kb)分为两段, 即5′端- SAR1+启动子(ubi)+GmDREB3和3′端-GmDREB3基因+NOS+SAR2, 对这两段分别进行扩增分析。用A1/C1或A2/C1引物(表1和图2-B)进行最小表达框5′端完整性检测, 用B1/D1引物(表1和图2-B)进行最小表达框3′端完整性检测。PCR反应条件为94℃ 8 min; 94℃ 45 s, 55℃ 30 s, 72℃ 3 min, 循环35次; 72℃ 10 min。

以小麦 puroindoline-b基因为内源参照基因, 利用real-time PCR方法^{[ 11]}确定转基因阳性植株中外源基因 GmDREB3的拷贝数。目的基因检测用引物为t₃-GmDREB3 (表1), 内标基因检测用引物为puroin- doline-b (表1)。反应体系包括正反向引物各0.5 μL、10 μL 2×SuperReal PreMix Plus、1 μL 50×Rox Reference Dye (TIANGEN, 北京)、2 μL cDNA模板和6 μL无RNase ddH₂O。将配置好的PCR体系置ABI 7500荧光定量PCR仪上, PCR反应程序为95℃ 15 min, 95℃ 15 s, 60℃ 20 s, 72℃ 32 s, 共40个循环。

2 结果与分析

2.1 pSGUS和pSHGmDREB3载体构建

用 KpnI和 SpeI双酶切pSGUS表达载体, 可得到3.0 kb载体骨架片段和6.6 kb目的片段(图3-A), 结果表明pSGUS表达载体构建正确。用 SacI和 BamH I双酶切pSGUS表达载体, 可得到2.0 kb的 GUS基因片段和7.6 kb载体骨架片段(图3-B), 表明pSGUS表达载体结构正确, 且可以通过 SacI和 BamH I双酶切位点将 GUS基因替换为 GmDREB3, 构建pSHGmDREB3表达载体。对pSGUS和pSHGmDREB3载体进行测序(结果未显示)验证, 比对分析表明序列完全正确。

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图3 pSGUS重组质粒的酶切A: KpnI和 SpeI双酶切pSGUS表达载体。M1: marker DL10000; 1: pSGUS质粒经 KpnI和 SpeI酶切; 2: pSGUS质粒; M2: marker III。B: SacI和 BamH I双酶切pSGUS表达载体。M: marker III; 1~2: pSGUS质粒经 SacI和 BamH I酶切; 3: pSGUS质粒。Fig. 3 Digested vector of recombinant plasmid pSGUSA: Vector pSGUS digested by KpnI and SpeI. M1: marker DL10000; 1: pSGUS digested by KpnI and SpeI; 2: pSGUS; M2: marker III. B: Vector pSGUS digested by SacI and BamH I. M: marker III; 1-2: pSGUS digested by SacI and BamH I; 3: pSGUS.

2.2 转 GUS基因小麦PCR检测

将含SAR序列的 GUS基因线性片段(pSGUS片段)与不含SAR序列的 GUS基因线性片段分别与 Bar基因线性片段共转化受体小麦科农199, 经过筛选后获得再生植株(图4-A), 再经壮苗培养(图4-B)和移栽(图3-C), 最终分别获得40株含SAR和31株不含SAR的再生植株。对T₀的转基因植株进行PCR检测, 结果31株不含SAR的转 GUS再生植株中, 5株为阳性, 进一步对来自这5个PCR阳性株系的10个T₁代单株进行PCR检测, 未获得阳性植株; 而含SAR的40株T₀代再生植株中, PCR检测有16株阳性植株, 从来自其中4个随机植株的18个T₁代单株中检测出15株PCR阳性植株(图5和表2)。

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	图4 GUS和pSGUS线性片段小麦转化过程A: 培养; B: 壮苗; C: 移栽。A: regeneration seedlings; B: enhancing seedlings; C: transplanted seedlings.Fig. 4 The transformation process of pSGUS and GUS linear fragments

