Application of AtGRF gene in improving genetic transformation efficiency of cucumber

Abstract

The application discloses application of AtGRF genes in improving cucumber genetic transformation efficiency and belongs to the technical field of genetic breeding. Overexpression vectors containing AtGRF1, AtGRF2, AtGRF3, AtGRF4, AtGRF5, AtGRF6, AtGRF7, AtGRF8 and AtGRF9 are respectively constructed and genetic transformation experiments are carried out, it is found that the overexpression vector of AtGRF9 improves the genetic transformation efficiency of cucumber, and the DsRed fluorescent marker gene of the transgenic plant can be stably inherited to the next generation and does not cause sterility due to gamete malformation; the overexpression AtGRF9 expression frame and AADA antibiotic elements are simultaneously loaded on the same transformation vector, and the genetic transformation efficiency of cucumber can be further improved to 16.53%. The application has important significance for cucumber gene function research, cucumber molecular breeding and acceleration of the breeding process of new cucumber varieties.

Description

TECHNICAL FIELD

[0001] The application relates to application of an AtGRF gene in improving cucumber genetic transformation efficiency and belongs to the technical field of genetic engineering and genetic breeding. BACKGROUND

[0002] Cucumber (Cucumis sativus L.) is an important horticultural crop in the Cucurbitaceae family and is one of the most widely planted important vegetables in the world. China is the main cucumber producing country, and the planting scale and yield are the largest in the world. In 2009, the genetic code of the cucumber genome was cracked, providing rich resources and information for the genetic breeding of cucumbers. In 2016, Chandrasekaran et al. first used the CRISPR / Cas9 gene editing technology for cucumbers, providing a feasible tool for using genomic big data to mine functional genes. Further use of genomic big data and gene editing tools for molecular breeding also requires efficient genetic transformation systems to cooperate. However, the genetic transformation of Cucurbitaceae crops has always been a difficult problem worldwide. Although there have been breakthroughs in recent years, there are still problems such as low transformation efficiency, difficult technology, time-consuming and labor-intensive, and the lack of an efficient genetic transformation and gene editing system is still one of the biggest technical bottlenecks restricting the biological research and molecular breeding of most Cucurbitaceae crops.

[0003] (1) Research progress of cucumber genetic transformation and gene editing

[0004] At present, the introduction of CRISPR / Cas9 gene editing elements into plant cells still mainly relies on the agrobacterium-mediated genetic transformation technology. Since Mohiuddin et al. first reported the cotyledon regeneration system of cucumber in 1997, researchers have tried to optimize it from different angles, but the genetic transformation efficiency of cucumber still remains at a very low level. In 2017, Hu et al. increased the genetic transformation efficiency of cucumber to about 0.1% by applying vacuum negative pressure to strengthen the agrobacterium infection depth, and based on this, they first realized gene editing of cucumber in China. In 2022, Xin et al. used physical methods such as ultrasonic waves and nanobrushes to create the best infection intensity and increased the genetic transformation efficiency of different genotypes of cucumber to 1.97%-5.18%. However, the use of nanobrushes has problems such as many technical details, high technical difficulty, time-consuming and labor-intensive, which makes it difficult to efficiently repeat the method in different research groups. In summary, most of the current reports on cucumber genetic transformation systems focus on the optimization of technical details, and have limited effect on further improving the genetic transformation efficiency.

[0005] (2) Research progress of regeneration-promoting genes in plant genetic transformation

[0006] Low regeneration efficiency is the main factor affecting the efficiency of cucumber genetic transformation. Recent studies have shown that overexpression of regeneration-promoting genes can significantly improve the efficiency of genetic transformation. Commonly used regeneration-promoting genes include GRF genes, WOX family genes, BBM family genes, and PLT family genes, which have been shown to significantly improve the efficiency of genetic transformation in various crops.

[0007] GRF (growth-regulating-factor) is a highly conserved transcription factor family in plants, mainly involved in the regulation of plant tissue or organ development. Arabidopsis AtGRF family contains 9 genes, except AtGRF9 with two WRC domains, other AtGRF only contains one WRC domain and one QLQ domain. Overexpression of AtGRF1, AtGRF2, AtGRF3 and AtGRF5 in Arabidopsis can improve leaf growth and development ability; overexpression of AtGRF7 and AtGRF9 can affect the development of pistil; AtGRF and GIFs transcription complex affect the development of stamen and anther meristem cells; overexpression of AtGRF1, AtGRF2 and AtGRF3 in Arabidopsis can regulate cotyledon development by controlling cell size.

[0008] Recently, some members of the GRF gene family have been found to improve the efficiency of genetic transformation. Studies have shown that overexpression of AtGRF5 and AtGRF6, AtGRF9 can promote the proliferation of transgenic rape callus cells; overexpression of AtGRF5 can not only promote the proliferation of transgenic soybean and sunflower cells and the formation of transgenic buds, but also overcome the genotype dependence and achieve the transformation of some difficult-to-transform sugar beet varieties; overexpression of ZmGRF5 can promote maize somatic embryogenesis transformation and obtain transgenic plants; overexpression of AtGRF5 in watermelon can improve the efficiency of genetic transformation to 24.73%; overexpression of endogenous GRF4-GIF1 complex in watermelon can achieve high-efficiency genetic transformation in various genotypes and overcome the genotype dependence to some extent; two pairs of GRF-GIF complexes were constructed in strawberry, and it was found that the genetic transformation efficiency of GRF5-GIF1 chimeric gene was 2.25 times higher than that of the control.

