转Cry1Ac-2A-gna基因甘蔗BCG-17转化体特异性检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2020-1206

摘要/Abstract

摘要：

转基因作物外源T-DNA插入的侧翼序列是转基因事件检测的关键组分,也是转基因作物生物安全评价和监管所必需提供的重要信息。为了明确抗虫转基因甘蔗BCG-17的分子特征和建立其事件特异性检测方法,推进其生物安全性评价工作及未来的监管工作,以其T2代为研究材料,通过Southern 杂交检测外源基因在甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立该转化体的高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BCG-17株系;经过3-4次的热不对称PCR扩增,分离到510 bp的T-DNA左侧翼序列和915 bp的右侧翼序列;以此为基础,分别设计左右侧翼检测引物,建立了BCG-17株系的转化事件特异性PCR检测方法,左侧翼的PCR检测方法特异性强、灵敏度高,检出极限为0.1%,相当于9个单倍体甘蔗基因组拷贝数。转基因株系BCG-17的分子特征及其转化事件特异性检测的完成,为该转基因甘蔗及其衍生产品的检测和身份识别提供了技术依据。

关键词: 抗虫转基因甘蔗, T-DNA侧翼序列, 转化事件特异性检测, 染色体步移

Abstract:

The flanking sequence of T-DNA inserted into the plant’s genome is a key component of transgenic event detection,and it is also important information that must be provided for biosafety evaluation and supervision of transgenic crops. In order to clarify the molecular characteristics of insect-resistant transgenic sugarcane BCG-17 and establish its event-specific detection method,as well as to promote its biosafety assessment and future regulatory work,the T2 generation of BCG-17 was used as the study material,and the copy number of foreign genes was detected by Southern hybridization,and the flanking sequence of the insertion site were isolated by the chromosome walking technology. An efficient and sensitive specific PCR method for detecting this transformant was established. The results showed that the foreign T-DNA inserted into the BCG-17 strain was a single copy. After 3-4 times amplification of thermal asymmetric nested PCR,the left flanking sequence was 510 bp and the right flanking sequence was 915 bp;and detection primers were designed respectively based on these sequences,then the transformation event-specific PCR detection system of BCG-17 strain was established. This method presented strong specificity and high sensitivity to the left flanking,and the limit of detection was 0.1%（approximates to 9 haploid genome copies）. The completion of the molecular characteristics and the establishment of the transformation event-specific detection in the transgenic strain BCG-17 provides a technical basis for the detection and identification of the transgenic sugarcane and its derivatives.

Key words: insect-resistant transgenic sugarcane, T-DNA flanking sequence, transformation event-specific detection, chromosome walking

冯翠莲, 万玥, 王俊刚, 冯小艳, 赵婷婷, 王文治, 沈林波, 张树珍. 转Cry1Ac-2A-gna基因甘蔗BCG-17转化体特异性检测方法的建立[J]. 生物技术通报, 2021, 37(5): 248-258.

FENG Cui-lian, WAN Yue, WANG Jun-gang, FENG Xiao-yan, ZHAO Ting-ting, WANG Wen-zhi, SHEN Lin-bo, ZHANG Shu-zhen. Establishment of a Transformant-specific Detection Method for Cry1Ac-2A-gna Transgenic Sugarcane BCG-17[J]. Biotechnology Bulletin, 2021, 37(5): 248-258.

图/表 12

表1 PCR DIG探针制备所用引物

Table 1 Primers used for PCR DIG probe preparation

探针 Probe	引物 Primer	引物序列 Primer sequence（5'-3'）	退火温度Tm/℃	探针长度 Probe length/bp
BG	BG-F	CCTCGTTAGACTCAACAGCA	58	857
BG	BG-R	GCTTTATCTTTCCAGCAGTAG	58	857
bar	bar2S	CATCGTCAACCACTACATCGAGACAAGC	60	450
bar	bar3A	CCACTACATCGAGACAAGCACGGTCAAC	60	450
sta	sta-F	GCCGCCGTCTAAAAAGGTGATG	60	682
sta	sta-R	GTCGCCCTCGGGTTCTGATTC	60	682
kan	kan-F	ATCGAAAAATACCGCTGCGTAAAAG	56	637
kan	kan-R	GGACGCAGAAGGCAATGTCATAC	56	637

图1 pNUBG植物表达载体酶切位点及探针位置

Fig.1 Enzyme digestion sites and probe positions of pNUBG plant expression vector

图2 pNUBG表达载体的T-DNA结构和特异性引物的位置

Fig. 2 T-DNA structure of pNUBG expression vector and the location of specific primers

