CN105296501B - Cotton plant event aC20-3 and primers and methods for detection thereof - Google Patents
Cotton plant event aC20-3 and primers and methods for detection thereof Download PDFInfo
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Abstract
The present invention provides cotton transformation event aC20-3 and its sequence, and reagents and methods for its detection that improve cotton fiber quality. Cotton plants carrying the aC20-3 event contained the event, i.e., the junction of the exogenous insertion DNA sequence and the cotton genomic DNA sequence, on chromosome D5. Using the exogenous inserted DNA sequence and the DNA sequences flanking the junction regions on the cotton genome, detection primers or probes can be designed for specific detection of the aC20-3 event. The detection method aiming at the aC20-3 event can provide a convenient means for tracking the specific gene insertion event for breeding application by utilizing the plant event, and the specific gene insertion event can be used as a molecular marker to improve the breeding work efficiency.
Description
Technical Field
The invention belongs to the field of plant molecular biology, in particular to the field of transgenic crop breeding in agricultural biotechnology research, and particularly relates to a cotton transformation event aC20-3 with improved cotton fiber quality and a special method for detecting the transformation event.
Background
The cotton cultivars are mainly upland cotton, and the superior fiber quality genes are mainly derived from diploid septoria cotton (fiber strength and fineness), abnormal cotton (fiber strength and fineness) and tetraploid sea island cotton (fiber strength and fineness) and the like.
The cotton fiber quality is mainly determined by the differentiation and development stage of cotton fiber cells, is complex and ordered processes, has a large amount of gene expression in different development stages and participates in the regulation and control of the development of the fiber cells, the cotton fiber cells are differentiated from single cells of ovule exocellum, are the model single cells with the fastest elongation and the most synthesized cellulose in higher plants, the differentiation and development processes can be divided into 4 stages of differentiation and protrusion of the fiber cells, rapid elongation of the fiber cells, synthesis and dehydration and maturation of secondary walls, and are complex processes of regulation and control of the joint expression of a plurality of genes, wherein the two stages of fiber elongation and secondary wall synthesis are partially overlapped and closely form the relationship between the development and the quality of the fibers.
At present, the idea of improving the quality of cotton fibers by a molecular biological means is mainly to introduce genes capable of regulating and controlling the synthesis of hormones, cellulose and polysaccharide in cotton bodies by a plant transgenic technology. John et al (1996) introduced acetyl CoA reductase gene (phaB) and PHB synthase gene (phaC) into upland cotton cultivars, and the heat-absorbing property and heat-conducting property of transgenic cotton were significantly increased although the fiber quality such as strength, length, etc. of the transgenic cotton were not significantly improved. John (1999) introduced genes iaaM and iaaH associated with auxin synthesis into conventional cotton, although the IAA content in transgenic cotton fibers was significantly increased, fiber length, fineness and strength were not significantly different from the control. Jiang et al (2011) transferred a cellulose synthase gene (GhSusA1) into cotton, and the result shows that the quality of the fiber of a cotton strain over-expressing the GhSusA1 gene is obviously higher than that of a non-transgenic strain.
Disclosure of Invention
The present inventors obtained a cotton line transgenic for event aC20-3 by transgenic methods. The transformation event has stable traits for improving the quality of cotton fiber. Representative seeds are preserved in China general microbiological culture Collection center, and the preservation numbers are as follows: CGMCC No. 8880.
The invention provides a cotton transformation event aC20-3 in the aspect, which is characterized in that the sequence is shown as SEQ ID No. 21 and consists of a T-DNA insertion sequence with 1118-6544bp, an upstream flanking cotton genome sequence with 1-1117bp and a downstream flanking cotton genome sequence with 6545-7386 bp.
In a second aspect, the invention provides nucleic acid constructs comprising the cotton transformation event aC 20-3.
In a third aspect the invention provides recombinant vectors comprising a T-D NA insertion sequence according to the aspect of the invention or a nucleic acid construct comprising cotton transformation event aC20-3 in embodiments the vector is the p2300-pE200-mCBD vector of figure 1 of the specification.
In a fourth aspect, the present invention provides recombinant cells comprising a T-DNA insert according to aspect of the present invention or a nucleic acid construct according to the second aspect of the present invention or a vector according to the third aspect of the present invention, wherein the recombinant cells are recombinant Agrobacterium cells.
