CN112662686A - Cloning method and application of Yunwu tribute tea CsPPO gene - Google Patents

Abstract

The invention discloses a cloning method and application of a Yunwu tribute tea CsPPO gene, belonging to the technical field of genetic engineering; the invention provides cloning of the Yunwu tribute tea CsPPO gene, construction of an overexpression vector of the Yunwu tribute tea CsPPO, genetic transformation of tobacco and other steps, and the transgenic tobacco is analyzed by utilizing fluorescent quantitative PCR (polymerase chain reaction), so that the expression quantity of the Yunwu tribute tea CsPPO is improved under low-temperature stress. When the CsPPO is over-expressed in the tobacco plants, the cold resistance of the plants is improved through analysis of the cold resistance physiological index measurement result. Compared with the prior art, the invention discloses a cloning method of the Yunwu tribute tea CsPPO for the first time, and lays a foundation for breeding stress-resistant varieties of tea trees and gene-assisted breeding.

Description

Cloning method and application of Yunwu tribute tea CsPPO gene

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a cloning method of a Yunwu tribute tea CsPPO gene and application thereof.

Background

The improved variety of Yunwu tribute tea [ Camellia Sinensis (L.) Kuntze var. niaowangensis Q.H.Chen ] is distributed in Yunwu mountain area of south of Guizhou province, Guizhou province. The Yunwu mountain has unique ecological environment advantages of an altitude of 1583.6m, cloud and fog turning around in the last year, clear spring water and stream staggering, and the unique ecological environment breeds the unique tea fragrance 'tender chestnut fragrance' of Yunwu tribute tea and is rich in organic germanium and tea polysaccharide. The cultivation and planting history of Yunwu tribute tea is long, recorded by Sheng Lu Yu of tea as early as Tang dynasty, Yuan Tai Ding (1325 gong Yuan) starts to go to the court tribute tea in two years, and becomes a royal treasure even in the light of Ming Qing, and Qianlong is one of eight famous teas in China. In recent years, the Guiding Xueya made from Yunwu tribute tea is connected with international gold prize and becomes international famous tea, which is the famous tea recorded in tribute tea tombstone at present in China.

The polyphenol oxidase PPO is a metalloprotease, plays an important role in the tea processing and manufacturing process, can be used for enzymatically oxidizing polyphenol substances in tea into key quality component substances such as theaflavin, thearubigins and the like, and has an important influence on the quality of black tea. According to the literature, polyphenol oxidase is mainly associated with stress resistance of plants. However, it is unclear whether polyphenol oxidase participates in the tea plant in resisting external stress or plays a role in a plant secondary metabolic pathway in the tea plant growth process. The most widely discussed physiological functions are the relationship between polyphenol oxidase and the disease resistance, insect resistance, photoprotection and photosynthesis of tea plants. Polyphenol oxidase oxidizes polyphenols to form quinones, which are then polymerized by nonenzymatic reaction, resulting in color change of plant body. This response plays an important role in the defense of plants against foreign insults. However, environmental factors have little influence on the PPO activity, such as extreme temperature, humidity and the like, so that the research on the expression analysis of polyphenol oxidase in tea trees under the condition of low-temperature stress has important theoretical and practical significance.

Disclosure of Invention

The invention aims to provide a cloning method of a Yunwu tribute tea CsPPO gene and application thereof, so as to solve the problems in the prior art.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides a cloning method of a Yunwu tribute tea CsPPO gene, which specifically comprises the following steps:

extracting total RNA of Yunwu tribute tea leaf, reverse transcribing to cDNA, using the reverse transcribed cDNA as template, and using primer

PPO–Forward3：GGCACATCCCAAACAAAATA，

PPO-Reverse2:：CCGCTCGAGGGAATCAAACTCAATATCGAGACCA，

And cloning and sequencing the amplified product by a TA cloning system, selecting No.4 and No. 15 clones as templates for segmented amplification, and selecting the No.4 cloned PCR product and the No. 15 cloned PCR product as templates for fusion PCR after the segmented amplification to obtain the Yunwu tribute tea CsPPO gene.

Further, the primer sequences for performing segmented amplification by using clone No.4 as a template are as follows:

PPO-Forward5:TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTCCACCATCAAGC，

PPO-Reverse3:AGCTTGGCAGTCTTACTACTCGA；

the primer sequence for segmented amplification by taking clone No. 15 as a template is as follows:

PPO-Forward2：TCGAGTAGTAAGACTGCCAAGCT，

PPO-Reverse5：ATCATCATCTTTGTAATCCCCGGGGGAATCAAACTCAATATCGAGACCA。

further, the PCR primers used in the fusion PCR amplification reaction are:

PPO-Forward5：TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTCCACCATCAAGC，

PPO-Reverse5：ATCATCATCTTTGTAATCCCCGGGGGAATCAAACTCAATATCGAGACCA。

the invention also provides application of the Yunwu tribute tea CsPPO gene in improving the cold resistance of plants, which is characterized in that the obtained Yunwu tribute tea CsPPO gene cloned by the cloning method of the Yunwu tribute tea CsPPO gene is integrated into the plants to construct transgenic plants, so that the Yunwu tribute tea CsPPO gene is expressed in the transgenic plants to improve the cold resistance of the plants, and the nucleotide sequence of the Yunwu tribute tea CsPPO gene is shown in SEQ ID NO. 11.

Further, the construction of the transgenic plant comprises the following steps:

(1) constructing a recombinant expression vector containing the CsPPO gene of the Yunwu tribute tea;

(2) identifying the recombinant expression vector constructed in the step (1) to ensure that the Yunwu tribute tea CsPPO gene exists in the expression vector;

(3) transforming the correct recombinant expression vector identified in step (2) into agrobacterium GV 3101;

(4) and (4) infecting the plant by the agrobacterium containing the recombinant expression vector obtained in the step (3), and finally expressing the CsPPO gene of the Yunwu tribute tea in the plant.

