CN112430584B

CN112430584B - Du pear ubiquitin ligase gene, encoding protein and application thereof in plant drought-resistant genetic improvement

Info

Publication number: CN112430584B
Application number: CN202011430369.8A
Authority: CN
Inventors: 黄小三; 王春孟; 戴雨沁; 董慧珍; 陈紫龄; 朱雨; 胡文洁; 罗婧; 张绍铃; 许鑫文; 王海旭
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2022-05-13
Anticipated expiration: 2040-12-07
Also published as: CN112430584A

Abstract

The invention provides a avocado ubiquitin ligase gene, a coded protein and application thereof in plant drought-resistant genetic improvement, belonging to the technical field of molecular biology. The amino acid sequence of the dutchmanspipe root ubiquitin ligase is shown as SEQ ID NO:1 is shown. A gene PbPUB21 nucleotide sequence for coding the pyrus betulaefolia ubiquitin ligase is shown as SEQ ID NO:2, respectively. The invention utilizes agrobacterium-mediated genetic transformation method to transform model plants, so that the avocado ubiquitin ligase gene is overexpressed in arabidopsis, and the obtained transgenic plants have the function of regulating and controlling the drought resistance of arabidopsis. Meanwhile, through the virus-induced gene PbPUB21, the drought resistance of the pear seedlings is reduced compared with that of a contrast wild type, and the fact that the pear ubiquitin ligase and the corresponding coding gene have the biological function of improving the drought resistance of plants is shown.

Description

Du pear ubiquitin ligase gene, encoding protein and application thereof in plant drought-resistant genetic improvement

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a avocado ubiquitin ligase gene, a coded protein and application thereof in plant drought-resistant genetic improvement.

Background

The pear is one of economic fruit tree species mainly cultivated in the world at present, and China is one of important origins of the pear and is the first major country where the yield of the pear is located all over the world. The pear is the third largest fruit in China, which is second to the apples and the oranges, and the planting area is particularly wide. Although the layout and the planning of the pear dominant producing areas in China greatly promote the rapid development of the pear industry, the pear producing areas are widely distributed in China, and a three-area and four-point producing area layout mode that the pear is planted from the northeast to the Guangxi and from the Yunnan to the Shandong is formed. The growth and development of pear trees are easily influenced by environmental factors such as geography, climate conditions and the like. The abiotic stress such as salt, drought and low temperature seriously affects the yield and quality of the pears, researches the molecular biology mechanism of the abiotic stress tolerance/resistance of plants, improves the abiotic stress tolerance (resistance) of crops, and is particularly important for ensuring the high and stable yield of the pears. Due to the problems of high genetic heterozygosity, long childhood period, incompatibility of most self-bred plants, uncertainty of breeding targets and the like in the breeding aspect of pears, the breeding progress of pears is slow, and the conventional breeding method cannot meet the requirements of modern pear industry cultivation on varieties. With the rapid progress of plant tissue culture technology and plant biotechnology, new plant varieties can be bred by methods such as tissue culture technology breeding, genetic transformation breeding, somatic cell hybridization and the like. The plant genetic engineering technology is utilized to improve the characters of the plants, and the varieties with strong resistance are cultured, so that the method has wide application and development prospect for pear stress-resistant breeding.

As a solid crop, plants are in an adverse growing environment throughout their life. Abiotic stresses such as drought, temperature change, high salt, radiation and nutrient loss seriously affect the growth, development and yield of plants. In order to effectively sense, respond to and adapt to these ever-changing environments, plants maximize avoidance of damage from abiotic stresses by precisely regulating their physiological changes (wuhuijia et al, 2006). When stress occurs, plants can respond to stress from molecular, cellular and physiological levels and can survive. At present, four approaches for plant abiotic stress signal regulation and control, including transcriptional regulation, post-transcriptional modification, epigenetic regulation and post-translational modification regulation and control, have been summarized by means of mutant screening, reverse genetics functional validation, transcriptome analysis, proteome analysis, metabolome analysis and the like (Hirayama et al, 2010). Posttranslational regulation including ubiquitination modification, SUMO modification, phosphorylation modification and the like (Hirayama et al, 2010) is a hot spot of the current research on the regulation mechanism of abiotic stress of plants.

The ubiquitin/26S proteasome system (UPS) is widely present in all eukaryotic cells, and its pathway of action is highly conserved (Wangqingmin et al, 2001). The 26S proteasome-mediated protein degradation pathway is one of the most prevalent and efficient protein degradation pathways in eukaryotes (McClellan et al, 2005). Ubiquitination of proteins requires the common involvement of multiple proteins, among which ubiquitin proteins Ub (ubiquitin), ubiquitin activating enzyme E1(ubiquitin activating enzyme), ubiquitin conjugating enzyme E2(ubiquitin conjugating enzyme), and ubiquitin ligase E3(ubiquitin ligand) are mainly involved. Ubiquitin proteins are covalently linked to substrate molecules as molecular tags for protein degradation, a process that requires the involvement of three different acting enzymes, ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2, ubiquitin ligase E3 (dawn et al, 2007). At least 6% of the genes in arabidopsis encode components in the 26S proteasome pathway, 16 of which encode ubiquitin proteins; there are 23 genes encoding 20S core protein particles; there are 31 genes encoding the 19S regulatory subunit protein; there are 2 genes encoding ubiquitin activating enzyme E1; 45 genes encode ubiquitin-conjugating enzyme E2; there are at least 1300 genes encoding ubiquitin ligase E3(Smalle et al, 2004), where ubiquitin ligase primarily determines substrate specificity.

Ubiquitin-proteasome systems are widely found in the cytoplasm and nucleus of eukaryotes, and they respond to abiotic stress by regulating the abundance of regulatory proteins, degrading misfolded and damaged proteins (guo fang, 2007). U-box region control Proteins (PUBs) play important roles in abiotic stress, including participation in regulating plant drought tolerance, low temperature tolerance, and salt stress response.

