CN116814652A

CN116814652A - Ganlong No.1 CcMYB4_LIKE gene and expression protein and application thereof

Info

Publication number: CN116814652A
Application number: CN202310874591.4A
Authority: CN
Inventors: 钟永达; 胥猛; 栾晓月
Original assignee: INSTITUTE OF BIOLOGICAL RESOURCES JIANGXI ACADEMY OF SCIENCES
Current assignee: INSTITUTE OF BIOLOGICAL RESOURCES JIANGXI ACADEMY OF SCIENCES
Priority date: 2023-07-17
Filing date: 2023-07-17
Publication date: 2023-09-29

Abstract

The application discloses a 'Ganlong No. 1' CcMYB4_LIKE gene and an expression protein and application thereof, belonging to the technical field of plant genetic engineering. The nucleotide sequence of the 'Ganlong No. 1' CcMYB4_LIKE gene is shown as SEQ ID NO.1, and the amino acid sequence of the expressed protein is shown as SEQ ID NO. 2. The application takes 7 tissues of Ganlong No.1 as materials, obtains the CcMYB4_LIKE gene of Ganlong No.1 through cloning, constructs a plant expression vector 35S based on the CcMYB4_LIKE, and transfers the CcMYB4_LIKE gene into Nanlin 895 poplar' and Arabidopsis thaliana to obtain transgenic plants. Compared with CK, the transgenic poplar has lighter leaf color and faster growth speed. In transgenic 'nan lin 895 poplar' plants, the 4CL and C4H gene expression levels were up-regulated. CHS, LDOX and DFR genes were down-regulated in transgenic Arabidopsis plants.

Description

Ganlong No.1 CcMYB4_LIKE gene and expression protein and application thereof

Technical Field

The application belongs to the technical field of plant genetic engineering, and particularly relates to a 'Ganman No. 1' CcMYB4_LIKE gene, an expression protein thereof and application thereof.

Background

Camphor tree (Cinnamomum camphora) is a representative tree species of the genus Cinnamomum (Lauraceae), and is mainly distributed in Jiangxi, fujian, taiwan, guangdong and Guangxi provinces in China. Camphor tree is a precious tree species in the country, has luxuriant branches and leaves, and has high material use, economy and ornamental value. The bark, root, branch, leaf and fruit of the plant are rich in secondary metabolites such as camphor and essential oil, and the plant provides an important raw material for the fields of spice essence, daily chemical industry and the like. In addition, the root, leaf and fruit of camphor tree are used as medicine to reach the analgesic, antioxidant and antibacterial effects, and the camphor tree extract may be used in resisting thrombosis, preventing arteriosclerosis and resisting cancer. At present, camphor tree can also be used for treating stomachache in Yunnan minority nationality areas. In addition, xu and the like find that the essential oil of the camphor tree leaves has better killing power on the anopheles lanuge larvae, and provide a new thought for inhibiting malaria transmission. The camphor tree has the capability of resisting air pollution such as sulfur dioxide, ozone, smoke dust and the like, can absorb various toxic gases, has stronger wind resistance, and is often planted as a street tree in the south area of the Yangtze river basin.

Camphor tree is rich in various plant secondary metabolites, such as flavonoid, terpenes and the like, and has high economic and research values. Wang Chunxia et al found that the main components of the pigment of camphor tree fruit were cyanidin-3-O-glucoside and cyanidin-3-O-diglycoside by liquid chromatography-mass spectrometry. Wen Chifu et al found that camphor tree fruit haematochrome has a strong ability to scavenge free radicals (DPPH). Chen Jiguang the second generation transcriptome sequencing was performed on camphor tree fruits at different periods, and as a result, differential genes were found to comprise structural genes of 4 PAL families, 1F 3'5' H family, 2 UFGT families and 1 UGAT family, and 11 MYB transcription factors and 22 bHLH transcription factors were found to be possibly related to anthocyanin. Zhong et al analyzed the color forming mechanism of the bark of Gantrong No.1 'using metabolome and transcriptome data, and found that pelargonidin, procyanidin and paeoniflorin were the main pigments that caused Gantrong No. 1' to appear red; in addition, 24 up-regulated differential genes were identified to be involved in anthocyanin biosynthesis, with 6 transcription factors (3 MYBs and 3 bhlh s) likely candidates for the 'gan-system number 1' anthocyanin synthesis pathway.

Disclosure of Invention

Aiming at the problems existing in the prior art, the technical problem to be solved by the application is to provide the CcMYB4_LIKE gene of the Ganman No. 1; another technical problem to be solved by the present application is to provide an expression protein of the ccmeyb4_like gene of the' gan system 1; the application also solves the technical problem of providing the application of the CcMYB4_LIKE gene of the' Gastrodia elata No.1 for improving the germplasm of the camphor tree.

In order to solve the technical problems, the technical scheme adopted by the application is as follows:

the nucleotide sequence of the CcMYB4_LIKE gene of Ganlong No.1 is shown as SEQ ID NO. 1.

