豌豆终止子<em> rbc-T</em>在转基因小麦研究中的应用

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王小婷, 黄锁, 徐兆师, 李连城, 马有志, 陈明, 闵东红. 豌豆终止子 rbc-T在转基因小麦研究中的应用 . 作物学报, 2017, 43(8): 1254-1258
WANG Xiao-Ting, HUANG Suo, XU Zhao-Shi, LI Lian-Cheng, MA You-Zhi, CHEN-Ming, MIN Dong-Hong. Application of Pisum sativum Terminator rbc-T in Wheat Transformation . ACTA AGRONOMICA SINICA, 2017, 43(8): 1254-1258 复制到剪切板

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作物学报编辑部

豌豆终止子 rbc-T在转基因小麦研究中的应用

王小婷^1,², 黄锁², 徐兆师², 李连城², 马有志², 陈明^2,^*, 闵东红^1,^*

¹西北农林科技大学农学院 / 旱区作物逆境生物学国家重点实验室, 陕西杨凌 712100

²中国农业科学院作物科学研究所 / 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作物遗传育种重点实验室, 北京100081

^* 通讯作者(Corresponding authors): 闵东红, E-mail: mdh2493@126.com, Tel: 13609123593; 陈明, E-mail: chenming02@caas.cn, Tel: 010-82108750

第一作者联系方式: E-mail: daisy19920829@foxmail.com

收稿日期:2017-01-20 接受日期:2017-04-20 网络出版日期:2017-04-27

基金:本研究由国家转基因生物新品种培育重大专项(2014ZX08002-003B, 2016ZX08002002, 2016ZX08002005)资助

摘要

目前常用的转基因载体元件大多为真菌或细菌来源, 引发对转基因食品安全性的担忧。本研究克隆了豌豆终止子 rbc-T, 并用其替换农杆菌终止子 nos-T构建转化载体, 利用基因枪法将携带Ubi启动子、 GUS基因和终止子 rbc-T组成的最小表达框片段转化普通小麦, 同时以带有 nos-T终止子的载体为对照, 经过PCR检测筛选获得T₂代稳定遗传的转基因小麦株系。GUS组织化学染色及酶活定量分析结果表明, 带有 nos-T及 rbc-T的转基因小麦中均能检测到 GUS基因不同程度的表达。因此认为, 豌豆终止子 rbc-T可以代替农杆菌终止子 nos-T用于小麦的转基因研究。

关键词: 胭脂碱合酶终止子( nos-T); 豌豆终止子( rbc-T); 转基因小麦; GUS报告基因

Application of Pisum sativum Terminator rbc-T in Wheat Transformation

WANG Xiao-Ting^1,², HUANG Suo², XU Zhao-Shi², LI Lian-Cheng², MA You-Zhi², CHEN-Ming^2,^*, MIN Dong-Hong^1,^*

¹College of Agronomy, Northwest A&F University / State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, China

² National Key Facility for Crop Gene Resource 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

Fund:This study was supported by the National Major Project for Developing New GM Crops (2014ZX08002-003B, 2016ZX08002002, 2016ZX08002005)

Abstract

The fungal- or bacterial-origin vector elements are commonly used in transgenic crops, which raise great concerns about the safety of GM foods. In the study, we isolated the pea ( Pisum sativum) terminator, rbc-T, and constructed a transformation vector by replacing the Agrobacteriumterminator nos-T with rbc-T.The minimum transformation fragment consisting of Ubiquitin promoter, GUS reporter gene and terminator rbc-T was transformed into common wheat by particle bombardment method. Meanwhile a minimum transformation fragment with the Agrobacterium terminator nos-T was used as the control. The T₂transgenic lines were regenerated after PCR validation of the transformation. In the transgenic lines carrying both kinds of terminators, GUS gene expression was detected by GUS histochemical staining and enzyme activity analysis, indicating that the pea terminator rbc-T is applicable as an alternative of the Agrobacterium terminator nos-T in transgenic wheat research.

Keyword: Nopaline synthase terminator ( nos-T); Pea terminator ( rbc-T); Transgenic wheat; GUS reporter gene

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终止子是给予RNA聚合酶转录终止信号的特殊DNA序列, 对于基因的表达调控具有重要作用。目前, 植物转化载体所使用的终止子主要是一些通用终止子, 常用的载体元件(如终止子、启动子等)大多为真菌或细菌来源^[1]。这些外源基因通过遗传转化整合进入植物基因组可能会增加人们对转基因植物安全性的担忧^[2]。因此, 开发利用植物来源的终止子元件就显得尤为重要。

