内含子编码蛋白Mg2+结合位点功能分析及验证

doi:10.13560/j.cnki.biotech.bull.1985.2020-0012

摘要/Abstract

摘要：

旨为筛选并构建II型内含子编码蛋白Mg²⁺结合位点突变体,验证该位点突变对II型内含子“归巢”效率的影响。利用生物信息学技术筛选关键位点,利用定点突变技术构建突变体,利用Targetron及蓝白斑计数法验证其“归巢”效率。结果显示,筛选到D308和D309两个位点是II型内含子编码蛋白Mg²⁺结合的核心催化位点,并成功构建该位点的三种突变体,包括两个单点突变体（D308A和D309A）和一个双点突变体（D308A/D309A）,大肠杆菌体内实验结果表明,三种突变体均完全失活了II型内含子的“归巢”功能。证实了II型内含子编码蛋白Mg²⁺结合位点是其发挥功能的核心催化位点。

关键词: Ⅱ型内含子, 内含子编码蛋白, Mg²⁺结合位点, Targetron, 反转录结构域

Abstract:

This work aims to screen and construct the mutants of having intron-encoded protein Mg²⁺ binding site,and to verify its effect on the “Retrohoming” efficiency of group II intron. Bioinformatics technology was used to screen key sites,site-directed mutation technology to construct mutants,and Targetron and blue-and-white spot counting methods to verify its “Retrohoming” efficiency. Results showed that 2 sites,D308 and D309,were identified as the core catalytic sites for group II intron-encoded protein Mg ²⁺ binding,and three mutants of these sites were successfully constructed,including two single-site mutants（D308A and D309A）and a double-site mutant（D308A/D309A）. The experimental results in Escherichia coli showed that all three mutants completely inactivated the “Retrohoming” function of group II intron. In conclusion,this work confirms that the Mg ²⁺ binding sites of group II intron-encoded protein is the core catalytic site of it playing function.

Key words: group II introns, intron-encoded protein, Mg²⁺ binding sites, Targetron, reverse transcription domain

崔古贞, 陈相好, 洪伟, 张峥嵘, 綦廷娜, 陈峥宏. 内含子编码蛋白Mg²⁺结合位点功能分析及验证[J]. 生物技术通报, 2020, 36(10): 165-172.

CUI Gu-zhen, CHEN Xiang-hao, HONG Wei, ZHANG Zheng-rong, QI Ting-na, CHEN Zheng-hong. Functional Analysis and Validation of Mg²⁺ Binding Sites of Intron-encoded Protein[J]. Biotechnology Bulletin, 2020, 36(10): 165-172.

图/表 7

图1 II型内含子“归巢”示意图 A：通过诱导Targetron载体表达后,获得内含子RNA及IEP,内含子RNA及IEP通过自我组装获得核糖核蛋白体复合物;B：核糖核蛋白体复合物通过碱基互补配对识别DNA靶位点;C：核糖核蛋白体复合物通过反式剪接插入到DNA靶位点其中一条链;D：核糖核蛋白体复合物利用其核酸酶活性切割DNA靶位点另一条链;E：核糖核蛋白体复合物利用其反转录酶活性以内含子RNA为模板,合成另一条链;F：机体启动DNA损伤修复机制,连接断端,从而实现II型内含子的“归巢”

表1 本研究用到的引物

Primers	Sequences（5'-3'）	Notes
lacZ-UF	CGGTCAATCCGCCGTTTGTTC	To detect the lacZ gene
lacZ-DR	GACCAGATGATCACACTCGG
IEP-F	ATCGAGGCTAGCGCTATATGCGTTGATG	To amplify the IEP fragment
IEP-R	CGTTCCAGATCTCCTTACTCGTA
D308A-F	TACGTCCGGTATGCGGCTGACTTCATTATCTCT	To mutate the D308A
D308A-R	AGCCGCATACCGGACGTATTTCAATACTTTATT
D309A-F	GTCCGGTATGCGGACGCTTTCATTATCTCTGTT	To mutate the D309A
D309A-R	AGCGTCCGCATACCGGACGTATTTCAATACTTT
D308A/D309A-F	TACGTCCGGTATGCGGCTGCTTTCATTATCTCTGTT	To mutate the D308A/D309A
D308A/D309A-R	AGCAGCCGCATACCGGACGTATTTCAATACTTTATT

表2 本研究用到的菌株及质粒

	Strains and plasmids	Features	Source or reference
Strains	E. coli DH5α	Clone strain,FˉlacZΔM15Δ（lacZYA-argF）relA1	TaKaRa
	E. coli BL21（DE3）	F-,ompT,hsdS（rBB-mB）,gal,dcm（DE3）	TaKaRa
Plasmids	pMD19T	TA clone Vector,Ampr	TaKaRa
	pSY7	Derived from pSY6,lacI,T7 promotor,Amp^r	[24-25]
	pSY7-lacZ-635s	Derived from pSY7,targeting the sense strand 635 site of lacZ in BL21（DE3）	[25]
	pSY7-lacZ-1063a	Derived from pSY7,targeting the antisense strand 1063 site of lacZ in BL21（DE3）	[25]

图2 突变型IEP蛋白构建及打靶效率检测利用PCR获得IEP的DNA产物,并与pMD19T载体连接后通过定点突变技术获得IEP反转录结构域关键催化位点突变的载体pMD19T-M-IEP;通过Nhe I和Bgl II双酶切质粒pMD19T-M-IEP及野生型Targetron载体pSY7-lacZ-635s/1063a,利用T4 DNA连接酶连接得到IEP关键氨基酸催化位点突变的Targetron载体pSY7-M-lacZ-635s/1063a;将突变型Targetron载体转入BL21（DE3）,IPTG诱导II型内含子的表达,菌落PCR结合蓝白斑筛选计数计算其“归巢”效率

图3 IEP蛋白生物信息学分析反转录结构域（RT）,成熟酶结构域（X）,DNA结构域（D）,核酸酶结构域（EN）,* Mg2+ 结合位点氨基酸

图4 突变型II型内含子功能验证 A：菌落PCR结构示意图;B：菌落PCR电泳图;M：8 000 bp DNA Marker;1：BL21-pSY7-lacZ-635s白色菌落;2：BL21-pSY7-lacZ-635s蓝色菌落;3：BL21-pSY7-M3-lacZ-635s蓝色菌落;4：BL21-pSY7-M4-lacZ-635s蓝色菌落;5：BL21-pSY7-M5-lacZ-635s蓝色菌落;6：BL21（DE3）;7：BL21-pSY7-lacZ-1063a白色菌落;8：BL21-pSY7-lacZ-1063a蓝色菌落;9：BL21-pSY7-M3-lacZ-1063a蓝色菌落;10：BL21-pSY7-M4-lacZ-1063a蓝色菌落;11：BL21-pSY7-M5-lacZ-1063a蓝色菌落;12：BL21（DE3）

图5 蓝白斑筛选及“归巢”效率比较突变 A：针对大肠杆菌lacZ-635s、lacZ-1063a位点,野生型II型内含子与突变型II型内含子通过IPTG诱导后蓝白斑筛选图;B：针对大肠杆菌lacZ-635s位点,IEP不同位点突变对II型内含子“归巢”效率的影响;C：针对大肠杆菌lacZ-1063a位点,IEP不同位点突变对II型内含子“归巢”效率的影响

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内含子编码蛋白Mg²⁺结合位点功能分析及验证

Functional Analysis and Validation of Mg²⁺ Binding Sites of Intron-encoded Protein

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