[0009] In addition, another limiting factor of cucumber genetic transformation system is the lack of highly efficient antibiotics as a screening agent. The screening agents currently used are mainly kanamycin (Kan) and herbicide (Basta), etc., which can only partially inhibit the growth of non-transformed seedlings, resulting in high false positive background and low genetic transformation rate. Therefore, screening more efficient antibiotics for cucumber genetic transformation system can further improve the efficiency of cucumber genetic transformation.

[0010] In summary, although there is now a cucumber genetic transformation technology system, but still face the transformation process cumbersome, labor and time-consuming and genetic transformation efficiency is low, etc. Promote the use of regeneration genes may further greatly improve the efficiency of cucumber genetic transformation provides a new opportunity. However, the current promotion of regeneration genes in Cucurbitaceae crops, especially in cucumber, is still relatively rare. Through the promotion of regeneration genes to improve the efficiency of cucumber genetic transformation, which can greatly speed up the process of cucumber gene function research and genetic improvement. SUMMARY

[0011] Due to the low efficiency of cucumber genetic transformation, it is difficult to carry out rapid and large-scale transformation, which greatly restricts the process of cucumber biological research and genetic improvement. Therefore, it is necessary to further develop related technologies to improve the efficiency of cucumber genetic transformation. In view of this, the present application screens AtGRF family genes with regeneration-promoting effects and uses spectinomycin resistance gene AADA as a screening marker to ultimately develop a method for improving the efficiency of cucumber genetic transformation.

[0012] In order to achieve the above purpose, the present application first studies the role of AtGRF gene in promoting the efficiency of cucumber genetic transformation. AtGRF family mainly has nine members, which are AtGRF1, AtGRF2, AtGRF3, AtGRF4, AtGRF5, AtGRF6, AtGRF7, AtGRF8 and AtGRF9. The CDS sequences thereof are constructed into overexpression vectors and subjected to genetic transformation experiments. The overexpression vector is simultaneously loaded with 35Spro:DsRed as a reporter gene, so that the positive transformation event can be monitored by DsRed fluorescence. In addition, the overexpression vector is also loaded with Basta herbicide resistance gene. It is found that except for AtGRF1, transgenic plants with healthy morphology are obtained by transforming the overexpression vectors of other AtGRF genes, among which overexpression of AtGRF7 and AtGRF9 has the most significant effect on improving the efficiency of cucumber genetic transformation, and the genetic transformation efficiency is 3.03% and 4.03%, respectively, which are much higher than the 0.43% of the empty vector control.

[0013] The T0 generation transgenic plants, fruits and T1 generation seeds overexpressing AtGRF7 and AtGRF9 genes are observed and counted. No significant difference is found between the transgenic plants and the control in terms of leaf development and plant type. The fruits of the transgenic plants can be observed to have DsRed red fluorescence. The seeds overexpressing AtGRF9 are full in shape and have no obvious difference with the seeds of wild type CU2, and the seeds can be observed to have DsRed red fluorescence. However, the seeds overexpressing AtGRF7 can be observed to have DsRed red fluorescence, but most of the seeds are deformed in shape, lack of nutrient filling inside, and have other developmental deformities. The T1 generation plants are obtained by seed germination, the T1 generation plants overexpressing AtGRF7 cannot be observed to have DsRed red fluorescence, indicating that the transgenic fragment containing DsRed cannot be inherited from the T0 generation plants to the T1 generation; on the contrary, the leaves and growth points of the T1 generation plants overexpressing AtGRF9 can be observed to have DsRed red fluorescence, indicating that the transgenic fragment can be stably inherited to the next generation.

[0014] The above results show that AtGRF9 can improve the genetic transformation efficiency of cucumber, and the seeds are fertile, and the transgenic fragment can be inherited to the next generation; overexpression of AtGRF7 significantly affects fertility, causes seed deformity, and the transgenic fragment cannot be inherited to the next generation. The present application finds that overexpression of the regeneration-promoting gene AtGRF9 can improve the genetic transformation efficiency of cucumber, which has important significance for carrying out cucumber gene function research and molecular breeding, providing a more efficient gene editing tool, and accelerating the process of cucumber new variety breeding.

[0015] The application effect of the spectinomycin resistance gene AADA gene in cucumber is further screened, and 125 mg / L of spectinomycin (Spe) is determined as the best screening concentration. On this basis, the overexpression vector loaded with the AADA gene and the AtGRF gene is subjected to genetic transformation experiment, so as to study the influence of the combination of overexpression of the AtGRF9 gene and the AADA antibiotic resistance gene on the genetic transformation of cucumber. It is found that compared with the transformation efficiency of 9.13% of the blank control containing only the AADA resistance gene, the combination of AtGRF9 and AADA antibiotic genes can further improve the genetic transformation efficiency of cucumber to 16.53%.

[0016] On this basis, the present application further provides a genetic transformation method of cucumber, which comprises the following steps: using the agrobacterium-mediated genetic transformation method to transform the expression vector containing the AtGRF9 gene into the genome of cucumber to obtain the cucumber transgenic plants expressing the AtGRF9 gene.

[0017] Further, the expression vector further contains the AADA antibiotic resistance gene.