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图5 T₀代转 GUS和pSGUS线性片段小麦PCR检测结果A: 转 GUS小麦PCR检测结果。1~2: 阳性植株; 3: 阴性对照; 4: 受体; 5: 质粒对照; M: DL2000。B: 转pSGUS小麦PCR检测结果; M: DL2000; 1~9阳性植株; 10: 阴性对照; 11: 受体; 12: 质粒对照。Fig. 5 Results of PCR detection of transgenic wheat plants transformed with GUS and pSGUS fragmentsA: PCR detection of GUS transgenic plants. 1-2: positive plants; 3: negative plant; 4: wild type; 5: plasmid control; M: DL2000. B: PCR detection of pSGUS transgenic plants. M: DL2000; 1-9: positive plants; 10: negative plant; 11: wild type; 12: plasmid control.

表2 T₀代和T₁代转 GUS片段和pSGUS片段小麦植株PCR检测结果 Table 2 Testing results of T₀and T₁generation transgenic wheat plants transformed with GUS and pSGUS fragments

2.3 GUS基因表达稳定性

对T₀代5株PCR检测为阳性的 GUS基因(不含SAR序列)植株进行GUS染色, 2株呈阳性; 对来自T₀代5个PCR阳性株系的10个T₁代单株进行GUS染色, 未获得阳性植株。对含SAR序列的T₀代16株阳性植株进行GUS染色, 获得15株阳性植株, 根部染色结果表明染色部位主要集中在根的幼嫩组织(图6-A)。随机选取来自T₀代4个PCR阳性株系的18个T₁代单株进行GUS染色, 其中15株为阳性植株。小麦胚乳和小花的染色结果表明胚乳全部染成蓝色(图6-B), 小花和颖壳基部均被染成蓝色(图6-C)。结果表明含SAR序列的 GUS片段比不含SAR序列的 GUS片段转化效率高, 而且含SAR序列的 GUS转基因小麦的遗传更加稳定。

受体为科农199。The recipient is Kenong 199.

2.4 转 GmDREB3基因小麦PCR检测及插入片段完整性检测

含SAR序列的 GmDREB3基因线性片段共轰击6045个幼胚(济麦22), 获得130株再生植株, PCR检测获得30株阳性植株(图7-A), 转化率0.50%。随机选取6株阳性植株进行RT-PCR分析, 其中5株为RT-PCR阳性(图7-B)。进一步分析RT-PCR呈阳性的5个株系插入片段的完整性, 引物A1/C1未检测到5′端全长序列, 引物A2/C1 (A2引物距SAR序列起始位点200 bp)检测出4株中扩增出3.6 kb的线性片段(图7-C)。用引物B1/D1对这4株扩增插入片段的3′端, 均检测到2.1 kb的线性片段(图7-D)。对5′端和3′端扩增出的线性片段测序验证, 结果经比对分析后均正确, 表明插入小麦基因组中的最小表达框的线性片段完整性良好。插入片段完整性检测结果(表3)显示, 5′末端SAR序列外侧有200 bp的缺失, 但不影响目的基因的稳定表达。

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图6 转pSGUS片段小麦不同部位不同时期 GUS报告基因表达检测结果A: T₀代转基因小麦根部染色; B: T₁代转基因小麦胚乳染色; C: T₁代转基因小麦小花染色。Fig. 6 Staining results of GUS reporter gene in different parts or at different stages of pSGUS transgenic wheat plantsA: root staining of T₀ plants; B: endosperm staining of T₁ plants; C: flowers staining of T₁ plants.

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图7 转 GmDREB3基因小麦分子检测结果A: PCR检测。M: DL2000; 1~6: 阳性株系; 7: 水对照; 8: 受体对照; 9: 质粒对照。B: RT-PCR检测。M: DL2000; 1~5: 阳性株系; 6: 水对照; 7: 受体对照; 8: 质粒对照。C: 插入片段5′端完整性检测。M: marker III; 1~4: 阳性株系; 5: 水对照; 6: 受体对照; 7: 质粒对照。D: 插入片段3′端完整性检测。M: marker III; 1~4: 阳性株系; 5: 水对照; 6: 受体对照; 7: 质粒对照。Fig. 7 Molecular detection results of GmDREB3 transgenic wheatA: PCR result. M: DL2000; 1-6: positive lines; 7: water control; 8: Jimai 22; 9: plasmid control. B: RT-PCR result. M: DL2000; 1-5: positive lines; 6: water control; 7: Jimai 22; 8: plasmid control. C: PCR result of 5′ terminal 5′ insertion fragment. M: Marker III; 1-4: positive lines; 5: water control; 6: Jimai 22; 7: plasmid control. D: PCR result of 3′ terminal of insertion fragment. M: marker III; 1-4: positive lines; 5: water control; 6: Jimai 22; 7: plasmid control.