The amino acid sequence of the expression protein of the CcMYB4_LIKE gene of Ganlong No.1 is shown as SEQ ID No. 2.

A vector containing the gene 'Ganlong No. 1' CcMYB4_LIKE.

The vector is a plant expression vector.

The plant expression vector is 35S, ccMYB4_LIKE.

Application of Ganlong No.1 CcMYB4_LIKE gene in lightening transgenic Nanlin 895 poplar leaves comprises the following steps:

1) Constructing a plant expression vector of a Ganlong No.1 CcMYB4_LIKE gene;

2) Transforming the constructed plant expression vector into 'nan lin 895 poplar' leaves;

3) Culturing and screening to obtain the 'nan lin 895 poplar' plant with the lightening leaf color.

Application of Ganlong No.1 CcMYB4_LIKE gene in accelerating growth rate of Nanlin 895 poplar plants.

The application of the Ganlong No.1 CcMYB4_LIKE gene in improving the expression level of anthocyanin synthesis related genes 4CL and C4H or reducing the expression level of anthocyanin synthesis related genes PAL, CHS, F3H, LDOX and DFR in transgenic Nanlin 895 poplar'.

The application of the Ganlong No.1 CcMYB4_LIKE gene in improving the expression level of anthocyanin synthesis related genes 4CL, C4H, PAL and F3H in transgenic Arabidopsis or reducing the expression level of anthocyanin synthesis related genes CHS, LDOX and DFR.

Compared with the prior art, the application has the beneficial effects that:

the application uses 7 tissues of ' Ganling Tong No.1 ' as materials, obtains ' CcMYB4_LIKE gene of ' Ganling No.1 ' through cloning, constructs a plant expression vector 35S based on the gene, and transfers the gene into ' Nanlin 895 poplar ' leaves and arabidopsis inflorescences to obtain transgenic plants. Compared with CK, the CcMYB4_LIKE transgenic plant has lighter leaf color and faster growth speed. When dicotyledonous leaves appear in Arabidopsis thaliana, both cotyledons of the control group are green, and stems are light green or white, and Arabidopsis thaliana transformed with the CcMYB4_LIKE gene also has a similar phenotype. In the CcMYB4_LIKE gene 'nan Lin 895 Yang' plant, other structural genes are down-regulated to express at different degrees except that the 4CL and C4H genes are up-regulated. In the arabidopsis plant transformed with the CcMYB4_LIKE gene, other structural genes are obviously up-regulated except CHS, LDOX and DFR genes.

Drawings

Fig. 1 is a agarose gel electrophoresis detection diagram of total RNA of different tissues of Ganlong No.1 camphor tree;

FIG. 2 is a graph showing qRT-PCR results of CcMYB4_LIKE in 7 tissues of the 'Ganlan No. 1' camphor tree;

fig. 3 is a diagram showing the cell viability of the protoplast of the camphorwood mesophyll of Ganna 1;

fig. 4 is a transformation chart of a 'gan tong 1' camphor leaf pulp protoplast;

fig. 5 is a graph showing the transformation efficiency of the protoplast of the camphorwood mesophyll of Ganna 1;

FIG. 6 is a diagram of a 35S CcMYB4_LIKE recombinant plasmid T-DNA region;

FIG. 7 is a graph showing the detection of the level of poplar DNA transformed with CcMYB4_LIKE gene;

FIG. 8 is a graph showing the expression level of the CcMYB4_LIKE gene in transgenic poplar (CK is non-transgenic poplar, T is transgenic poplar);

FIG. 9 is a phenotype diagram of a CcMYB4_LIKE transgenic poplar;

FIG. 10 is a semi-quantitative assay of transgenic Arabidopsis plants;

FIG. 11 is a graph showing the expression level of the CcMYB4_LIKE gene in transgenic Arabidopsis;

FIG. 12 is a chart of phenotype observations of transgenic Arabidopsis dicotyledonous phase;

FIG. 13 is a CcMYB4_LIKE transgenic Arabidopsis phenotype map;

FIG. 14 is a graph showing the relative expression levels of the downstream structural genes of transgenic poplar;

FIG. 15 is a graph showing the relative expression level of the downstream structural gene of transgenic Arabidopsis thaliana.

Detailed Description

The present application will be further described with reference to specific embodiments for the purpose of making the objects, technical solutions and advantages of the present application more apparent. Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art.

The experimental material 'Tongshi No. 1' and the control plant from the same half-sibling family are derived from the institute of biological resources of academy of sciences of Jiangxi province and are used for the study through tender branch cuttage.

The root, flower, phloem, leaf, xylem and stem tip of 'gan-co-1' were collected in month 4 of 2020, and the fruits were collected in month 11 of 2020, for a total of 7 tissues.

