转GbVe1基因在棉花基因组中的整合与定位分析

doi:10.11963/1002-7807.ctzzbl.20190105

摘要/Abstract

摘要：

【目的】明确转GbVe1基因棉花的插入位点序列特征。【方法】Southern杂交筛选低拷贝基因插入的转基因棉花株系,以hiTAIL-PCR（Polymerase chain reaction）获取其T-DNA侧翼序列,然后根据获得的T-DNA侧翼序列设计特异PCR引物,验证插入位点的准确性。【结果】Southern杂交候选了T-DNA低拷贝插入的3个转基因棉花株系,hiTAIL-PCR分离到RB端侧翼序列（119~1 018 bp）、LB端侧翼序列（243~516 bp）; 侧翼序列的AT碱基含量在63%以上。转基因株系7/100826-152和12/100826-393插入位点都位于Gohir.D01G157600.1内含子上。转基因株系1/w-ch14插入位点分别位于Gohir.D01G157600.1内含子和A12染色体的基因间隔区中。T-DNA在Gohir.D01G157600.1内含子的插入事件造成了21 bp碱基的基因组序列缺失。T-DNA到侧翼序列的PCR产物证明Gohir.D01G157600.1上的插入位点真实可靠。【结论】hiTAIL-PCR获取了转GbVe1基因棉花的T-DNA侧翼序列,提供了T-DNA插入位点位于Gohir.D01G157600.1基因内含子的特异性检测引物。

关键词: 侧翼序列; GbVe1; 棉花; 插入位点

Abstract:

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.

Key words: flanking sequence; GbVe1; Gossypium hirsutum; insertion site

陈天子,凌溪铁,杨郁文,张保龙. 转GbVe1基因在棉花基因组中的整合与定位分析[J]. 棉花学报, 2019, 31(1): 1-11.

Chen Tianzi,Ling Xitie,Yang Yuwen,Zhang Baolong. The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)[J]. Cotton Science, 2019, 31(1): 1-11.

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链接本文: http://journal.cricaas.com.cn/Jweb_mhxb/CN/10.11963/1002-7807.ctzzbl.20190105

http://journal.cricaas.com.cn/Jweb_mhxb/CN/Y2019/V31/I1/1

图/表 8

图1

表1

本研究中所用的引物"

引物名称 Primer name	引物序列 Sequences of primers	引物位置 Location of primers
LAD1-1	5'-ACGATGGACTCCAGAGCGGCCGC(G/C/A)N(G/C/A)NNNGGAA-3'	无
LAD1-2	5'-ACGATGGACTCCAGAGCGGCCGC (G/C/T)N(G/C/T)NNNGGTT-3'	无
LAD1-3	5'-ACGATGGACTCCAGAGCGGCCGC (G/C/A)(G/C/A)N(G/C/A)NNNCCAA-3'	无
LAD1-4	5'-ACGATGGACTCCAGAGCGGCCGC (G/C/T)(G/A/T)N(G/C/T)NNNCGGT-3'	无
AC1	5'-ACGATGGACTCCAGAG- 3'	无
LB35STR-1	5'- AAAACCAAAATCCAGTACTAAAATCCAG -3'	LB端35S polyA(5'->3') 41-14 bp,距LB 105 bp
LB35STR-2	5' -CGCTCATGTGTTGAGCATATAAGAAACCCT- 3'	LB端35S polyA(5'->3') 124-95 bp,距LB 186 bp
LB35STR-3C1	5'-ACGATGGACTCCAGTCCGGCCAATAATGTGTGAGTAGTTCCCAGATAAGGG-3'	LB端35S polyA(5'->3') 176-147 bp,距LB 238 bp
LB35STR-4	5' -CTCCATAATAATGTGTGAGTAGTTCCCAGATAAGG -3'	LB端35S polyA(5'->3') 182-148 bp,距LB 239 bp
RB-2a	5'-GTAATGCATGACGTTATTTATGAGATGGGTT- 3'	RB端nos 终止子,距RB 175 bp
RB-0a	5'-GGCAATAAAGTTTCTTAAGATTGAATCCTGT- 3'	RB端nos 终止子,距RB 272 bp
RBGUS-4C	5'-ACGATGGACTCCAGTCCGGCCCAAGCTAGCCACCACCACCACCACCAC- 3'	GUS CDS(5'->3') 1816-1842 bp,距RB 380 bp
RBGUS-2	5' -GCGTTGGCGGTAACAAGAAAGGGAT- 3'	GUS CDS(5'->3') 1694-1718 bp,距RB 501 bp
GUS-1F	5'-CAGCGCGAAGTCTTTATACCGAAAGGTTGGGCAGG- 3'	GUS CDS(5'->3') 432-457 bp
GUS-1R	5'-ACTGCTTTTTCTTGCCGTTTTCGTCGGTAATCACC- 3'	GUS CDS(5'->3') 690-656 bp
GUS-2F	5'-GCCGGGAATGGTGATTACCGACGAAAACGGCAAGA- 3'	GUS CDS(5'->3') 649-681 bp
GUS-2R	5'-GACATGCGTCACCACGGTGATATCGTCCACCCAG- 3'	GUS CDS(5'->3') 794-761 bp
Gbvdr-1F	5'-GGTTGTTGCGTCAACTTGAAAGCAGCTTCAGCTAC- 3'	Gbve1 CDS(5'->3') 126-160 bp
Gbvdr-1R	5'-CCCGTAGCTGATATATTTACTCCATCAAGATAGAG- 3'	Gbve1 CDS(5'->3') 618-584 bp
Gbvdr-2F	5'-CTTTGAAGGGCGAATACCAGAAGTCATTGGAAC- 3'	Gbve1 CDS(5'->3') 2 645-2 879 bp
Gbvdr-2R	5'-CAAATCCCACCCCCAGGAATATGAACAGCCAATCT- 3'	Gbve1 CDS(5'->3') 3 059-3 025 bp
nptii-1F	5'-TCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATG- 3'	nptii CDS(5'->3') 36-70 bp
nptii-1R	5'-GCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGG- 3'	nptii CDS(5'->3') 197-164 bp
R4	5'-GTATTTGGGGCTTTTTTGGACGAATCATTTCCGGC - 3'	见图4示意
R2	5'-GGCAATATATAAACTGGTACATCTCTACCATCATTTT- 3'	见图4示意
R1	5'-AATATTTTGTGCTTTGAGCCGCAAAGCTGCATAAT - 3'	见图4示意
S11	5'-AAGCTATCCAAATTGGCCAACCTTGCAATGACCTC- 3'	见图4示意
S21	5'-TATTAGTTCACCTACTTTGCAACCAACACTCACT- 3'	见图4示意