表3 转 GmDREB3小麦插入片段完整性检测结果 Table 3 Test of inserting fragments in GmDREB3 transgenic plants

2.5 Real-time PCR确定转 GmDREB3基因小麦的拷贝数

利用定量PCR的方法确定转 GmDREB3基因小麦的拷贝数, 目的基因 GmDREB3的标准曲线相关性较高(图8), 起始模板数的对数值(lg C_0,GmDREB3)与 C_t值之间的相关方程为 C_t = -3.35 × lg C_0,GmDREB3 31.60。内标基因 puroindoline-b的标准曲线(图8) R²高达0.9879, 起始模板数的对数值(lg C_0,pur)与 C_t值之间的相关方程为 C_t = -3.15 × log C_0,pur + 32.09。选取6株阳性植株提取基因组DNA, 进行定量PCR分析。将获得的 GmDREB3和 puroindoline-b的 C_t值(表5)及各自标准曲线的斜率( S)和截距( I)代入方程

计算出样品起始模板数的比值^{[ 11]}。由于小麦中 puroindoline-b基因为2拷贝, 用 GmDREB3起始模板数与 puroindoline-b起始模板数的比值乘以2, 得到转基因植株中目的基因 GmDREB3的拷贝数。结果显示, 转 GmDREB3基因的6个株系中, 目的基因的拷贝数为1~3 (表4)。

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	图8 GmDREB3基因和 puroindoline-b基因的标准曲线 C_t: 阈值循环数; lg C₀: 起始模板浓度对数。Fig. 8 Standard curves of GmDREB3 and puroindoline-bgenes C_t: Threshold value of cycle number; lg C₀: Logarithm value of initial template concentration.

表4 GmDREB3和 puroindoline-b基因的 C_t值及 GmDREB3 基因拷贝数估算 Table 4 C_t values of GmDREB3 and puroindoline-b genes and estimation of copy number of GmDREB3gene in transgenic lines