A fifth aspect of the invention provides a primer pair for detecting a cotton transformation event according to aspect of the invention, consisting of a primer that specifically recognizes the T-DNA insert sequence according to aspect of the invention and a second primer that specifically recognizes any flanking sequence according to aspect of the invention in embodiments , the primer is selected from SEQ ID NO: 22 or 23 and the second primer is selected from SEQ ID NO: 14 or 16 in embodiments , the second primer is selected from SEQ ID NO: 25 or 19 in embodiments the primer is selected from SEQ ID NO: 18 or 24.
In a sixth aspect, the present invention provides methods for identifying an aC20-3 transgenic event in a biological sample of cotton, comprising:
(a) extracting a DNA sample from a cotton biological sample to be identified;
(b) performing PCR amplification using the primer set according to claim 6 or 7 using the extracted DNA sample as a template;
(c) detecting the PCR amplification product, if the length of the amplification product is times the length between the PCR primer pair sequences as set forth in SEQ ID NO. 21, indicating the presence of the aC20-3 transgenic event in the cotton biological sample.
The seventh aspect of the invention provides methods for preparing transgenic cotton, culturing the cotton seeds or other tissues containing the transformation event of the aspect of the invention, comprising the steps of crossing the cotton material containing the transformation event material of the aspect of the invention with other cotton breeding materials, carrying out step backcross to obtain a new material containing the transformation event of the aspect of the invention, and screening and identifying in progeny populations by using the method of the sixth aspect of the invention during crossing and backcross to confirm the existence of the transformation event of the aspect of the invention.
In an eighth aspect, the invention provides the use of a transformation event according to aspect of the invention, a nucleic acid construct according to aspect second of the invention, a recombinant vector according to aspect third of the invention, a recombinant cell according to aspect fourth of the invention, a method according to aspect sixth or seventh of the invention for improving cotton fiber quality, for plant breeding and as a molecular marker.
The CBD gene (GenBank: AAA23218.1) encoding the CBD protein utilized in the present invention was derived from Clostridium cellulovorans (Clostridium cellulovorans), which is a constituent of a cellulase complex purified from Clostridium cellulovorans by O.Shoseyov in 1990, the complex having a cellulase activity on microcrystalline fibers. Experiments have shown that removal of CBDs from the scaffolding structure of cellulase or cellulosome greatly reduces the enzyme activity. Functional studies of CBDs by shpigel found that CBDs enhanced the elongation of the prunes persica l pollen tube in vitro; increase the activity of A.xylinum cellulose synthetase and promote the synthesis of cellulose. Wherein the SP sequence is derived from 24 bases at the N end of the Arabidopsis thaliana CEL1 gene and is a leader peptide sequence. The invention utilizes a fiber specificity promoter EVO200 to drive SP + mCBD gene, utilizes an agrobacterium-mediated method to transform cotton, obtains a single copy transformation event aC20-3 with cotton fiber clothing, obviously improved specific strength and micronaire value by screening, and obtains cotton genome sequences at the left side and the right side of the insertion event by utilizing a molecular biological means.
The high-quality cotton transformation event of the mCBD gene created by the invention can not only obviously improve the quality of cotton fibers, has stable heredity, single copy integration and clear molecular characteristics of integrated flanking sequences, and has important application value in production and breeding, but also can conveniently aggregate different commercial transformation events in a hybridization aggregation mode due to the unique detection method.
Drawings
FIG. 1 shows the construction scheme of plant expression vector p2300-pE 200-mCBD.
FIG. 2 shows the results of an experiment using Southern hybridization to detect the copy number of transgenic event aC 20-3. 1, carrying out enzyme digestion on plasmid containing mCBD gene by Hind III; 2, transformation event aC20-3 genomic DNA piece digested with EcoR I; 3, Hind III digested transformation event aC20-3 genomic DNA; marker, lambda DNA digested by EcoR I/Hind III;
fig. 3 is a diagram of Right Border (RB) flanking sequences.
Fig. 4 is a diagram of Left Border (LB) flanking sequences.
FIG. 5 is a schematic representation of the insertion sequence and identifying primers for the aC20-3 event.