In arabidopsis, overexpression of 2 PUB genes PUB22 and PUB23 made them more susceptible to salt stress and drought stress, and their ubiquitination disrupted the 26S proteasome cascade, leaving plants susceptible to the environment (Cho S K et al, 2008). Likewise, the PUB genes PUB46 and PUB48 also exert positive regulatory effects in drought stress resistance (Adler G et al, 2017). In rice, the ZFRGI gene negatively regulates plant drought resistance (fankemin et al, 2014). The potato U-box type E3 ligase StPUB27 contains 66 genes, and overexpression of the genes increases the stomata of leaves, increases the conductivity and reduces the stomata opening, which indicates that StPUB27 can regulate the plant resistance through protein modification. Meanwhile, StPUBl7 negatively regulates the drought resistance of plants (Thoho et al, 2018). VaPUB is U-box type E3 ligase in Vitis amurensis, and overexpression of VaPUB in Arabidopsis can improve the expression of a positive control gene in an ICE-CBF cold reaction path, so that the cold resistance and the salt tolerance of plants (Jieli, 2017) are obviously improved. In Arabidopsis thaliana, the U-box type E3 genes AtPUB18, AtPUB19 and AtPUB31 all positively regulated their salt tolerance. And in salt stress, double mutants of atpubl8 and atpubl9 show higher sensitivity. In addition, Arabidopsis thaliana also contains a salt tolerance negative regulator gene AtPUB30 (Lilong, 2016; Bergler J et al, 2011). However, reports of gene cloning and function verification are not found in pears, and literature reports of applying the gene to transform plants to improve drought resistance are not found.

Disclosure of Invention

In view of the above, the present invention aims to provide a avocado ubiquitin ligase gene, a coding protein and applications thereof in plant drought resistance genetic improvement.

The invention provides a avocado ubiquitin ligase, wherein the amino acid sequence of the avocado ubiquitin ligase is shown as SEQ ID NO:1 is shown.

The invention provides a gene PbPUB21 for coding the avocado ubiquitin ligase, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO:2, respectively.

The invention provides a primer pair for amplifying the gene PbPUB21, which comprises a nucleotide sequence shown as SEQ ID NO:3 and the nucleotide sequence of the forward primer is shown as SEQ ID NO:4, or a reverse primer as shown in figure 4.

The invention provides a recombinant plant expression vector containing the gene PbPUB 21.

Preferably, the gene PbPUB21 is inserted on the basis of a plant expression vector pCAM 1300-GFP.

Preferably, the multiple cloning site of the plant expression vector pCAM1300-GFP into which the gene PbPUB21 is inserted includes Xba I and BamHI.

The invention provides application of the pyrus betulaefolia ubiquitin ligase, the gene PbPUB21, the primer pair or the recombinant plant expression vector in drought resistance of plants.

The invention provides application of the pyrus betulaefolia ubiquitin ligase, the gene PbPUB21, the primer pair or the recombinant plant expression vector in drought-resistant plant breeding.

The invention provides application of the pyrus betulaefolia ubiquitin ligase, the gene PbPUB21, the primer pair or the recombinant plant expression vector in construction of drought-resistant transgenic plants.

Preferably, the plant comprises a herbaceous plant or a woody plant; more preferably, the herbaceous plant comprises arabidopsis; the woody plant comprises pyrus betulaefolia.

The invention provides a avocado ubiquitin ligase, wherein the amino acid sequence of the avocado ubiquitin ligase is shown as SEQ ID NO:1 is shown. According to the invention, an agrobacterium-mediated genetic transformation method is utilized to transform a model plant (arabidopsis thaliana), so that the avocado ubiquitin ligase gene is overexpressed in the arabidopsis thaliana, and biological function verification shows that the avocado ubiquitin ligase and the corresponding coding gene PbPUB21 provided by the invention have the function of regulating and controlling the drought resistance of the arabidopsis thaliana. Meanwhile, through virus-induced gene PbPUB21 silencing (VIGS) of the birch pear seedlings, the obtained silencing strain is verified by biological function to have reduced drought resistance compared with a contrast wild type, and the existence of the birch pear ubiquitin ligase and the corresponding coding gene thereof has the biological function of improving the plant drought resistance.

Drawings

FIG. 1 is a schematic technical flow chart of the construction method of a transgenic drought-resistant plant according to the present invention;

FIG. 2 is a schematic representation of the expression of the gene encoding PbPUB21 under dehydration (FIG. 2-A), low temperature (FIG. 2-B), high salt (FIG. 2-C) and abscisic acid (FIG. 2-D) stresses in example 2;

FIG. 3 is the subcellular localization of the PbPUB21 gene in example 3, wherein; FIG. 3-A, 35s: imaging of GFP gene (control) in dark field, uv light, bright field, where the image (right-most) is the image after superposition; FIG. 3-B, PbPUB21-GFP imaging in dark field, UV light, bright field, where the image (rightmost) is the superimposed image;

FIG. 4 is an identification chart of a transgenic PbPUB21 Arabidopsis plant in example 4, and FIG. 4-A is a PCR identification of Arabidopsis T0 generation transgenic plant using gene specific primers, wherein M: marker, +: plasmid, -: wild-type plants, #4 to # 8: the transgenic strains, FIG. 4-B is the semi-quantitative PCR detection of the mRNA level of T1 generation Arabidopsis thaliana, and FIG. 4-C is the overexpression analysis of RT-PCR identification of T1 generation transgenic Arabidopsis thaliana plants;

FIG. 5 shows the determination of phenotype and physiological index after dehydration treatment of the PbPUB21 transgenic Arabidopsis lines (#4 and #5) and non-transgenic plants (WT) in example 4 of the present invention, wherein FIG. 5-A shows the phenotype before and after dehydration treatment of 120min for a 20-day-old Arabidopsis plant; measuring the water loss rate (5-B), the conductivity (5-C) and the malonaldehyde content (5-D) of the dehydrated plants;

FIG. 6 is the phenotype and physiological index measurements before and after drought treatment and after 3 days of rehydration of the transgenic PbPUB21 gene lines (#4 and #5) and non-transgenic plants (WT) in example 4 of the present invention, wherein FIG. 6-A is a phenotype graph of a 30-day-old Arabidopsis plant after 14 days of drought treatment and 3 days of rehydration; FIG. 6-B is a phenotype plot of the corresponding fluorescent chlorophyll; FIGS. 6-C to 6-E show the results of the electric conductivity, malondialdehyde content and Fv/Fm after drought treatment in this order;