Example 1

1. Extraction of Total RNA

Extracting total RNA of 7 tissues (root, flower, phloem, leaf, xylem, stem tip and fruit) of 'Ganman No. 1' by using RNAprepPure polysaccharide polyphenol total RNA extraction kit (Tiangen, china). The specific method comprises the following steps:

475 mu L of lysate SL and 25 mu L of beta-mercaptoethanol are added into a 1.5mLRNase-Free centrifuge tube to prepare a premix; taking fresh sample or plant tissue sample stored in-80deg.C refrigerator, rapidly adding liquid nitrogen, immediately grinding to obtain powder, taking about 100mg of ground sample, rapidly adding into premix, and immediatelyVortex shaking 30s, centrifuging at 12,000rpm for 2min; the supernatant was transferred to a filter column, again centrifuged at 12,000rpm for 2min, after which the supernatant was carefully aspirated into a new 1.5ml rnase-Free centrifuge tube; adding 0.4 times of absolute ethyl alcohol with the volume of the supernatant, uniformly mixing, transferring to an adsorption column, centrifuging at 12,000rpm for 30s, pouring out waste liquid in a collecting pipe, and placing the adsorption column in the collecting pipe again; adding 350 mu L deproteinized liquid RW1 into an adsorption column, centrifuging at 12,000rpm for 30s, and pouring out waste liquid; taking out the prepared 10 mu LDNaseI from the refrigerator at the temperature of minus 20 ℃, adding 70 mu LRDD and gently mixing; suspending the prepared 80 mu LDNaseI working solution in the center of an adsorption film of an adsorption column (the gun head needs to avoid touching the adsorption film), and standing for 15min at room temperature; adding 350 mu L deproteinized liquid RW1 into the adsorption column again, centrifuging at 12,000rpm for 30s, and pouring out waste liquid; adding 500 μl of rinsing liquid RW (ethanol added) to the adsorption column, centrifuging at 12,000rpm for 30s, pouring out the waste liquid, and placing the adsorption column in a collection tube again, performing 2 times; ) Centrifuging again at 12,000rpm for 2min, placing the adsorption column into a new 1.5mLRNase-Free centrifuge tube; drop-in 20 mu LRNase-FreeddH to the middle of the adsorption film at the bottom of the adsorption column ₂ O, standing at room temperature for 2min, transferring into a low-temperature centrifuge, and centrifuging at 12,000rpm for 2min; re-sucking out the solution at the bottom of the centrifuge tube, vertically suspending, dripping into an adsorption column, standing at room temperature for 2min, transferring into a low-temperature centrifuge, centrifuging at 12,000rpm for 2min, immediately performing reverse transcription on the obtained RNA solution, and preserving the rest RNA solution in an ultralow-temperature refrigerator at-80 ℃ for later use.

The concentration and purity of the total RNA extracted was determined using a nanodrop2,000c uv-vis spectrophotometer (ThermoFisher, USA) (OD 260/280=1.8-2.1), and the RNA extracted was separated using 1% agarose gel electrophoresis, after which it was observed under a uv imaging system to determine its integrity and the presence of contamination.

As shown in FIG. 1, all RNA sample strips are clear, which indicates that the integrity of the total RNA of camphor tree is good, and the quality of the total RNA of camphor tree meets the requirement of further experiments.

The total plant RNA was reverse transcribed into cDNA using the cDNA reverse transcription Prime0script TMRTMastermix kit (TaKaRa, japan), the PCR reaction system was: 5×PrimeScriptRTMasterMix4μL、TotalRNA(500ng/μL)2μL、RNaseFreeddH ₂ O14 mu L, totalvolume mu L. The PCR amplification procedure was: 37 ℃ for 15min;85 ℃,5s.

2. MYB protein sequences provided in Arabidopsis thaliana and Populus delbrueckea were queried and downloaded from TAIR and JGI databases, respectively, and primers for CcMYB4_LIKE on both sides of the CDS sequence were designed using Oligo7 according to the previously published camphor tree genome database of the subject group (T.F.Shen, H.R.Qi, X.Y.Luan, et al Thechrommeome-levelgenomesequence of the camphormaster video sequence of the plant biosystemsystem [ J ]. Plant Biotechnol.J.,2022,20,2,244-246 ]), as follows:

CcMYB4_LIKE-F：5’-ATGAGGAAACCTTGCTGTGAT-3’，

CcMYB4_LIKE-R：5’-TCAATTGAAGAGAAGAAGAGT-3’。

3. using PrimeMax DNA Polymerase (TaKaRa, japan) clone a target gene, and the reaction system is as follows:

Forward Primer(10μM)	1μL
		Reverse Primer(10μM))	1μL
Template(cDNA)	2μL
		Prime STAR Max Premix(2×)	25μL
ddH ₂ O	21μL
		Total Volume	50μL

the PCR amplification procedure is as follows:

the PCR amplified products were detected using 1.5% agarose gel electrophoresis, and a single and clear gel band was recovered for recovery and purification.

