CN107034204B - 一种特异性重组酶系统及其应用 - Google Patents
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Abstract
本发明公开了一种特异性重组酶，该重组酶由Cre酶中1‑244 aa的N端片段和Cre酶中245‑343 aa的C端片段重组构成的。该重组酶系统的构建方法是：1）构建含有可表达Cre酶N端片段1‑244 aa的表达载体a；2）构建含有可表达Cre酶C端片段245‑343 aa的表达载体b；3）将表达载体a和表达载体b转入到同一宿主中进行表达。该重组酶系统避免了完整的Cre酶转入宿主体内引起的毒害作用，并且其在植株体内有较高的重组率，提高了植物转基因的效果。
Description
技术领域
本发明涉及生物工程领域，特别涉及一种特异性重组酶系统及其应用。
背景技术
杂种优势是提高作物产量的有效手段。从亲本自交系转基因性状纳入杂交基因组，可能会更好的改善外源基因的表达。例如，在亲本中没有活性或者有活性的基因在杂交后代中可能被激活或者失活，可诱导/可阻扼启动子可用来实现这个和目的，但用于多个外源基因时十分冗余。另一方面，位点特异性重组是另一种更高效的选择。
Cre-lox位点特异性重组酶系统被广泛应用于真核细胞进行特异的DNA重排。有343个氨基酸残基的Cre重组酶可以催化34bp的lox位点可逆的重组反应(Ow & Medberry,1995)。对于同向或者反向的lox位点，重组反应可使其间的DNA序列删除或者倒置，如果两个lox位点位于不同的分子当中，当至少其中一个分子为环形时可发生共整合，当两个分子皆为线性时发生易位。通过将两个无功能的Cre蛋白组合在一起形成一个有功能的重组酶（如通过杂交）是十分有用处的。但是这种常规的位点特异性重组酶系统存在着重组率低的缺点，因此发现一种新的重组系统或者重组方式是十分有必要的。
发明内容
本发明的目的在于公开一种特异性重组酶系统及其应用。
本发明所采取的技术方案是：
一种特异性重组酶，由Cre酶中1-244 aa的N端片段和Cre酶中245-343 aa的C端片段重组构成的。
一种特异性重组系统的构建方法，其步骤是：
1）构建含有可表达Cre酶N端片段1-244 aa的表达载体a；
2）构建含有可表达Cre酶C端片段245-343 aa的表达载体b；
3）将表达载体a和表达载体b转入到同一宿主中进行表达。
优选的，表达载体a是由载体pETDuet和Cre酶N端片段1-244 aa的表达序列构成的。
优选的，表达载体a带有筛选抗性基因。
优选的，表达载体b是由载体pRSFDuet和Cre酶C端片段245-343aa的表达序列构成的。
优选的，表达载体b带有筛选抗性基因。
特异性重组系统的构建方法在制备转基因植物中的应用。
优选的，所述植物为拟南芥，棉花，水稻，小麦，玉米，水稻。
特异性重组系统的构建方法在制备转基因微生物中的应用。
优选的，所述微生物为大肠杆菌，酿酒酵母，毕赤酵母。
本发明的有益效果是：该重组酶系统避免了完整的Cre酶转入宿主体内引起的毒害作用，并且其在植株体内有较高的重组率，提高了植物转基因的效果。
附图说明
图1中（a）为大肠杆菌和植物转化载体，PT7= T7 病毒启动子；P35S=CaMV 35S启动子；TT7= T7 病毒终止子； Tnos = nos终止子; hpt=潮霉素抗性基因; gus=β-葡萄糖醛酸酶基因；luc=萤火虫荧光素酶基因; a-f=使用的引物；p1和p2是Southern杂交实验中使用的探针（b）为原生质体瞬时转化的荧光强度测定，数值为平均值±标准差，n=9. *p <0.05, Dunnett’s t检验；（c）为8个F1代株系的Southern杂交结果M= DNA标准品，单位千碱基对； 3C=pCambia-3C-luc株系, 3N= pCambia-3N株系, W=野生型对照组；（d）为4个单拷贝F1株系的Southern杂交结果。
具体实施方式
实验例1 设计重组切割位点
分别按照以下位点设计重组切割位点，它们分别是：Asn96 (位点1)、Val116 (位点2)、Lys244 (位点3)和Asp298 (位点4)。在位点1切割可以分成1N (1-96 aa) 和 1C(97-343 aa)这两个N端和C端部分，在位点2切割可以分成2N (1-116 aa)和2C (117-343aa)这两个N端和C端部分，在位点3切割可以分成3N (1-244 aa)和3C (245-343 aa)这两个N端和C端部分，在位点4切割可以分成4N (1-298 aa)和4C (299-343 aa)这两个N端和C端部分。
设计第五种切割方式aa1-192/aa181-343（ATG起始密码子加在aa181之前）作为正对照(5N/5C)。
实验例2 设计重组切割位点
每一个N端和C端片段分别用pETDuet和pRSFDuet来表达，其中，pETDuet-N带有氨苄青霉素抗性基因，pRSFDuet-C带有卡那霉素抗性基因（图1a）。第三种质粒pACYCDuet-inv有氯霉素抗性，可用于检测位点特异性重组是否发生，这个质粒中潮霉素抗性基因插入在两个反向lox之间，如果重组反应发生，可以用PCR检测出来。用完整的Cre重组酶的在pACYCDuet-inv中表达作正对照。
将载体转入大肠杆菌进行表达。转化pETDuet-Cre正对照菌落检测到了重组反应，并且pETDuet空载没有检测到重组反应。共同转化N-Cre和 C-Cre来检测重组酶活性，一共检测了11种组合，正对照5N/5C、1N/1C、2N/2C、3N/3C、4N/4C，以及有重叠部分的组合2N/1C、3N/1C、3N/2C、4N/1C、4N/2C、4N/3C，完整Cre重组菌落占90±10%、正对照5N/5C重组菌落占67±19%，3N/3C重组菌落占75±13%，2N/1C重组菌落占63±13%。