小麦是重要的粮食作物, 转基因技术的应用有助于小麦品种改良和种质创新, 而关于植物终止子在转基因小麦中的应用研究鲜有报道。本研究选取常用报告基因GUS^{[3, 4]}为目的基因, 构建基因枪遗传转化表达载体, 采用玉米的Ubiquitin启动子及豌豆的终止子rbc-T, 以nos-T终止子为对照, 利用基因枪法转化普通小麦, 对获得的后代阳性植株进行GUS组织染色及蛋白酶活性分析, 结果证明豌豆终止子(rbc-T)在小麦体内发挥正常的转录终止活性, 且与农杆菌胭脂碱合酶终止子(nos-T)功能无明显差异。豌豆终止子rbc-T在小麦转基因研究中的应用将对小麦基因转化方法、转化效率及转基因小麦生物安全的研发和产业化产生一定的促进作用。

1 材料与方法

1.1 引物设计及载体构建

利用双元载体pKM24KH (GenBank登录号为HM036220)^[5]分别构建携带豌豆终止子rbc-T和农杆菌终止子nos-T的载体pGUS+rbc-T和pGUS+nos-T, pGUS+ rbc-T的结构和构建过程如图1。

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	图1 pGUS+rbc-T载体的构建Fig. 1 Construction of vector pGUS+rbc-T

利用在线工具(https://www.ncbi.nlm.nih.gov/tools/ primer-blast/)设计GUS序列特异性引物(F: 5′ -TTAACCAC AAACCGTTCTACTTTAC-3′ ; R: 5′ -AATCGCCGCTTTGG ACATA-3′ ), 以及豌豆终止子rbc-T扩增引物(F: 5′ -GTTC GAGTATTATGGCATTG-3′ ; R: 5′ -GTGTTTTACTCCTCAT ATTAAC-3′ )。引物合成及测序由北京奥科鼎盛生物科技有限公司完成。

利用限制性内切酶Hind III和Sac I酶切pGUS+nos-T去掉农杆菌终止子nos-T, 1%琼脂糖凝胶检测酶切产物并回收。从pKM24KH扩增豌豆终止子rbc-T, PCR产物切胶、纯化并送测序, 将测序正确的豌豆终止子片段与已回收的载体片段连接转化大肠感受态Top10, 选取阳性菌液送测序, 测序正确后提取质粒。用Kpn I和Spe I酶切pGUS+rbc-T, 将整个SAR片段从载体骨架上分离作为最小表达框片段, 1%琼脂糖凝胶检测酶切产物并回收, -20℃保存。

1.2 转基因小麦的获得及其DNA制备

参照徐琼芳等^[6]描述的基因枪转化方法和培养体系, 用pGUS+rbc-T和pGUS+nos-T转化受体小麦科农199, 对T₀代移栽成活的小麦取样进行PCR检测, 将阳性转基因株系进行温室加代培养, 经过逐代筛选并扩繁, 目前已获得稳定遗传的T₂代转基因小麦株系8个, 其中5个携带农杆菌胭脂碱合酶终止子(nos-T), 3个携带豌豆终止子(rbc-T)。以正常生长1周的T₂代转基因小麦为材料, 采用CTAB法提取植物样品DNA, 参照Lipp等^[7]描述的方法, 并稍作改动。

1.3 组织化学染色

按Jefferson等^[8]描述的方法对T₂代稳定遗传的转基因小麦株系的种子和幼苗进行GUS组织化学染色分析。取5粒小麦种子置于10 mL离心管中, 分别加入GUS检测液至完全浸没种子, 于37℃过夜避光孵育(约8 h), 无水乙醇洗脱2~3次, 肉眼观察种子染色情况, 并照相记录。将15粒小麦种子于6 cm培养皿中发芽, 长出幼根后移至水培环境中生长1周左右, 取两叶期幼苗置于50 mL离心管中, 加入检测液, 于37℃过夜(约8 h)避光孵育, 无水乙醇洗脱2~3次, 肉眼观察幼苗染色情况并照相。每个材料3次重复。

1.4 GUS蛋白酶活性分析

参考Kevan等^[9]所报道的方法分析GUS蛋白酶活性, 试验步骤略作改动。生长2周左右的幼苗取样后分作两组, 一组为整株用锡箔纸包裹, 经液氮速冻后保存于-80℃冰箱, 用于不同转基因株系间的比较; 另一组先按根、茎、叶分样, 再用锡箔纸包裹, 液氮速冻后于-80℃冰箱保存, 用于比较同一株系不同组织中的酶活性。试验均设3个生物学重复。

2 结果与分析

2.1 转基因植株的获得

以科农199为转化受体, 农杆菌终止子nos-T轰击1000个幼胚, 豌豆终止子rbc-T轰击1200个幼胚。前者共获得再生植株52株, 移栽成活36株, 对成活再生植株进行PCR检测共获得T₀代的阳性转基因植株12株, 阳性率为33.33%; 后者共获得再生植株56株, 移栽成活28株, 对成活再生植株进行分子检测获得T₀代阳性转基因6株, 阳性率为21.43%。对T₁代转基因株系进行检测, 已获得8个稳定转化株系, 其中携带农杆菌终止子(nos-T)的转基因小麦株系5个, 携带豌豆终止子(rbc-T)的转基因株系3个, 温室扩繁至T₂代用于后续试验研究。