[0018] Further, the specific steps of the genetic transformation are as follows:

[0019] 1) Construct an expression vector containing the AtGRF9 gene, and the expression vector also contains an AADA antibiotic resistance gene;

[0020] 2) Use the expression vector constructed in step 1) to transform Agrobacterium;

[0021] 3) Use cucumber cotyledon as explants, use the transformed Agrobacterium to infect the explants, and induce differentiation in the medium containing antibiotics, to obtain the cucumber transgenic plants expressing the AtGRF9 gene.

[0022] Further, the antibiotic is spectinomycin.

[0023] Further, the concentration of the spectinomycin is 125 mg / L.

[0024] The nucleotide sequence of the AtGRF9 gene is shown in SEQ ID NO: 1.

[0025] The expression vector, recombinant bacteria, transgenic cucumber plants derived from the AtGRF9 gene, and the application of these derivatives in improving the genetic transformation efficiency of cucumber all belong to the protection scope of the present application.

[0026] For more detailed technical solutions, refer to the specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 : Analysis of the conserved domain of AtGRF family genes.

[0028] Figure 2 : Analysis of the protein motif encoded by AtGRF family genes.

[0029] Figure 3 : Phylogenetic tree of AtGRF family genes.

[0030] Figure 4 : Analysis of the relative expression amount of overexpressed AtGRF genes. A is the overexpressed AtGRF7 strain; B is the overexpressed AtGRF9 strain.

[0031] Figure 5 : Bright field and fluorescence images of regenerated shoots of the blank vector and AtGRF vector.

[0032] Figure 6 : Bright field and fluorescence images of wild type CU2 cucumber fruits and cucumber fruits overexpressing AtGRF genes.

[0033] Figure 7: Bright field and fluorescence images of seeds and T1 plants of wild type CU2 and overexpression AtGRF gene plants.

[0034] Figure 8 : Plasmid map of pAAtGRF9. DETAILED DESCRIPTION

[0035] The application will be further described in detail below with specific examples.

[0036] The application utilizes the regeneration promoting gene to improve the genetic transformation efficiency of cucumber, and provides a more efficient genetic transformation system. The most effective AtGRF gene is screened by using the agrobacterium-mediated genetic transformation method, and the transgenic plants are obtained.

[0037] pBAtGRF1, pBAtGRF2, pBAtGRF3, pBAtGRF4, pBAtGRF5, pBAtGRF6, pBAtGRF7, pBAtGRF8 and pBAtGRF9 are constructed by using AtGRF1, AtGRF2, AtGRF3, AtGRF4, AtGRF5, AtGRF6, AtGRF7, AtGRF8 and AtGRF9 respectively. The sequence numbers of the nine genes are as follows:

[0038] AtGRF1: AT2G22840.1

[0039] AtGRF2: AT4G37740.1

[0040] AtGRF3: AT2G36400.1

[0041] AtGRF4: AT3G52910.1

[0042] AtGRF5: AT3G13960.1

[0043] AtGRF6: AT2G06200.1

[0044] AtGRF7: AT5G53660.1

[0045] AtGRF8: AT4G24150.1

[0046] AtGRF9: AT2G45480.1

[0047] Then the overexpression vectors are constructed by gene synthesis. The application constructs the overexpression vectors pBAtGRF1, pBAtGRF2, pBAtGRF3, pBAtGRF4, pBAtGRF5, pBAtGRF6, pBAtGRF7, pBAtGRF8, pBAtGRF9 of AtGRF1, AtGRF2, AtGRF3, AtGRF4, AtGRF5, AtGRF6, AtGRF7, AtGRF8, AtGRF9 genes, and introduces them into CU2 cucumber germplasm by the method of Agrobacterium-mediated genetic transformation. The vector plasmids all contain 35Spro: DsRed red light reporter gene expression frame, and are loaded with herbicide resistance gene Basta.

[0048] After stable genetic transformation experiment, the overexpression vectors containing AtGRF genes all obtain transgenic plants with healthy morphology except AtGRF1, wherein the overexpression of AtGRF7 and AtGRF9 is most significant in improving the genetic transformation efficiency of cucumber, and the genetic transformation efficiency reaches 3.03% and 4.03% respectively, which is much higher than 0.43% of the empty vector control. The experimental results show that the overexpression vectors containing AtGRF7 and AtGRF9 genes improve the genetic transformation efficiency of cucumber to a certain extent.

[0049] Meanwhile, the T0 generation transgenic plants, fruits and seeds and T1 generation seedlings are observed and counted. It is found that most of the seeds overexpressing AtGRF7 present shape shrinkage and developmental deformity; although the seeds overexpressing AtGRF7 present DsRed red fluorescence can be observed, the fluorescence cannot be observed in T1 generation seedlings, indicating that the transgenic fragment containing DsRed cannot be inherited to the next generation. The overexpression of AtGRF9 does not affect fertility, and the seeds develop normally, and DsRed fluorescence can be detected in T1 generation seedlings, indicating that the transgenic fragment can be normally inherited to the next generation. The above results show that the overexpression of AtGRF9 can significantly improve the genetic transformation efficiency of cucumber, and the transgenic fragment can be successfully inherited to the next generation. In summary, AtGRF9 can be used for optimizing the genetic transformation system of cucumber and improving the genetic transformation efficiency.