4. The fragment of interest was recovered and purified using the axypepdna gel recovery kit (Axygen, usa), the experimental procedure was as follows:

cutting the gel containing the target DNA, and weighing the gel; adding corresponding volume of BufferDE-A (V), mixing, and heating in a water bath kettle at 75 ℃ until the gel is completely melted; adding 0.5V volume of BufferDE-B, and uniformly mixing to obtain a mixed solution; transferring the mixed solution into a preparation tube, centrifuging at 11,000rpm for 1min, pouring out the waste liquid and putting the preparation tube back into a centrifuge tube again; 500 μLBuferW 1 was added to the preparation tube, centrifuged at 11,000rpm for 30s, the waste liquid was decanted and the preparation tube was returned to the centrifuge tube; then 700 μLBuferW 2 (with the specified volume of ethanol added) was added to the preparation tube, centrifuged at 12,000rpm for 30s, the waste liquid was decanted and the preparation tube was returned to the centrifuge tube; repeating the previous step; centrifuging the empty preparation tube at 12,000rpm for 30s; transferring the preparation tube into a 1.5mL centrifuge tube provided by the kit, suspending and dripping 20 mu L of heated deionized water in the center of the preparation film, and standing for 2min at room temperature; centrifuging at 11,000rpm for 1min to elute the DNA; and re-sucking the solution at the bottom of the centrifuge tube, vertically suspending and dropwise adding the solution at the right center of an adsorption film of the adsorption column, standing for 2min at room temperature, and centrifuging at 11,000rpm for 1min to improve the recovery rate.

Recovered DNAAfter concentration and mass measurement by NanoDrop2000c, recombinant ligation was performed to that provided by the full gold companyZero Cloning Vector carrier, the reaction system is as follows:

the reaction procedure is as follows:

25℃	15min
		16℃	∞

the ligation product is transformed into an escherichia coli competent strain Trans1-T1 to realize amplification of a target gene, and the specific method is as follows:

taking out competent strain Trans1-T1 of Escherichia coli stored in-80deg.C refrigerator, immediately placing above ice box, melting, directly adding 5 μl of the connecting product into competence, gently mixing, and standing on ice for 30min; placing the sample in a water bath kettle at 42 ℃, carrying out heat shock for 30s, immediately inserting the sample into an ice box, and carrying out ice bath for 2min; 700. Mu.L of LB liquid medium is added into the sample, and the sample is placed on a shaking table and cultured for 1h at a constant temperature of 37 ℃ and 200 rpm; centrifuging at 4,000rpm for 5min, sucking about 550 mu L of liquid in an ultra-clean workbench, gently scattering and uniformly mixing the residual bacterial liquid by a pipetting gun, transferring the residual and uniformly mixed bacterial liquid into LB solid culture medium with ampicillin (Amp, 100 mg/mL) resistance, and uniformly smearing the LB solid culture medium by using a sterilized coating rod; after the plating is completed, the plate is inversely buckled in a constant temperature incubator at 37 ℃ for culturing for about 12 hours.

PCR was performed using the bacterial liquid as a template and 2×RapidTaqMastermix from Nanjinouzan Biolabs, and the reaction system was as follows:

2×RapidTaqMasterMix	10μL
		Forward Primer(10μM)	1μL
Reverse Primer(10μM))	1μL
		bacterial liquid	1μL
ddH ₂ O	7μL
		Total Volume	20μL

The reaction procedure was as follows:

the PCR products were detected by 1% agarose gel electrophoresis. Samples with clear partial strips and consistent fragment sizes are picked up and sent to the biological engineering (Shanghai) company for sequencing. According to the sequencing result, the CcMYB4_LIKE is determined to be positioned on chromosome 4, the nucleotide sequence of the CcMYB4_LIKE is shown as SEQ ID NO.1, and the gene length is 2,266bp; the CDS sequence is shown as SEQ ID NO.2, and the length is 684bp.

Example 2

The root, flower, phloem, leaf, xylem and stem tip of 'gan-co-1' were collected in month 4 of 2020, and the fruits were collected in month 11 of 2020, for a total of 7 tissues. RNA of the 7 tissues of Ganlong No.1 was extracted and reverse transcribed into cDNA, and the specific method was as shown in example 1. Specific expression of the ccmeyb4_lie gene in Phloem (Phloem), flower (flow), leaf (Leaf), xylem (Xylem), root (Root), fruit (Fruit) and Stem tip (Stem tip) tissues was detected using qRT-PCR. The primer sequences are shown below:

QCcMYB4_LIKE_F：5’-TGAGGCCAGACCTGAAGAG-3’，

QCcMYB4_LIKE_R：5’-CCAGCGATCAACGACCATC-3’

the qRT-PCR reaction system is as follows:

the reaction procedure was as follows:

as a result, ccMYB4_LIKE showed the highest expression level in flowers and the lowest expression level in xylem, as shown in FIG. 2.