Further, the construction method of the recombinant expression vector in the step (1) comprises the following steps:

the Yunwu tribute tea CsPPO gene and an expression vector CaMV35S subjected to double enzyme digestion by XbaI and SmaI, namely E3-FLAG (PBI121) form a recombinant expression vector in a homologous recombination and enzyme digestion connection mode.

Further, the expression vector CaMV35S comprises E3-FLAG (PBI121) which comprises: kan⁺Resistance gene, multiple cloning site, CaMV35S strong promoter, and 3 × FLAG tag.

Further, the specific steps of transforming the recombinant expression vector to agrobacterium GV3101 in step (3) are as follows:

adding the recombinant expression vector into a centrifugal tube containing agrobacterium GV3101, carrying out ice bath for 40min, freezing for 1min by liquid nitrogen, carrying out heat shock for 5min at 37 ℃, adding 500 mu L of an antibiotic-free LB liquid culture medium into the centrifugal tube, carrying out shake cultivation at 160rpm at 28 ℃ for 2h, coating the bacterial liquid into an antibiotic-containing LB solid culture medium, standing at 28 ℃ for 2d, screening and carrying out PCR identification to obtain the recombinant engineering bacteria.

Further, the antibiotics contained in the LB solid medium are kanamycin, gentamicin and rifampicin.

Further, the specific steps of infecting the plant with agrobacterium containing the recombinant expression vector in the step (4) are as follows:

(1) preparing a sterile explant;

(2) carrying out propagation on agrobacterium containing the recombinant expression vector to obtain a bacterial liquid;

(3) and (3) placing the explant prepared in the step (1) into the agrobacterium liquid expanded and propagated in the step (2), and finally obtaining a transgenic plant containing the Yunwu tribute tea CsPPO gene after co-culture, bud induction differentiation, rooting and resistant plant screening.

The invention discloses the following technical effects:

the invention clones polyphenol oxidase gene CsPPO from Yunwu tribute tea by utilizing the fusion PCR amplification technology, can be used for the recombinant expression and gene transfer of the CsPPO, and lays a foundation for the breeding of stress-resistant varieties of tea trees and gene-assisted breeding.

Drawings

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

FIG. 1 shows the PCR identification result of pEASY-Blunt Simple cloning vector;

FIG. 2 shows the result of the CsPPO segmented PCR amplification identification of Yunwu tribute tea

FIG. 3 shows the result of PCR amplification identification of Yunwu tribute tea CsPPO fusion;

FIG. 4 shows Blast results of CsPPO homology analysis of Yunwu tribute tea;

FIG. 5 shows the results of double-restriction electrophoresis of a CaMV35S E3-FLAG (PBI121) target vector;

FIG. 6 shows the PCR identification result of Escherichia coli DH5 alpha;

FIG. 7 shows the result of PCR identification of Agrobacterium GV 3101;

FIG. 8 is a flow chart of the transgene process;

FIG. 9 is a detection electrophoretogram of a genetic transformation strain of tobacco; from left to right, the lanes are: a first row: marker, positive control, negative control, line 1, line 2, line 3, line 23, line 24, line 4, line 5, line 6, line 7, line 8, line 9, line 10, line 11, line 12, line 13, line 14, line 15, line 16, line 17, line 18, line 19; a second row: marker, line 20, line 21, line 22;

FIG. 10 shows the relative expression of CsPPO in transgenic tobacco;

FIG. 11 shows the results of the analysis of the antioxidant activity of transgenic tobacco.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1 cloning method of Yunwu tribute tea polyphenol oxidase gene CsPPO

1. Sample source

The tested material was an annual seedling of Yunwu tribute tea, which was obtained from the tea industry office of Yunwu Town agricultural office of Guizhou province, Guidingcounty, and the material was uniformly planted in plastic pots, with 1 plant per pot. And (4) carrying out normal management in a sunlight greenhouse under natural illumination, and selecting plants with good health conditions and consistent growth vigor for test treatment when the plants grow to 70-80cm in height.

Synthesis of cDNA

After selecting young leaves, extracting total RNA of the Yunwu tribute tea leaves by a CTAB + Trizol method, and performing reverse transcription operation by using a reverse transcription Kit (TURE script 1st Stand cDNA SYNTHESIS Kit) of Aidlab company:

a20. mu.L reaction was used: 1000ng total RNA, 4. mu.L 5 XT Reaction Mix, 0.8. mu.L Rondam primer/oligodT, 0.8. mu.L TURescript H-RTase/RI Mix, and finally RNase Free dH₂O to 20. mu.L.

The reverse transcription reaction conditions were as follows:

42℃ 40min

65℃ 10min

after the reaction, the cDNA was obtained and stored at-80 ℃.

3. Design and amplification of Yunwu tribute tea CsPPO primer

Using Yunwu tribute tea cDNA as a template, designing primers PPO-Forward 3 and PPO-Reverse2 according to a published polyphenol oxidase gene sequence (GenBank accession number: FJ597757) for PCR amplification, wherein the used primer sequences are as follows:

PPO-Forward 3 (shown as SEQ ID NO. 1): GGCACATCCCAAACAAAATA the flow of the air in the air conditioner,

PPO-Reverse2 (shown as SEQ ID NO. 2): CCGCTCGAGGGAATCAAACTCAATATCGAGACCA, respectively;

the PCR reaction system (50. mu.L) was: template-1. mu.L, PPO-Forward 3 (10. mu.M) -1. mu.L, PPO-Reverse2 (10. mu.M) -1. mu.L, Fastpfu enzyme-1. mu.L, dNTPs (2.5mM) -4. mu.L, 5 XFastpfu Buffer) -10. mu.L, ddH₂O-32μL。

The PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 52 deg.C for 30s, and extension at 72 deg.C for 2min and 15s for 34 cycles, and extension at 72 deg.C for 5 min.

