山新杨蔗糖合酶基因PCR介导的重组现象研究

doi:10.12302/j.issn.1000-2006.202009006

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南京林业大学学报（自然科学版） ›› 2021, Vol. 45 ›› Issue (3): 79-86.doi: 10.12302/j.issn.1000-2006.202009006

山新杨蔗糖合酶基因PCR介导的重组现象研究

葛宝柱(), 徐强, 陈赢男^*()

南京林业大学,南方现代林业协同创新中心,林木遗传与生物技术省部共建教育部重点实验室, 江苏南京 210037

收稿日期:2020-09-02 修回日期:2021-02-22 出版日期:2021-05-30 发布日期:2021-05-31
通讯作者: 陈赢男
基金资助:
国家自然科学基金项目(32071795);国家自然科学基金项目(31800562);江苏高校优势学科建设工程资助项目(PAPD)

The phenomenon of PCR-mediated recombination by using SUS genes of Populus davidiana×P. bolleana

GE Baozhu(), XU Qiang, CHEN Yingnan^*()

Co-Innovation Center for the Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics and Biotechnology of Ministry of Education, Nanjing Forestry University, Nanjing 210037, China

Received:2020-09-02 Revised:2021-02-22 Online:2021-05-30 Published:2021-05-31
Contact: CHEN Yingnan

摘要/Abstract

摘要：

【目的】山新杨(Populus davidiana×P. bolleana)是林木基因工程育种基础和应用研究的良好材料;以山新杨蔗糖合酶基因(PdbSUS)为例研究聚合酶链式反应(polymerase chain reaction,PCR)过程介导的重组现象,在此基础上区分每一个PdbSUS基因的两种单倍型,为后续研究提供准确的序列信息,并为判断木本植物基因真实的单倍型遗传信息提供参考。【方法】分别采集番茄(Lycopersicon esculentum)和山新杨新鲜嫩叶,以番茄基因组为内参,利用流式细胞仪测定山新杨基因组大小及其倍性水平。参考毛果杨(P. trichocarpa)蔗糖合酶序列设计引物,分别以山新杨基因组DNA和cDNA为模板进行同源克隆,将PCR扩增产物回收纯化,连接测序载体并转化大肠杆菌感受态细胞,挑取阳性克隆进行菌液PCR验证,将整合有外源片段的转化子进行一代测序。利用SnapGene及DNAMAN软件对测序结果进行分析比对。【结果】山新杨为二倍体植物,获得了7个PdbSUS基因的全长序列,并分别在基因组和转录水平鉴定出每个基因的两种单倍型序列。同时,检测到PCR介导的重组现象并通过限制性酶切多态性(RFLP)进行了验证。在测序的209个克隆中,发现有18个含有嵌合产物,占比8.6%,不同基因产生嵌合产物的概率为0~33.3%。检测到的嵌合产物均来自同一个位点的不同等位基因之间发生重组,未发现不同蔗糖合酶基因之间发生重组的现象。【结论】PCR介导的重组是PCR反应过程中能够衍生重组分子的一种高频事件,该现象可能是由于引物延伸不完全或聚合酶模板转换导致。在利用PCR技术克隆木本植物或其他基因组杂合度较高物种的基因时,需识别产物中重组分子才能够准确区分单倍型的遗传信息。

关键词: 山新杨, 蔗糖合酶基因, PCR介导重组, 模板转换, 嵌合产物, 重组分子

Abstract:

【Objective】 Populus davidiana × P. bolleana (Shanxin poplar) is an ideal material for basic and applied research in tree genetic engineering. This study aimed to determine the ploidy level of Shanxin poplar, reveal the phenomenon of PCR-mediated recombination via a case study of PdbSUS genes, and define two haplotypes of each gene to provide precise sequence information for subsequent genetic engineering studies and serve as a reference for cloning and determining the haplotypes of genes in woody plants. 【Method】 Fresh young leaves of tomato (Lycopersicon esculentum) and Shanxin poplar were separately collected. The genome size and ploidy level of Shanxin poplar were analyzed by influx flow cytometry, using the tomato genome as an internal reference. Primers designed based on the sequences of sucrose synthase (SUS) genes in P. trichocarpa, were used to amplify the full-length genomic DNA and the cDNA sequences of SUS genes in Shanxin poplar. The PCR amplicons were visualized on 1% agarose gels by GelRed staining, then appropriate bands were excised from the gels and purified using the AxyPrep ^TM DNA Gel Extraction Kits. All purified products were separately ligated to the pEASY-Blunt vector and transformed into Escherichia coli strain DH5α competent cells. Colonies of E. coli were randomly selected from individual LB agar plates containing kanamycin and screened for target fragments by using colony PCR. Independent bacterial clones harboring the recombinant plasmids were sequenced using the Sanger method, then the sequences for each gene were aligned and analyzed using DNAMAN (version 8) and SnapGene (version 1.1.3) software with default parameters. 【Result】 Shanxin poplar is a diploid plant. Seven PdbSUS genes were obtained, and the haplotypes of each gene were identified at the genomic and transcriptional levels. Recombination mediated by PCR was verified by analyzing restriction fragment length polymorphisms (RFLP). Among the 209 sequenced clones, 18 harbored chimeric products, accounting for 8.6%, and the frequency of chimera generation in individual genes ranged from 0 to 33.3%. All chimeric products were recombinants derived from heterozygous alleles located at the same locus of homologous chromosomes. Chimeras of different PdbSUS genes were undetectable. 【Conclusion】 The present findings indicated that PCR-mediated recombination is not rare, but is rather a high-frequency event leading to PCR-derived recombinants, which might arise via incomplete primer extension or polymerase template switching. Thus, chimeric products should be differentiated from parental haplotypes when cloning genes from woody plants or other species with highly heterozygous genomes by using PCR.