Example 3

1. The target gene fragment is inserted into a pJIT166-GFP transient expression vector by using a seamless cloning method, and the primer sequences are as follows:

CcMYB4_LIKE-GFP-F：

5’-CACCATGGCGTGCAGGTCGACATGAGGAAACCTTGCTGTGAT-3’，

CcMYB4_LIKE-GFP-R：

5’-GCCCTTGCTCACCATGGATCCATTGAAGAGAAGAAGAGTTGG-3’。

the purified ccmeyb4_LIKE-GFP vector plasmid was analyzed and sequenced to confirm the success of fusion vector construction.

2. The recombinant plasmid with correct sequencing is transformed into agrobacterium EHA105, and the specific method is as follows:

extracting plasmid from the bacterial liquid with correct sequencing; taking out EHA105 from the ultralow temperature refrigerator at-80 ℃ to be competent, and putting the EHA105 into an ice box immediately after partial melting by using finger temperature; adding 200ng of plasmid into the solution in an ultra-clean workbench, gently flicking the bottom of a centrifuge tube to mix the plasmid and the centrifuge tube uniformly; immediately standing in an ice box for 25min after uniform mixing, immediately quick-freezing with liquid nitrogen for 5min, then placing the ice box into a water bath kettle at 37 ℃ for water bath for 5min, taking out the bacterial liquid after water bath completion, and rapidly inserting the bacterial liquid into the ice box for 5min; in an ultra clean bench, 700. Mu.L of LB liquid medium was added to the centrifuge tube and mixed well. Resuscitating in a temperature-controlled shaking table at 28deg.C and 200rpm for 2-3 hr; taking out the resuscitated bacterial liquid, and centrifuging at 3,000rpm for 3min; sucking out about 550 mu L of liquid in an ultra-clean workbench, gently scattering and mixing the residual bacterial liquid by a pipetting gun, transferring the residual bacterial liquid into LB solid medium with resistance to rifampicin (rif, 20 mu g/mL) and kanamycin sulfate (kana, 50 mu g/mL), and uniformly smearing the residual bacterial liquid in the LB medium by a sterilized coating rod; after the coating is finished, the flat plate is reversely buckled in a constant temperature incubator at 28 ℃ for 48 hours; and (3) delivering the bacterial liquid to a company for detection, and placing the agrobacterium with correct detection into an ultralow temperature refrigerator at-40 ℃ for preservation.

3. Protoplast isolation was performed using young leaves of 60d camphor tree tissue culture seedling plants, which required that the leaves were light green and the back surface was free of powdery mildew to reduce the effect of secondary metabolites, as follows:

3 different concentrations of mannitol (DM), 20mM MES,50mM KCl, and the remainder ddH were added to different 10mL tubes ₂ Supplementing O, mixing, and placing into a water bath kettle at 70 ℃ for 5min; respectively adding 3 kinds of cellulase and pectase with different concentrations into a test tube, mixing, and placing into a water bath kettle with the temperature of 55 ℃ for 10min; after the temperature of the mixture is reduced to room temperature,adding 0.1% BSA into the mixture, and slowly and uniformly mixing the mixture; 10mM CaCl was added ₂ Filtering enzyme solution into a 6-hole plate by using a water-based filter membrane with the diameter of 0.45nm after uniformly mixing; taking a camphor tree tissue culture seedling leaf blade which is good in growth state and flat, cutting the camphor tree tissue culture seedling leaf blade into strips with the length of about 0.5mm (the strips are cut off if the leaf blade is too large), and immediately soaking the cut leaf blade in prepared enzyme solution; placing the 6-hole plate into a paper box with a cover, culturing in dark for 4 hours at 28 ℃, taking out the paper box every 2 hours, and manually and gently shaking for 1-2min; after 4 hours, the 6-well plate is taken out, and an equal volume of W5 solution is added into the enzyme solution to terminate the reaction; pre-cooling 50mL round bottom centrifuge tubes in an ice box for 5min, and completely screening the obtained solution into the 50mL centrifuge tubes by using a 200-mesh cell screen; precooling the centrifugal machine to 4 ℃, setting the acceleration and deceleration to 4 at 900rpm, and centrifuging for 7min; taking out the centrifuge tube, standing in an ice box for 2min to thoroughly precipitate suspended protoplast, and discarding the supernatant; adding 1-2mL of W5 solution into the centrifuge tube, and stirring the bottom of the test tube by gently rotating the palm so as to resuspend the protoplast; after mixing, 8. Mu.L of the solution was pipetted from the center of the centrifuge tube onto a 0.1mm blood cell counting plate using a shear gun head, the protoplast viability was determined using 0.02% FAD and its status and quantity were observed under a fluorescence microscope (ZEISS, germany).

Protoplast yield (per gram) = (protoplast concentration x protoplast suspension volume)/fresh weight of material used to prepare protoplasts.

Protoplast viability = number of protoplasts that fluoresce/total number of protoplasts observed x 100%.

