Establishment of a Transformant-specific Detection Method for Cry1Ac-2A-gna Transgenic Sugarcane BCG-17

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

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

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

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

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

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

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

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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