Cloning and sequencing the PCR product by TA cloning system

M13-F and M13-R (carried by pEASY-Blunt Simple cloning vector) are used as primers, a target gene fragment containing enzyme cutting sites XbaI and XhoI is obtained through PCR amplification, a T4 DNA ligase is used for inserting the target gene into the pEASY-Blunt Simple vector to construct a cloning vector, and the DNA sequence is determined through the sequence.

The PCR primer sequence is as follows:

M13-F (shown as SEQ ID NO. 3): TGACCGGCAGCAAAATG, respectively;

M13-R (shown as SEQ ID NO. 4): GTCCTTTGTCGATACTG, respectively;

the PCR system (50. mu.L) was: template-1. mu.L, M13-F (10. mu.M) -1. mu.L, M13-R (10. mu.M) -1. mu.L, Fastpfu enzyme-1. mu.L, dNTPs (2.5mM) -4. mu.L, 5 XFastpfu Buffer) -10. mu.L, ddH 2O-32. mu.L.

The PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 2min and 15s for 34 cycles, and extension at 72 deg.C for 5 min.

The ligation reaction system (10. mu.L) was: pEASY-Blunt Simple Vector 1. mu.L, PCR product 4. mu.L, T4 DNA ligase 5. mu.L, 4 ℃ overnight reaction.

As shown in FIG. 1, PCR identification results of pEASY-Blunt Simple cloning vector show that the No.4 clone and the No. 15 clone are accurate, and the sequencing result analysis shows that the first half segment sequence (1-940bp) of the No.4 clone (shown as SEQ ID NO. 5) and the second half segment sequence (900 plus 1791bp) of the No. 15 clone (shown as SEQ ID NO. 6) are accurate, so that primers are designed and amplified in segments by taking the No.4 clone and the No. 15 clone as templates, and then fusion PCR is carried out.

4. Fusion PCR amplification of Yunwu tribute tea CsPPO

(1) And (3) carrying out segmented amplification by using clone No.4 as a template, wherein the sequence of the used primers is as follows:

PPO-Forward5 (shown as SEQ ID NO. 7) TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTCCACCATCAAGC;

PPO-Reverse3 (shown as SEQ ID NO. 8) AGCTTGGCAGTCTTACTACTCGA;

the PCR amplification system (50. mu.L) was: template-1. mu.L, PPO-Forward5 (10. mu.M) -1. mu.L, PPO-Reverse3 (10. mu.M) -1. mu.L, Fastpfu enzyme-1. mu.L, dNTPs (2.5mM) -4. mu.L, 5 XFastpfu Buffer) -10. mu.L, ddH₂O-32μL。

The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, extension at 72 deg.C for 1min and 15s for 34 cycles, extension at 72 deg.C for 5min, and final storage at 12 deg.C for 10 min.

(2) And (3) carrying out segmented amplification by using clone No. 15 as a template, wherein the sequence of the used primers is as follows:

PPO-Forward2 (shown as SEQ ID NO. 9): TCGAGTAGTAAGACTGCCAAGCT, respectively;

PPO-Reverse5 (shown as SEQ ID NO. 10): ATCATCATCTTTGTAATCCCCGGGGGAATCAAACTCAATATCGAGACCA, respectively;

the PCR amplification system (50. mu.L) was: template-1. mu.L, PPO-Forward2 (10. mu.M) -1. mu.L, PPO-Reverse5 (10. mu.M) -1. mu.L, Fastpfu enzyme-1. mu.L, dNTPs (2.5mM) -4. mu.L, 5 XFastpfu Buffer) -10. mu.L, ddH 2O-32. mu.L.

The PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 1min and 15s for 34 cycles, extension at 72 deg.C for 5min, and final storage at 12 deg.C for 10 min.

The results are shown in FIG. 2, and the analysis of the sequencing results shows: the PCR product of clone No.4 is 940bp and the PCR product of clone No. 15 is 891bp, and the PCR product gel is recovered and then used as a template to perform fusion PCR.

(3) The fusion PCR amplification is specifically divided into two steps:

the first step is as follows: the fusion PCR pre-system (47. mu.L) was: mu.L dNTPs (2.5mM), 10. mu.L 5 XFast Pfu Buffer, 1. mu.L clone PCR product No.4, 1. mu.L Template clone PCR product No. 15, 31. mu.L ddH₂O；

The PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 55 deg.C for 30s, and extension at 72 deg.C for 2min and 15s for 5 cycles, extension at 72 deg.C for 5min, and storing at 12 deg.C for 10 min.

The second step is that: after the first step is finished, taking out the reaction tube and adding a primer to obtain a final system;

the primer sequences used were:

PPO-Forward5 (shown as SEQ ID NO. 7): TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTC CACCATCAAGC, respectively;

PPO-Reverse5 (shown as SEQ ID NO. 10): ATCATCATCTTTGTAATCCCCGGGGGAATCAAACTCAATATCGAGACCA, respectively;

the fusion PCR final system (50. mu.L) is: adding 1 mu L of PPO-Forward5, 1 mu L of PPO-Reverse5 and 1 mu L of Fast Pfu enzyme into a fusion PCR pre-system (47 mu L);

the PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 2min and 15s for 34 cycles, extension at 72 deg.C for 5min, and final storage at 12 deg.C for 10 min. After the reaction is finished, agarose gel electrophoresis is carried out to identify PCR products, gel cutting is carried out at the same time, fusion fragments are recovered, sequencing is carried out, and as shown in figure 3, gel recovery and purification are carried out to obtain 1740bp target fragments.