Key words: Populus davidiana × P. bolleana (Shanxin poplar), sucrose synthase gene, PCR-mediated recombination, template-switching, chimeric product, recombinant molecule

中图分类号:

S722

葛宝柱,徐强,陈赢男. 山新杨蔗糖合酶基因PCR介导的重组现象研究[J]. 南京林业大学学报（自然科学版）, 2021, 45(3): 79-86.

GE Baozhu, XU Qiang, CHEN Yingnan. The phenomenon of PCR-mediated recombination by using SUS genes of Populus davidiana×P. bolleana[J].Journal of Nanjing Forestry University (Natural Science Edition), 2021, 45(3): 79-86.DOI: 10.12302/j.issn.1000-2006.202009006.

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项目 item		基因名称 gene name		引物 primer (5'-3')	预期产物长度/bp expected amplicon length		延伸时间/min extending time
基因组DNA全长 genomic DNA full length		PdbSUS1		F: GAATAGTGTGGTTTCTCTG	4 998		4.0
		PdbSUS1		R: CCGTGTTTCCTCCATTTCTTCAGTT	4 998		4.0
		PdbSUS2		F: CCTTTTGTTATGGAAACCTATTG	5 570		4.5
		PdbSUS2		R: GGAGGATTTGTAAAGAACAGTGC	5 570		4.5
		PdbSUS3		F: AGTGACAGTTTCCCAAT	6 000		5.5
		PdbSUS3		R: TGTGATAGTGCTGCGTCTGA	6 000		5.5
		PdbSUS4		F: GACCCTTTTCTGTTATCATTCGTT	4 727		4.5
		PdbSUS4		R: ATCAGCCTAGTTAGTTTCCTTTCTTT	4 727		4.5
		PdbSUS5		F: CGTATCAGGATTTCCAGCAGAGG	3 187		3.0
		PdbSUS5		R: ATACAATGTGCTCCCGATGAAAT	3 187		3.0
		PdbSUS6		F: TTGCGAGCACGGAAAGGA	4 541		4.5
		PdbSUS6		R: GAGGAGAGAAGAAGCCGC	4 541		4.5
		PdbSUS7		F: TCAGGGTGCCATTTAGGGTAGA	2 263		2.0
		PdbSUS7		R: GAGCAGGTGAAAGTAGAGGA	2 263		2.0
项目 item	基因名称 gene name		引物 primer (5'-3')			预期产物长度/bp expected amplicon length	延伸时间/min extending time
cDNA全长 cDNA full length	PdbSUS1-c		F: ATGGCTGAACGTGCTCTTAC			2 613	2.5
	PdbSUS1-c		R: TCACTCCTTAGTCAAAGGAA			2 613	2.5
	PdbSUS2-c		F: ATGGCTGCACTTACTCGT			2 412	2.5
	PdbSUS2-c		R: TTACTCGATAGTCAAAGGAACAGAATC			2 412	2.5
	PdbSUS3-c		F: ATGGCAAACCCTAAGCTTGA			2 436	2.5
	PdbSUS3-c		R: TTAATGCCGGTCATCGATTG			2 436	2.5
	PdbSUS4-c		F: ATGGCTTCTGCACCAGTCCT			2631	2.5
	PdbSUS4-c		R: TTATGGACTCCAGCCTTCATCTCTG			2631	2.5
	PdbSUS5-c		F: ATGAGGTTGCTTTCTGAATCACTTCAA			1 737	2.0
	PdbSUS5-c		R: CTACTTTGTCTGCTGCTTCCTG			1 737	2.0
	PdbSUS6-c		F: ATGGCAACCTTGAAGAGGTCTGA			2 472	2.5
	PdbSUS6-c		R: TTAAGTAACGTCAGGTGGCGC			2 472	2.5
	PdbSUS7-c		F: ATGGCTGGTAAACTTGGGGT			1 251	1.0
	PdbSUS7-c		R: TTAAGCTCCGAAAAACTTCTGCCA			1 251	1.0

基因名称 gene name	预期产物长度/bp expected amplicon length	实际产物长度/bp actual amplicon length		总测序克隆数 total number of sequenced clones	含嵌合产物克隆数 number of clones with chimeric products	单倍型数目 number of genotypes
基因名称 gene name	预期产物长度/bp expected amplicon length	单倍型Ⅰ Haplotype Ⅰ	单倍型Ⅱ Haplotype Ⅱ	总测序克隆数 total number of sequenced clones	含嵌合产物克隆数 number of clones with chimeric products	单倍型数目 number of genotypes
PdbSUS1	4 998	3 955	3 935	20	3	5
PdbSUS2	5 570	4 511	4 510	12	4	4
PdbSUS3	6 000	5 463	5 462	13	0	2
PdbSUS4	4 727	4 695	—	16	0	1
PdbSUS5	3 187	3 186	3 189	14	3	4
PdbSUS6	4 541	4 543	4 556	16	0	2
PdbSUS7	2 263	2 274	2 274	13	2	4
PdbSUS1-c	2 613	2 418	2 418	17	0	2
PdbSUS2-c	2 412	2 412	2 412	10	2	4
PdbSUS3-c	2 436	2 436	2 436	8	1	3
PdbSUS4-c	2 631	2 769	2 769	19	0	1
PdbSUS5-c	1 737	1 923	1 923	18	2	4
PdbSUS6-c	2 472	2 661	2 583	18	0	2
PdbSUS7-c	1 251	1 251	1 251	15	1	3
总数total number	—	—	—	209	18	—

山新杨蔗糖合酶基因PCR介导的重组现象研究

The phenomenon of PCR-mediated recombination by using SUS genes of Populus davidiana×P. bolleana

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