As a result, as shown in Table 1, when the DM concentration was 0.56M, the cellulase concentration was 2.2%, and the pectinase concentration was 0.7%, the protoplast isolation efficiency was the highest, and the morphology was intact. When the concentration of DM is less than 0.6M, the number of protoplasts digested is reduced; the quality of the enzymatic hydrolysis is poor when the DM concentration is higher than 0.6M, and most protoplasts are broken or stuck. When the enzyme concentration is not proper, the phenomenon of reduced enzymolysis efficiency or poor quality can also occur. In a 5mL system, about 1.2X10 were obtained from 100mg leaf tissue ⁶ And (3) protoplasts.

As shown in FIG. 3, the survival rate of the produced protoplast is higher than 95% by FDA staining and identification, and the survival rate is 63.3% by FDA staining and identification after 72 hours of dark culture, and the method obtains the camphor tree mesophyll protoplast with high yield and high quality.

TABLE 1 extraction efficiency of protoplast from camphor tree in different combinations

Factors of	DM concentration (M)	Cellulase (%)	Pectase (%)	Yield (. Times.10) ⁶ Personal/gram)
					1	0.52	2.00	0.40	0.50
2	0.52	2.20	0.60	0.39
					3	0.52	2.40	0.70	0.31
4	0.52	2.60	0.80	0.27
					5	0.56	2.00	0.60	1.20
6	0.56	2.20	0.40	0.43
					7	0.56	2.40	0.80	0.46
8	0.56	2.60	0.70	0.31
					9	4-0.576	2.00	0.70	0.67
10	0.576	2.20	0.80	0.24
					11	0.576	2.40	0.40	0.32
12	0.576	2.60	0.60	0.86
					13	0.6	2.00	0.80	0.36
14	0.6	2.20	0.70	0.50
					15	0.6	2.40	0.60	0.98
16	0.6	2.60	0.40	0.25

4. 0.2M DM and 0.1M CaCl were added to 10mL sterile centrifuge tubes ₂ 30% PEG 4000, ddH remaining ₂ O is filled up; sucking 100 mu L of the shaken protoplast, adding the protoplast into a sterile 2mL centrifuge tube, adding 1 mu g of GFP fusion expression vector plasmid, lightly stirring the tube by hand for mixing, adding 110 mu L of the solution obtained in the step 2), immediately lightly stirring the tube for mixing, and standing for 2-5min at room temperature; adding 500 mu L of W5 solution, stirring and mixing uniformly to terminate the reaction; centrifuging at 1,000rpm for 5min at 4deg.C, and removing supernatant; 100. Mu.L of WI (4 mM MES, pH=5.7, 0.6mM DM,20mM KCl) was added, gently stirred and mixed, and left to stand at room temperature for 10min; dark culturing for 16-20h at room temperature; counting the number of the camphor tree protoplast by using a 0.1mm blood cell counting plate; mu.L of the solution was aspirated and observed under a fluorescence microscope (ZEISS, germany).

As shown in FIG. 4, GFP fluorescence was clearly detected in the cytoplasm and nucleus of the mesophyll protoplast of camphor tree, and the same phenomenon was observed in the constructed GFP fusion vector 35S: ccMYB4_LIKE-GFP

The results are shown in Table 2 and FIG. 5, and the conversion efficiency was tested for different concentrations of PEG solution and conversion times, and was higher when converted for 20min using 15% PEG, and 40%.

TABLE 2 influence of different factors on the conversion efficiency of the protoplasts of the mesophyll of camphor tree

Factors of	PEG concentration (%)	Conversion time (min)	Conversion efficiency (%)
				1	15	10	0
2	30	10	10
				3	40	10	5
4	15	15	15
				5	30	15	20
6	40	15	0
				7	15	20	40
8	30	20	10
				9	40	20	0

Example 4

1. Plasmid extraction and construction of over-expression vector

Activating the strain containing the pH35GS carrier on LB solid medium containing Spc resistance, and culturing at 37 ℃ for 12 hours; selecting a monoclonal colony, culturing in an LB liquid culture medium with the same resistance at 250rpm for 4-6 hours, and detecting bacterial liquid; the overexpression vector pH35GS was linearized using XhoI and KpnI-HF (NewEnglandBiolabs, USA), the reaction system is as follows:

the reaction procedure is as follows:

using PrimeMax DNAPolymerase (TaKaRa, japan) the target gene was amplified, and the primer sequences were as follows:

35S::CcMYB4_LIKE-F：

5’-GGGGACTCTAGAATAGGTACCATGAGGAAACCTTGCTGTGAT-3’，

35S::CcMYB4_LIKE-R：

5’-CCTCAGCTACTTAGGCTCGAGTCAATTGAAGAGAAGAAGAGT-3’。

the reaction system is as follows:

the amplification procedure is as follows:

after 1% agarose gel electrophoresis verification, the target fragment is recovered, and a seamless cloning kit Clon produced by Novain company is usedII One Step Cloning Kit recombinant plasmid was constructed and the reaction system was as follows:

linearization carrier	X mu L (0.02X length)
		Insertion fragment	Y mu L (0.04X length)
5×CEII Buffer	4μL
		Exnase II	2μL
ddH ₂ O	Up to 20μL

The reaction procedure is:

37℃	30min
		4℃	∞

the product was transformed into Trans1-T1 PhageResistantank chemical lycomplete cell competence, and part of the positive bacteria test solution was sent to the biological engineering (Shanghai) company for sequencing. And (5) preserving the bacterial liquid with correct sequencing to an ultralow temperature refrigerator at the temperature of minus 40 ℃.