5. Sequence and homology analysis

Sequencing results show that the total length of the cloned polyphenol oxidase gene CsPPO sequence is 1740bp, as shown in SEQ ID NO.11, 580 amino acids are coded, and the molecular weight of the protein is 64.397 KD. And GenBank accession number: the disclosure in MT 134047.1.

As shown in FIG. 4, the Blast results of homology analysis with other species showed that the homology of Yunwu tribute tea polyphenol oxidase gene (CsPPO) sequence with tea plant (Camellia sinensis) was 96.72% at the highest, followed by 78.79% of Camellia nitidissima (Camellia nitidissima), 74.81% of Camellia sinensis (Camellia ptilophylla) and the rest was essentially Camellia.

Example 2CsPPO overexpression vector construction and in vitro recombinant expression

1. Construction of expression vectors

Carrying out double digestion (double digestion XbaI/SmaI) on the E3-FLAG (PBI121) target vector (14 kb);

the 20. mu.L double enzyme digestion system of the plasmid vector is:

mu.L of Sma I, 1. mu.L of Xba I, 2. mu.L of 10 Xfast Digest Green Buffer, 10. mu.L of vector plasmid, and 6. mu.L of ddH₂O；

After mixing, the mixture was digested at 4 ℃ overnight. Agarose gel electrophoresis, and gel cutting recovery after correct identification, as shown in FIG. 5. The constructed expression vector CaMV35S is E3 XFLAG (PBI121) containing Kan⁺Resistance gene, multiple cloning site, CaMV35S strong promoter, and 3 × FLAG tag (protein level identification).

2. Ligation of target Gene to plasmid vector

The homologous recombination connecting system is as follows:

mu.L of PPO-F5/R5 PCR fusion product fragment (125ng), 8. mu.L of PBI 121-FLAG (48ng) (XbaI/SmaI), 10. mu.L of 2 × Smaless Cloning Mix, and 1.5. mu.L of ddH in step 4 of example 1₂O; the system was mixed well and ligated for 25min at 50 ℃.

Adding the ligation product into a centrifuge tube containing 100 mu L of escherichia coli competence DH5 alpha, carrying out ice bath for 30min, carrying out heat shock at 42 ℃ for 45s, adding the centrifuge tube into 500 mu L of antibiotic-free LB liquid culture medium, and culturing at 37 ℃ and 160rpm for 1 h; after the culture, the bacterial liquid is centrifuged for 1min, the supernatant is taken and evenly coated on LB solid culture medium containing Kan (100 mug/ml) and Amp (100 mug/ml), and the plate is placed in an incubator at 37 ℃ for overnight culture. Plaque PCR identification was performed and followed by sequencing, using primers for PCR:

35S-proForward (shown as SEQ ID NO. 12): GACGCACAATCCCACTATCC

PPO-Forward5 (shown as SEQ ID NO. 7): TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTCCACCATCAAGC, respectively;

PPO-Reverse3 (shown as SEQ ID NO. 8): AGCTTGGCAGTCTTACTACTCGA, respectively;

the PCR reaction system (50. mu.L) was: template-1. mu.L, 35S-proForward/PPO-Forward 5 (10. mu.M) -1. mu.L, PPO-Reverse3 (10. mu.M) -1. mu.L, Fastpfu enzyme-1. mu.L, dNTPs (2.5mM) -4. mu.L, 5 XFastpfu Buffer-10. mu.L, ddH₂O-32μL。

The PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 60 deg.C for 30s, and extension at 72 deg.C for 1min and 15s for 34 cycles, and extension at 72 deg.C for 5 min.

As shown in fig. 6; the results showed that the strains containing the objective plasmid E.coli DH 5. alpha. were Nos. 13 and 15.

3. Agrobacterium transformation of expression vectors

After the plasmid construction is completed, the plasmid is transformed into agrobacterium GV3101 for stable expression, and the steps are as follows:

adding CaMV35S:: PPO-3 FLAG (PBI121) recombinant plasmid into a centrifuge tube containing 100 uL agrobacterium GV3101, ice-cooling for 40min, freezing for 1min with liquid nitrogen, heat-shocking for 5min at 37 ℃, adding 500 uL antibiotic-free LB liquid medium into the centrifuge tube, culturing for 2h at 28 ℃ and 160rpm of a shaker, and spreading the bacterial solution on kanamycin (Kan 121)⁺LB solid medium of 50. mu.g/ml) + rifampicin (Rif 50. mu.g/ml) + gentamicin (Gent 50. mu.g/m), plates were stood in an incubator at 28 ℃ for 2 d; selecting bacterial plaque to carry out bacterial liquid PCR identification:

the primers, system and reaction procedure used for PCR were the same as in step 2 above.

As shown in FIG. 7, the results showed that the strain containing the desired plasmid, Agrobacterium GV3101, was: 13-2, 13-3, 13-4, 13-5, 13-8 and 13-9.

Example 3 study on transformation of tobacco by Yunwu tribute tea CsPPO overexpression vector

Preparation of tobacco Material

1. Obtaining of sterile seedlings

Cleaning SR tobacco seeds with sterile water, sterilizing with 75% ethanol for 30s, sterilizing with sodium hypochlorite solution (effective chlorine of 10%) for 5min, washing with sterile water for 3-5 times, sucking water from the seed surface with sterile filter paper, and inoculating to MS culture medium (pH 5.8).

