CN108034756B - Method for identifying tea tree varieties by using tea tree cytochrome P450 - Google Patents
Method for identifying tea tree varieties by using tea tree cytochrome P450 Download PDFInfo
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Abstract
The invention discloses a method for identifying tea tree varieties by using tea tree cytochrome P450, which comprises the following steps: picking one bud of a tea tree to be identified, one leaf of the tea tree to be identified, and extracting total RNA; reverse transcribing the total RNA into a cDNA template for qRT-PCR, and designing a primer for the qRT-PCR according to the nucleotide sequences of CYP71A26 and CYP71B34, wherein the nucleotide sequences of CYP71A26 and CYP71B34 are respectively shown as SEQ ID No. 1 and SEQ ID No. 2; and (3) carrying out qRT-PCR by using the cDNA template and the primers, detecting and calculating the relative expression ratio of cytochrome P450 genes CYP71A26 and CYP71B34 in the same tea plant variety, and distinguishing the tea plant variety according to the relative expression ratio. Compared with the prior art, the method has the advantages of simple and convenient operation, high identification efficiency, accurate identification result and the like.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a method for identifying tea tree varieties by using tea tree cytochrome P450.
Background
Tea (Camellia sinensis [ L. ] O.Kuntze) belongs to perennial evergreen woody plant, and is one of important economic crops in China. At present, more than 100 national varieties are approved, and more provincial varieties are approved in each tea-producing province. Therefore, it is necessary to grasp a method for discriminating between varieties of tea trees, and to discriminate the genuineness of the variety when the tea trees are introduced and cultivated, thereby preventing the mixture between varieties. The traditional method for identifying the variety of the tea tree comprises the following steps: s1, observing tree phases, wherein the tree phases comprise tree types and tree postures: the tree types include shrub, small arbor and arbor, and the tree postures are developed, semi-developed and upright; s2, observing the leaf, wherein the contents include the size of the leaf, the shape of the leaf, the uplifting property of the leaf surface, the growth angle of the leaf, the color of the leaf and the like; in addition, the amount, shape, size, early and late germination period and the like of tricholoma matsutake are also used as reference bases for identifying tea varieties. Therefore, the traditional tea tree variety identification method is complex, and accurate identification of tea trees can be carried out only by having certain perceptual knowledge and long-time experience accumulation.
Fuyun No. 6, golden kwan-yin, red cinnamon and yellow denier are representative varieties in various tea areas of Fujian, the planting area is wide particularly in the eastern Fujian area, but 4 varieties are difficult to distinguish rapidly in morphology, and the molecular biology technology has the characteristics of objectivity, accuracy, rapidness and the like, and can accurately and reliably identify the varieties. Different tea varieties have different abilities against pests, among which the most relevant to resistance is cytochrome P450(cytochrome P450). P450 is a superfamily gene of plants, the largest family of which is the CYP71 family. It has been proved that the CYP71 family genes play an important role in the synthesis of plant secondary metabolites, such as phytohormones (phytohorones), phenylalanines (phenylanapanids), terpenes (terpenes), lignins (lignins), flavonoids (flavanones) and alkaloids (alkaloids), and these substances can be used as toxins or insecticides to make plants resistant to insects and pathogenic bacteria, so that the plants which are in place are protected from the harm of external pathogenic bacteria, insects, and the harsh environment. Therefore, tea varieties with different resistance capabilities have independent P450 expression characteristics. Therefore, the method has great significance for identifying the tea tree varieties by using the tea tree cytochrome P450, and particularly has great significance for identifying the tea tree varieties which are morphologically difficult to distinguish quickly, such as Fuyun No. 6, Jinguanyin, Dangui, Huangdan and the like.
Disclosure of Invention
The invention aims to provide a method for quickly and accurately identifying tea tree varieties.
A method for identifying tea plant varieties by using tea plant cytochrome P450 comprises the following steps:
s1, picking one bud and one leaf of the tea tree to be identified, and extracting total RNA;
s2, reverse transcribing the total RNA into a cDNA template for fluorescent quantitative real-time polymerase chain reaction (qRT-PCR), and designing a qRT-PCR primer according to the nucleotide sequences of CYP71A26 and CYP71B34, wherein the nucleotide sequences of the CYP71A26 and CYP71B34 are respectively shown as SEQ ID No. 1 and SEQ ID No. 2;
s3, carrying out qRT-PCR by using the cDNA template and the primers, detecting and calculating the relative expression ratio of cytochrome P450 genes CYP71A26 and CYP71B34 in the same tea plant variety, and distinguishing the tea plant variety according to the relative expression ratio.