FIG. 6 shows the results of cotton samples from the aC20-3 event and Ji Cotton 14 amplified with the primer pairs RBcheck51 '/GSP 2-mCBD 32', RBcheck52 '/GSP 2-mCBD 32', and LBcheck31 '/GSP 2-NPTII 52', LBcheck32 '/GSP 2-NPTII 52', respectively. M, marker, lambda DNA/EcoR I + Hind III; 1,3: the RBcheck51 '/GSP 2-mCBD 32' amplifies aC20-3 event and Ji cotton 14, and a positive amplification band is 1767 bp; 2,4: the RBcheck52 '/GSP 2-mCBD 32' amplifies aC20-3 event and Ji cotton 14, and a positive amplification band is 1535 bp; 5,6: LBcheck31 '/GSP 2-NPTII 52' amplifies aC20-3 event and Ji cotton 14, positive amplification band is 3197 bp; 8,7: LBcheck32 '/GSP 2-NPTII 52' amplifies aC20-3 event and Ji cotton 14, and positive amplification band is 3152 bp.
Detailed Description
As used herein, a "transformation event" refers to the joining of an exogenous insertion DNA sequence to a specific cotton genomic DNA sequence, a "transformation event" is not plant cells or plants that are the vector in which the transformation event is present, and the core characteristic of the transformation event is the characteristic DNA sequences joining the exogenous insertion sequence formed by the insertion of the exogenous gene at a specific site in the plant genome to the specific cotton genomic sequence.
Lines of plants with a certain transformation event are referred to as lines of the same name. For example, a plant line having an aC20-3 transformation event is referred to as an aC20-3 line.
Examples
The invention is further illustrated in connection with the following non-limiting examples.
Example 1 plant expression vector construction
Because the gene for coding the cellulose binding domain CBD (cellulose binding domain) is derived from clostridium cellulovorum and has different GC content and codon preference from cotton genome, the base composition is changed according to the cotton codon preference, and an SP and CBD gene fusion sequence (the sequence is shown as SEQ ID No: 1) is synthesized by an artificial synthesis method, wherein the SP sequence is from 24 bases at the N end of the Arabidopsis CEL1 gene and is a peptide guide sequence. The fusion sequence was artificially synthesized from the whole gene by Takara corporation, and the new gene was named SP + mCBD. A plant binary expression vector pCAMBIA2300 (purchased from Changsheng biotechnology, Limited liability company in Beijing ancient China) is selected as a plant expression vector, and a Pnos promoter is used for replacing the NPTII gene and the 35S promoter of a double enhancer so as to reduce the expression of the NPTII protein in plants. A promoter EVO200 (shown as SEQ ID No: 2) from the specific expression of the early elongation stage of cotton fibers is selected to drive the expression of the fusion gene, and Tnos is used as a terminator.
With primers SEQ ID NO: 3 and SEQ ID NO: 4 Pnos was amplified using a plant expression vector PBI121 (available from TaKaRa, North China, ocean technologies, Inc.) as a template, using PrimeSTAR HS DNA polymerase from TaKaRa. 50 μ l PCR reaction: 10 μ l of 5 XPS Buffer, 3 μ l of 2.5mM dNTP, 1.0 μ l of PBI121, 1.0 μ l of PrimeSTAR, 10 μ M of primer SEQ ID NO: 3 and SEQ ID NO: 4 of 2.0. mu.l each, and 31. mu.l of double distilled water. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 33 cycles. The fragment was ligated to pCAM BIA2300(promega, T4 ligase cassette) by EcoR I, BamHI digestion to obtain pCAM BIA 2300-1.
With primers SEQ ID NO: 5 and SEQ ID NO: 6 Tnos was amplified using PBI121 as a template, and PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μ l PCR reaction: 10 μ l5 XPS Buffer, 3 μ l 2.5mM dNTP, 1.0 μ l PBI121, 1.0 μ l PrimeSTAR, 10 μ M primer SEQ ID NO: 5 and SEQ ID NO: 6 of 2.0. mu.l each, and 31. mu.l of double distilled water. And (3) PCR reaction conditions: pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 10min after 33 cycles. The obtained pCAMBIA2300-2 is obtained by the enzyme digestion and connection of Sac I and EcoR I to pCAMBIA2300-1(promega T4 ligase box)
The cotton EVO200 promoter (refer to ZM-06838 in the code of ZM-06838) DNA is amplified by using primers SEQ ID NO: 7 and SEQ ID NO: 8 and using African cotton (national Cotton Medium library, acquisition Unit, China Cotton institute, Japan Cotton institute, USA ) as a template to extract cotton genomic DNA, east agricultural science, 2008(5) the method in 14-16 is used to extract cotton DNA. PrimeSTAR HS DNA polymerase using TaKaRa 50. mu.l PCR reaction system 10. mu.l 5 XPS Buffer, 3. mu.l 2.5mM dNTP, 1.0. mu.l cotton DNA, 1.0. mu.l PrimeSTAR, 10. mu.M primers SEQ ID NO: 7 and SEQ ID NO: 8 are each 2.0. mu.l, and 31. mu.l double distilled water are used to prepare PCR reaction conditions of pre-denatured 5min at 94 ℃, denatured 30s at 94 ℃, 30s annealed at 58 ℃, 30s extended at 72 ℃, 33 cycles are extended, 10min is connected to BmIII-2300 pCA by enzyme digestion, and the DNA is connected to obtain a DNA sequence obtained by the method of BmCIA-2300 pCA (AMmCA) and the method of the BD-2300 pCA is used to synthesize BD-200.
