阅读说明：本技术 一种利用种子荧光快速鉴定转基因拟南芥的方法 (Method for rapidly identifying transgenic arabidopsis by using seed fluorescence ) 是由 罗丽娟 李敏 刘秦 吴远航 刘攀道 蒋凌雁 陈银华 于 2019-12-30 设计创作，主要内容包括：本发明属于生物技术领域,公开了一种利用种子荧光快速鉴定转基因拟南芥的方法,通过构建的pOGT载体构建重组基因载体,重组基因载体携带外源基因导入拟南芥中后,转基因拟南芥种子能够观察到荧光,与野生型拟南芥区分开来。本发明公开的pOGT植物表达载体体系避免了抗生素或除草剂对发育缺陷型植株的危害,摆脱了对无菌工作环境和大面积种植空间的需求,缩短了获得转基因植株的时间和成本。(The invention belongs to the technical field of biology, and discloses a method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence. The pOGT plant expression vector system disclosed by the invention avoids the damage of antibiotics or herbicides to development defective plants, gets rid of the requirements on sterile working environment and large-area planting space, and shortens the time and cost for obtaining transgenic plants.)

1. A method for rapidly identifying transgenic arabidopsis by using seed fluorescence is characterized by comprising the following steps:

(1) pro amplification from Arabidopsis genome_OLE1Cloning an OLE1 fragment into a pA7-GFP vector to obtain a recombinant vector pA7-OLE 1-GFP;

(2) cloning Pro by using recombinant vector pA7-OLE1-GFP as template_OLE1OLE1-GFP fragment; pro to be cloned_OLE1The OLE1-GFP fragment is inserted into a pCXSN vector to obtain a recombinant vector pCXSN-OLE 1-GFP;

(3) mutant recombinant vector pCXSN-OLE 1-Pro on GFP_OLE1Obtaining pOGT vector from XcmI site;

(4) and (4) constructing a recombinant gene vector by adopting the pOGT vector in the step (3), transferring the recombinant gene vector into a competent cell, selecting correct monoclonal infection arabidopsis thaliana to obtain a transgenic arabidopsis thaliana seed, and observing fluorescence in the transgenic arabidopsis thaliana seed.

2. The method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence according to claim 1, wherein the method comprises the following steps: in the step (1), the construction method of the recombinant vector pA7-OLE1-GFP comprises the following steps: designing and synthesizing a specific primer pOLE1-F, pOLE1-R according to the Arabidopsis OLE1 gene and a promoter sequence thereof, and cloning a promoter of the OLE1 gene and an OLE1 gene (Pro) by taking an Arabidopsis genome as a template_OLE1OLE 1); digesting the pA7-GFP vector by SalI and XhoI restriction endonucleases, recovering 4591bp linearized pA7-GFP fragment, and cloning to obtain Pro_OLE1Cloning the OLE1 fragment into a pA7-GFP vector to obtain a recombinant vector pA7-OLE 1-GFP; the specific primer pOLE1-F, pOLE1-R has the sequences as follows: ACGAACGATACTCGAGCACCCTACTTAGATCAACACATA, and TCACTAGTACGTCGACAGTAGTGTGCTGGCCACC.

3. The method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence according to claim 1, wherein the method comprises the following steps: in the step (2), the construction method of the recombinant vector pCXSN-OLE1-GFP comprises the following steps: according to Pro_OLE1Comprises designing an OLE1-GFP sequence to synthesize a pair of specific primers pOGT-F, pOGT-R, cloning Pro by using pA7-OLE1-GFP plasmid as a template and pOGT-F, pOGT-R as a primer_OLE1OLE1-GFP fragment; HindIII enzyme cuts the pCXSN carrier, and recovers 10802bp linearization pCXSN large fragment; pro by homologous recombinase_OLE1The OLE1 shows that the GFP fragment is inserted between Hind III restriction sites of the pCXSN vector, and then Escherichia coli DB3.1 competent cells are transformed, so that the pCXSN-OLE1-GFP recombinant vector is obtained.