3 讨论

随着转基因植物产业化的发展, 人们对转基因作物的安全性更加关注, Fu等^{[ 5]}成功地用含目的基因的线性表达框转化水稻, 获得了不含载体骨架序列的转基因植株。Nguyen等^{[ 12]}共转化 CRY1AC基因、 GNA基因和标记基因 hpt的线性表达框, 发现与转化环状质粒对照的转基因植株相比, 转化线性表达框的转基因植株可以积累更多的目标蛋白, 但二者在效率上没有显著差异。Fu等^{[ 5]}和Nguyen等^{[ 12]}都发现, 转化线性表达框的转基因植株都含有1~2个目的基因拷贝, 基因重排现象发生较少。Wang等^{[ 8]}利用电击法将含SAR序列的 CAT基因环形质粒转化到藻类细胞中, 阳性克隆中CAT的表达水平比不含SAR序列的克隆提高4.5倍, 且CAT表达水平的提高并不依赖于 CAT基因拷贝数的升高。Vain等^{[ 13]}分析转GUS质粒T₀和T₁代阳性植株中GUS的表达活性, 发现含SAR序列的转基因水稻植株中, GUS的表达更稳定, 这也说明SAR序列可以提高环形质粒转基因植株中目的基因的表达稳定性。本研究发现, 含SAR序列的 GUS基因线性表达框在T₀代的转化率为1.86%, 高于0.49%的不含SAR序列的T₀代转化率(表2)。跟踪检测T₁代GUS的表达活性, 发现不含SAR序列的阳性植株均检测不到 GUS基因的表达活性, 含SAR序列阳性植株在T₁代仍有83.33%植株显示为GUS阳性(表2和图6)。这表明SAR序列不仅可以提高基因枪法的转化率, 也可以提高最小表达框转基因阳性植株中目的基因的稳定表达。前人研究认为2个SAR之间可以形成位置效应, 提高目的基因表达的稳定性^{[ 7]}。针对这个特性在最小表达框两端分别加上SAR序列, 可以阻止邻近染色质区的顺式调控元件对环内基因影响, 而提高目的基因的稳定。这种结构可以克服植物基因组对外来基因的影响, 因为SAR使外源基因形成独立的转录单位, 使其不受周围基因的影响, 避免位置效应引起的基因失活^{[ 14, 15]}。van der Geest等^{[ 16]}发现, SAR能提高转基因烟草植株中目的基因的总表达水平, 同时可以降低转基因系间的差异, 而基因枪转化时, 转基因在受体中常以多拷贝形式存在, 整合模式较为复杂, 更易导致转基因同源依赖性沉默, SAR有可能通过减弱基因沉默所造成的负影响来提高转基因的表达效率, 促进目的基因的稳定遗传和稳定表达。鉴于SAR序列在植物遗传转化中的优点, 本研究将含SAR序列的 GmDREB3基因转化到济麦22中, 分析T₁代阳性植株中目的基因 GmDREB3的转录水平和其线性表达框的完整性, 发现阳性植株中 GmDREB3均可以正常转录, 且除线性表达框5′末端的200 bp丢失外, 其余序列均完整存在。分析转 GmDREB3基因阳性植株的拷贝数, 发现阳性植株均为1~3个拷贝(表4)。此结果证明如果采用不带有SAR序列的线性表达框进行基因枪转化, 线性表达框两端的序列可能会在转化过程中受到损伤, 这种损伤可能会影响转化片段的基因表达及遗传稳定性, 这可能就是我们转化不带有SAR序列的GUS表达框比转化带有SAR序列的表达框的遗传稳定性差的原因, 同时, 带有SAR序列的GUS最小表达框转化效率高于不带有SAR序列的表达框转化效率可能也与线性表达框的缺失相关, 具体的原因需要进一步鉴定。Allen等^{[ 17]}发现, 烟草的SAR序列与酵母的SAR序列相比, 具有更强的结合核基质的亲和力。本研究中选取的是烟草的SAR序列, 是否存在更好的SAR序列资源, 需进一步研究。

4 结论

以科农199为受体, 与不带有SAR序列的最小表达框比较, 加 SAR序列的最小表达框pSGUS转化效率明显高, 并且所转化线性片段中 GUS基因可以稳定遗传和表达。以大豆抗旱基因 GmDREB3为目的基因进行转化, 分子检测结果表明目的基因可以稳定表达, 而且插入片段基本完整, 转 GmDREB3基因株系含有较低拷贝数(1~3个)。因而认为改进的最小表达框转化技术与原技术相比可以提高小麦基因枪法转化效率, 减少目的基因的拷贝数, 提高目的基因表达和遗传的稳定性, 增加插入片段的完整性。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。

参考文献

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1998

3.297

0.0

. 1998, 97:684-695

The influence of auxins on transformation of wheat and Tritordeum and analysis of transgene integration patterns in transformants

Barro F , Cannell M , Lazzeri P , Barcelo P

Although it is possible to transform wheat, broad application of the technology is limited because of the low overall efficiency and the lack of reliability of the technique. In addition there is little published data on transgene integration patterns and inheritance in wheat. We have generated a population of transgenic wheat and tritordeum lines under different auxin regimes and show that, under the conditions described, the presence of picloram results in higher transformation efficiencies than the presence of 2,4-D. Molecular analysis shows low-copy numbers and simple integration patterns to be prevalent in the transgenic lines. Mendelian inheritance of transgenes inT₁ progeny was observed for the majority of lines.

... Barro等^[1]和Cannell等^[2]分别对基因枪转化获得的6株转基因阳性植株进行Southern-blot分析, 发现这些阳性植株的子二代中各有3株带有氨苄抗性基因bla ...