FIG. 7 shows histochemical staining analysis H of the strains (#4 and #5) transformed with PbPUB21 gene and wild-type (WT) plants after drought treatment in example 4 of the present invention₂O₂And O^2-A graph of accumulated measurements; FIG. 7-A is a graph of reactive oxygen histochemical staining of untransformed plants and two transgenic lines after drought treatment of 30-day old Arabidopsis plants for 14 days, with Diaminobenzidine (DAB) and Nitrotetrazole (NBT) for H, respectively₂O₂(on the figure) and O^2-(below the figure) dyeing is carried out; and measured H in the sample₂O₂(FIG. 7-B) and anti-O^2-(FIG. 7-C) content;

FIG. 8 is a graph showing the results of determination of phenotypic and physiological indicators of drought-treated 15 days for PbPUB 21-encoding gene-silenced seedling lines of Du pear (pTRV2-1, pTRV2-2 and pTRV2-3) and wild-type plants (WT) in example 5, wherein FIG. 8-A is a phenotype graph of potted plants growing normally (before treatment) and 15 days after drought treatment; FIG. 8-B are fluorescent chlorophyll photographs of 3 silencing lines and wild type plants before and after treatment, FIG. 8-C is a graph of conductivity measurement after drought treatment, and FIG. 8-D is a graph of malondialdehyde measurement after drought treatment; FIG. 8-E shows the real-time quantitative PCR analysis of the expression level of the coding gene of PbPUB21 in different viral silencing strains of Pyrus pyrifolia; FIG. 8-F is a graph of Fv/Fm results before and after drought treatment;

FIG. 9 shows H-drought-treated Du pear seedling lines (pTRV2-1, pTRV2-2 and pTRV2-3) and wild type plants (WT) encoding gene-silenced PbPUB21 in example 5 of the present invention₂O₂And O^2-Measurement of accumulationDetermining a result graph; FIGS. 9-A and 9-B are H in the sample, respectively₂O₂And resistance to O^2-And (4) content.

Detailed Description

The invention provides a avocado ubiquitin ligase, wherein the amino acid sequence of the avocado ubiquitin ligase is shown as SEQ ID NO:1 is shown. The amino acid sequence of SEQ ID NO: the avocado ubiquitin ligase shown in 1 codes 444 amino acids, has an isoelectric point of 6.01 and a molecular weight of 49.81 KDa.

The invention provides a gene PbPUB21 for coding the dutchmanspipe root ubiquitin ligase, wherein the nucleotide sequence of the coding gene is shown as SEQ ID NO:2, respectively. Experiments show that with the prolonging of the processing time, the expression quantity of the coding gene shows an increasing trend within 9 hours, the expression quantity begins to decline after 12 hours, and the coding gene is influenced by drought stress, so that a drought resisting mechanism is responded, and the pyrus betulaefolia realizes the drought resisting function; after the PbPUB 21-transferred overexpression pure line is treated by dehydration stress, the water loss degree of leaves of a transgenic line is obviously lower than that of untransformed plants in phenotype. Meanwhile, the strain of the over-expressed PbPUB21 gene effectively enhances the active oxygen scavenging capacity of the transgenic plant, thereby improving the drought resistance of the plant.

In the present invention, the cloning method of the gene PbPUB21 encoding the Du pear ubiquitin ligase preferably comprises the following steps:

extracting RNA of the pyrus betulaefolia;

reverse transcribing the RNA to obtain cDNA;

and carrying out PCR amplification by using the cDNA as a template and using a forward primer F1 and a reverse primer R1 to obtain the avocado ubiquitin ligase gene PbPUB 21.

In the present invention, the sequence of the forward primer F1 is shown as SEQ ID NO. 3 (5'-atgggtttaggctggagaagga-3') and the sequence of the reverse primer R1 is shown as SEQ ID NO. 4 (5'-aaacgaccttctaatactcttgaaatctacag-3'). The source of the primer pair is not particularly limited, and it may be synthesized by biosynthetic companies well known in the art. In the examples of the present invention, the primer pair was synthesized by Shanghai Bioengineering Co., Ltd. The system used for PCR amplification preferably comprises, per 50. mu.L: 10 μ L of 5 XQ 5 reaction buffer, 10mu.L of mM dNTP, 12.5. mu.L of 10. mu.M forward primer F, 2.5. mu.L of 10. mu.M reverse primer, 0.5. mu.L of template cDNA, 0.5. mu.L of Q5 high fidelity DNA polymerase, dd H₂O make up to 50. mu.L. The PCR amplification procedure preferably comprises pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles.

The type of the plant expression vector of the present invention is not particularly limited, and plant expression vectors known in the art may be used. In the embodiment of the invention, the gene PbPUB21 is preferably inserted on the basis of a plant expression vector pCAM 1300-GFP. The multiple cloning site of the plant expression vector pCAM1300-GFP into which the gene PbPUB21 is inserted preferably includes XbaI and BamHI.

In the present invention, the method for constructing the recombinant plant expression vector preferably comprises the steps of:

A. carrying out enzyme digestion on the gene PbPUB21 obtained by amplification and the vector respectively by using the same endonuclease; obtaining a gene PbPUB21 fragment containing a cohesive end and a linear plasmid;

B. and connecting the gene PbPUB21 fragment containing the cohesive end with a linear plasmid to obtain a recombinant plant expression vector containing the gene PbPUB 21.

In the present invention, the type of the endonuclease is selected according to the type of the vector, and the corresponding endonuclease is selected according to the position of insertion of the exogenous fragment of the vector. In the present example, when the plant expression vector is pCAM1300-GFP, the endonucleases used include Xba I and BamHI. The enzyme digestion conditions are not particularly limited, and the enzyme digestion conditions known in the art can be adopted, for example, the enzyme digestion is carried out for 3-4 hours at 37 ℃. The method of ligation is not particularly limited in the present invention, and a ligation method known in the art may be used, for example, ligation using T7 ligase; the ligation conditions are preferably 37 ℃ for 12 h. Following ligation, the invention also preferably identifies the ligation product. Preferably, the ligation product is transformed into an escherichia coli strain, the escherichia coli strain is cultured and screened, the obtained bacterial colony is subjected to amplification culture and plating, and bacterial liquid obtained after single bacterial colony is selected for bacterial colony PCR. The primers for colony PCR amplification are a forward primer F1(SEQ ID NO:3) and a reverse primer R1(SEQ ID NO: 4).