4. The method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence according to claim 1, wherein the method comprises the following steps: in the step (3), the recombinant vector pCXSN-OLE1-GFP is mutated to generate Pro_OLE1The XcmI site method adopts the specification of a QuickMutationTM gene site-directed mutagenesis kit to mutate Pro_OLE1The XcmI site mutation method comprises the following steps of carrying out point mutation on a primer PM-XcmI-F, PM-XcmI-R, wherein the sequence of the PM-XcmI-F is as follows: TCGGTCTTGGTCACACAAGGAACTCTCTGGTAA, respectively;

the PM-XcmI-R has the sequence as follows:

TTACCAGAGAGTTCCTTGTGTGACCAAGACCGA。

5. the method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence according to claim 1, wherein the method comprises the following steps: in the step (4), the construction method of the recombinant gene vector comprises the following steps: xcm I enzyme digestion pOGT vector, and large fragment is recovered to obtain T vector; adding adenine deoxyribonucleotide at the tail end of the exogenous gene, and then connecting the exogenous gene into a T vector by using T4 ligase; the ligation product is transferred into an Escherichia coli strain Trans1-T1 to obtain a recombinant gene vector.

6. The method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence according to claim 1, wherein the method comprises the following steps: the gene of the recombinant gene vector is a cassava MeEXPA30 gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

7. The method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence as claimed in claim 6, wherein the method for transferring the cassava MeEXPA30 gene into arabidopsis thaliana is as follows: xcm I enzyme digestion pOGT vector, and recovery of the large fragment to obtain a T vector; adding adenine deoxyribonucleotide to the tail end of an exogenous gene MeEXPA30, then connecting the exogenous gene MeEXPA30 into a T vector by using T4 ligase, and transferring a ligation product into an escherichia coli strain Trans1-T1 by a heat shock method; after overnight culture at 37 ℃, obtaining a pOGT-MeEXPA30 recombinant vector; the vector pOG-MeEXPA30 was transferred into competent cells and infected with Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana seeds.

8. The method for rapidly identifying transgenic arabidopsis thaliana by using seed fluorescence according to claim 1 or 7, wherein: the method for infecting arabidopsis thaliana adopts a flower soaking method.

9. The method for rapid identification of transgenic Arabidopsis thaliana using seed fluorescence according to claim 1 or 7, wherein the competent cell is Agrobacterium tumefaciens GV 3101.

10. A recombinant gene vector for the fluorescent identification of transgenic Arabidopsis seeds, wherein said recombinant gene vector is the pOGT vector of claim 1.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly identifying transgenic arabidopsis by using seed fluorescence.

Background

The emergence of transgenic Arabidopsis greatly facilitates the study of plant gene functions. Screening transgenic Arabidopsis thaliana is an important step for identifying positive transgenic plants. Selection of transgenic plants generally requires a selectable marker gene, which is often an antibiotic resistance or herbicide resistance gene. This screening method has provided us with convenience for a long time, but obtaining transgenic Arabidopsis thaliana by this method has some problems that are difficult to avoid: first, while transgenic Plants carry selectable markers such as antibiotic or herbicide resistance genes, developmental-defective Plants resulting from transgenes may be very sensitive to antibiotics or herbicides [ Toxic Reactivity of Wheat (Triticum aestivum) Plants to herbicide isoprotron. yin XL, Jiang L, Song NH, Yang h.agr Food chem.2008; 56(12) 4825-4831 ]; second, conventional selectable markers require aseptic working conditions, bulk plant throughput, and plant growing space. These factors bring difficulties to some extent for the establishment of transgenic Arabidopsis lines.

A large number of oil body proteins (OLESs) are distributed in Arabidopsis seeds. OLE1(At4g25140) is a kind of oil body protein, which is present throughout the development period of the seed to ensure the normal germination of the seed [ A novel rollers for oleoresins in free distance of oils in Arabidopsis, plant, Shimada TL, Tomoo S, Hideyuki T, Fukao Y, Hara-Nishimura I.2010; 55(5):798-809.]. The pCXSN plasmid is a commercial vector and can be used for constructing a plant over-expression vector. The pCXSN Vector has a segment of ccdB lethal Gene, and is transformed into a common Escherichia coli strain (such as DH5a and Trans1-T1) to cause strain death, Xcm I sites of pVS1 StaA and KanR genes are mutated, the ccdB lethal Gene on the pCXSN Vector can be cut off after the Xcm I site is cut by restriction enzyme Xcm I to form a T Vector, and the common Escherichia coli strain can be directly transformed after the Gene is connected [ AVERARTLE Zero Back T-Vector System for Gene Cloning and functional genomics.Chen SB, Songkumarn P, Liu JL, Wang GL.plant Physiol.2009; 150(3):1111-1121.].