2.2 PCR检测阳性植株

以携带不同终止子的8个转基因小麦株系的DNA为模板进行PCR扩增, 在两种终止子背景下均成功检测到大小为585 bp的GUS基因片段(图2), 阴性对照中均无目标带, 说明通过基因枪法转化最小表达框可以将目标片段整合到小麦基因组中。

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图2 PCR检测T₂代转基因小麦中GUS基因
M: DL2000 marker. 1~5: 携带农杆菌终止子(nos-T)的转基因小麦株系; 6~8: 携带豌豆终止子(rbc-T)的转基因小麦株系; H₂O和KN (小麦受体科农199)为阴性对照。Fig. 2 GUS detection in T₂transgenic wheat by PCR
M: DL2000 marker; 1-5: transgenic wheat lines carrying Agrobacterium terminator (nos-T); 6-8: transgenic wheat lines carrying pea terminator (rbc-T); both H₂O and KN (Kenong 199) are negative control.

2.3 GUS酶活分析

为了鉴定豌豆终止子(rbc-T)在转基因小麦体内能否正常发挥功能, 随机取8个转基因小麦株系种子于试管内进行染色, 结果8个株系的种子均有不同程度的着色, 染色部位均集中于种胚、胚乳及腹沟, 从染色的深浅程度可以看出不同转基因株系间GUS表达水平存在差异(图3)。带有不同终止子的转基因小麦幼苗GUS染色结果相似, 染色部位均集中于根部, 由染色的深浅可以看出, GUS在根部的表达量较高, 而在叶片及茎的表达较少(图4)。

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图3 不同T₂代转基因小麦株系种子GUS染色结果
L1~L5: 携带农杆菌终止子(nos-T)的转基因小麦种子; L6~L8: 携带豌豆终止子(rbc-T)的转基因小麦种子。Fig. 3 GUS staining of seeds from different T₂ transgenic wheat lines
L1-L5: transgenic wheat seeds carrying Agrobacterium terminator (nos-T); L6-L8: transgenic wheat seeds carrying pea terminator (rbc-T).

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图4 不同T₂代转基因小麦株系幼苗GUS染色结果
L1~L5: 携带农杆菌终止子(nos-T)的转基因小麦幼苗; L6~L8: 携带豌豆终止子(rbc-T)的转基因小麦幼苗。Fig. 4 GUS staining of seedlings from different T₂ transgenic wheat lines
L1-L5: transgenic wheat seedlings carrying Agrobacterium terminator (nos-T); L6-L8: transgenic wheat seedlings carrying pea terminator (rbc-T).

GUS酶活分析发现不同转基因小麦株系GUS酶活高低不同, 尤其是携带农杆菌终止子(nos-T)的转基因小麦株系间差异较大, 甚至达到极显著水平; 携带豌豆终止子(rbc-T)的株系间变化不明显, 且整体维持在一个稳定的表达水平(图5-a); 在5个携带农杆菌终止子(nos-T)的转基因小麦中, GUS在根中的表达量均高于茎和叶, 茎和根部的GUS酶活在L1、L2和L4株系间达到极显著差异水平, 而在叶中无明显差异; 在携带豌豆终止子(rbc-T)的转基因小麦中, 根、茎、叶中的GUS酶活差异较小, 且叶中略高(图5-b)。这与图4中GUS组织化学染色结果不尽相同, 推测可能是取样时间不同造成的。

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图5 不同T₂代转基因小麦株系GUS酶活比较
(a)不同转基因小麦株系的GUS酶活; (b)同一转基因小麦株系不同组织的GUS酶活。L1~L5: 携带农杆菌终止子(nos-T)的转基因小麦株系; L6~L8: 携带豌豆终止子(rbc-T)的转基因小麦株系。误差线表示标准误(n = 3), 误差线上不同字母表示株系间差异显著(P< 0.01)。Fig. 5 GUS activity comparison of T₂ transgenic wheat lines
(a) GUS activities in different transgenic wheat lines; (b) GUS activities in different tissues of the same transgenic wheat line. L1-L5: transgenic wheat lines carrying Agrobacterium terminator (nos-T); L6-L8: transgenic wheat lines carrying Pisum sativum terminator (rbc-T). Error bars represent standard errors (n = 3) and different letters above the error bar indicate significant difference among transgenic lines (P< 0.01).