In the present invention, the method for drought resistance of the plant preferably comprises the following steps:

s1, carrying out PCR amplification by using cDNA of the pyrus betulaefolia as a template and adopting the primer pair to obtain a PCR product;

s2, inserting the PCR product into a plant expression vector to construct a recombinant plant expression vector;

and S3, transferring the recombinant plant expression vector into plant tissues through agrobacterium mediation, and performing induction culture to obtain a recombinant plant.

The method for PCR amplification, construction of recombinant plant expression vector and Agrobacterium-mediated transfer into plant tissue is not specifically limited, and the same construction method is adopted for transgenic plants known in the art. The plant preferably comprises a herbaceous plant or a woody plant; in the examples of the present invention, arabidopsis thaliana is used as a representative of herbaceous plants, and pyrus betulaefolia is used as a representative of woody plants for illustration, but the present invention should not be construed as being limited to the scope of the present invention.

In the invention, the gene PbPUB21 is overexpressed in the constructed recombinant plant, so that the dehydration resistance and drought resistance of the recombinant plant are obviously enhanced compared with those of an untransformed plant (WT). The over-expression of the PbPUB21 gene can effectively enhance the active oxygen scavenging capacity of transgenic plants, thereby improving the drought resistance of the plants. After the PbPUB21 gene in the pyrus betulaefolia is transiently silenced by utilizing a virus-mediated VIGS technology, the drought resistance of a strain for silencing the PbPUB21 gene is obviously reduced compared with that of a contrast wild type. Therefore, the invention also provides application of the pyrus betulaefolia ubiquitin ligase, the gene PbPUB21, the primer pair or the recombinant plant expression vector in constructing drought-resistant transgenic plants.

In the invention, the application in the breeding of the drought-resistant plant preferably comprises the steps of taking the gene PbPUB21 as a screening marker or carrying out amplification and identification on bred offspring by adopting the primer pair, and taking the bred offspring of the high-expression gene PbPUB21 as a breeding target.

The durian ubiquitin ligase gene, the encoded protein and the application thereof in the drought-resistant genetic improvement of plants provided by the invention are described in detail below with reference to the examples, but the genes and the encoded protein are not to be construed as limiting the scope of the invention.

Example 1

Cloning method of birchleaf pear PbPUB21 gene full-length cDNA

A ubiquitin ligase gene PbPUB21 is screened from the pyrus betulaefolia, a primer is designed according to the sequence of the PbPUB21 gene and premier 5.0, and the full length of the pyrus betulaefolia is amplified by an RT-PCR method. The detailed steps are as follows: first strand cDNA synthesis was performed according to the instructions for the TIANGEN reverse transcription kit. The resulting first strand cDNA was used for PCR amplification of the PbPUB21 gene. The total reaction volume of PCR amplification is 50 mu l, wherein 1 mu l of pyrus betulaefolia cDNA, 2.5 mu l of each of upstream and downstream primers, 1 mu l of Taq DNA polymerase, 25 mu l of Buffer solution and sterilized ddH₂O18. mu.l. The reaction procedure for PCR amplification was as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 90s, and extension at 72 ℃ for 90s, and 35 cycles of extension at 72 ℃ for 10min after the completion of the cycles. After the PCR product which generates a single band after the amplification is subjected to 1% agarose gel electrophoresis, a target band is cut off, and the specific target band is recovered according to the using instruction steps of the AxyPrep gel recovery kit.

The recovered and purified solution was ligated with pEASY-T1 vector in a total volume of 10. mu.l, wherein 5. mu.l buffer and 4.5. mu.l PCR-purified product were ligated at 25 ℃ for 30min and transformed into E.coli competent DH 5. alpha. by heat shock method, PCR-verified and sequenced with the gene sequence primers of interest (done by Shanghai Biotechnology, Ltd.). The sequencing result is shown in SEQ ID NO. 2 (atgggtttaggctggagaaggaggagaaccggccatcgcgccgcgaaaaaccagccaggggacctcgacatggaagtcctgatcccggaccattttctctgcccgatatcgcttgatttgatgaaggatccggtcacattgtcatccggaataacctacgatagacagagcatcgacacttggctcgaaggtgggaactttacttgccctgtgacaaatcaggtgttgagaagctttgatcagattcccaaccatagtctcagaaaaatgattcaagaatggtgcaccgaaaacaagcgctacggaatcgaacgtgttccaacgccgcgagttcctgttatgccgatcgaggtctctgagaatcttttcagtttagcgggctcagctcgaagtttggatcaccaagggtgcttggaatatgtgcgcagaatcaagaattgggccgctgagagtgagcgaaacaagcggtgcatattggagaacggggctgcatctgtactctctgccgcctttgattcttttgcgggcgattgcgaaaagaaaaatgcagttgtgtgcgaagaattactctctgcgttgagttggatgcttccagctttcgatgaagaatcacacaaatacttgggatcgaaggcatcgttgcgttgcatggtttggtttctcaaggaaaccgatgatcttttggtcaagcaaaactcccttctagcattgaaacagcttctttcttgcgatgatcacactaaacatgctgaagcgttggcggaaattggtggggtgaatgacattctgttcaagctcatcagagagaaaatttcgccggcgattaccaaggcatctctgatggtagttttctacttgatttcatcatcttcttccgccggtgagaagatcaagttatcatatttggagatgggattggtttctgtgttgctggaaatgcttgtggactcggaaaggggcgtaagcgagggggcattggcggttatcgacagtctttgtgattgcgaagaagggagggaaaaggcgtacgctgatgcgctaaccataccggttttagtgaagaaaattttgaggatatcggaaatggcgactgagtattcgatttcagcgatttggaagctgtgtaaatgtgcaagcaggcaagaagaaagagtgctggttgaggcccttcaagtcggtacatttcagaagctcttgttggttttacaggttcgttgcggggatgatcacacgaaggagaagacgaacgagcttctgaagctcttgaatccttacagagctggattggaatgcattgagtctgtagatttcaagagtattagaaggtcgttttga), and the amino acid sequence obtained by codon transformation is shown in SEQ ID NO. 1 (MGLGWRRRRTGHRAAKNQPGDLDMEVLIPDHFLCPISLDLMKDPVTLSSGITYDRQSIDTWLEGGNFTCPVTNQVLRSFDQIPNHSLRKMIQEWCTENKRYGIERVPTPRVPVMPIEVSENLFSLAGSARSLDHQGCLEYVRRIKNWAAESERNKRCILENGAASVLSAAFDSFAGDCEKKNAVVCEELLSALSWMLPAFDEESHKYLGSKASLRCMVWFLKETDDLLVKQNSLLALKQLLSCDDHTKHAEALAEIGGVNDILFKLIREKISPAITKASLMVVFYLISSSSSAGEKIKLSYLEMGLVSVLLEMLVDSERGVSEGALAVIDSLCDCEEGREKAYADALTIPVLVKKILRISEMATEYSISAIWKLCKCASRQEERVLVEALQVGTFQKLLLVLQVRCGDDHTKEKTNELLKLLNPYRAGLECIESVDFKSIRRSF).