表2 巢式PCR反应所需要的特异性引物

Table 2 Specific primers required for nested PCR reactions

名称 Name	引物序列（5'-3'） Sequence（5'-3'）	Tm/℃
Lsp1	CCATCGTCAACCACTACATCGAGACA	62
Lsp2	GCCCCTGGAAGGCACGCAA	66
Lsp3	CGTGGTCGCTGTCATCGGGCT	65
Lsp4	CTTCAAGCACGGGAACTGGCAT	64
Rsp1	GCCCTAAAAAGATAAAACTGTAGAGTCCTGT	64
Rsp2	GCGCTTTATATGATTCTCTAAAACACTGATAT	60
Rsp3	CTTCAAACAAGTGTGACAAAAAAAATATGT	59
Rsp4	GTGACTGGGAAAACCCTGGCGTT	62

表3 BCG-17事件特异性PCR检测引物

Table 3 Primers for BCG-17 event-Specific PCR detection

名称 Name	引物序列 Primer sequence（5'-3’）	Tm/℃	产物长度 Fragment length/bp
LS132 LA797	CGCTCATGTGTTGAGCATATAAGAAA	58	665
LS132 LA797	GCAATCCCACGGACCCACAA	58	665
LS132 LA791	CGCTCATGTGTTGAGCATATAAGAAA	60	659
LS132 LA791	CCACGGACCCACAACTGTCTTTT	60	659
LS040 LA791	GTCCTGCCCGTCACCGAGATTT	61	751
LS040 LA791	CCACGGACCCACAACTGTCTTTT	61	751
RS139 RA467	GTGACTGGGAAAACCCTGGCGTT	56	328
RS139 RA467	CTGATGGAAGAGTGGTCAAAAGATGT	56	328
RS139 RA526	GTGACTGGGAAAACCCTGGCGTT	57	387
RS139 RA526	GCTTCACACTTGTTGGACAGGATTT	57	387
RS006 RA467	CTTCAAACAAGTGTGACAAAAAAAATATGT	55	461
RS006 RA467	CTGATGGAAGAGTGGTCAAAAGATGT	55	461

表4 BCG-17和pNUBG质粒Southern Blot杂交带大小的理论值

Table 4 Theoretical values of Southern Blot hybridization zone size in BCG-17 and pNUBG

探针 Probe	BCG-17		pNUBG
探针 Probe	限制性内切酶 Restriction enzyme	杂交带长度 Hybrid band length/bp	限制性内切酶 Restriction enzyme	杂交带长度 Hybrid band length/bp
BG	BamHI	2300	Kpn I	11225
BG	Nde I	≥4650	Kpn I	11225
bar	BamHI	1830	Kpn I	1823
bar	EcoRV	≥1000	Kpn I	1823
sta	Kpn I	/	Kpn I	11225
kan	Kpn I	/	Kpn I	11225

图3 转基因甘蔗BCG-17 Southern Blot分析 A:BG探针杂交;B:bar探针杂交;C:sta探针杂交;D:kan探针杂交。M：Marker;P：Kpn I酶切质粒;N：Nde I酶切非转基因甘蔗基因组;1：BamH I酶切;2：EcoRⅤ酶切;3：Nde I酶切;4：Hind Ⅲ 酶切

Fig. 3 Southern Blot analysis of transgenic sugarcane BCG-17 A: BG prob. B: Bar probe. C:sta probe. D: kan probe. M: DNA marker for southern blot, P: Kpn I enzyme digest plasmid, N: Nde I enzyme digest Non-GM sugarcane genome, 1: BamHI enzyme digestion, 2: EcoRⅤ enzyme digestion, 3: Nde I enzyme digestion, 4: Hind Ⅲ enzyme digestion

图4 BCG-17左右侧翼序列的巢式PCR扩增结果 A:左侧翼序列扩增;B:右侧翼序列扩增。M：DNA Marker;1：1st产物;2：2nd产物3：3rd产物;4：4th产物

Fig. 4 Nested PCR amplification of left and right flank sequence of BCG-17 A: Left flanking sequence. B: Right flanking sequence. M:DNA marker,1: Amplification for 1st, 2: Amplification for 2nd, 3: Amplification for 3rd,4: Amplification for 4th

图5 BCG-17左侧翼序列及拼接位置特征 A：BCG-17左侧翼序列;下划直线（大写字母）：T-DNA载体序列(511~840 bp), 其余：甘蔗基因组序列。B：T-DNA拼接位置特征;pUNBG：包括T-DNA左边界在内的载体序列;箭头：T-DNA转入甘蔗基因组时的断裂位置,箭头左边为载体非T-DNA区;红色序列:T-DNA左边界核心序列;下划波浪线：T-DNA转入甘蔗基因组时缺失序列;小写字母：BCG-17甘蔗基因组序列;方框：BCG-17与pUNBG的重叠载体序列