[0050] In view of the above results, the amino acid sequences of the nine genes of AtGRF family are analyzed, as shown in Table 1, the nine members all contain highly conserved QLQ and WRC domains. Figure 1

[0051] Figure 2 ​​As shown, all members of the AtGRF family contain both Motif 1 and Motif 2, indicating that they share similar motif types. Annotation of Motif 1 and Motif 2 revealed that Motif 1 belongs to the WRC domain, while Motif 2 belongs to the QLQ domain. Notably, only AtGRF3 and AtGRF4 contain Motif 4, and only AtGRF9 contains two WRC domains. The similarity of conserved motifs and gene structures among AtGRF family members further supports the accuracy of the phylogenetic tree.

[0052] The protein sequence of Arabidopsis thaliana AtGRF gene was downloaded from NCBI website, and the phylogenetic tree was constructed using MEGA7.0 software (Kumar et al 2016) and ML Tree with the default system parameters. Figure 3 As shown, AtGRF1 and AtGRF2 are in one branch, AtGRF3 and AtGRF4 are in one branch, AtGRF5 and AtGRF6 are in one branch, AtGRF7 and AtGRF8 are in one branch, and AtGRF9 is in a separate branch.

[0053] Furthermore, amino acid sequence comparison analysis was performed on AtGRF family members AtGRF1, AtGRF2, AtGRF3, AtGRF4, AtGRF5, AtGRF6, AtGRF7, and AtGRF8 with AtGRF9, respectively. It was found that AtGRF3 and AtGRF7 had the highest similarity with AtGRF9, about 48%. Secondly, the similarity between AtGRF1, AtGRF4, AtGRF6 and AtGRF9 was about 44%, the similarity between AtGRF2, AtGRF5 and AtGRF9 was about 42%, and the similarity between AtGRF8 and AtGRF9 was about 30%. The similarity results also further supported the accuracy of the evolutionary tree.

[0054] Example 1 Agrobacterium-mediated genetic transformation of cucumber

[0055] 1. Construction of AtGRF family overexpression vector

[0056] The vector pBSE403DsR in the laboratory was recovered after enzyme digestion with Hind III. The gene sequence of pAtUBI10: AtGRF5 and hspT terminator was cloned into the above-mentioned enzyme-digested vector to construct the vector pBAtGRF5. Then the vector pBAtGRF5 was recovered after enzyme digestion with BsrGI and BamHI, and other AtGRF genes were cloned into the enzyme-digested pBAtGRF5 vector through 5' BsrGI and 3' BamHI to successfully construct the overexpression vectors pBAtGRF1, pBAtGRF2, pBAtGRF3, pBAtGRF4, pBAtGRF6, pBAtGRF7, pBAtGRF8 and pBAtGRF9 by replacing AtGRF5. The overexpression vector also contains a 35Spro:DsRed red fluorescent protein independent expression frame for subsequent tracking and monitoring of positive transformation events. The overexpression vector also contains the herbicide resistance gene Basta, which corresponds to the addition of 2 mg / L herbicide (PPT) in the differentiation medium as a screening agent.

[0057] The vector construction was entrusted to Jinweizhi Biotechnology Co., Ltd. The specific steps are as follows:

[0058] Vector enzyme digestion: first, the pBAtGRF5 vector was double-digested with BsrGI and BamHI enzymes at 37°C overnight, and then inactivated at 80°C for 10 minutes.

[0059] Ligation: the gene-synthesized target fragment pAtUBI10: AtGRF5-hspT was connected with the pBAtGRF5 backbone vector fragment recovered after double enzyme digestion using a kit for homologous recombination. The homologous recombination connection reaction system was prepared and reacted in a PCR instrument at 37°C for 30 min. The reaction system is shown in Table 1.

[0060] Table 1 Homologous recombination connection system

[0061]

[0062] Transformation of E. coli: the connected product was transformed into E. coli by heat shock method. The specific steps are as follows: the E. coli competent cells were taken out from the -80°C refrigerator, 10 μL of the connected product was added to 100 μL of the E. coli competent cells, and then reacted in a 42°C water bath for 45 s, and then reacted on ice for 5 min. Then 700 μL of LB liquid medium without antibiotics was added in a clean bench, and then placed in a 37°C shaker at 200 r / min for 30 min. Then 100 μL of the uniform liquid was spread on LB solid medium (Kan resistance, 50 mg / L) plate, and then the plate was sealed and cultured in a 37°C incubator overnight.

[0063] Colony PCR: Single colony was picked up from the LB solid medium containing Kan resistance with sterilized toothpick or gun head and transferred into 1 mL liquid LB medium containing Kan resistance in a centrifuge tube. After 37°C 200 r / min shaking, colony PCR was performed. After the PCR reaction, 1% agarose gel electrophoresis was performed for identification. After the band size was consistent with the expected size, the company was sent for sequencing, and then the sequence alignment was performed using Geneious software. Whether the obtained gene coding region sequence is consistent with the reference CDS sequence.