Example 2

qRT-PCR analysis of PbPUB21 encoding gene under different stress conditions

To analyze the PbPUB21 gene in Du pear against dehydration, low temperature, high salt and abscisic acid (ABA) stressThe expression pattern of the gene encoding PbPUB21 was analyzed by Real-time PCR. RNA was extracted using the Plant Total RNAI Kit Plus from Chengdu-Fuji Biotechnology Ltd, and the first strand of DNA was synthesized according to the manual of the TANGEN reverse transcription Kit. The reaction system of 10. mu.l was as follows: mu.l 2 × SYBR Premix ExTaq, 0.1. mu.l cDNA, 0.4. mu.l primer (SEQ ID NO:5, 5'-GCCAGGGGACCTCGACATGGAAGTCC-3' and SEQ ID NO:6, 5' -CGATCGGCATAACAGGAACTCGCGGCG-3), 4.5. mu.l ddH₂And O. The reaction procedure of real-time quantitative PCR using Tublin as internal reference primer (sequence of primer pair is SEQ ID NO:7, 5'-TGGGCTTTGCTCCTCTTAC-3' and SEQ ID NO:8, 5'-CCTTCGTGCTCATCTTACC-3') is shown in Table 1.

TABLE 1 real-time quantitative PCR procedure

The results of the qRT-PCR are shown in FIG. 2. FIG. 2 is a schematic diagram of the expression of a PbPUB21 encoding gene under the stress of dehydration, low temperature, high salt and abscisic acid (ABA), wherein FIG. 2-A is a sample of a birch pear seedling (non-transgenic) dehydrated at corresponding time points at room temperature, and the relative expression amount of the encoding gene is analyzed by real-time quantitative PCR; FIG. 2-B is the expression pattern of seedlings of Pyrus betulaefolia at different time points under low temperature treatment at 4 ℃; FIG. 2-C shows the relative expression level of the coding gene in the Du pear seedlings treated with 200mM NaCl solution, sampled at corresponding time points, and analyzed by real-time quantitative PCR; FIG. 2-D shows the relative expression of the coding gene of the seedlings of Pyrus betulaefolia, which were sampled at corresponding time points under the treatment of 100. mu. MABA solution, and analyzed by real-time quantitative PCR; as can be seen from FIG. 2-A, the expression level of the coding gene showed an increasing trend within 9 hours as the treatment time was prolonged, and the expression level began to decrease after 12 hours. This shows that the coding gene is affected by drought stress, so that the drought-resistant mechanism is responded, and the drought-resistant function of the pyrus betulaefolia is realized.

Example 3

Subcellular localization of PbPUB21 Gene

XbaI and BamHI cleavage sites were added before and after the gene sequence based on the nucleotide sequence of PbPUB21 gene and pCAM1300-GFP vector map, respectively. The plasmid extracted from the target gene with the correct sequencing result is used as a template, and the plasmid is amplified by using a primer (SEQ ID NO:9:5'-GAGAACACGGGGGACTCTAGAATGGGTTTAGGCTGGAGAAGGA-3', SEQ ID NO: 10: 5'-GCCCTTGCTCACCATGGATCCAAACGACCTTCTAATACTCTTGAAATCTACAG-3') added with an enzyme cutting site. The PCR procedure used was: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 60s, extension at 72 ℃ for 90s, and 35 cycles; extension at 72 ℃ for 10 min. The stop codon TAG was removed 3' of the gene in order to allow fusion of the gene with GFP. After the PCR product was electrophoresed through 1% agarose gel, the objective band was recovered by using a gel kit. The pCAM1300-GFP vector plasmid was digested with XbaI and BamHI restriction enzymes, digested at 37 ℃ for 4 hours, purified and recovered. The pCAM1300-GFP vector after enzyme digestion and the gel recovered PbPUB21 fragment are connected by recombinant ligase, are connected for 30min at 37 ℃ and are transformed into escherichia coli competence DH5 alpha. And detecting the transformed bacterial liquid by using PCR, sending the bacterial liquid with positive PCR identification to a company for sequencing, extracting the plasmid of the bacterial liquid with the correct sequencing result, and naming the obtained recombinant vector as pCAM1300-GFP-PbPUB 21.

Agrobacterium-mediated transient transformation of tobacco: the pCAM1300-GFP-PbPUB21 recombinant vector was transformed into Agrobacterium-containing competent GV3101, and the activated Agrobacterium containing the recombinant plasmid was shake-cultured in LB liquid medium containing 505mg/mL kanamycin and 50mg/mL rifampicin at 250rpm and 28 ℃ with shaking. Centrifuged at 6000rpm for 10min and stained with an staining solution (per 100mL included 10mL 100mM MgCl₂，10mL 100mM MES，75μL200 mMAS，ddH₂O make up 100mL) was resuspended to OD₆₀₀Is 0.8. And incubating at room temperature for 3-4 h, injecting the tobacco, removing the injector of the needle head, and injecting the staining solution from the back of the tobacco leaves until the leaves are water-stained. Culturing the injected tobacco in dark for 24h, then culturing in a phytotron at 25 ℃ for 48-72 h, observing, and cutting the leaves into 1cm²Small square, and using syringe negative pressure method to stain the leaf cell nucleus DAPI, used to mark the cell nucleus, and to make slides. Photographs were taken using an inverted laser scanning confocal microscope (Zeiss LSM 780).