1999

3.297

0.0

. 1999, 99:772-784

A population of wheat and Tritordeum transformants showing a high degree of marker gene stability and heritability

Cannell M , Doherty A , Lazzeri P , Barcelo P

The stability and heritability of three marker genes was investigated in a population of twelve independent transgenic cereal lines (six wheat and six tritordeum). Integration patterns, inheritance of structural transgenes and inheritance of expression were analysed in the T₀ and T₁ generations for all 12 lines. Transmission and expression were analysed in the T₂ generation for 9 lines and in the T₃ generation for the six wheat lines. Inheritance of integration patterns was highly stable, and transmission of the transgenes and inheritance of their expression followed Mendelian ratios in the majority of lines. A gradual reduction inuidA expression was observed over three generations, which was not accompanied by a similar reduction inbarexpression. Some unexpected phenomena associated with transgene inheritance were also observed and are discussed.

... Barro等^[1]和Cannell等^[2]分别对基因枪转化获得的6株转基因阳性植株进行Southern-blot分析, 发现这些阳性植株的子二代中各有3株带有氨苄抗性基因bla ...

... 此外, 载体骨架序列中会存在一些潜在的重组热点序列, 引起外源基因不同程度的重排、断裂, 导致遗传不稳定^[2] ...

1996

9.737

0.0

. 1996, 93:7966-7972

The recombination signals for adeno-associated virus site-specific integration

Linden R M , Winocour E , Berns K I

The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.

... 质粒的复制原点会促进复制介导的基因重排^[3] ...

1999

4.001

0.0

. 1999, 291:29-46

Palindromic sequences and A+T-rich DNA elements promote illegitimate recombination in Nicotiana tabacum

Müller A E , Kamisugi Y , Grüneberg R , Niedenhof I , Hörold R J , Meyer P

Illegitimate recombination is the prevailing molecular mechanism for the integration of recombinant DNA into the genome of most eukaryotic systems and the generation of deletions by intrachromosomal recombination. We developed a ?selectable marker system to screen for intrachromosomal illegitimate recombination events in order to assess the sequence and structure-specific requirements for illegitimate recombination in tobacco. In 12 illegitimate recombination products analysed, we found that all deletion termini localise to sites of palindromic structures or to A+T-rich DNA elements. All deletion termini showed microhomologies of two to six nucleotides. In three plants, the recombination products contained filler-DNA or an inversion of an endogenous segment. Our data strongly suggest that illegitimate recombination in plants is mediated by a DNA synthesis-dependent process, and that this mechanism is promoted by DNA regions that can form palindromic structures or facilitate DNA unwinding.

... 载体骨架序列中富含A-T的序列会导致DNA片段的多聚化, 从而引起外源基因的多拷贝插入^[4] ...

2000

2.754

0.0

Transgenic Res. 2000, 9(1):11 - 19 DOI:10.1023/A:1008993730505

Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns

1.Molecular Biotechnology Unit, John Innes Centre
2.Biotechnology Research Center
3.Cuu Long Delta Rice Research Institute (CLRRI)
4.Tamil Nadu Agricultural University
5.Zhejiang University
6.Molecular Biotechnology Unit, John Innes Centre

Whole plasmids are used in bothAgrobacterium-mediated transformation and direct DNA transfer, generally leading to the integration of vector backbone sequences into the host genome along with the transgene(s). This is undesirable, as vector backbone sequences often have negative effects on transgene or endogenous gene expression, and can promote transgene rearrangements. We, therefore, bombarded rice tissue with two constructs: a plasmid containing thebar gene, and a linear DNA fragment isolated from the same plasmid, corresponding to the minimalbar gene expression cassette (promoter, open reading frame and terminator). We recovered phosphinothricin-resistant plants from both experiments, showing that the selectable marker was efficiently expressed. Transformation with such constructs resulted in predominantly 'simple' integration events (one or two bands on Southern blots), producing low-copy-number transgenic plants with a low frequency of transgene rearrangements. Conversely, transformation with supercoiled or linearized whole plasmids generated plants with 'complex' integration patterns, that is, higher copy numbers and frequent transgene rearrangements. We monitored transgenic lines through to the R4 generation and observed no silencing in plants carrying minimal constructs. We also carried out experiments in which rice tissue was simultaneously bombarded with minimal linearhpt andgusA cassettes. We observed robust GUS activity in hygromycin-resistant plants, confirming co-expression of the selectable and nonselectable markers. Furthermore, the efficiency of cotransformation using minimal constructs was the same as that using supercoiled plasmid cointegrate vectors.