Example 2 genetic transformation of upland cotton (Gossypium hirsutum):
the method utilizes an agrobacterium-mediated genetic transformation method to transform the hypocotyl of Ji cotton 14 by using a vector containing the mCBD gene.
Agrobacterium LBA4404 harboring the p2300-pE200-mCBD vector was picked up, inoculated into LB liquid medium containing 50mg/L kanamycin (kanamycin, km), 50mg/L rifampicin (rifampicin, rif) and 50mg/L streptomycin (streptomycin, S/Sm), and cultured overnight at 28 ℃ in a shaking dark to log phase of bacterial growth. Preparing bacterial liquid: diluting the bacterial liquid by LB or YEB liquid culture medium according to the ratio of the culture medium to 1: 50-1: 100, then carrying out shake culture for 4-6 h, and diluting the bacterial liquid to the OD600 value of 0.8-1.0.
The transgenic acceptor material is Ji cotton 14, sterile seedling hypocotyls growing for 3-4 days are taken, cut into sections of 0.6-0.8 cm, soaked for 10-15 min, the hypocotyl sections are taken out, a co-culture medium (MSB + KT 0.1mg/L +2, 4-D0.1mg/L) is placed into the co-culture medium (MSB + KT 0.1mg/L +2, 4-D0.1mg/L + Kan50mg/L) for 20-30 days for subculture times, the transgenic acceptor material is transferred into a callus propagation medium (MSB + KT 0.1mg/L +2, 4-D0.05mg/L + Kan50mg/L) after 90 days, the transgenic acceptor material is transferred into a callus propagation medium (MSB + KT 0.1mg/L +2, 4-D0.05mg/L + Kan 50/L) for 20-30 days for subculture times, after embryogenic callus growth, the embryogenic callus propagation medium is transferred into a callus propagation medium (MSB + KT propagation medium) (MSB + KT 0.50 mg/L + 50 + SH 50mg/L), the transgenic plantlets are grafted for germination, and the transgenic plantlets are grafted for 50mg days, and the transgenic plantlets are grafted, and the transgenic plantlets are obtained, and the transgenic plant.
Example 3 screening and identification of transgenic material of T1 generation:
215 single plants of 41 strains of cotton of an aC series of a T1 generation are harvested, clothing determination and fiber inspection are carried out on the harvested cotton, the statistical analysis is further carried out on the result , and a transformation event aC20-3 is obtained through screening.
TABLE 1
Example 4 transgenic event aC20-3 copy number detection
The Southern hybridization technique was used to detect the copy number of transgenic event aC 20-3.