[0064] 2. Agrobacterium transformation

[0065] The plasmid was transformed into Agrobacterium competent cells by freeze-thaw method. The specific steps are as follows: the expression vector plasmid returned by Genewiz Biotechnology Co., Ltd. was transformed into Agrobacterium EHA105 (Agrobacterium strain EHA105 was purchased from Shanghai Weidi Biotechnology Co., Ltd.). 2 μL plasmid was taken in a super-clean bench, and 100 μL of EHA105 Agrobacterium competent cells were added and mixed by flicking. After mixing, it was placed in ice water for 5 min, frozen in liquid nitrogen for 1 min, and then quickly placed in a 37°C water bath for 5 min. 800 μL of LB medium was added, and it was cultured at 28°C, 220 rpm on a shaking table for 2 h. It was evenly coated on the solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin (Rif), and cultured in a 28°C incubator for two days in the dark. Single colony was selected and cultured in LB+Kana+Rif liquid for 12 h. After shaking, positive identification was performed, and the positive Agrobacterium liquid was stored in a 4°C refrigerator for short-term use. For long-term use, an equal volume of 50% sterilized glycerol was added, mixed well, and stored in a -80°C refrigerator. It was used for stable genetic transformation in the later stage.

[0066] 3. Cucumber genetic transformation experiment

[0067] The germplasm CU2 was selected for genetic transformation.

[0068] (1) Sowing: Take the CU2 cucumber seeds with full grains and uniform size, soak in warm water at 55°C for more than half an hour, and remove the seed coat. In a super-clean bench, first wash with 75% alcohol for less than 30 s, then soak in 0.3% NaClO solution for 15 min, shake gently during the process. After disinfection, rinse with sterile water for 5 times. The disinfected seeds were transferred to the prepared seed germination medium. Place in a 28-degree dark culture for 24 h. When the seed coat begins to be removed and the cotyledon appears clear vascular ridge line, the explant can be cut.

[0069] (2) Agrobacterium infection: Pick a positive Agrobacterium EHA105 single colony and place it in 2 mL of LB liquid medium containing 50 mg / L Kan and 50 mg / L Rif, shake at 28°C and 220 rpm overnight. Then dilute it at a ratio of 1:500 and add it to fresh 50 mL of LB liquid medium (containing Kan and Rif) and culture it at 28°C and 220 rpm overnight. The next day, when the bacterial liquid grows to OD 600 When the OD value was 0.4-0.8, the Agrobacterium was collected by centrifugation at 6000 rpm for 8 min, and the Agrobacterium was resuspended in IM liquid medium and diluted to OD 600 0.2 is reserved. Take the germinated seeds and, in a clean bench, discard approximately 1 / 3 of the distal cotyledons, remove the embryonic axis, and separate the two cotyledons, forming a U-shaped wound at the proximal end of each cotyledon. This will yield the explants. Place the explants in the prepared resuspension and sonicate at 50W for 30 seconds. In the clean bench, remove the syringe plunger and add the sonicated cotyledon explants and resuspension to the barrel of a 20mL syringe. Gently insert the plunger and push the plunger forward until it reaches the 10mL mark. Seal the syringe needle hole with a rubber stopper and slowly and forcefully pull the plunger backward to the 20mL mark, holding for 1.5 minutes to achieve the desired vacuum pressure. Gently release the plunger and allow it to slowly return to the 10mL mark. Repeat the vacuum pressure application for another 1.5 minutes.

[0070] (3) Co-cultivation: After infection, the explants were spread on filter paper to lightly absorb the attached bacterial liquid and then transferred to the co-cultivation medium padded with two layers of filter paper. After sealing, the co-cultivation was carried out at 23°C in the dark for four days. The luminescence of DsRed was observed using a handheld fluorescent protein observation lamp LUYOR-3415RG dual-wavelength fluorescent protein excitation light source UV lamp to evaluate the infection efficiency.

[0071] (4) Differentiation and regeneration: Wash the explants 7-8 times with sterile water, dry the liquid attached to the surface with sterilized absorbent paper, and insert the explants obliquely on the recovery medium. After 7 days of recovery culture, transfer them to the differentiation medium and subculture them every two weeks. After culturing under light for four weeks, use a handheld fluorescent protein observation lamp to select cotyledons that show DsRed fluorescence and subculture them into tissue culture bottles containing regeneration medium or elongation medium.

[0072] (5) Identification of positive buds: Positive plants were detected using a handheld fluorescent protein observation lamp LUYOR-3415RG dual-wavelength fluorescent protein excitation light source UV lamp. According to the instructions for use of the fluorescent protein observation lamp, turn on the red light source to excite fluorescence and use the corresponding red filter to detect whether red fluorescence is expressed.

[0073] The medium formula used in the experiment is as follows:

[0074] Seed germination medium: 4.43 g / L MS + 30 g / L sucrose + 3.5 g / L phytagel + 2 mg / L 6-BA + 1 mg / L ABA, pH 5.85-5.90;

[0075] IM liquid medium: 4.43 g / L MS + 30 g / L sucrose + 2 mg / L 6-BA + 1 mg / L ABA + 2.5 M MES + 80 mg / L As, pH 5.85-5.90;

[0076] Co-culture medium: 4.43 g / L MS + 30 g / L sucrose + 3.5 g / L phytagel + 2 mg / L 6-BA + 1 mg / L ABA + 2.5 M MES + 80 mg / L As + 150 mg / L DTT, pH 5.85-5.90;

[0077] Recovery medium: 4.43 g / L MS + 30 g / L sucrose + 9 g / L agar + 2 mg / L 6-BA + 1 mg / L ABA + 200 mg / mL TMT, pH 5.85-5.90;

[0078] Differentiation medium: 4.43 g / L MS + 30 g / L sucrose + 3.5 g / L phytagel + 2 mg / L 6-BA + 1 mg / L ABA + 200 mg / mL TMT, pH 5.85-5.90; 2 mg / L herbicide (PPT) is added as a screening agent;

[0079] Rooting medium: 4.43 g / L MS + 30 g / L sucrose + 3.5 g / L phytagel + 200 mg / mL TMT, pH 5.85-5.90.