综上, 利用基因枪转化法可以有效地将带有豌豆终止子rbc-T的GUS最小表达框转入植物体内, rbc-T在转基因小麦中可以发挥功能, 且与常用的nos-T相比没有明显效果差异。

3 讨论

终止子是能使mRNA脱离RNA聚合酶和模板DNA 的短序列。目的基因的表达, 需要可诱导的强启动子, 而基因的关闭则需要可靠的终止子, 因此, 基因工程在很大程度上依赖目的基因转录的有效启动和终止。目前, 基因工程载体构建元件中常用的终止子序列多为非植物来源, 对植物转基因生物安全存在一定地隐患, 且种类较少, 如果在复杂系统中重复使用同样的终止子序列, 可能会因为同源重复而消失, 因此迫切需要克隆其他物种, 特别是植物来源的终止子来适应植物基因工程技术的发展。

关于植物转化载体元件的研究已有较多报道。Voigt等^[10]构建了包括582个大肠杆菌终止子在内的文库, 并从中筛选鉴定得到了近40个强终止子序列; 张发云等^[11]克隆了毛白杨的叶绿体16SrRNA基因启动子Prrn、psbA基因启动子PpsbA及其终止子TpsbA; 金双侠等^[12]获得了棉花叶绿体psbA基因启动子、终止子; 钟山等^[13]分别以鲤鱼β -actin基因终止子和生长激素基因终止子构建载体, 利用显微注射技术研究这2种不同基因终止子在促进鲤鱼生长方面的效应差异。

本研究采用玉米Ubiquitin启动子和豌豆终止子(rbc-T), 以GUS为报告基因构建最小表达框片段转化普通小麦, 通过一系列实验证明豌豆终止子rbc-T在植物表达载体里能够有效地发挥其终止功能, 为以后利用最小表达框片段转化载体进行其他作物的转基因研究奠定基础, 促进植物转基因植物产品研发和产业化。

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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[1]	李军, 瞿绍洪. 用无终止子的RFP报告基因捕获水稻终止子研究. 浙江农业学报, 2012, 24: 758-763 Li J, Qu S H. PolyA trapping by using terminatorless RFP reporter gene in rice. Acta Agric Zhejiangensis, 2012, 24: 758-763 (in Chinese with English abstract) [本文引用:1]
[2]	陈贞. 转基因作物的多重PCR检测体系的研究. 暨南大学硕士学位论文, 广东广州, 2011 Chen Z. Multiplex PCR for Detection of Genetically Modified Crops. MS Thesis of Jinan University, Guangzhou, China, 2011 (in Chinese with English abstract) [本文引用:1]
[3]	焦改丽, 孟钊红, 聂安全, 南芝润, 张换样, 李俊峰, 王娇娟, 赵俊侠, 李燕娥, 郭三堆. 花椰菜花叶病毒(CaMV)35S启动子在转基因棉花中的表达. 作物学报, 2004, 30: 1135-1139 Jiao G L, Meng Z H, Nie A Q, Nan Z R, Zhang H Y, Li J F, Wang J J, Zhao J X, Li Y E, Guo S D. Expression of CaMV35S promoter in transgenic cotton. Acta Agron Sin, 2004, 30: 1135-1139 (in Chinese with English abstract) [本文引用:1]
[4]	牛东东, 郝育杰, 荣瑞娟, 韦汉福, 兰金苹, 史佳楠, 魏健, 李雪姣, 杨烁, 奚文辉, 武鹏程, 刘丽娟, 吴琳, 刘斯奇, 尹长城, 刘国振. 转基因水稻中GUS蛋白质的检测及其表达特征. 中国农业科学, 2014, 47: 2715-2722 Niu D D, Hao Y J, Rong R J, Wei H F, Lan J P, Shi J N, Wei J, Li X J, Yang S, Xi W H, Wu P C, Liu L J, Wu L, Liu S Q, Yin C C, Liu G Z. Detection of GUS protein and its expression pattern in transgenic rice plants. Sci Agric Sin, 2014, 47: 2715-2722 (in Chinese with English abstract) [本文引用:1]
[5]	Dipak K S, Nrisingha D, Indu B M. pSiM24 Is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants. PLoS One, 2014, 9: e98988. [本文引用:1]
[6]	徐琼芳, 李连城, 陈孝, 马有志, 叶兴国, 张增艳, 徐惠君, 辛志勇. 基因枪法获得GNA转基因小麦植株的研究. 中国农业科学, 2001, 34: 5-8 Xu Q F, Li L C, Chen X, Ma Y Z, Ye X G, Zhang Z Y, Xu H J, Xin Z Y. Study on the obtaining of transgenic wheats with GNA alien gene by biolistic particle. Sci Agric Sin, 2001, 34: 5-8 (in Chinese with English abstract) [本文引用:1]
[7]	Lipp M, Brodmann P, Pietsch K, Pauwels J, Anklam E. IUPAC collaborative trial study of a method to detect genetically modified soybeans and maize in dried powder. JAOAC Int, 1999, 82: 923-928 [本文引用:1]
[8]	Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6: 3901-3907 [本文引用:1]
[9]	Kevan M A G, Angela T M, Stanislav V, Karel B, Richard J I, Jill S G. Analysis of genetically modified plant gene expression using GUS fluorimetry. Mol Biotechnol, 2000, 14: 235-239 [本文引用:1]
[10]	Chen Y J, Liu P, Alec A K N, Jennifer A N B, Kevin C, Todd P, Christopher A V. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods, 2013, 10: 659-664 [本文引用:1]
[11]	张发云, 尹维波, 胡赞民. 毛白杨叶绿体基因启动子和终止子的克隆及其功能鉴定. 农业生物技术学报, 2007, 15: 439-445 Zhang F Y, Yin W B, Hu Z M. Cloning and function identification of the promoters and terminator of Populus tomentosa chloroplast genes. J Agric Biotechnol, 2007, 15: 439-445 (in Chinese with English abstract) [本文引用:1]
[12]	金双侠, 韩杰, 刘小云, 刘冠泽, 王一娴, 唐文鑫, 张献龙. SDS-蛋白酶法分离棉花cpDNA及psbA基因启动子、终止子克隆. 分子植物育种, 2007, 5: 683-689 Jin S X, Han J, Liu X Y, Liu G Z, Wang Y X, Tang W X, Zhang X L. Chroloplast DNA extraction in cotton by SDS-proteinase method and cloning of the promoter and terminator of psbA gene. Mol Plant Breed, 2007, 5: 683-689 (in Chinese with English abstract) [本文引用:1]
[13]	钟山, 罗大极, 吴刚, 徐婧, 汪亚平, 朱作言. 两种不同终止子在转基因鲤鱼中的促生长效应. 遗传, 2009, 31: 831-836 Zhong S, Luo D J, Wu G, Xu J, Wang Y P, Zhu Z Y. The growth promoting activity of two different kinds of reconstructed terminators in transgenic common carp. Hereditas (Beijing), 2009, 31: 831-836 (in Chinese with English abstract) [本文引用:1]