The preparation of the sample comprises the steps of taking 4.0g of tender plant tissue of an aC 20-3T 0 generation of transformation event, extracting plant genome DNA, grinding into powder in liquid nitrogen, preheating extraction Buffer solution 15ml in 65 ℃ water bath, adding the powder into the extraction Buffer solution after grinding into uniform powder, oscillating and mixing uniformly, carrying out 45min in 65 ℃ water bath, shaking uniformly for 2-3 times, fully cracking, adding 1/3 volumes of 5mol/L KAc, reversing and mixing uniformly up and down, carrying out ice bath for about 2-h3, 4 ℃, 12000rpm, centrifuging for 10min, taking supernatant, adding 1/5 volumes of 5% CTAB Buffer, reversing and mixing uniformly up and down, carrying out 65 ℃ water bath for about 20min, cooling to room temperature, adding equal volumes of chloroform/isoamyl alcohol (24: 1), extracting for 3 times, carrying out centrifugation for 5min at room temperature 00rpm, extracting to twice if interface, taking supernatant, adding 2/3 volumes of isopropanol, fully reversing and mixing uniformly up and down, placing 10min at room temperature, carrying out centrifugation for 12000 min, removing supernatant, washing for 5min, carrying out precipitation with 70% ethanol, adding water, carrying out precipitation for twice, adding 10min, carrying out precipitation for dry precipitation at room temperature, adding 30 ℃ and adding RN5 min, adding ethanol, adding 30.20 rpm, adding RN5, adding water, dissolving precipitation for precipitation, carrying out precipitation for twice (30) and carrying out precipitation, adding precipitation for twice, adding 30 ℃ and carrying out precipitation, adding precipitation2Dissolving O, measuring the concentration and purity of DNA by using an ultraviolet spectrophotometer, taking 100-200 mu g of DNA, respectively using restriction enzymes EcoR I and Hind III, digesting overnight, then precipitating with alcohol, and using a proper amount of ddH2Dissolving O, adding into 0.8% agarose gel, and electrifying at 1V/cm voltageThe swimming was carried out overnight. Transferring to a nylon membrane in vacuum, and carrying out ultraviolet crosslinking and fixing.
Preparing a probe: the plasmid containing mCBD gene is used as a template, and SEQ ID No: 9 and SEQ ID No: 10 is a primer, a PCR method is utilized to prepare a high zinc labeled specific probe, and the PCR system is as follows:
PCR procedure: 94.0 deg.C for 5 min; 30 cycles: 94.0 ℃ for 30s, 53.0 ℃ for 30s, and 72.0 ℃ for 30 s; 72.0 ℃ for 5 min.
The hybridization detection comprises placing nylon membrane into hybridization tube, adding hybridization solution (10ml/100 cm)2) Prehybridization is carried out for 3-4 h at 65 ℃; denaturing DIG labeled probe (25ng/ml) at 95 ℃ for 10min, quickly putting into ice water, and cooling for 10min to completely change; the denatured probe was quickly added to the hybridization tube (3.5ml/100 cm)2membrane), mixing, and hybridizing at 65 ℃ overnight (>10h) In that respect And taking out the nylon membrane, and washing the membrane. 30ml of 2 XSSC/0.1% SDS were washed at room temperature for 2X 5min with shaking. The membrane was washed at 50 ℃ for 2X 15min with 0.1 XSSC/0.1% SDS and then washed 5min with 20ml of wash buffer. After hybridization and rigorous washing, soaking the membrane in a washing buffer solution for 1-5 min; incubating for 30min in 20-30 ml of confining liquid; incubating in 10ml of antibody solution for 30 min; washing with 20-30 ml of washing liquid for 2 × 15 min; balancing in 15ml of detection solution for 2-5 min; 20ml of chromogenic substrate (NBT/BCIP) is prepared in the dark and stands for color development; washing the membrane with 50ml of sterile water or TE for 5min to stop color development, and photographing for storage. The detection results are shown in FIG. 2, and the two groups of single-enzyme digestion hybridization results show that the target gene is inserted in a single copy in the transformation event aC 20-3.
Example 5 side sequence analysis
Sample preparation: 2.5 mu g of DNA is taken and digested by Stu I for 6-8 hours respectively, and is dissolved in a proper amount of water after being precipitated and purified by alcohol.
Design synthesis butt joint:
genome Walker Adaptor +, sequence of SEQ ID No. 11, SEQ ID No. 12)
The genome Walker Adaptor + and the genome Walker Adaptor + were mixed in equal amounts, and the mixture was incubated at 70 ℃ for 10 minutes and then slowly cooled to room temperature. Mu.l of the digested and purified DNA was added to 1.9. mu.l of genome Walker adapter (25. mu.M), 1.6. mu.l of 10 Xligation buffer, 0.5. mu. l T4DNA ligase (6 units/. mu.l), incubated overnight at 16 ℃ to stop the reaction, incubated at 70 ℃ for 5min, and 72. mu.l of TE (10/1, pH 7.5) was added to each tube and shaken at a low speed for 5-10 sec.