The results are shown in FIG. 3. FIG. 3 is a diagram showing the result of subcellular localization of PbPUB21 gene, wherein; FIG. 3-A, 35s imaging of GFP gene (control) in dark field, UV light, bright field, with the rightmost image superimposed; 3-B, PbPUB21-GFP imaging in dark field, UV light, bright field, with the right-most image being superimposed; the cell location map can be used for obtaining the location of the PbPUB21 gene in the cell nucleus.

Example 4

Genetic transformation of Arabidopsis thaliana

1. The plant transformation vector used was the recombinant vector pCAM1300-GFP-PbPUB21 constructed in example 3.

2. The agrobacterium-mediated genetic transformation of arabidopsis thaliana was as follows:

(1) and (3) agrobacterium culture: taking Agrobacterium tumefaciens bacteria liquid stored in an ultra-low temperature refrigerator, streaking on a plate added with LB (lysogeny broth) with 50mg/L kanamycin and 50mg/L rifampicin, culturing the streaked bacteria liquid in an incubator at 28 ℃ for 36-48 h, scraping streaked bacterial plaque, adding liquid MS (2.37g/LMS +50g/L sucrose +0.1mg/L IBA, pH value 5.8) culture medium, and carrying out shaking culture at 28 ℃ for 30min until the bacteria liquid concentration reaches OD (OD)₆₀₀When the dyeing rate is 0.8-1.0, surfactant silwet L-77200 mul/L is added for dip dyeing.

(2) Dip dyeing: taking a wild type arabidopsis thaliana plant with a stem height of about 10cm, removing all seed pods, then inversely placing the plant in a glass bottle containing the prepared agrobacterium tumefaciens bacterial liquid, vacuumizing, maintaining the pressure of 0.05Mpa for 5min, and placing the plant in a dark side for 24 h.

(3) Culturing: cultivating the plant to seed according to conventional method, and harvesting mature seed (T)₀Generation).

3. Screening of transgenic Positive seedlings

The PbPUB21 transgenic Arabidopsis thaliana T was obtained according to the above method₀Generation of seed, mixing T₀Sterilizing the surfaces of the generation seeds, uniformly spreading the generation seeds on an MS selective culture medium containing 50mg/L hygromycin and 50mg/L timentin, culturing the generation seeds under the illumination of 16h/d at the temperature of 22 ℃, selecting plants with fast growth and longer root length after one week of growth, transplanting the plants into sterilized nutrient soil, and growing for a period of time.

3.1 transgenic Arabidopsis DNA extraction method

Obtaining the PbPUB21 transgenic arabidopsis thaliana according to the method, extracting DNA from each arabidopsis thaliana, and designing a primer gene inner primer to carry out PCR amplification to identify positive seedlings.

(1) An appropriate amount of arabidopsis thaliana leaves was ground to powder with liquid nitrogen, and then 500. mu.l of CTAB (100mmol/L of a 65 ℃ pre-heated solution of LTris-HCl pH 8.0, 1.5mmol/L of NaCl, 50mmol/L of EDTA pH 8.0, 2% w/v CTAB, fully dissolved in a 65 ℃ water bath for further use) and 10. mu.l of beta-mercaptoethanol were added and mixed well.

(2) Heating in 65 deg.C water bath for 30min, taking out every 10min, slightly turning upside down, and mixing; centrifuging at 10000g for 10min at normal temperature; collecting supernatant, adding 500 μ l chloroform isoamyl alcohol (chloroform: isoamyl alcohol volume ratio is 24: 1), reversing and mixing;

(3) centrifuging at 10000g for 10min, collecting supernatant 450 μ l to a new 1.5ml centrifuge tube, adding isopropanol 450 μ l, and mixing by turning upside down;

(4)10000g, centrifuging for 10min, discarding the supernatant, rinsing with 1mL of 75% ethanol aqueous solution for 2 times, centrifuging for 10min under the condition of 10000g, completely removing ethanol, and placing on a superclean bench for ventilation drying until the DNA is colorless and transparent;

(5) adding 50 μ l of ultrapure water, placing in an incubator at 65 ℃ for dissolving for 40min, and performing gel detection.

3.2 Positive transgenic plant detection

And carrying out PCR amplification by adopting a primer gene specific primer and a vector downstream primer. The reaction procedures and systems are shown in tables 2 and 3, respectively. PCR was performed using gene forward and vector reverse primers (SEQ ID NO:11, 5'-GCGATGATCACACTAAACATGCT-3' and SEQ ID NO:12, 5'-CGTCGTCCTTGAAGAAGATG-3') to amplify fragments of the expected size in the selected transgenic lines, indicating that they were positive transgenic lines.

TABLE 2 PCR reaction procedure

TABLE 3 PCR reaction System

Independently sampling each strain, extracting RNA of pear seedling samples of a control group and an experimental group, detecting the complete structure of the pear seedling samples by glue running, determining the concentration of the pear seedling samples by using Nanodrop (the concentration is 200-1000 ng/mu l), adjusting the total amount of the RNA to 3 mu g, carrying out reverse transcription to obtain cDNA, and carrying out qPCR amplification by using an AtActin gene of arabidopsis thaliana as an internal reference gene, wherein the concentration is shown in figure 4-B. The nucleotide sequence of the AtActin primer is as follows: AtActin forward primer: 5'-GGTGTCATGGTTGGTATGGGTC-3' (SEQ ID NO: 15); AtActin reverse primer: 5'-CCTCTGTGAGTAGAACTGGGTGC-3' (SEQ ID NO: 16).