2012

0.0

... 目前, 植物转化载体所使用的终止子主要是一些通用终止子, 常用的载体元件(如终止子、启动子等)大多为真菌或细菌来源^[1] ...

2011

0.0

... 这些外源基因通过遗传转化整合进入植物基因组可能会增加人们对转基因植物安全性的担忧^[2] ...

2004

0.0

焦改丽, 孟钊红, 聂安全, 南芝润, 张换样, 李俊峰, 王娇娟, 赵俊侠, 李燕娥, 郭三堆. 花椰菜花叶病毒(CaMV)35S启动子在转基因棉花中的表达. 作物学报, 2004, 30: 1135-1139

Jiao G

, Meng Z

, Nie A

, Nan Z

, Zhang H

, Li J

, Wang J

, Zhao J

, Li Y

, Guo S

. Expression of CaMV35S promoter in transgenic cotton. Acta Agron Sin, 2004, 30: 1135-1139 (in Chinese with English abstract)

The CaMV 35S promoter is the most widely used promoter for driving transgene expression in plants. It is effective driving transgene expression in both dicots and monocots. It is usually classified as constitutive promoter. However, some reports suggest that it is not expressed in all cell and tissu

Institute of Cotton Research, Shanxi Academy of Agricultural Sciences, Yuncheng 044000, Shanxi

利用GUS作为报告基因，通过GUS组织化学定位法检测棉花转化愈伤组织、体细胞胚，R0代棉花根、茎、叶、花器官以及正在发育的胚GUS基因表达情况，详细阐述了CaMV 35S启动子在棉花细胞中的表达轮廓。结果表明，在愈伤组织细胞有丝分裂和增殖过程中GUS基因能稳定表达并遗传给后代细胞；在根、茎、叶细胞中检测到GUS表达活性。在

... 本研究选取常用报告基因GUS^[3,4]为目的基因, 构建基因枪遗传转化表达载体, 采用玉米的Ubiquitin启动子及豌豆的终止子rbc-T, 以nos-T终止子为对照, 利用基因枪法转化普通小麦, 对获得的后代阳性植株进行GUS组织染色及蛋白酶活性分析, 结果证明豌豆终止子(rbc-T)在小麦体内发挥正常的转录终止活性, 且与农杆菌胭脂碱合酶终止子(nos-T)功能无明显差异 ...