[0080] 4. Expression analysis of AtGRF gene in transgenic plants

[0081] qRT-PCR experiment and expression analysis: The overexpression AtGRF7, AtGRF9 transgenic plants were used as the experimental group, and wild type CU2 was used as the negative control. The AtGRF7, AtGRF9 gene was used as the target gene, and the CsActin gene was used as the internal reference gene for qRT-PCR. The relative expression of AtGRF7, AtGRF9 gene was calculated. The leaf of overexpression strain was randomly selected to extract RNA. RNA extraction used TransZol (Beijing Zoonking Biotechnology Co., Ltd.) according to the instructions to extract RNA, and reverse transcription was performed according to the instructions of HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) reverse transcription kit (Nanjing Novogene Bioinformatics Technology Co., Ltd.). The cDNA product was stored at -20℃.

[0082] The qRT-PCR probe primers RT-AtGRF4-F, RT-AtGRF4-R, RT-AtGRF7-F, RT-AtGRF7-R, RT-AtGRF9-F, RT-AtGRF9-R (primer sequences are shown in Table 2) were designed, and the above cDNA was used as the template, and the concentration was diluted to 200 ng / μL. The reaction system was configured according to Table 3, and the qRT-PCR was performed according to the program of Table 4. The data after the reaction was processed by GraphPad Prism.

[0083] Table 2 qRT-PCR primers

[0084]

[0085] Table 3 qRT-PCR reaction system

[0086]

[0087]

[0088] Note: When amplifying the target gene and the internal reference gene, each sample was repeated 4 times.

[0089] Table 4 qRT-PCR amplification program

[0090]

[0091] The results of the RT-qPCR quantitative experiment analysis of the expression of AtGRF7, AtGRF9 in transgenic plants are shown in Table 5. Figure 4As shown, the expression of AtGRF7 in the 6 lines overexpressing AtGRF7 was up-regulated by 213-fold, 146-fold, 142-fold, 174-fold, 55-fold and 130-fold, respectively, compared with the wild type; the expression of AtGRF9 in the 8 lines overexpressing AtGRF9 was up-regulated by 323-fold, 743-fold, 593-fold, 332-fold, 43-fold, 59-fold, 13-fold and 53-fold, respectively. In summary, AtGRF7 and AtGRF9 were up-regulated to different degrees in the transgenic plants.

[0092] 5. Effect evaluation of overexpression of AtGRF for improving the efficiency of genetic transformation of cucumber

[0093] DsRed fluorescence observation was performed on the obtained regenerated shoots by means of a hand-held fluorescence observation lamp to track positive transformation events. The genetic transformation efficiency of the empty vector control without the regeneration-promoting gene was only 0.43% (Table 5), and most of the obtained regenerated shoots were transgenic negatives, and no DsRed fluorescence was observed. In the overexpression vectors of the AtGRF family genes, only overexpression of AtGRF1 failed to obtain healthy regenerated shoots, and the other family members could obtain regenerated shoots expressing DsRed fluorescence. Figure 5 Among them, the overexpression vectors containing AtGRF7 and AtGRF9 most significantly improved the efficiency of genetic transformation of cucumber, and the genetic transformation efficiencies were 3.03% and 4.03%, respectively (Table 5).

[0094] Table 5 Effect of AtGRF gene on the efficiency of genetic transformation of cucumber

[0095]

[0096]

[0097] Note: Transgenic plants: regenerated shoots with complete and healthy plant morphology. Genetic transformation efficiency = (number of transgenic plants / total number of explants) x 100%.

[0098] 6. Reproductive genetic performance of transgenic positive plants

[0099] Afterwards, we further observed the growth and development of the transgenic T0 generation plants overexpressing AtGRF7 and AtGRF9. The results showed that there was no significant difference in leaf and plant type between the transgenic plants and the control. The transgenic plants overexpressing AtGRF7 and AtGRF9 genes could normally bloom, and the fruits were normal after self-pollination. Compared with the white-green fruits of wild type CU2, the self-pollinated fruits of the transgenic plants were pink due to the overexpression of the DsRed gene. Then, we used a handheld fluorescent protein lamp to observe the DsRed fluorescence of the obtained fruit materials. No DsRed red fluorescence was observed in the wild type control fruits, but the fruits of the transgenic plants could be observed to have DsRed red fluorescence. Figure 6

[0100] Subsequently, we found that the seeds of AtGRF9 overexpressing plants were plump, and there was no obvious difference in shape between the seeds of wild type CU2 and AtGRF9 overexpressing plants. However, the seeds of AtGRF7 overexpressing plants were mostly shriveled, lacked nutrient filling, and were deformed. Further DsRed fluorescence observation of the seeds using a handheld fluorescent protein lamp showed that the seed coat of wild type CU2 seeds had no DsRed red fluorescence, while the seed coat of AtGRF7 and AtGRF9 overexpressing seeds had DsRed red fluorescence.