实验例3 通过瞬时转化实验在植物中重建Cre活性
用完整的Cre重组酶作为对照，并通过聚乙二醇介导的拟南芥原生质体转化，将载体转入拟南芥原生质体二十小时后，检测位点特异性重组的活性。将完整的Cre重组酶的活性认定为1，2N/1C 和5N/5C组合只有27%和69%的Cre重组酶 活性，而3N/3C组合是完整Cre重组酶活性的2.5倍（图1b）。
实验例4
在转基因拟南芥植株中检测重组酶活性。用pCambia-3N和pCambia-3C-luc两个分别含有N-Cre和C-Cre双元载体（图1a）分别转化拟南芥，得到pCambia-3N和pCambia-3C-luc的单拷贝植株，将两种株系进行杂交，在F1代植株中，通过PCR初筛，筛选到8个株系，使用EcoRI (E) 和BstEII (B)限制性内切酶酶切基因组DNA，使用p1作为探针，对这8个备选株系进行Southern杂交实验，检测到2.5 kb的条带，说明发生了重组反应，出现4.3 kb的条带说明没有发生重组反应，两个loxP之间的条带没有删除，出现其他杂带说明基因组中可能还有其他不完整的T-DNA插入（图1c），然后我们选取出现2.5 kb单一条带的4个完全实现删除的株系，使用SphI (S)酶切，使用p2作为探针，进行确认，出现大于1.6 kb的单一条带，确认这4株都为位点特异性删除的转基因株系（图1d），以上实验说明N-Cre和C-Cre的重组酶表达系统可用于转基因植物的基因删除。
Claims (10)
1.一种特异性重组酶，其特征在于，所述重组酶由Cre酶中1-244 aa的N端片段和Cre酶中245-343 aa的C端片段重组构成的。
2.一种特异性重组系统的构建方法，其步骤是：
1）构建含有可表达Cre酶N端片段1-244 aa的表达载体a；
2）构建含有可表达Cre酶C端片段245-343 aa的表达载体b；
3）将表达载体a和表达载体b转入到同一宿主中进行表达。
3.根据权利要求2所述的特异性重组系统的构建方法，其特征在于，所述表达载体a是由载体pETDuet和Cre酶N端片段1-244 aa的表达序列构成的。
4.根据权利要求3所述的特异性重组系统的构建方法，其特征在于，所述表达载体a带有筛选抗性基因。
5.根据权利要求2所述的特异性重组系统的构建方法，其特征在于，所述表达载体b是由载体pRSFDuet和Cre酶C端片段245-343aa的表达序列构成的。
6.根据权利要求5所述的特异性重组系统的构建方法，其特征在于，所述表达载体b带有筛选抗性基因。
7.权利要求2所述的特异性重组系统的构建方法在制备转基因植物中的应用。
8.根据权利要求7所述的应用，其特征在于，所述植物为拟南芥、棉花、水稻、小麦、玉米或水稻。
9.权利要求2所述的特异性重组系统的构建方法在制备转基因微生物中的应用。
10.根据权利要求9所述的应用，其特征在于，所述微生物为大肠杆菌、酿酒酵母或毕赤酵母。
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| CN107034204B true CN107034204B (zh) | 2019-08-16 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1413586A1 (en) * | 2002-10-21 | 2004-04-28 | Centre National De La Recherche Scientifique (Cnrs) | Regulation of the Cre recombinase using a dissociation/re-association system of said recombinase |
| CN103146818A (zh) * | 2013-02-18 | 2013-06-12 | 西南大学 | 一种改造重组酶Cre的方法及其在植物中的应用 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1413586A1 (en) * | 2002-10-21 | 2004-04-28 | Centre National De La Recherche Scientifique (Cnrs) | Regulation of the Cre recombinase using a dissociation/re-association system of said recombinase |
| CN103146818A (zh) * | 2013-02-18 | 2013-06-12 | 西南大学 | 一种改造重组酶Cre的方法及其在植物中的应用 |
Non-Patent Citations (2)
| Title |
|---|
| Cre重组酶结构与功能的研究进展;王立霞等;《生物工程学报》;20020923;第18卷(第5期);第531-534页 * |
| Recombination System Based on Cre α Complementation and Leucine Zipper Fusions;Seidi, A等;《APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY》;20081111;第158卷(第2期);第334-341页 * |
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