2014

0.0

牛东东, 郝育杰, 荣瑞娟, 韦汉福, 兰金苹, 史佳楠, 魏健, 李雪姣, 杨烁, 奚文辉, 武鹏程, 刘丽娟, 吴琳, 刘斯奇, 尹长城, 刘国振. 转基因水稻中GUS蛋白质的检测及其表达特征. 中国农业科学, 2014, 47: 2715-2722

Niu D

, Hao Y

, Rong R

, Wei H

, Lan J

, Shi J

, Wei

, Li X

, Yang

, Xi W

, Wu P

, Liu L

, Wu

, Liu S

, Yin C

, Liu G

. Detection of GUS protein and its expression pattern in transgenic rice plants. Sci Agric Sin, 2014, 47: 2715-2722 (in Chinese with English abstract)

【Objective】 The objective of the current research is to establish a method for detecting GUS protein in transgenic rice via immunoassay and reveal the expression pattern of GUS protein driven by cauliflower mosaic virus (CaMV) 35S promoter.【Method】Genomic DNA isolated from E. coli was used as template to amplify Gus gene, the PCR product was cloned into pET30a vector, sequencing verified recombinant was transferred into E. coli expression strain BL21, recombinant protein was induced by adding IPTG and His-tag beads purified recombinant protein was used as immunogen to generate monoclonal antibody. The GUS protein-specific antibody was selected using western blot analysis. Broadford assay was used to quantify recombinant GUS protein, a standard curve of GUS protein concentration versus the signal intensity of western blot analysis was generated and the quantification of GUS protein in rice leaves was carried out by comparing its intensity with the standard curve. Total proteins were isolated from different tissues at different developmental stages, including shoot and root at seedling stage, stem, node sheath, cushion, upper, middle and lower part of leaves at tillering stage, stem, stalk, sheath, cushion, leaves, spike (1 cm, 2 cm, 10 cm and 20 cm in length, respectively) at booting state, stem, stalk, sheath, leaves and spike at flowering stage, stem, leaves, seed at different days after pollination (10 d, 20 d, 30 d and 40 d, respectively) at mature stage, embryo, endosperm and lemma at milky mature stage, whole seed, embryo, endosperm and lemma of mature seed, leaves and roots at different developmental stages. SDS-PAGE separated total proteins were detected by GUS-specific antibody to detect the abundance of GUS protein.【Result】An anti-GUS monoclonal antibody with high specificity was obtained (clone number #27), specific band was detected by western blot analysis using the antibody for GUS protein in transgenic rice or recombinant GUS protein, while no visible background signal could be detected. The lower limit for recombinant GUS protein detection was about 4 ng, and GUS protein could be detected in about 2.5% single grain sample of transgenic rice. The abundance of GUS protein in transgenic rice leaves at different stages was pretty stable, while its abundance in roots was decreased dramatically, specifically, the abundance of GUS protein in 5-leaf stage was one third less than in that at 3-leaf stage, and it was undetectable at 6-leaf stage. GUS protein accounted for about 0.02 ‰ of the fresh weight in rice leave at seedling stage. Further more, GUS protein was constitutively expressed in almost all tested samples in transgenic rice except the roots after tillering stage. However, slight different in abundance was detected among tissues, e. g. the abundance in stems and lemmas at booting and flowering stages was lower than that in leaves.【Conclusion】A applicable immunoassay method was established for GUS protein detection in transgenic rice, which is specific to the abundance of GUS protein while independent of enzymatic activity, about 0.6 mg sample is needed for the detection and results obtained from different laboratories using same reference protein can be compared. The constitutive expression of GUS protein driven by CaMV 35S promoter in transgenic rice was demonstrated.

1、河北农业大学生命科学学院，河北保定 071001； 2、北京华大蛋白质研发中心有限公司，北京 101318； 3、中国科学院北京基因组研究所基因组科学与信息重点实验室，北京 100101