[0101] Further seed germination and DsRed fluorescence observation of the one-leaf-one-heart stage T1 generation seedlings using a handheld fluorescent protein lamp showed that the wild type CU2 plants had no DsRed red fluorescence, while the leaves and growth points of the T1 generation plants overexpressing AtGRF9 could be observed to have DsRed red fluorescence, indicating that the transgenic fragment containing DsRed was successfully inherited to the next generation. However, no DsRed red fluorescence was observed in the T1 generation plants overexpressing AtGRF7, indicating that the transgenic fragment contained in the T0 generation plants was not inherited to the T1 generation. Figure 7 As to why the seed coat of AtGRF7 overexpressing plants could be observed to have DsRed red light, it is because the seed coat is developed from the mother ovule cells by mitosis, and thus the fluorescence observed in the seed coat reflects the gene expression of the T0 generation mother. The seed embryo and T1 plants are formed by double fertilization of the mother egg cell and father sperm cell after meiosis. We speculate that overexpression of AtGRF7 leads to gamete or embryo arrest, resulting in the failure of the transgenic fragment to be inherited to the T1 generation.

[0102] ​In summary, overexpression of AtGRF7 can increase the genetic transformation efficiency of cucumber to 3.03%, but due to the influence of fertility, the transgenic fragments cannot be stably inherited to the offspring, and its practical application value is relatively low. Overexpression of AtGRF9 can increase the genetic transformation efficiency of cucumber to 4.03%, and does not affect fertility, and the transgenic fragments can be stably inherited, so it can be applied to optimize the genetic transformation vector, and used to improve the genetic transformation efficiency of cucumber.

[0103] Example 2 AtGRF9 combined with antibiotic gene AADA for cucumber genetic transformation experiment

[0104] 1. Vector construction

[0105] Vector enzyme digestion: first use Xba I enzyme and Mlul enzyme to double enzyme cut pASE403DsR vector containing AADA resistance gene, 37°C overnight, 80°C inactivation for 10 minutes, the reaction system is shown in Table 6.

[0106] Table 6 Xba I and Mlul double enzyme cutting reaction system

[0107]

[0108] Gene cloning and recovery: use Geneious software to design cloning primers (cloning primers are shown in Table 7), synthesized by Beijing Genesky Biotechnology Co., Ltd., and the primer is added with the upstream and downstream adapter primer sequence of pASE403DsR vector. Use 2x Phanta Flash Master Mix (Dye Plus) to perform high-fidelity PCR reaction system to amplify the CDS of the target gene. The PCR amplification reaction system and procedure are shown in Table 8 and Table 9.

[0109] Table 7 Cloning primers

[0110]

[0111] Table 8 High-fidelity PCR reaction system of target gene

[0112]

[0113] Table 9 PCR amplification procedure

[0114]

[0115] After the PCR reaction program is completed, 5 μL of the product is taken for 1% agarose gel electrophoresis, and after electrophoresis, the band size is observed on the gel instrument whether it is consistent with the size of the target fragment. The expected band is cut and then the gel recovery kit of AxyPrep company is used for gel recovery.

[0116] Ligation: Then the target fragment AtGRF9 glue recovery product and double enzyme cut pASE403DsR vector were connected by homologous recombination using ClonExpress II One Step Cloning Kit (Novagen) kit, and the homologous recombination connection reaction system was prepared. After 37°C reaction for 30 min in PCR instrument, the reaction system was shown in Table 10.

[0117] Table 10 Homologous recombination connection system

[0118]

[0119] Transformation of E. coli: The connected product was transformed into E. coli DH5α by heat shock method, and the specific steps were as follows: E. coli DH5α competent cells were taken out from the-80°C refrigerator, 10 μL of the connected product was added to 100 μL of E. coli competent cells, and then it was reacted at 42°C water bath for 45 s, and then it was reacted on ice for 5 min. Then 700 μL of antibiotic-free LB liquid medium was added in the clean bench, and then it was placed in a 37°C shaker at 200 r / min for 30 min. Then 100 μL of it was uniformly coated on the LB solid medium (Kan resistance, 50 mg / L) plate, and then it was sealed and placed in a 37°C incubator for overnight culture.

[0120] Colony PCR: The single colony was picked up with a sterilized toothpick or gun head from the overnight culture of the LB solid medium containing Kan, and then it was placed in a 1 mL centrifuge tube containing Kan-containing liquid LB medium. After shaking at 37°C and 200 r / min, colony PCR identification was performed. After the PCR reaction, 1% agarose gel electrophoresis was performed for identification. After the band size was consistent with the expected size, it was sent to Genescript Biotech Co., Ltd. for sequencing, and then the sequence alignment was performed using Geneious software. Whether the obtained AtGRF9 gene coding region sequence was consistent with the reference CDS sequence was determined.

[0121] Plasmid extraction: After the correct sequence alignment, the TIANprep MiniPlasmid Kit II DP103 was used to extract the plasmid. The plasmid map containing the AADA gene and the AtGRF9 gene is shown in Figure 8 .

[0122] 2. Agrobacterium transformation

[0123] The Agrobacterium transformation method was consistent with that of Example 1.

[0124] 3. Cucumber genetic transformation method by adding spectinomycin concentration (Spe) for screening

[0125] The genetic transformation was performed on the germplasm CU2. (1) sowing (2) Agrobacterium infection (3) co-cultivation were consistent with Example 1.

[0126] (4) Differentiation and regeneration: the explants were washed with sterile water for 7-8 times, the surface liquid was absorbed with sterilized absorbent paper, and the explants were obliquely inserted into the recovery medium. After 7 days of recovery culture, the explants were transferred to the differentiation medium containing 125 mg / L of spectinomycin (Spe), and subcultured once every two weeks. After four weeks of culture under light, the cotyledons containing DsRed fluorescent buds were selected using a handheld fluorescent protein observation lamp, and subcultured into tissue culture bottles containing regeneration medium or elongation medium.