As shown in FIG. 6, the 12 monoclonal colonies can amplify specific bands with obvious specificity and correct positions, which indicates that the construction of the overexpression vector 35S for over-expressing the target MYB gene is successful.

2. Transformation of 'nan Lin 895 Yang'

The 4 th and 5 th stretched leaves of 30d 'nan lin 895 poplar' were taken. Cutting a wound along the edge of the blade, and cutting the blade into square blade discs with the side length of about 1 cm; spreading leaf discs on MS differentiation medium, and culturing in dark for 2d; the remaining specific transformation method is disclosed in an earlier published article (Chen Caihui. Excavation and function research of camphor tree essential oil biosynthesis related genes [ D ]. Nanjing forestry university, 2019.) of the present laboratory on transformation of 'mountain new poplar'. Tim (200. Mu.g/mL) +kana (30. Mu.g/mL) was added to the medium during the selection; when adventitious buds grow on the edge of the leaf, the leaf is transferred to a strong seedling culture medium containing Tim (200 mug/mL) +kana (30 mug/mL) for culturing stems; when the resistant buds grow out by about 1cm, they are cut off and transferred into WPM medium containing Tim (300 mug/mL) +kana (30 mug/mL) for rooting culture; and (3) weaving a plant formed by rooting an adventitious bud into a plant line.

After three transgenic operations, 47 transgenic CcMYB4_LIKE gene lines are finally obtained, and the negative control group has no survival line finally. 15 strain transgenic poplars and non-transgenic control poplars were randomly selected using the upstream primer p35sf (5'-AGGAAGGTGGCTCCTACAAATGCCATC-3') and the downstream primer 35S:: ccMYB4_LIKE-R at the DNA level

(5'-CCTCAGCTACTTAGGCTCGAGTCAATTGAAGAGAAGAAGAGT-3') amplification of the desired fragment.

As a result, as shown in FIG. 7, after detection by agarose gel electrophoresis, it was found that CK was not amplified, and of 15 random strains, 8 strains overexpressed the CcMYB4_LIKE gene were present.

The expression level of the target gene in 3 transgenic 'nan lin 895 poplar' lines and non-transgenic plants was detected by qRT-PCR using QCCMYB4_LIKE_F (5'-TGAGGCCAGACCTGAAGAG-3') and QCCMYB4_LIKE_R (5'-CCAGCGATCAACGACCATC-3') primers, the reference gene used was EFl a, and the expression level of the target gene in different transgenic lines was statistically calculated by the 2-DeltaCT method relative to the reference gene.

As a result, as shown in FIG. 8, the expression level of CcMYB4_LIKE in the non-transgenic poplar was very low, and the expression level of CcMYB4_LIKE relative to the reference gene was several hundred to several tens of thousands times, wherein the highest expression level was the T54 line, demonstrating that the target gene was successfully integrated into the genome of the recipient plant.

As a result, as shown in FIG. 9, the CcMYB4_LIKE transgenic plant has no obvious color change in the whole growth process, but compared with CK, the leaf color of the CcMYB4_LIKE transgenic plant is lighter, and the growth speed is faster.

3. Transformation of Arabidopsis thaliana

Culturing Agrobacterium with recombinant plasmid (50 mg/Lkana and 20mg/Lrif in culture medium) at 28deg.C and 200rpm for 4-6 hr to OD value of 0.8-1.0; 500mL of an infestation liquid containing 50mL of 5% (wt/vol) g sucrose and 40. Mu.L of 0.02% (vol/vol) of surfactant Silwet L-77 was prepared; centrifuging the fungus liquid at 4000rpm for 10min, and pouring out the supernatant; gently suspending with an infection liquid and then using the suspension for infection of arabidopsis thaliana; soaking inflorescences which are not fully developed into the invasion dye liquor for 30-40s; culturing in dark at 23-25deg.C for 20 hr, and culturing in light environment with illumination period of 16/8 hr; once every 10d or so until the flowering phase is finished. The harvested seeds were sown in 1/2MS medium containing 60. Mu.g/mLkana for resistance selection.

Three lines of Arabidopsis were randomly tested for RNA levels using the upstream primer p35sf (5'-AGGAAGGTGGCTCCTACAAATGCCATC-3') and the downstream primer 35S: ccMYB4_LIKE-R (5'-CCTCAGCTACTTAGGCTCGAGTCAATTGAAGAGAAGAAGAGT-3').