2. Agrobacterium propagation

The Agrobacterium of step 3 of example 2 was streaked on LB solid medium (50mg/L kanamycin +50mg/L gentamicin +50mg/L rifampicin), after two days at 28 ℃ single colonies were picked and inoculated into 3ml LB-containing liquid medium (50mg/L kanamycin +50mg/L gentamicin +50mg/L rifampicin), and shake-cultured at 28 ℃ and 220rpm at constant temperature overnight to OD₆₀₀Centrifuging at 5000rpm for 10min at 0.6, collecting thallus, and re-suspending the collected thallus with MS liquid culture medium to OD₆₀₀＝0.4。

(II) transforming tobacco with engineering bacteria

1. Preparation of explants

After 1 month, sterile and tender tobacco leaves are taken, edges and main veins are removed, and the tobacco leaves are cut into small pieces of 0.5 multiplied by 0.5.

2. Agrobacterium infection and co-cultivation

Placing the cut explant in the resuspended Agrobacterium liquid, gently shaking for 10min, blotting the liquid on the surface of the explant with sterile filter paper, and culturing in the dark for 2-3 days in a co-culture medium (MS +6-BA 1mg/L, pH 5.8).

3. Bud induced differentiation

After co-cultivation, the explants were transferred to shoot-inducing differentiation medium (MS +6-BA 1mg/L + Timentin 300mg/L + Kan 100mg/L, pH 5.8). Sprouting was induced and the medium was changed every two weeks.

4. Rooting and resistant plant screening

When the resistant bud grows to 2cm, cutting the resistant bud, transferring the resistant bud into a rooting culture medium (MS + Timentin 300mg/L + NAA 0.1mg/L + Cef 250mg/L, pH 5.8), when a complete plantlet is formed, cutting part of leaves, extracting genome DNA by using a TransDirect Plant Tissue PCR Kit, and detecting a positive Plant by using a gene specific primer;

FIG. 8 shows a flow chart of the transgenic process.

(III) verification of transgenic tobacco

1. And (3) extracting the genome DNA of the positive plant obtained in the step (II) by using a kit, wherein the specific operation steps are as follows:

taking 5mg or 0.5cm²The left and right plant tissue samples were cut into pieces and added with 40. mu.L PD1 Buffer, and then mixed by aspiration or vortexing. Incubate at 95 ℃ for 10 min. Adding 40. mu.l PD2 Buffer, mixing uniformly, and directly using as template to carry out PCR or storing at 4 ℃ or-20 ℃.

The PCR primer sequences used were:

2300-K-F (shown as SEQ ID NO. 13) ACCGTAAAGCACGAGGAAG;

2300-K-R (shown as SEQ ID NO. 14) GTTGTCACTGAAGCGGGAA;

the PCR reaction system is as follows: 3 mul sample to be tested, 0.5 mul 2300-K-F, 0.5 mul 2300-K-R, 5 mul 2 Xeasy Taq Mix, and ddH₂Supplementing 10 mu l of O;

the PCR reaction program is: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 58 deg.C for 30s, and extension at 72 deg.C for 1min and 15s for 34 cycles, and extension at 72 deg.C for 5 min.

The result is shown in FIG. 9, the electrophoresis chart of the tobacco genetic transformation strain detection shows that the length of the PPO-PBI 121 amplification fragment is 489bp, and the result shows that the tobacco transformation by the Yunwu tribute tea CsPPO overexpression vector is successful.

(IV) expression analysis of CsPPO under transgenic tobacco low-temperature stress

The transgenic tobacco is transferred into an illumination incubator at the test 1d, the temperature of the illumination incubator is set to be 4 ℃, the illumination intensity is l0000lux, the relative humidity is 50%, and the photoperiod is 16h/8h day and night. Functional leaves were selected for fluorescent quantitative PCR analysis at 1d, 3d, 5d, 7d, 9d of stress treatment, with wild type as control.

The experimental procedure for cDNA synthesis was the same as in example 1, step 2.

Real-time PCR reaction procedure

The fluorescent quantitative PCR program is:

Step1-95℃----3min

Step2-95℃----10s

Step3-58℃----30s+plate read

Go to step2，39cycles

the fluorescent quantitative PCR system comprises:

5μL

Green Supermix(1×)、0.5μL Forward primer(200nM)、0.5Reverse primer(200nM)、1μL cDNA(N/A)、3μL ddH₂O(N/A)。

the primer synthesis information is shown in table 5 below:

TABLE 5

3. Test sample result detection

The calculation of the relative expression amount of the target gene in each sample is automatically carried out by the soft qPCRsoft3.2 of the instrument, and is simultaneously calculated manually by adopting a Pfaffl method, wherein the formula is as follows:

actin is used as an internal reference and 2 is utilized^-△△CtThe relative expression level of the gene in each sample was calculated.

4. Results and analysis

The results are shown in FIG. 10: the polyphenol oxidase gene is overexpressed in the transformed tobacco, the expression level of the polyphenol oxidase gene in the transgenic tobacco body rises under the condition of low temperature stress at 4 ℃, the trend that the expression level rises firstly and then falls is shown along with the prolonging of time, the highest expression level is reached when the tobacco is treated at the 5 th day at low temperature, and the highest expression level is 3 times that of a control group, so that the polyphenol oxidase gene gradually makes regulation and control response under the low temperature stress.

Example 4 assay of antioxidase Activity of transgenic tobacco under Low temperature stress

Under low temperature stress, a protective enzyme antioxidant defense system is generated in a plant body, the system mainly comprises SOD, POD, PPO, CAT and the like, and the substances have synergistic action to remove active oxygen free radicals in the plant body, so that the generation and the elimination of the free radicals in the plant body are in a balanced state, and the effects of regulating membrane permeability, increasing membrane structure and functional stability are achieved. Therefore, the protective enzyme activity is closely related to the cold resistance of plants.

1. Materials and methods

(1) Materials same as example 3 step (IV)

(2) Method of producing a composite material

SOD activity was measured by a Nitro Blue Tetrazolium (NBT) inhibition photoreduction method; POD activity was measured by guaiacol method; CAT activity is measured by adopting an ultraviolet absorption method; PPO activity was measured by the catechol method.