The brightness of bands amplified by AtActin is consistent, which indicates that the concentration of reverse transcription cDNA is the same, then PbPUB21 specific primers (SEQ ID NO:5 and SEQ ID NO:6) and AtActin gene of Arabidopsis are used for qRT-PCR detection, the expression level of the strain to be detected is analyzed, and 2 plants (#4 and #5) with higher expression level are selected as over-expression positive strains according to the expression level of PbPUB21 gene. FIG. 4-A shows the identification of Arabidopsis thaliana T by PCR using gene-specific primers₀A transgenic plant, wherein M: marker, +: plasmid, -: wild-type plants, #4- # 8: transgenic lines, FIG. 4-B is T₁Semi-quantitative PCR detection of Arabidopsis thaliana mRNA level, FIG. 4-C is RT-PCR identification of transgenic Arabidopsis thaliana T₁And (5) carrying out overexpression analysis on the generation plants.

Example 5

Transient transformation of birchleaf pear seedlings

1. Virus-induced gene silencing (VIGS) vector construction

Viral silencing vectors were constructed according to the method of example 4. The double enzyme cutting sites of the viral silencing vector pTRV2 are XbaI and Smal, the coding gene of PbPUB21 is amplified and inserted into the middle of the two enzyme cutting sites on the vector by using primer 5.0 software to design upstream and downstream primers (SEQ ID NO:13, 5'-AAGGTTACCGAATTCTCTAGATGATCCCGGACCAGTTTCTC-3' and SEQ ID NO:14, 5'-TGTCTTCGGGACATGCCCGGGATTGGCATAACAGGAACTCGC-3') according to the general principle of primer design, so that a recombinant vector pTRV2-PbPUB21 is obtained and is transformed into the competence of agrobacterium GV 3101.

2. Virus-induced gene silencing of birch pear seedlings

(1) And (3) agrobacterium culture: taking the Agrobacterium tumefaciens bacterial liquid stored in an ultra-low temperature refrigerator to perform shaking culture for 12h in LB liquid culture medium containing 50mg/L kanamycin and 50mg/L rifampicin at 28 ℃ and 220 rpm. The cultured bacterial liquid was centrifuged at 6000g for 10min to collect the cells, and the cells were treated with an infecting solution (10mM MgCl. RTM.)₂10mM MES, 200mM acetosyringone, pH 5.6) to OD₆₀₀＝0.8～1.0；

(2) Inducing with bacterial liquid: placing the bacterial liquid with the adjusted OD value in dark, and inducing at the normal temperature of 100rpm for 4 hours;

(3) pear seedling injection: the control group was prepared by mixing pTRV1 and pTRV2 bacterial solutions at a ratio of 1:1, and the experimental group was prepared by mixing pTRV1 and pTRV2-PbPUB21 bacterial solutions at a ratio of 1:1, and the seedlings of birch pear seedlings with consistent growth status and good health status were injected for 35 days.

3. Identification of virus-induced gene silencing inhibition positive vaccine

Treating the pear seedlings after injection in the dark at normal temperature for 12h, then recovering normal culture for 5 days, independently sampling each strain, extracting RNA of pear seedling samples of a control group and an experimental group, detecting the complete structure of the pear seedling samples by glue running, adjusting the total amount of the RNA to 3 mu g and then carrying out reverse transcription to obtain cDNA (shown in figure 5) after the concentration of the RNA is determined by using Nanodrop (the concentration of the RNA is 200-1000 ng/mu l), and carrying out qPCR amplification by using Tublin genes (SEQ ID NO:7 and SEQ ID NO:8) of pears as internal reference genes.

The brightness of bands amplified by Tublin is consistent, which indicates that the concentration of reverse transcription cDNA is the same, then a PbPUB21 specific primer and a pear internal reference primer Tublin are used for qRT-PCR detection, and the expression quantity of a strain to be detected, namely a PbPUB21 specific primer (SEQ ID NO:5 and SEQ ID NO:6), is analyzed.

According to the expression level of the PbPUB21 gene, three plants with lower expression levels are selected as virus silencing positive strains, and are named as pTRV2-1, pTRV2-2 and pTRV 2-3.

The results are shown in FIG. 8. FIG. 8-E shows the real-time quantitative PCR analysis of the expression level of the gene encoding PbPUB21 in different virus-silenced strains of Pyrus pyrifolia.

Example 6

Resistance identification of PbPUB21 transgenic resistant plants

1. Drought resistance analysis of transgenic Arabidopsis plants

The seeds of arabidopsis untransformed plants (WT) and the transformed PbPUB21 overexpression pure lines (#4, #5) harvested in the same batch are sterilized and sowed in a common MS basic culture medium, 20-day-old seedlings of each line are taken, the seedlings are cut off, the roots of the seedlings are placed on a culture dish for natural dehydration for 120 minutes, the fresh weight is measured every 20 minutes, and the water loss rate is calculated by taking the materials before dehydration as a reference; and the conductivity at the last time point (120 minutes of dehydration) was measured. The results show that both #4 and #5 lost water slower than WT during 120min water loss (FIG. 5-B), that after 120min water loss #4 and #5 had lower conductivity than WT (FIG. 5-C), and that MDA content was also lower than wild type (FIG. 5-D), and that the leaf water loss was significantly lower in 2 transgenic lines phenotypically than in untransformed plants (FIG. 5-A).

30d large WT, #4 and #5 were watered normally for 3 days before beginning water management for drought stress. The results showed that after stopping watering for 10 days, the lower leaf parts #4 and #5 wilted, and WT began to die at this time; the photosynthetic efficiency of the two transgenic overexpression lines (#4 and #5) was more significant than that of WT (FIG. 6-B), and 3 days after rehydration, both transgenic overexpression lines (#4 and #5) grew normally while only one WT survived (FIG. 8-F); after 14 days of drought, both the malondialdehyde content (FIG. 6-D) and conductivity (FIG. 6-C) of lines #4 and #5 were lower than WT; whereas Fv/Fm of the overexpression lines was significantly higher than WT (FIG. 6-E). The test results show that the dehydration resistance and drought resistance of the Arabidopsis plant with the transferred PbPUB21 gene are obviously enhanced compared with the untransformed plant (WT).