As a result, as shown in FIG. 10, the bands were bright, single and approximately correctly positioned, except for WT (control group) after detection by agarose gel electrophoresis.

The expression level of the target gene in 3 transgenic Arabidopsis lines and non-transgenic plants is detected by qRT-PCR, the primers used are QCCMYB4_LIKE_F (5'-TGAGGCCAGACCTGAAGAG-3') and QCCMYB4_LIKE_R (5'-CCAGCGATCAACGACCATC-3'), the reference gene used is an action, and the expression level of the target gene in different transgenic lines relative to the reference gene is counted by a 2-delta CT method.

As a result, as shown in FIG. 11, the relative expression level of CcMYB4_LIKE was thousands of times, and the strain with the highest relative expression level was T2, which demonstrated that the target gene was successfully integrated into the genome of the recipient plant.

As a result, as shown in FIG. 12, when the dicotyledonous leaves of Arabidopsis thaliana appeared, both cotyledons of WT were green, and the stem was pale green or white, and Arabidopsis thaliana transformed with the CcMYB4_LIKE gene had a similar phenotype.

The results are shown in FIG. 13, where premature white lesions appear on mature leaves of CcMYB4_LIKE over-expressed transgenic Arabidopsis, suggesting reduced levels of hydroxycinnamic acid derivatives. From The studies (L.Tamagnone, A.Merida, A.Parr, et al, the AmMYB308 and AmMMYB 330 Transcription Factors from Antirrhinum Regulate Phenylpropanoid and Lignin Biosynthesis in Transgenic Tobacco [ J ]. The Plant Cell,1998,10,2,135-154.) (H.Jin, E.Cominelli, P.Bailey, et al, transmission repression by AtMYB4 controls production of UV-protecting sunscreens in Arabidopsis [ J ]. The EMBO Journal,2000,19,22,6150-6161.), we speculated that CcMYB4_LIKE overexpression reduced The manner in which Arabidopsis reduced The steady-state transcription levels of genes such as C4H, 4CL1 and CAD, and that transgenic plants developed more trichomes.

4. To verify if ccmeyb4_LIKE is linked to anthocyanin regulation, the expression levels of structural genes on anthocyanin biosynthesis pathways in transgenic 'nanlin 895 poplar' and CK, transgenic arabidopsis and WT (including PAL, C4H and 4CL in the first three stages of the phenylpropane metabolic pathway, two EBGs: CHS and F3H and two LBGs: LDOX and DFR) were verified.

As a result, as shown in FIG. 14, in the CcMYB4_LIKE transgenic 'nan Lin 895 Yang' plant, other structural genes were down-regulated to different degrees except for the 4CL and C4H genes.

As a result, as shown in FIG. 15, in the CcMYB4_LIKE transgenic Arabidopsis plants, the structural genes were significantly up-regulated except for CHS, LDOX and DFR genes.

Claims

The nucleotide sequence of the CcMYB4_LIKE gene of Ganlong No.1 is shown as SEQ ID No. 1.
2. The' ccmeyb4_like gene of claim 1, wherein the amino acid sequence of the expressed protein is shown in SEQ ID No. 2.
3. A vector comprising the' ccmeyb4_like gene of claim 1.
4. The vector of the' ccmeyb4_like gene of claim 3, wherein said vector is a plant expression vector.
5. The vector of the 'CcMYB4_LIKE gene of claim 4, wherein the plant expression vector is 35S:' CcMYB4_LIKE.
Application of the Ganlong No.1 ' CcMYB4_LIKE gene in lightening the color of transgenic ' nan Lin 895 Yang ' leaves.
7. The use of the ' ccmeyb4_like gene of ' ccmeyb 1 ' of claim 6 for lightening transgenic ' nanlin 895 poplar ' leaves, comprising the steps of:

1) Constructing a plant expression vector of a Ganlong No.1 CcMYB4_LIKE gene;

2) Transforming the constructed plant expression vector into 'nan lin 895 poplar' leaves;

3) Culturing and screening to obtain the 'nan lin 895 poplar' plant with the lightening leaf color.
Application of the Ganlong No.1 ' CcMYB4_LIKE gene in accelerating the growth rate of ' nan Lin 895 Yang ' plants.
Application of the Ganlong No.1 ' CcMYB4_LIKE gene in improving the expression level of anthocyanin synthesis related structural genes 4CL and C4H in transgenic ' nan lin 895 poplar ' or reducing the expression level of anthocyanin synthesis related structural genes PAL, CHS, F3H, LDOX and DFR.
The application of the Ganlong No.1 CcMYB4_LIKE gene in improving the expression level of anthocyanin synthesis related structural genes 4CL, C4H, PAL and F3H in transgenic Arabidopsis or reducing the expression level of anthocyanin synthesis related structural genes CHS, LDOX and DFR.