【目的】建立转基因水稻中GUS蛋白质的免疫学检测方法，并了解花椰菜花叶病毒（CaMV）35S启动子驱动的GUS蛋白质在转基因水稻中的表达特征。【方法】以细菌基因组DNA为模板，PCR扩增GUS基因后克隆到表达载体pET30a中，测序验证的重组子转入大肠杆菌表达菌BL21中，IPTG诱导获得重组表达的GUS蛋白质，用HIS-tag beads纯化后作为免疫原免疫小鼠制备GUS蛋白质特异的抗体，通过免疫印迹分析筛选高特异性的单克隆抗体，用Broadford法对重组的GUS蛋白质进行定量，对不同浓度的GUS蛋白质进行免疫印迹分析，绘制检测GUS蛋白质的标准曲线，通过与标准曲线的比较对水稻叶片中GUS蛋白质进行定量分析。提取不同时期、不同部位的水稻总蛋白质，包括苗期的地上部、地下部，分蘖期的茎、茎节、叶鞘、叶枕、叶片上部、叶片中部和叶片下部，孕穗期的茎、穗轴、叶鞘、叶枕、叶片、幼穗（长度分别为1、2、10和20 cm），开花期的茎、穗轴、叶鞘、叶片、穗子，成熟期的茎、叶片、授粉后不同时期的种子（分别为授粉后10、20、30和40 d）、乳熟期的胚、胚乳和颖壳、成熟种子的全种子、胚、胚乳和颖壳以及不同时期的叶片和根部材料等。SDS-PAGE分离后用抗体检测其GUS蛋白质的丰度。【结果】筛选获得了高特异性的抗GUS单克隆抗体（编号为#27），用该抗体检测转基因水稻中及重组的GUS蛋白质均呈现特异条带，没有可见的背景信号，用本研究建立的免疫印迹方法对重组GUS蛋白质的检测下限约为4 ng，可检出转基因水稻单粒大米2.5%样品中（约0.6 mg）的GUS蛋白质。在不同时期的转基因水稻叶片中GUS蛋白质的表达丰度基本稳定，而在水稻根部的GUS丰度随生长急剧减少，5叶期根中的表达量不到3叶期的三分之一，到6叶期检测不到GUS蛋白质。在水稻苗期叶片中，GUS蛋白质约占鲜重的0.02‰。另外，除分蘖期以后的根部之外，GUS蛋白质几乎在所有的水稻组织部位中呈组成型表达，只是不同组织中的表达量略有差异，如在孕穗期和开花期的茎及颖壳中的表达量较低。【结论】建立了具有应用价值的对转基因水稻中GUS蛋白质丰度检测的免疫印记方法。该方法特异性高、样品用量少、不依赖于GUS蛋白质的酶活性、测定结果易于在不同实验室间比较。证明了35S启动子驱动的GUS蛋白质在转基因水稻中基本呈组成型表达。

2014

0.0

... 1 引物设计及载体构建利用双元载体pKM24KH (GenBank登录号为HM036220)^[5]分别构建携带豌豆终止子rbc-T和农杆菌终止子nos-T的载体pGUS+rbc-T和pGUS+nos-T, pGUS+ rbc-T的结构和构建过程如图1 ...

2001

0.0

徐琼芳, 李连城, 陈孝, 马有志, 叶兴国, 张增艳, 徐惠君, 辛志勇. 基因枪法获得GNA转基因小麦植株的研究. 中国农业科学, 2001, 34: 5-8

Xu Q

, Li L

, Chen

, Ma Y

, Ye X

, Zhang Z

, Xu H

, Xin Z

. Study on the obtaining of transgenic wheats with GNA alien gene by biolistic particle. Sci Agric Sin, 2001, 34: 5-8 (in Chinese with English abstract)

中国农业科学院作物育种栽培研究所!农业部作物遗传育种重点实验室

以小麦品种京 411作为基因枪转化的靶材料 ,取其未成熟胚诱导愈伤组织 ,经过 10 d左右培养后 ,用含有雪花莲凝集素 (Galanthus nivalis agglutinin ,GNA)和 bar基因的质粒 p BI12 1- 2轰击 80 0个胚性愈伤组织 ,在含有4mg/ L Basta溶液的培养基上进行筛选 ,分化及生根培养 ,获得 6 7棵再生植株。田间涂抹 Basta溶液 (5 0、75 m g/ L)检测和接麦蚜实验 ,提取转基因植株基因组 DNA,用扩增 GNA基因的引物经 PCR扩增 Southern杂交 ,结果表明利用基因枪转化已从 T2 代获得了 8株含有编码 bar/ GNA基因的转基因植株。

... 2 转基因小麦的获得及其DNA制备参照徐琼芳等^[6]描述的基因枪转化方法和培养体系, 用pGUS+rbc-T和pGUS+nos-T转化受体小麦科农199, 对T₀代移栽成活的小麦取样进行PCR检测, 将阳性转基因株系进行温室加代培养, 经过逐代筛选并扩繁, 目前已获得稳定遗传的T₂代转基因小麦株系8个, 其中5个携带农杆菌胭脂碱合酶终止子(nos-T), 3个携带豌豆终止子(rbc-T) ...

1999

0.0

... 以正常生长1周的T₂代转基因小麦为材料, 采用CTAB法提取植物样品DNA, 参照Lipp等^[7]描述的方法, 并稍作改动 ...

1987

0.0

... 3 组织化学染色按Jefferson等^[8]描述的方法对T₂代稳定遗传的转基因小麦株系的种子和幼苗进行GUS组织化学染色分析 ...

2000

0.0

... 4 GUS蛋白酶活性分析参考Kevan等^[9]所报道的方法分析GUS蛋白酶活性, 试验步骤略作改动 ...

2013

0.0

... Voigt等^[10]构建了包括582个大肠杆菌终止子在内的文库, 并从中筛选鉴定得到了近40个强终止子序列 ...