表1

图2

图3

表2

表3

图4

图5

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片段^a Fragments^a	长度^b Length^b /bp	T-DNA^c T-DNA^c	未知序列^d Unknown sequence^d /bp	侧翼序列 ^e Flanking sequence^e /bp
1	1 300	N	605,1 015	0
2	450	Y	119,216	119,216
3	1 200	Y	1 018	1 018
4	470	Y	180	180
5	1 500	Y	1 207	1 207
6	1 000	Y	780	780
7	1 200	Y	1 018	1 018
8	1 200	Y	1 008,1 010	1 008,1 010
9	1 200	Y	1 007,1 009	1 007,1 009
10	750	Y	513,516	513,516
11	250	N	0	0
12	250	N	0	0
13	700	Y	516	516
14	250	N	0	0
15	700	Y	243,516	243,516
16	500	N	0	0

检索项 Query id	目标项 Subject id	相同率 Identity /%	比对长度 Alignment length /bp	错配Mismatches /bp	空缺 Gap opens /bp	检索项起始位置 Query start position /bp	检索项终止位置 Query end position /bp	目标项起始位置 Subject start position /bp	目标项终止位置Subject end position /bp	E	分值 Bit score
15-1	D01 G. hirsutum_er	100	243	0	0	57	299	53 897 337	53 897 095	1.39E-120	439
15-1	A01 G. hirsutum_er	84.364	275	11	2	57	299	90 666 064	90 665 790	2.69E-85	322
15-2	D01 G. hirsutum_er	99.612	516	2	0	238	753	53 897 337	53 896 822	0	922
15-2	A01 G. hirsutum_er	90.328	548	21	2	238	753	90 666 064	90 665 517	0	769
8-1	D01 G. hirsutum_er	99.008	1 008	8	2	59	1 065	53 898 365	53 897 359	0	1 766
8-1	A01 G. hirsutum_er	89.877	1 057	49	6	59	1 065	90 667 134	90 666 086	0	1 449
8-2	D01 G. hirsutum_er	99.307	1 010	6	1	56	1 064	53 898 368	53 897 359	0	1 786
8-2	A01 G. hirsutum_er	89.906	1 060	48	6	56	1 064	90 667 137	90 666 086	0	1 456
9-1	D01 G. hirsutum_er	99.603	1 007	4	0	59	1 065	53 898 365	53 897 359	0	1 799
9-1	A01 G. hirsutum_er	90.255	1 057	45	6	59	1 065	90 667 134	90 666 086	0	1 467
9-2	D01 G. hirsutum_er	99.504	1 009	3	1	59	1 067	53 898 365	53 897 359	0	1 795
9-2	A01 G. hirsutum_er	90.35	1 057	46	5	59	1 067	90 667 134	90 666 086	0	1 474