... 鉴于环状质粒的载体骨架序列在遗传转化中存在的问题, Fu等^[5]用包含启动子、编码框和终止子的DNA最小表达框轰击水稻愈伤组织并获得阳性植株, Southern-blot结果显示转基因植株含1~2个外源基因拷贝, 转基因引起的重排反应频率较低 ...

... 3 讨论随着转基因植物产业化的发展, 人们对转基因作物的安全性更加关注, Fu等^[5]成功地用含目的基因的线性表达框转化水稻, 获得了不含载体骨架序列的转基因植株 ...

... Fu等^[5]和Nguyen等^[12]都发现, 转化线性表达框的转基因植株都含有1~2个目的基因拷贝, 基因重排现象发生较少 ...

2006

0.0

. 2006, 28:695-698

Inheritance of the foreign gene 1Ax 1 in transgenic wheat (Triticum aestivum L. ) with gene cassettes lacking vector backbone sequences

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

为了研究无载体框架序列转基因小麦中转基因表达框的遗传规律，选育稳定表达的转基因株系，利用基因枪介导1Ax1基因的最小表达框转化得到了转基因小麦，对其后代转基因植株中1Ax1基因表达框的遗传进行了分析，结果表明：无载体框架结构的1Ax1基因在转基因后代中稳定遗传，在T1代中呈现3:1的分离，遵从孟德尔遗传模式；SDS－PAGE分析表明其中部分转基因后代分离出新的高分子量蛋白亚基分子量略低于1Dx5亚基。

... 姚琴等^[6]利用基因枪法进行小麦最小表达框转化, 虽获得了阳性植株, 但没有进一步验证目标基因表达的稳定性 ...

2004

0.0

0.3175

... 核基质结合区SAR (scaffold attachment region)可作为DNA的边界元件与核基质结合, 使两个SAR之间的基因片段被界定成一个独立的染色质环状结构, 从而阻挡邻近染色质区的作用与影响, 提高外源基因的稳定性^[7,8] ...

... 前人研究认为2个SAR之间可以形成位置效应, 提高目的基因表达的稳定性^[7] ...

2007

3.425

0.0

Appl Microbiol Biot. 2007, 76(3):651 - 657 DOI:10.1007/s00253-007-1040-7

Increased expression of transgene in stably transformed cells ofDunaliella salina by matrix attachment regions

Tianyun Wang(12) Lexun Xue(1) Weihong Hou(1) Baosheng Yang(2) Yurong Chai(1) Xiang Ji(1) Yafeng Wang(1)

1.Zhengzhou University Laboratory for Cell Biology, The First Affiliated Hospital 40 Daxue Road Zhengzhou Henan 450052 People’s Republic China
2.Xinxiang Medical College Department of Biochemistry and Molecular Biology Xinxiang Henan 453003 People’s Republic China

Nuclear matrix attachment regions (MARs) are known to bind specifically to the nuclear scaffold and are thought to influence expression of the transgenes. In our previous studies, a new deoxyribonucleic acid fragment isolated fromDunaliella salina could bind to the nuclear matrix in vitro and had the typical characteristics of MARs. In this study, to investigate effects of MARs on expression of transgenes in the stably transformed cells ofD. salina, expression vectors with and without MARs, which contained chloramphenicol acetyltransferase (CAT) reporter gene driven byD.salina ribulose 1,5-bisphosphate carboxylase/oxygenase promoter, were constructed and delivered, respectively, into cells ofD. salina by electroporation. Twenty stably transformed colonies ofD. salina were randomly picked out, and CAT gene expression was assayed. The results showed that the CAT enzyme of the colonies ofD.salina transformed with the expression vector containing MARs averaged out about 4.5-fold higher than those without MARs, while the transgene expression variation among individuals of transformants decreased threefold. The CAT enzyme in the stably transformed lines was not significantly proportional to the gene copy numbers, suggesting that the effects of MARs on transgene expression may not be through increasing the transgene copy numbers.