2. Analysis of antioxidant enzyme Activity under Low temperature stress

As shown in FIG. 11, the activities of SOD, POD, CAT and PPO increased first and then decreased with the increase of stress time, and the contents were higher than those of the control, and the peak values were 1.07 times, 4.72 times, 1.34 times and 2.84 times of the control, respectively, and the peak value appeared at the 5 th day of the low-temperature stress treatment. The result shows that the cold resistance of the plant can be enhanced by improving the activity of the antioxidant enzyme and eliminating excessive active oxygen free radicals in a short time, and the activity of the antioxidant enzyme is reduced as the cold damage is aggravated and the active oxygen such as superoxide free radicals in the plant body is increased along with the prolonging of the low-temperature stress time. In order to resist low-temperature harm, the activity of polyphenol oxidase of the plant is enhanced through biological regulation, and as the stress time is prolonged, the accumulation of phenolic substances begins to be reduced, and the activity of the polyphenol oxidase is also reduced. The result is consistent with the change rule of the fluorescent quantitative PCR result of the transgenic tobacco CsPPO under low temperature stress.

The invention discloses a cloning method of Yunwu tribute tea polyphenol oxidase gene CsPPO, which constructs an overexpression vector CaMV35S:: PPO-3. FLAG (PBI121) for researching the function of the gene and obtains a transgenic positive tobacco plant. After the stress at the low temperature of 4 ℃, the expression quantity of the polyphenol oxidase gene is increased and reaches the highest value when the 5 th day is treated, which is 3 times of that of a control group. Analysis of the determination result of the activity of the antioxidant enzyme of the transgenic tobacco shows that the CsPPO has the function of improving the cold resistance of the transformed plants.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Sequence listing

<110> biological research institute of Guizhou university, Guizhou province

<120> cloning method of Yunwu tribute tea CsPPO gene and application thereof

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ggcacatccc aaacaaaata 20

<210> 2

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ccgctcgagg gaatcaaact caatatcgag acca 34

<210> 3

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

tgaccggcag caaaatg 17

<210> 4

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gtcctttgtc gatactg 17

<210> 5

<211> 1710

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgtaagcgcg ggatcttcag agattggcac atcccaaaca aaatatgaat tctcttccac 60

catcaagcac caccattccc acccattctt catgtgcatt tttcaaaaac acattccaag 120

ttcccacaat tggaaagagc aagcattgct ttaaagtatc atgcaaggcc atagatggtg 180

atacaaatga aaaagcccca aatccaaatc caccaaagct cgatagaaga aatatgctta 240

ttggcttagg aggctacggc gccgccacca tcgccggaaa cccaccagcc accgcttctc 300

cgatctcccc accggacgta accaaatgcg gccccgccga actaccctcc ggcgcaaaac 360

ccaccaactg ttgtccccca aaatcgacaa aaatcatcga tttcaagctt ccgccgccgc 420

caaccaccgt gagagtccga cccgccgtcc atttagccga tgacgagtac atagccaagt 480

tctcaaaagc acttgctcta atgaaagcac ttcccgaaga cgaccctcgt agcttcactc 540

aacaagccaa tgttcactgc gcttattgcg acggagctta tcaccaagtc ggcttcccag 600

acctcgatct tcaagtccac aactcatggc tcttcttccc ctatcatcga tactacctct 660

acttcttcga aaagattttg ggaaaactaa tcggcgatcc gagtttcgca atccctttct 720

ggaattggga ttccccgatc ggaatgaaca tgccgccgat ctacgccgat cctaaatcgc 780

cgatgtacga tcggctccgc gacgccaagc accagccgcc gatcacgatc gacctcgact 840

acaatttaac tgattcgaaa agcccttctg cgaagctcat ttcgaacaat ctcactatta 900

tgtatagaca agttgtttcg agtagtaaga ctgccaagct ctttttggga agcgcttatc 960

gggctgggga tgaacccgac ccgggagctg gggctttgga gaatattccc cacgggccag 1020

ttcatatatg gtgcggggat cggacccaac ccaatataga ggatatgggg aacttgtatt 1080

catcgggtcg ggacccgata tttttcggcc atcatgggaa tgtggatcgg atgtggacca 1140

tatggaagag tttgggtagg cgggatttta ccgacccgga ttggttggat tccgggttct 1200

tgttttatga tgagaatgct cagcctgtca gagtgaaggt caaggattgt ttggacatga 1260

caaagctagg gtatgtatac caagacgtgg acatcccatg gctgaaaagt cggccaaccc 1320

ctcgcgtctc aaaactttcc cggaaaacaa acaaggccgg cgtagccaag gcggccgaga 1380

cacaaccaac cttcccaaca aaactcgaca aggtggtgcg agcgacggtt cagagaccga 1440

agaaatcaag aagcaagaaa gagaaagaag aagaagaaga aatactagtg atcgatggga 1500

tcgaggtcga aagagatcaa tttgtgaagt tcgatgtgtt cattaatgac ggagatgagc 1560

cagtgatcgg accggagaac tcggagttcg ccggaagctt tgtgaatgtg ccacataggc 1620

ataagcatgg gaagaagatg aagacttgct tgaggttagg gatgagtgag ttgttggagg 1680

actaatcgtc gaacggcagg cgtaaataag 1710

<210> 6

<211> 1791

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ggacgcgcgc atcttcagag attggcacat cccaaacaaa atatgaattc tcttccacca 60