Disclosure of Invention

The invention takes pCXSN plant expression vector as a vector framework and Pro_OLE1OLE1 GFP seed fluorescent protein gene was used as a selection marker gene to develop a novel plant expression vector and was named pOGT (plasmid of OLE1-GFP T-vector). The pOGT vector is cut by Xcm I enzyme and then becomes a T vector, an exogenous PCR fragment can be cloned into the pOGT vector through TA cloning, the obtained recombinant vector is introduced into arabidopsis thaliana, and the screening of transgenic arabidopsis thaliana is completed by utilizing seed fluorescence.

The technical scheme of the invention is realized as follows:

a method for rapidly identifying transgenic arabidopsis by using seed fluorescence comprises the following steps:

(1) pro amplification from Arabidopsis genome_OLE1Cloning an OLE1 fragment into a pA7-GFP vector to obtain a recombinant vector pA7-OLE 1-GFP;

(2) cloning Pro by using recombinant vector pA7-OLE1-GFP as template_OLE1OLE1-GFP fragment; pro to be cloned_OLE1The OLE1-GFP fragment is inserted into a pCXSN vector to obtain a recombinant vector pCXSN-OLE 1-GFP;

(3) mutant recombinant vector pCXSN-OLE 1-Pro on GFP_OLE1Obtaining pOGT vector from XcmI site;

(4) the method comprises the following steps of constructing a recombinant gene vector by adopting a pOGT vector, transferring the recombinant gene vector into a competent cell, selecting correct monoclone, infecting arabidopsis thaliana to obtain a transgenic arabidopsis thaliana seed, and observing fluorescence of the transgenic seed.

Further, in the step (1), the construction method of the recombinant vector pA7-OLE1-GFP is as follows: designing and synthesizing a specific primer pOLE1-F, pOLE1-R according to the Arabidopsis OLE1 gene and a promoter sequence thereof, and cloning a promoter of the OLE1 gene and an OLE1 gene (Pro) by taking an Arabidopsis genome as a template_OLE1OLE 1); digesting the pA7-GFP vector by SalI and XhoI restriction endonucleases, recovering 4591bp linearized pA7-GFP fragment, and cloning to obtain Pro_OLE1Cloning the OLE1 fragment into a pA7-GFP vector to obtain a recombinant vector pA7-OLE 1-GFP; the specific primer pOLE1-F, pOLE1-R has the sequences as follows:

ACGAACGATACTCGAGCACCCTACTTAGATCAACACATA, and TCACTAGTACGTCGACAGTAGTGTGCTGGCCACC.

Further, in the step (2), the construction method of the recombinant vector pCXSN-OLE1-GFP comprises the following steps: according to Pro_OLE1In which a pair of specific primers pOGT-F and pOGT-R are synthesized by designing an OLE1-GFP sequence, and Pro is cloned by using pA7-OLE1-GFP plasmid as a template and pOGT-F, pOGT-R as a primer_OLE1OLE1-GFP fragment; HindIII enzyme cuts the pCXSN carrier, and recovers 10802bp linearization pCXSN large fragment; pro by homologous recombinase_OLE1The OLE1 comprises the steps of inserting a GFP fragment between Hind III enzyme cutting sites of a pCXSN vector, and then transforming escherichia coli DB3.1 competent cells to obtain a pCXSN-OLE1-GFP recombinant vector; the sequences of the specific primers pOGT-F and pOGT-R are respectively as follows: GCAGGCATGCAAGCTTCACCCTACTTAGATCAACACAT, respectively; pOGT-R has the sequence: GGCCAGTGCCAAGCTTCTTGTACAGCTCGTCCATG are provided.

Further, in the step (3), the recombinant vector pCXSN-OLE 1-Pro on GFP is mutated_OLE1The XcmI site of (1) is obtained by mutating Pro in the instruction of Quick mutation (TM) gene site-directed mutagenesis kit (Biyotime Co., Ltd.)_OLE1The XcmI site mutation method comprises the following steps of carrying out point mutation on a primer PM-XcmI-F, PM-XcmI-R, wherein the sequence of the PM-XcmI-F is as follows: TCGGTCTTGGTCACACAAGGAACTCTCTGGTAA, respectively; the PM-XcmI-R has the sequence as follows: TTACCAGAGAGTTCCTTGTGTGACCAAGACCGA are provided.

Further, in the step (4), the construction method of the recombinant gene vector comprises: xcm I enzyme digestion pOGT vector, and large fragment is recovered to obtain T vector; adding adenine deoxyribonucleotide at the tail end of the exogenous gene, and then connecting the exogenous gene into a T vector by using T4 ligase; the ligation product is transferred into an Escherichia coli strain Trans1-T1 to obtain a recombinant gene vector.