[0127] (5) Identification of positive buds: the positive plants were detected using a handheld fluorescent protein observation lamp LUYOR-3415RG dual-wavelength fluorescent protein excitation light source ultraviolet lamp. According to the instructions of the fluorescent protein observation lamp, the red light source was turned on to excite fluorescence, and a corresponding red filter was used to detect whether red fluorescence was expressed.

[0128] The formula of the cucumber tissue culture medium used in the experiment is as follows:

[0129] The seed germination medium, IM liquid medium, co-cultivation medium, recovery medium, and rooting medium were consistent with Example 1.

[0130] Differentiation medium: 4.43 g / L MS + 30 g / L sucrose + 3.5 g / L plant gel + 2 mg / L 6-BA + 1 mg / L ABA + 200 mg / mL TMT + 125 mg / L Spe, pH 5.85-5.90.

[0131] 4. Evaluation of genetic transformation effect

[0132] Subsequently, the vector pASAtGRF9 constructed to overexpress AtGRF9 gene and combined with AADA antibiotic gene was used for cucumber genetic transformation experiment. 125 mg / L spectinomycin was used for transgenic positive selection during differentiation, and the effect on cucumber genetic transformation was observed and counted. The vector plasmid contains a 35Spro:DsRed red light reporter gene expression frame, which can be used to detect positive transgenic events.

[0133] DsRed fluorescent observation of the transformed explants was performed using a handheld fluorescent protein observation lamp, and it was found that positive regenerated buds containing red fluorescent protein expression could be obtained. The application effect of overexpression of AtGRF9 combined with AADA in cucumber transformation was statistically analyzed in detail, as shown in Table 11. The results showed that the combination of AtGRF9 and AADA antibiotic resistance gene could significantly improve the genetic transformation efficiency of cucumber to 16.53%.

[0134] Table 11 Effect of using AtGRF9 in combination with AADA antibiotic on genetic transformation of cucumber

[0135]

[0136] 5. Reproductive genetic performance of transgenic positive plants

[0137] Similarly, using a handheld fluorescent protein lamp to observe the DsRed fluorescence of fruit material of transgenic plants obtained by transforming the pASE403DsR-AtGRF9 vector (AtGRF9+AADA), DsRed red fluorescence can be observed in the selfed fruits of the transgenic plants, while no DsRed red fluorescence is observed in the fruits of wild type plants.

[0138] Subsequently, seed germination was performed on the fruits, and it was found that under the condition of overexpression of AtGRF9 and AADA antibiotic resistance genes in combination, the seed morphology was plump, and there was no obvious difference with the seeds of wild type CU2. Subsequently, using a handheld fluorescent protein lamp to observe the DsRed fluorescence of seed coat, seed embryo and T1 generation seedlings, no DsRed red fluorescence was observed in the seed coat, seed embryo and plants of wild type CU2, while DsRed red fluorescence was observed in the seed coat, seed embryo and T1 generation seedlings of the combination of overexpression of AtGRF9 and AADA genes. This indicates that the combination of AtGRF9 and AADA can stably inherit the transgenic fragments in the transgenic plants to the offspring.

[0139] In summary, the present study found that overexpression of AtGRF9 can improve the genetic transformation efficiency of cucumber, and the transgenic fragments can be stably inherited to the offspring; under the selection condition of 125 mg / L spectinomycin, overexpression of AtGRF9 gene in combination with AADA antibiotic resistance gene can significantly improve the genetic transformation efficiency of cucumber to 16.53%, and the obtained transgenic plants can be stably inherited.

[0140] SEQUENCE LIST EXPLANATION:

[0141] SEQ ID NO: 1: Gene sequence of AtGRF9;

[0142] SEQ ID NO: 2: Gene sequence of AtGRF7;

[0143] SEQ ID NO: 3, 4: Upstream and downstream primer sequences for cloning AtGRF7 gene;

[0144] SEQ ID NO: 5, 6: Upstream and downstream primer sequences for cloning AtGRF9 gene.

Claims

1. AtGRF Gene or its encoded protein, or containing said AtGRF Application of gene expression vector or recombinant bacteria in improving cucumber genetic transformation efficiency, AtGRF The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

2. A method for improving the efficiency of cucumber genetic transformation, characterized in that The following steps are involved: The method of genetic transformation mediated by Agrobacterium is used to transform the AtGRF The expression vector of the gene is transformed into the genome of cucumber to obtain the expression of the AtGRF Genetic cucumber varieties.

3. The method for improving the genetic transformation efficiency of cucumber as claimed in claim 2, wherein: The expression vector also contains AADA Resistance genes.

4. The method for improving the genetic transformation efficiency of cucumber according to claim 3, wherein: The specific steps of the genetic transformation are as follows: 1) Build the AtGRF Gene expression vector, the expression vector also contains spectinomycin resistance gene AADA ; 2) Transform Agrobacterium using the expression vector constructed in step 1); 3) Using cucumber cotyledons as explants, infecting the explants with Agrobacterium from step 2) and inducing differentiation in a medium containing 125 mg / L spectinomycin to obtain the expression of the AtGRF Transgenic cucumber plants.

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