... 采用来自真核蓝藻SAR序列构建的载体, 能够提高转基因的表达水平, 增强外源基因表达的稳定性^[8] ...

... Wang等^[8]利用电击法将含SAR序列的CAT基因环形质粒转化到藻类细胞中, 阳性克隆中CAT的表达水平比不含SAR序列的克隆提高4 ...

1993

8.987

0.0

... 从酵母中扩增到SAR序列, 并在最小表达框一端或两端加上SAR序列, 采用基因枪法转化烟草悬浮细胞, 结果加SAR序列能提高目的基因的表达, 而且两端加SAR序列比对照(无SAR序列的最小表达框)目的基因的表达量高20倍以上^[9] ...

1992

8.987

0.0

... 利用来自烟草中的SAR序列, 同样可以增强目的基因的表达^[10] ...

2004

2.453

0.0

Plant Mol Biol Rep. 2004, 22(3):289 - 300 DOI:10.1007/BF02773139

Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay withHMG I/Y as an endogenous reference gene

Haibo Weng(12) Aihu Pan(3) Litao Yang(1) Chengmei Zhang(3) Zhili Liu(1) Dabing Zhang(23)

1.Nanjing University Department of Biological Science and Technology 22 Hankou Road 210093 Nanjing PR China
2.Shanghai Jiao Tong University School of Life Science and Biotechnology 800 Dongchuan Road 200240 Shanghai PR China
3.Agro-biotech Research Center, Shanghai Academy of Agricultural Sciences Shanghai Key Laboratory of Agricultural Genetics and Breeding 2901 Beidi Road 201106 Shanghai PR China

In transgenic plants, the number of transgene copies can greatly influence the level of expression and genetic stability of the target gene. Transgene copy numbers are estimated by Southern blot analysis, which is laborious and time-consuming, requires relatively large amounts of plant materials, and may involve hazardous radioisotopes. Here we report the development of a sensitive, convenient real-time PCR technique for estimating the number of transgene copies in transgenic rapeseed. This system uses TaqMan quantitative real-time PCR and comparison with a novel, confirmed single-copy endogenous reference gene, high-mobile-group protein I/Y (HMG I/Y), to determine the numbers of copies of exogenous β-glucuronidase (GUS) and neomycin phosphotransferase II (nptII) genes. TheGUS andnptII copy numbers in primary transformants (T₀) were calculated by comparing threshold cycle (C_T) values of theGUS andnptII genes with those of the internal standard,HMG I/Y. This method is more convenient and accurate than Southern blotting because the number of copies of the exogenous gene could be directly deduced by comparing itsC_T value to that of the single-copy endogenous gene in each sample. Unlike other similar procedures of real-time PCR assay, this method does not require identical amplification efficiencies between the PCR systems for target gene and endogenous reference gene, which can avoid the bias that may result from slight variations in amplification efficiencies between PCR systems of the target and endogenous reference genes.

... 以小麦puroindoline-b基因为内源参照基因, 利用real-time PCR方法^[11]确定转基因阳性植株中外源基因GmDREB3的拷贝数 ...

... 计算出样品起始模板数的比值^[11] ...

2002

2.852

0.0

Mol Breeding. 2002, 9(4):231 - 244 DOI:10.1023/A:1020333210563

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

1.University of Durham, Crop Protection Group
2.John Innes Centre
3.University of Newcastle, Crop Protection Group

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin ( delta

-endotoxin fromBacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac andgna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressinggna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressingcry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.

... Nguyen等^[12]共转化CRY1AC基因、GNA基因和标记基因hpt的线性表达框, 发现与转化环状质粒对照的转基因植株相比, 转化线性表达框的转基因植株可以积累更多的目标蛋白, 但二者在效率上没有显著差异 ...