tcaagcacca ctatttccac ccattcttca tgtgcatttt tcaaaaacac attccaaatt 120

cccacaattg gaaagagcaa gcattgcttt aaactatcag gcaacgccat agatagtgat 180

acaaatgaaa aaaccccaaa tccaaatcca ccaaagctgg atagaagaaa catgcttatt 240

ggcttaggag gctacggcgc cgccaccctc gccggaaacc caccagccac cgcttctccg 300

gtctccccgg acttaaccaa atgccgcccc tccgaactag cccccggcga aatatccacc 360

aactgttgtc ccccaaaatc gacaaaaatc atcgatttca agcttccgcc gccgccaact 420

accgcgagag tccgacccgc cgcccattta gtcgactgcg attacatagc caagttctca 480

gacgcacttg ctctaatgaa agcacttccc aatgacgacc cacgtagctt cactcaacaa 540

gccaatgttc actgcgctta ttgcgacgga gcttatgacc attttgaagt ccacaagtca 600

tggctcttct tcccctttca tcgatactac ctctacttct tcgaaaagat tttgggaaaa 660

ctaatcggcg atccgagttt cgcaatccct ttctggaatt gggattcccc gatcggaatg 720

aacatgccgc cgatctacgc cgatcctaaa tcgccgatgt acgatcggct ccgcgacgcc 780

aagcaccagc cgccgatcac gatcgacctc gactacaatt taactgattc gaaaagccct 840

tctgcgaagc tcatttcgaa caatctcact attatgtata gacaagttgt ttcgagtagt 900

aagactgcca agctcttttt gggaagcgct tatcgggctg gggatgaacc cgacccggga 960

gctggggctt tggagaatat tccccacggg ccagttcata tatggtgcgg ggatcggacc 1020

caacccaata tagaggatat ggggaacttg tattcatcgg gtcgggaccc gatatttttc 1080

ggccatcatg ggaatgtgga tcggatgtgg accatatgga agagtttggg taggcgggat 1140

tttaccgacc cggattggtt ggattccggg ttcttgtttt atgatgagaa tgctcagcct 1200

gtcagagtga aggtcaagga ttgtttggac atgacaaagc tagggtatgt ataccaagac 1260

gtggacatcc catggctgaa aagtcggcca acccctcgcg tctcaaaact ttcccggaaa 1320

acaaacaagg ccggcgtagc caaggcggcc gagacacaac caaccttccc aacaaaactc 1380

gacaaggtgg tgcgagcgac ggttcagaga ccgaagaaat caagaagcaa gaaagagaaa 1440

gaagaagaag aagaaatact agtgatcgat gggatcgagg tcgaaagaga tcaatttgtg 1500

aagttcgatg tgttcattaa tgacggagat gagccagtga tcggaccgga gaactcggag 1560

ttcgccggaa gctttgtgaa tgtgccacat aggcataagc atgggaagaa gatgaagact 1620

tgcttgaggt tagggataag tgagttgttg gaggacttgg aagctgaaga tgatgagagt 1680

gttttggtta ctctggtccc aagaaagggt tctgatgctt tgatcattgg tggtctcgat 1740

attgagtttg attccctcga gcggaatcgt cgaacggcag gcgtcaagac t 1791

<210> 7

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tggagagaac acgggggact ctagaatgaa ttctcttcca ccatcaagc 49

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

agcttggcag tcttactact cga 23

<210> 9

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tcgagtagta agactgccaa gct 23

<210> 10

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atcatcatct ttgtaatccc cgggggaatc aaactcaata tcgagacca 49