Fig. 5 Left flanking sequence of BCG-17 and the character of junction region A: The left flanking sequence of BCG-17. The underlined sequences are T-DNA (511-840 bp),the rest are the sugarcane genome sequence. B: The character of junction region. pUNBG: vector sequences including the left border of T-DNA. The arrow, the break position when the T-DNA was transferred into the sugarcane genome, the left side of the arrow is non-T-DNA region, the red sequences,the core sequence of left T-DNA border, the wavy line, the missing sequence when the T-DNA was transferred into the sugarcane genome, lowercase letters, the left flanking sequence of BCG-17 sugarcane genome, square, overlapping vector sequence of BCG-17 and pUNBG

图6 BCG-17右侧翼序列及拼接位置特征 A：BCG-17右旁侧序列;下划直线（大写字母）：T-DNA载体序列(1-318 bp), 其余（小写字母）：甘蔗基因组序列。B：T-DNA拼接位置特征;pUNBG：包括T-DNA右边界在内的载体序列;箭头：T-DNA转入甘蔗基因组时的断裂位置,箭头右边为载体非T-DNA区;红色序列:T-DNA右边界核心序列;下划波浪线：T-DNA转入甘蔗基因组时缺失序列;BCG-17：右侧翼序列,小写字母为甘蔗基因组序列;方框：BCG-17与pUNBG的重叠载体序列

Fig. 6 Right flanking sequence of BCG-17 and the character of junction region A: The right flanking sequence of BCG-17. The underlined sequences are T-DNA (1-318 bp),the rest are the sugarcane genome sequence. B: The character of junction region. pUNBG: vector sequences including the right border of T-DNA. The arrow: the break position when the T-DNA was transferred into the sugarcane genome, the right side of the arrow is non-T-DNA region, the red sequences, the core sequence of right T-DNA border. The wavy line: the missing sequence when the T-DNA was transferred into the sugarcane genome. BCG-17: The obtained right flanking sequence;lowercase letters, sugarcane genome, square,overlapping vector sequence of BCG-17 and pUNBG

图7 甘蔗BCG-17转化事件特异性PCR检测 A: 左、右侧翼特异性引物筛选,M：DNA Marker;1-3：右侧引物RS139/RA467、RS139/RA526、RS006/RA467扩增的PCR产物;4-6：左侧引物LS132/LA797、LS132/LA791、LS040/LA791扩增的PCR产物。B：转Cry1c-2A-gna基因甘蔗bar基因PCR检测;C：转Cry1c-2A-gna基因甘蔗左侧翼事件特异性PCR检测(引物LS040/LA791);D：转Cry1c-2A-gna基因甘蔗右侧翼事件特异性PCR检测(引物RS139/RA526 ),其中M：DNA Marker;1：表达载体质粒pUNBG;2：非转基因甘蔗ROC22;3-10：分别为转Cry1c-2A-gna基因甘蔗BCG-1、BCG-2、BCG-4、BCG-17、BCG-32、BCG-35、BCG-36和BCG-41

Fig. 7 Transformation event-specific PCR detection of the BCG-17 sugarcane 170A: Screening of event-specific primers for left and right flanking. M: DNA marker. 1-3: PCR products amplified by right primers RS139/RA467, RS139/RA526, RS006/RA467. 4-6: PCR products amplified by left primersLS132/LA797, LS132/LA791, LS040/LA791. B: PCR detection of bar gene in transgenic sugarcane transformed with Cry1c-2A-gna gene. C: Event-specific PCR detection of the left flanking in transgenic sugarcane transformed with Cry1c-2A-gna gene(primer pairs: LS040/LA791 ). D: Event-specific PCR detection of the right flanking in transgenic sugarcane transformed with Cry1c-2A-gna gene(primer pairs:RS139/RA526). M: DNA marker. 1: Expression vector plasmid pUNBG. 2: Non-GM sugarcane ROC22. 3-10：transgenic sugarcane transformed with Cry1c-2A-gna gene BCG-1, BCG-2, BCG-4, BCG-17, BCG-32, BCG-35, BCG-36和BCG-41 respectively

图8 左右侧翼特异性引物对PCR检测限度的测定 A:左边界特异性引物LS040/LA791对检测限度的测定;B: 右侧翼特异性引物RS139/RA526对检测限度的测定;M：DNA Marker;1-8: 转基因甘蔗BCG-17的DNA含量分别为100 %、50 %、10 %、1 %、0.5 %、0.1 %、0.05 %和0 %（非转基因甘蔗ROC22）

Fig. 8 Limitation of event-specific detection by use of specific primer pairs of left and right flanking sequences A: Limitation of event-specific detection with primer pairs of left flanking（LS040/LA791）. B: Limitation of event-specific detection with primer pairs of right flanking（RS139/RA526）. M: DNA marker. 1-8: The DNA content of transgenic sugarcane BCG-17 is 100 %, 50 %, 10 %, 1 %, 0.5 %, 0.1 %, 0.05 % and 0 % (non-GM sugarcane ROC22), respectively

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