RB terminal sequence analysis Using Clontech genome WalkerTMThe Universal kit utilizes primers AP1 and GSP1-mCBD31 '(sequences are SEQ ID No: 13 and SEQ ID No: 14 respectively), a connecting product is used as a template to carry out th round of amplification, 7 rounds of amplification are carried out, wherein the amplification comprises the steps of 94 ℃ 25S, 72 ℃ 6min, 32 rounds of amplification, 94 ℃ 25S, 67 ℃ 6min, rounds of amplification and heat preservation at 67 ℃ for 7 min, the PCR product is diluted by 50 times, and then, a second round of PCR amplification is carried out by using AP2 and GSP2-mCBD 32' (sequences are shown as SEQ ID No: 15 and SEQ ID No: 16), the PCR program comprises the steps of 5 rounds of PCR amplification, 94 ℃ 25S, 72 ℃ 5min, 20 rounds of amplification, 94 ℃ 25S, 67 ℃ 5min and rounds of amplification and heat preservation at 67 ℃ for 10min, and the product is recycled and sequenced.
The RB end sequence analysis of the transformation event aC20-3 is shown in figure 3, so that a 2506bp nucleotide sequence (the sequence is shown as SEQ ID No: 17) is obtained together, the sequence comprises a1 bp-1117 bp cotton genome sequence, the 1118 bp-1348 bp vector sequence between RB and EVO200 promoter, the 1349 bp-2368 bp EVO200 promoter sequence and the 2369 bp-2506 bp CBD gene partial sequence.
Description of the sequence:
1-1117 Cotton DNA
1118 1348 RB and a promoter
1349-2368 EVO200 promoter
2369 part of 2506 CBD Gene sequence
LB terminal sequence analysis, according to the obtained cotton flanking sequence, using published diploid cotton (Gossypiumraimonindii) D genome sequence (http:// www.phytozome.net/cotton. php) to carry out comparative analysis, designing downstream primer LBcheck31 ' (SEQ ID NO: 18), using aC20-3 genome as template, carrying out PCR amplification by using LBcheck31 ' and GSP2-NPTII52 ' (shown as SEQ ID NO: 18, SEQ ID NO: 19), wherein the PCR program is 35 cycles: 94 ℃ 25S, 67 ℃ 5min, and finally cycles, then carrying out heat preservation at 67 ℃ for 10min, and product recovery and sequencing.
The LB terminal sequence of aC20-3 event is analyzed as shown in FIG. 4, 3198bp nucleotide sequence (sequence is shown as SEQID No: 20) is obtained, including NPTII sequence at 1 bp-376 bp, Tnos sequence at 377 bp-585 bp, carrier sequence between Tnos terminator and LB at 586 bp-632 bp, reverse complementary sequence of partial mCBD gene at 633 bp-1080 bp, EVO200 reverse complementary sequence at 1081 bp-2130 bp, reverse complementary sequence of carrier between RB and EVO200 promoter at 2131 bp-2361 bp, and cotton genome sequence at 2362 bp-3198 bp.
Description of the sequence:
1-37b NPTll
377-585 Tnos
vector sequence between 586-632 Tnos terminator and LB
633-Asa 1080 part mCBD gene reverse complementary sequence
1081-2130 promoter EVO200 reverse complement sequence
2131 reverse complement of the vector between the 2361 RB and the EVO200 promoter
2362 Alternaria 3198 Cotton genome sequence
From the above results, one skilled in the art can readily derive the DNA sequence characteristic of transformation event aC20-3 (SEQ ID NO: 21), as shown below, with the T-DNA insert shown in underlined sections and the flanking cotton genomic DNA sequence of the insert shown in non-underlined sections.
Example 6 transformation event detection
Amplification was performed to detect the aC20-3 event using a pair of DNA primers consisting of a primer that specifically recognizes the T-DNA insert of the invention and a second primer that specifically recognizes any flanking sequence of the insert the aC20-3 insert and identifying primers are shown in FIG. 5. for example, when the primer is RBcheck51 ' (SEQ ID NO: 22) or RBcheck52 ' (SEQ ID NO: 23), the second primer can be GSP1-mCBD31 ' (SEQ ID NO: 14), and when the primer is LBcheck31 ' (SEQ ID NO: 18), the second primer is LBcheck32 ' (SEQ ID NO: 24).