<210> 11

<211> 1740

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

atgaattctc ttccaccatc aagcaccacc attcccaccc attcctcatg tgcatttttc 60

aaaaacacat tccaaattcc cacaattgga aagagcaagc attgctttaa actatcaggc 120

aacgccatag atagtgatac aaatgaaaaa accccaaatc caaatccacc aaagctcgat 180

agaagaaata tgcttattgg cttaggaggc tacggcgccg ccaccctcgc cggaaaccca 240

ccagccaccg cttctccgat ctccccaccg gacgtaacca aatgcggccc cgccgaacta 300

ccctccggcg cgaaacccac caactgttgt cccccaaaat cgacaaaaat catcgatttc 360

aagcttccgc cgccgccaac caccgtgaga gtccgacccg ccgtccattt agccgatgac 420

gagtacatag ccaagttctc aaaagcactt gctctaatga aagcacttcc cgaagacgac 480

cctcgtagct tcactcaaca agccaatgtt cactgcgctt attgcgacgg agcttatcac 540

caagtcggct tcccagacct cgatcttcaa gtccacaact catggctctt cttcccctat 600

catcgatact acctctactt cttcgaaaag attttgggaa aactaatcgg cgatccgagt 660

ttcgcaatcc ctttctggaa ttgggattcc ccgatcggaa tgaacatgcc gccgatctac 720

gccgatccta aatcgccgat gtacgatcgg ctccgcgacg ccaagcacca gccgccgatc 780

acgatcgacc tcgactacaa tttaactgat tcgaaaagcc cttctgagaa gctcatttcg 840

aacaatctca ctattatgta tagacaagtt gtttcgagta gtaagactgc caagctcttt 900

ttgggaagcg cttatcgggc tggggatgaa cccgacccgg gagctggggc tttggagaat 960

attccccacg ggccagttca tatatggtgc ggggatcgga cccaacccaa tatagaggat 1020

atggggaact tgtattcatc gggtcgggac ccgatatttt tcggccatca tgggaatgtg 1080

gatcggatgt ggaccatatg gaagagtttg ggtaggcggg attttaccga cccggattgg 1140

ttggattccg gattcttgtt ttatgacgag aatgctcagc ttgttaaagt gaaggtcagg 1200

gattgtttgg acacgacaaa gctaggatat gtctaccaag aagtggacat tccatggctg 1260

aaatctcggc caacccctcg cgtctcaaaa ctttcccgga aaacaaacaa ggccggcgta 1320

gccaaggcgg ccgagacaca accaaccttc ccaacaaaac tcgacaaggt ggtgcgagcg 1380

acggttcaga gaccgaagaa atcaagaagc aagaaaaaga aagaagaaga agaagaagaa 1440

atactagtga tcgatgggat cgagttcgaa agagatcaat ttgtgaagtt cgatgtgctc 1500

attaatgacg gaaacgagcc ggtgatcgga ccggaaaact cggagttcgc cggaagcttt 1560

gtgaatgtac cacatgggca taagcatggg aagaagactg agacttgctt gaggttaggg 1620

ataagtgagt tgttggagga cttggaagct gatgatgatg agagtatttt ggttactttg 1680

gccccaagaa agggttgtga tgctttgatc attggtggtc tcgatattga gtttgattcc 1740

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gacgcacaat cccactatcc 20

<210> 13

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

accgtaaagc acgaggaag 19

<210> 14

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gttgtcactg aagcgggaa 19

<210> 15

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

actggtgtta tggttggtat gg 22

<210> 16

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

gctcaatggg atacttcaag gt 22

<210> 17

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacccaaccc aatatagagg at 22

<210> 18

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gcctacccaa actcttccat a 21

Claims (10)

1. A method for cloning a Yunwu tribute tea CsPPO gene is characterized by comprising the following steps:

extracting total RNA of Yunwu tribute tea leaf, reverse transcribing to cDNA, using the reverse transcribed cDNA as template, and using primer

PPO–Forward3：GGCACATCCCAAACAAAATA，

PPO-Reverse2:：CCGCTCGAGGGAATCAAACTCAATATCGAGACCA，

And cloning and sequencing the amplified product by a TA cloning system, selecting No.4 and No. 15 clones as templates for segmented amplification, and selecting the No.4 cloned PCR product and the No. 15 cloned PCR product as templates for fusion PCR after the segmented amplification to obtain the Yunwu tribute tea CsPPO gene.

2. The cloning method according to claim 1,

the primer sequence for carrying out segmented amplification by taking clone No.4 as a template is as follows:

PPO-Forward5:TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTCCACCATCAAGC，

PPO-Reverse3:AGCTTGGCAGTCTTACTACTCGA；

the primer sequence for segmented amplification by taking clone No. 15 as a template is as follows:

PPO-Forward2：TCGAGTAGTAAGACTGCCAAGCT，

PPO-Reverse5：ATCATCATCTTTGTAATCCCCGGGGGAATCAAACTCAATATCGAGACCA。

3. the cloning method according to claim 3, wherein the PCR primers used in the fusion PCR amplification reaction are:

PPO-Forward5：TGGAGAGAACACGGGGGACTCTAGAATGAATTCTCTTCCACCATCAAGC，

PPO-Reverse5：ATCATCATCTTTGTAATCCCCGGGGGAATCAAACTCAATATCGAGACCA。

4. the application of the Yunwu tribute tea CsPPO gene in improving the cold resistance of plants is characterized in that the obtained Yunwu tribute tea CsPPO gene cloned by the cloning method of the Yunwu tribute tea CsPPO gene in claim 1 is integrated into plants to construct transgenic plants, so that the Yunwu tribute tea CsPPO gene is expressed in the transgenic plants to improve the cold resistance of the plants, and the nucleotide sequence of the Yunwu tribute tea CsPPO gene is shown in SEQ ID NO. 11.

5. The use according to claim 4, wherein said constructing a transgenic plant comprises the steps of:

(1) constructing a recombinant expression vector containing the CsPPO gene of the Yunwu tribute tea;

(2) identifying the recombinant expression vector constructed in the step (1) to ensure that the Yunwu tribute tea CsPPO gene exists in the expression vector;

(3) transforming the correct recombinant expression vector identified in step (2) into agrobacterium GV 3101;

(4) and (4) infecting the tobacco plants by the agrobacterium containing the recombinant expression vector obtained in the step (3), and finally expressing the CsPPO gene of the Yunwu tribute tea in the tobacco plants.

6. The use of claim 5, wherein the recombinant expression vector of step (1) is constructed by the following steps:

the Yunwu tribute tea CsPPO gene and an expression vector CaMV35S subjected to double enzyme digestion by XbaI and SmaI, namely E3-FLAG (PBI121) form a recombinant expression vector in a homologous recombination and enzyme digestion connection mode.

7. The use of claim 6, wherein the expression vector CaMV35S E3-FLAG (PBI121) comprises: kan⁺Resistance gene, multiple cloning site, CaMV35S strong promoter, and 3 × FLAG tag.

8. The use of claim 5, wherein the step (3) of transforming the recombinant expression vector into Agrobacterium GV3101 comprises the following steps:

adding the recombinant expression vector into a centrifugal tube containing agrobacterium GV3101, carrying out ice bath for 40min, freezing for 1min by liquid nitrogen, carrying out heat shock for 5min at 37 ℃, adding 500 mu L of an antibiotic-free LB liquid culture medium into the centrifugal tube, carrying out shake cultivation at 160rpm at 28 ℃ for 2h, coating the bacterial liquid into an antibiotic-containing LB solid culture medium, standing at 28 ℃ for 2d, screening and carrying out PCR identification to obtain the recombinant engineering bacteria.

9. The use as claimed in claim 8, wherein the antibiotics contained in the LB solid medium are kanamycin, gentamicin and rifampicin.

10. The use according to claim 5, wherein the specific steps of infecting the plant with Agrobacterium containing the recombinant expression vector in step (4) are:

(1) preparing a sterile explant;

(2) carrying out propagation on agrobacterium containing the recombinant expression vector to obtain a bacterial liquid;

(3) and (3) placing the explant prepared in the step (1) into the agrobacterium liquid expanded and propagated in the step (2), and finally obtaining a transgenic plant containing the Yunwu tribute tea CsPPO gene after co-culture, bud induction differentiation, rooting and resistant plant screening.

Images