2007

0.0

... 张发云等^[11]克隆了毛白杨的叶绿体16SrRNA基因启动子Prrn、psbA基因启动子PpsbA及其终止子TpsbA ...

2007

0.0

金双侠, 韩杰, 刘小云, 刘冠泽, 王一娴, 唐文鑫, 张献龙. SDS-蛋白酶法分离棉花cpDNA及psbA基因启动子、终止子克隆. 分子植物育种, 2007, 5: 683-689

Jin S

, Han

, Liu X

, Liu G

, Wang Y

, Tang W

, Zhang X

. Chroloplast DNA extraction in cotton by SDS-proteinase method and cloning of the promoter and terminator of psbA gene. Mol Plant Breed, 2007, 5: 683-689 (in Chinese with English abstract)

采用SDS-蛋白酶法提取了棉花的叶绿体DNA.琼脂糖电泳及紫外分光光度分析表明,所提取的叶绿体DNA质量高.用所提取的叶绿体DNA作为模板进行RAPD扩增,获得了清晰的扩增图谱.同时以所提取的叶绿体DNA作为模板,以psbA基因启动子、终止子序列设计引物,成功地扩增出预期为312 bp和382 bp的目标片段.将目标片段克隆测序,经Blantn分析,结果表明上述片段正是psbA基因启动子、终止子序列.该方法与传统的提取方法相比省时、高效、无污染,为进一步开展棉花叶绿体基因工程奠定了良好基础.

... 金双侠等^[12]获得了棉花叶绿体psbA基因启动子、终止子 ...

2009

0.0

钟山, 罗大极, 吴刚, 徐婧, 汪亚平, 朱作言. 两种不同终止子在转基因鲤鱼中的促生长效应. 遗传, 2009, 31: 831-836

Zhong

, Luo D

, Wu

, Xu

, Wang Y

, Zhu Z

. The growth promoting activity of two different kinds of reconstructed terminators in transgenic common carp. Hereditas (Beijing), 2009, 31: 831-836 (in Chinese with English abstract)

The transgenic promoter directly affects the activity of transgenic gene. Choice of terminator has been proved to affect the activity of transgenic gene. Since the relevant theoretical research is rare, using the terminator of transgenic gene or the promoter gene to construct the transgenic vector has not come to the conclusion. In order to construct a better transgenic vector to nurture “all fish” growth hormone transgenic fish which has a rapid growth character, two different transgenic vectors were constructed separately by the terminator of β-actin gene and the terminator of growth hormone gene. After micro-injection, we obtained the P0 generation of “all fish” transgenic fish farming groups. Comparing the activities of the two different transgenic vectors, the weight distribution of the breeding groups from growth hormone gene terminator transgenic vector felt into a normal distribution; however, the weight distribution of the breeding groups originated from β-actin gene terminator exhibited a non-normal mode and in a rightist trend. The average weight of GH (Growth hormone ) gene terminator transgenic groups was significantly higher than that of the β -actin gene terminator transgenic groups. Inter-estingly, a fast-growth transgenic carp was found from a mixed farming group, which was positive for the growth hormone gene terminator. This result showed that “all fish” GH gene transgenic carp could grow fast, and the transgenic gene could inherit from generation to generation. Our results suggest that the growth hormone gene terminator transgenic vector has stronger growth-promoting activity than the β-actin gene terminator transgenic vector.

1. State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 , China; 2. Department of Genetics, College of Life Science, Wuhan University, Wuhan 430072 , China; 3. Department of Genetics, Basic Medical School of Wuhan University, Wuhan 430071 , China

转基因构建体中启动子的选择会直接影响转植基因的活性, 近年来有研究表明转基因构建体中终止子的选择会一定程度地影响转植基因的活性。为了更好地筛选转基因构建体和培育快速生长的转“全鱼”生长激素(Growth hormone, GH)基因鱼, 文章用鲤鱼β-actin基因终止子和生长激素基因终止子分别构建了转基因构建体, 显微注射得到转“全鱼”GH基因鱼P0代养殖群体, 比较两种不同终止子构建体的活性。统计分析发现, 生长激素基因终止子构建体的养殖群体的体重频率呈正态分布且平均体重显著高于β-actin基因终止子构建体的养殖群体, β-actin基因终止子构建体的养殖群体的体重频率呈右倾趋势的非正态分布。值得一提的是在混合养殖组中得到一条生长最为快速的鲤鱼证实为转基因阳性且为生长激素基因终止子构建体的转基因鲤鱼。该结果表明转“全鱼”生长激素基因鲤鱼可快速生长, 并能将转植基因向下代遗传。实验结果提示生长激素基因终止子构建体比β-actin基因终止子构建体表现的促生长活性要强。

... 钟山等^[13]分别以鲤鱼#cod#x003b2 ...