The results of amplifying aC20-3 strain and Ji cotton 14 cotton sample by using the primer pairs RBcheck51 '/GSP 2-mCBD 32', RBcheck52 '/GSP 2-mCBD 32' and LBcheck31 '/GSP 2-NPTII 52', LBcheck32 '/GSP 2-NPTII 52' are shown in FIG. 6. M, marker, lambda DNA/EcoR I + HindIII; 1,3: the RBcheck51 '/GSP 2-mCBD 32' amplifies cotton and Ji cotton 14 which combine aC20-3 transformation events, and a positive amplification band 1767 bp; 2,4: the RBcheck52 '/GSP 2-mCBD 32' is amplified to combine aC20-3 transformation event cotton and Ji cotton 14, and a positive amplification band is 1535 bp; 5,6: LBcheck31 '/GSP 2-NPTII 52' amplifies and combines aC20-3 transformation event cotton and Ji cotton 14, and a positive amplification band is 3197 bp; 8,7: LBcheck32 '/GSP 2-NPTIl 52' amplifies the positive amplification band 3152bp of the combination of aC20-3 transformation event cotton and Ji cotton 14. It is shown that cotton with the aC20-3 transformation event had this insertion event, while the Ji cotton 14 did not.
Example 9 chromosomal mapping
According to the obtained cotton flanking sequences, the cotton DNA sequences flanking the exogenous insertion sequence of the transformation event aC20-3 are highly homologous with the sequences on the D5 chromosome by using the published genome sequence (http:// www.phytozome.net/cotton. php) of the D chromosome of diploid cotton (Gossypium raimonidii) through comparative analysis, so that the integration site of the exogenous DNA insertion sequence in the AC20-3 event is positioned on the 5 th chromosome of the receptor tetraploid cotton.
Claims (8)
1. The sequence of the conjugant of the exogenous insertion DNA sequence and the specific cotton genome DNA sequence is shown as SEQ ID No: 21 consisting of a T-DNA insert of 1118-6544bp, an upstream flanking cotton genomic sequence of 1-1117bp and a downstream flanking cotton genomic sequence of 6545-7386 bp.
nucleic acid construct comprising the sequence SEQ ID No. 21.
recombinant vector comprising the T-DNA insert of claim 1 or the nucleic acid construct of claim 2.
recombinant cell comprising the T-DNA insert of claim 1 or the nucleic acid construct of claim 2 or the vector of claim 3, said recombinant cell being a recombinant Agrobacterium cell.
5. A primer pair for detecting an adapter of the foreign insert DNA sequence of claim 1 to a specific cotton genomic DNA sequence consisting of an th primer specifically recognizing the T-DNA insert sequence of claim 1 or the upstream flanking cotton genomic sequence and a second primer specifically recognizing the T-DNA insert sequence of claim 1 or the downstream flanking cotton genomic sequence,
the th primer is selected from SEQ ID NO. 22 or 23, the second primer is SEQ ID NO. 14, or
The th primer is SEQ ID NO. 18, and the second primer is SEQ ID NO. 24.
A method for identifying adapters of the exogenous insert DNA sequence of claim 1 to a specific cotton genomic DNA sequence in a cotton biological sample, comprising (a) extracting a DNA sample from the cotton biological sample to be identified;
(b) performing PCR amplification using the primer pair of claim 5 using the extracted DNA sample as a template;
(c) detecting the PCR amplification product, if the length of the amplification product is times the length between the sequence of the PCR primer pair shown in SEQ ID NO. 21, indicating the presence of an adapter of the foreign insert DNA sequence and the specific cotton genomic DNA sequence in the cotton biological sample.
A method of producing transgenic cotton of the species , comprising growing cotton seeds or other tissues containing an adapter of the exogenous insertion DNA sequence of claim 1 and a specific cotton genomic DNA sequence.
8. Use of an adapter of an exogenous insertion DNA sequence according to claim 1 and a specific cotton genomic DNA sequence, a nucleic acid construct according to claim 2, a recombinant vector according to claim 3, a recombinant cell according to claim 4, a method according to claim 6 or claim 7 for improving cotton fiber quality, for plant breeding and as a molecular marker.
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Denomination of invention: Cotton plant event aC20-3 and primers and methods for its detection Effective date of registration: 20231129 Granted publication date: 20200131 Pledgee: China Construction Bank Co.,Ltd. Shenzhen Branch Pledgor: BIOCENTURY TRANSGENE (CHINA) Co.,Ltd. Registration number: Y2023980068376 |