Furthermore, the gene of the recombinant gene vector is a cassava MeEXPA30 gene, and the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

Further, the method for transferring the cassava MeEXPA30 gene into arabidopsis thaliana comprises the following steps: xcm I enzyme digestion pOGT vector, and recovery of the large fragment to obtain a T vector; adding adenine deoxyribonucleotide to the tail end of an exogenous gene MeEXPA30, then connecting the exogenous gene MeEXPA30 into a T vector by using T4 ligase, and transferring a ligation product into an escherichia coli strain Trans1-T1 by a heat shock method; after overnight culture at 37 ℃, obtaining a pOGT-MeEXPA30 recombinant vector; the vector pOG-MeEXPA30 was transferred into competent cells and infected with Arabidopsis thaliana to obtain transgenic Arabidopsis thaliana seeds.

Further, the method of infecting Arabidopsis thaliana was selected as the flower-soaking method.

Further, the competent cells used were Agrobacterium tumefaciens GV 3101.

The invention has the beneficial effects that:

by adopting the pOGT vector, exogenous gene fragments can be cloned into the pOGT vector, after the recombinant gene vector is introduced into arabidopsis thaliana, fluorescence can be observed from transgenic arabidopsis thaliana seeds, and the screening of transgenic arabidopsis thaliana can be realized by utilizing the fluorescence of the seeds. Compared with the traditional screening technology, the pOGT plant expression vector system disclosed by the invention avoids the harm of antibiotics or herbicides to development defective plants, gets rid of the requirements on sterile working environment and large-area planting space, shortens the time and cost for obtaining transgenic plants to a certain extent, and provides more choices for researchers engaged in Arabidopsis transformation work.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a schematic diagram of the construction scheme of pOGT vector, in which the 16bp upstream sequence from the vector is indicated by lower case letters in the amplification primers.

FIG. 2 is cloning Pro_OLE1Gel electrophoresis of OLE1 fragment, wherein 1 is Arabidopsis OLE1promoter, and the PCR amplification product of OLE1 gene.

FIG. 3 is a gel electrophoresis of the vector pA7-GFP digested with XhoI and SalI, in which 2 and 3 are the products of digestion with pA7-GFP plasmids SalI and XhoI.

FIG. 4 gel electrophoresis of colony PCR of 5 random single clones with primers pOLE1-F and pOLE1-R, wherein 4-8 are 5 pA7:: GFP:: OLE1:: promoter:: OLE1 recombinant vector PCR product.

FIG. 5 shows PCR amplification of Pro_OLE1Gel electrophoresis of an OLE1-GFP fragment, wherein 9 is Pro_OLE1Comprises OLE1-GFP fragment, HindIII enzyme cutting pCXSN carrier product 10-11, pCXSN-OLE1-GFP recombinant carrier 12-13.

FIG. 6 is a gel electrophoresis of pOGT recombinant vector.

FIG. 7 is a fluorescent and molecular assay of pOGT transgenic Arabidopsis seeds in which (a-e) bright field images; (f-j) identical object image under fluorescent microscope; (a, f) Col-0 (wild type) Arabidopsis seeds; (b, g) T₀Generation of pOGT transgenic Arabidopsis seeds; (c, h) T₁Generation of pOGT transgenic Arabidopsis seeds; (d, i) T₀Generation pCXSN-OLE1-GFP transgenic Arabidopsis seeds; (e, j) T₁Generation pCXSN-OLE1-GFP transgenic Arabidopsis seeds.

FIG. 8 shows T before and after mutation₀、T₁And (5) comparing the fluorescence ratios of the generation seeds.

FIG. 9 is a graph showing a comparison of the relative expression levels of GFP in wild type, after point mutation and before point mutation transgenic Arabidopsis thaliana. FIG. 10 is a gel electrophoresis of transgenic Arabidopsis genomic DNA.

FIG. 11 is a schematic diagram of the construction of pOGT-MeEXPA30 vector.

FIG. 12 shows wild type Arabidopsis thaliana seed and T₀、T₁A picture of Arabidopsis thaliana seed, pOGT-MeEXPA 30. FIG. 13 is the ratio of fluorescent seeds to total number of seeds in each random view under the microscope.

FIG. 14 is T₁PCR characterization gel electrophoresis of pOGT-MeEXPA30 transgenic Arabidopsis thaliana.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.