2. Transgenic arabidopsis plant reactive oxygen species accumulation assay

In transgenic Arabidopsis lines, lower conductivity and lower malondialdehyde content indicate that they may have a greater ROS resistance than WT. It is necessary to identify the amount of ROS accumulated in the plant. Activity oxygen histochemical staining of untransformed plants and two transgenic lines after drought treatment of 30-day-old Arabidopsis plants for 14 daysColor chart, using Diaminobenzidine (DAB) and Nitrotetrazole (NBT) to respectively react with H₂O₂(upper part of FIG. 7-A) and O^2-(lower part of FIG. 7-A) dyeing; it was evident that WT stained more than 2 transgenic lines. After 14 days of drought stress, the hydrogen peroxide content, the superoxide anion resistance content, in the tissues was determined, the hydrogen peroxide content of the wild line type was significantly higher than that of the 2 transgenic lines (FIG. 7-B), and the superoxide anion resistance free radical content in the tissues of the 2 transgenic lines was higher than that of the control line (FIG. 7-C). These evidence suggests that transgenic tobacco lines accumulate less residual reactive oxygen species and are less cell damaging than control lines after drought stress. Further shows that the over-expressed PbPUB21 gene can effectively enhance the active oxygen scavenging capacity of transgenic plants, thereby improving the drought resistance of the plants.

Example 7

Resistance identification of PbPUB21 virus-silenced plants

1. Drought resistance analysis of virus-silenced birchleaf pear seedlings

In order to identify whether the birch pears with virus-silenced PbPUB21 genes are related to drought stress, a control line and a virus-silenced line are subjected to drought treatment, wherein the drought treatment adopts a natural drying control method, materials required by experiments are placed in a 26 ℃ daylight lamp culture room, the illumination intensity is 20500lux, the air temperature is 44.0%, the birch pears are subjected to natural drought water control, and a picture is taken and sampled when the birch pears have phenotypes.

The phenotype and physiological indexes of the PbPUB21 gene virus-silenced birchleaf pear silent strains (pTRV2-1, pTRV2-2 and pTRV2-3) and a control plant (WT) after 15 days of drought treatment at room temperature are measured. Wherein: FIG. 8-A is the phenotype after drought treatment at room temperature for 15 days. FIG. 8-B is a photograph of fluorescent chlorophyll after drought treatment at room temperature for 15 days; FIG. 8-C is a conductivity measurement after a dry drought treatment at room temperature for 15 days. FIG. 8-F is a graph of Fv/Fm results before and after drought treatment, with lower Fv/Fm indicating greater plant damage. The results show that during the treatment, viral vector pTRV2 silencing the PbPUB21 gene reduced its resistance under drought conditions.

2. Virus silencing birch pear seedling active oxygen accumulation analysis

After drought treatment, the active oxygen accumulation of the control plants and the virus silencing strains is analyzed, as shown in figure 9-A and figure 9-B, after the pear is subjected to drought stress for 15 days, the hydrogen peroxide content and the superoxide anion content in the tissues of the pear are measured, the hydrogen peroxide content of 3 silencing strains is obviously higher than that of the control strains, and the superoxide anion free radical content in the tissues of the three silencing strains is lower than that of the control strains. These evidences indicate that silent tobacco strains accumulate more residual reactive oxygen species in vivo after being subjected to drought stress and cause greater cell damage than control lines. It is shown that silencing of the PbPUB21 gene by the viral vector pTRV2 reduces the ability to scavenge ROS under drought conditions.

Comprehensive analysis shows that the functions of PbPUB21 transferred into arabidopsis thaliana and PbPUB21 transiently silenced by using a virus-mediated VIGS technology transferred into pyrus betulaefolia are identified, and the following test results are found: compared with a control wild type, the transgenic PbPUB21 overexpression strain in arabidopsis thaliana has greatly improved drought resistance. Hydrogen peroxide (H) in transgenic Arabidopsis thaliana₂O₂) And superoxide anion (O)^2-) The content of the active oxygen is lower than that of a wild type, the active oxygen residue in a plant body is lower, the cell damage is less, and the oxidation resistance is stronger; compared with a control wild type, the drought resistance of a strain which transiently silences PbPUB21 in the pyrus betulaefolia by using a virus-mediated VIGS technology is obviously reduced. Hydrogen peroxide (H) in pTRV2-PbPUB21 transgenic Du-pear strain₂O₂) And superoxide anion (O)^2-) The content of the strain is higher than that of the wild strain, the active oxygen residue in the plant body is higher, the cell damage is larger, and the oxidation resistance is poorer. These results show that the over-expressed PbPUB21 gene can effectively enhance the active oxygen scavenging ability of transgenic plants, and the active oxygen scavenging ability of plants with the gene silenced is reduced, which indicates that PbPUB21 can improve the drought resistance of plants.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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Claims

1. Du pear ubiquitin ligase and genePbPUB21Or the application of a recombinant plant expression vector in drought resistance of plants, wherein the amino acid sequence of the pyrus betulaefolia ubiquitin ligase is shown as SEQ ID NO:1 is shown in the specification;

the genePbPUB21The nucleotide sequence of the gene for coding the pyrus betulaefolia ubiquitin ligase is shown as SEQ ID NO:2 is shown in the specification;

the recombinant plant expression vector comprises the genePbPUB21The recombinant plant expression vector of (1).

2. Du pear ubiquitin ligase and genePbPUB21Or the application of the recombinant plant expression vector in drought-resistant plant breeding, wherein the amino acid sequence of the pyrus betulaefolia ubiquitin ligase is shown as SEQ ID NO:1 is shown in the specification;

3. Du pear ubiquitin ligase and genePbPUB21Or the application of the recombinant plant expression vector in constructing drought-resistant transgenic plants, wherein the amino acid sequence of the pyrus betulaefolia ubiquitin ligase is shown as SEQ ID NO:1 is shown in the specification;

4. The use according to any one of claims 1 to 3, wherein the gene is inserted into a plant expression vector pCAM1300-GFPPbPUB21Obtaining the recombinant plant expression vector.

5. The use according to any one of claims 1 to 3, wherein the plant comprises a herbaceous plant or a woody plant.

6. The use of claim 5, wherein said herbaceous plant comprises Arabidopsis thaliana; the woody plant comprises pyrus betulaefolia.