... Fu等^[5]和Nguyen等^[12]都发现, 转化线性表达框的转基因植株都含有1~2个目的基因拷贝, 基因重排现象发生较少 ...

1999

6.16

0.0

. 1999, 18(3):233-242

Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny

Philippe Vain^1,*,Barbara Worland¹,Ajay Kohli¹,John W. Snape¹,Paul Christou¹,George C. Allen²,William F. Thompson²

¹ John Innes Centre, Colney Lane, Norwich NR4 7UH, UK, and
² Department of Botany, North Carolina State University, Raleigh, NC 27695, USA
^**For correspondence (fax +44 1603 502241; e-mailvain@bbsrc.ac.uk).

To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing agusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T₀), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non-expressing lines compared to the population of plants without MARs. However, variation in β-glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T₁), transgene expression levels were significantly correlated with those of the T₀ parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals.

... Vain等^[13]分析转GUS质粒T₀和T₁代阳性植株中GUS的表达活性, 发现含SAR序列的转基因水稻植株中, GUS的表达更稳定, 这也说明SAR序列可以提高环形质粒转基因植株中目的基因的表达稳定性 ...

1996

6.535

0.0

... 这种结构可以克服植物基因组对外来基因的影响, 因为SAR使外源基因形成独立的转录单位, 使其不受周围基因的影响, 避免位置效应引起的基因失活^[14,15] ...

1998

11.047

0.0

. , :null-null

1994

6.16

0.0

. 1994, 6(3):413-423

The β-phaseolin gene is flanked by matrix attachment regions

Apolonia H.M. van der Geest¹,Gerald E. Hall Jr.^2,†,Steven Spiker²,Timothy C. Hall^1,*

¹ Institute of Developmental and Molecular Biology and Department of Biology, Texas As&M University, College Station, TX 77843-3155, USA
² Department of Genetics, North Carolina State University, Raleigh, NC 27695, USA
^* *For correspondence (fax + 1 409 8624098).

The β-phaseolin promoter yields high levels of spatially and temporally regulated expression in transgenic plants. It is shown that elements within 5′ and 3′ regions flanking the coding sequence bind strongly and specifically to matrix preparations from tobacco nuclei. Both of these matrix attachment regions (MARs) are A/T-rich and contain topoisomerase II, Abox, T-box and MAR motifs frequently found in MARs inDrosophila and other organisms. The β-phaseolin 5′ MAR is located on a 1047 bp fragment with a core element of 673 bp (positions −1096 to −424), that includes a 55 bp region (−682 to −628) previously shown to function as an enhancer. A 1205 bp fragment (positions +1879 to +3083) 3′ of the phaseolin coding region also showed strong and specific binding to nuclear matrix preparations and contained consensus MAR motifs. A β-phaseolin promoter-GUS fusion construct (p1470/phas) that included the MAR elements exhibited higher expression levels and lower plant-to-plant variability in stably transformed tobacco plants than did a similar construct lacking MAR sequences. Seeds of plants containing two intact copies of the insert had higher expression levels than did those of plants containing a single copy. These findings, which are the first to report a plant gene being flanked both 5′ and 3′ by MARs, are in accordance with previous observations of increased DNase I sensitivity associated with the onset of phaseolin transcription in bean and with the consistently high and position-independent expression observed in transgenic plants from the β-phaseolin promoter. The results suggest that the native β-phaseolin gene is located on a small chromatin loop that functions as an active and independent domain.

... van der Geest等^[16]发现, SAR能提高转基因烟草植株中目的基因的总表达水平, 同时可以降低转基因系间的差异, 而基因枪转化时, 转基因在受体中常以多拷贝形式存在, 整合模式较为复杂, 更易导致转基因同源依赖性沉默, SAR有可能通过减弱基因沉默所造成的负影响来提高转基因的表达效率, 促进目的基因的稳定遗传和稳定表达 ...

1996

8.987

0.0

... Allen等^[17]发现, 烟草的SAR序列与酵母的SAR序列相比, 具有更强的结合核基质的亲和力 ...