The invention has the beneficial effects that: the technical scheme of the invention utilizes molecular biology technology, and can accurately and reliably identify the tea tree variety compared with the traditional field morphology identification by establishing the ratio of two representative subfamily cytochrome genes in the same variety. The method has accurate and reliable identification result, greatly reduces errors caused by identification by an empirical sensory method, improves the identification efficiency of tea varieties, is beneficial to distinguishing the true and false of the varieties when the tea trees are introduced and cultivated, and avoids the mixing of the varieties.
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following description is given in detail with reference to the embodiments.
A method for identifying tea plant varieties by using tea plant cytochrome P450 comprises the following steps:
s1, picking one bud and one leaf of the tea tree to be identified, and extracting total RNA;
s2, reverse transcribing the total RNA into a cDNA template for qRT-PCR, and designing a primer for the qRT-PCR according to the nucleotide sequences of CYP71A26 and CYP71B34, wherein the nucleotide sequences of CYP71A26 and CYP71B34 are respectively shown as SEQ ID No. 1 and SEQ ID No. 2;
s3, carrying out qRT-PCR by using the cDNA template and the primers, detecting and calculating the relative expression ratio of cytochrome P450 genes CYP71A26 and CYP71B34 in the same tea plant variety, and distinguishing the tea plant variety according to the relative expression ratio.
From the above description, the beneficial effects of the present invention are: the technical scheme of the invention utilizes molecular biology technology, and can accurately and reliably identify the tea tree variety compared with the traditional field morphology identification by establishing the ratio of two representative subfamily cytochrome genes in the same variety. The method has accurate and reliable identification result, greatly reduces errors caused by identification by an empirical sensory method, improves the identification efficiency of tea varieties, is beneficial to distinguishing the true and false of the varieties when the tea trees are introduced and cultivated, and avoids the mixing of the varieties.
Further, the primer should satisfy the following conditions:
c1, the length of the primer is between 18bp and 23 bp;
c2, the total length of the fluorescent quantitative product is between 100bp and 150 bp;
c3, the primer annealing temperature TM value is between 58 ℃ and 62 ℃;
c4, the base distribution of the primer is uniform, T/C and A/G continuous structures are avoided, and the content of G + C in the primer is between 45 and 55 percent;
c5, primer itself, did not present 4 consecutive complementary bases.
Preferably, the length of the quantitative amplification primers for CYP71A26 and CYP71B34 do not differ significantly.
Further, the sequence of the upstream primer of CYP71A26 is as follows: 5'-TAGGCAACGGCAAGCAATAC-3' (SEQ ID No:3), the sequence of the downstream primer is: 5'-GCAATGGAATAGGAGGATGTAGG-3' (SEQ ID No: 4).
Further, the sequence of the upstream primer of CYP71B34 is as follows: 5'-GCTTCCTGCTCTCATCATCCT-3' (SEQ ID No:5), the sequence of the downstream primer is: 5'-TGCCGAGTTGGTGGAGGTT-3' (SEQ ID No: 6).
From the above description, the beneficial effects of the present invention are: the method is more accurate and reliable for the tea plant variety which is difficult to identify depending on the traditional field shape; although the shape difference of the four varieties of tea trees is not large, the relative expression ratio difference is obvious, and the identification of 4 varieties of Fuyun No. 6, golden kwan-yin, red cinnamon and Huangdan can be quickly and accurately realized by only utilizing a single variety sample to carry out self double-gene comparison from the perspective of double genes by adopting the method.
Further, the tea tree to be identified in the step S1 is a 1-year tea tree with uniform growth vigor.
Preferably, during the identification process, 3 tree buds per tea tree are selected for repeated tests.
Further, in the step S1, the picked tree buds are frozen by liquid nitrogen and stored at-80 ℃ for later use.
Further, in step S1, the operation steps of extracting total RNA are as follows:
s101, grinding tea tree buds to be identified in liquid nitrogen into powder, adding a lysis solution, and vibrating and uniformly mixing after vortexing;
s102, centrifuging the tea tree bud solution after the operation;
s103, transferring the supernatant obtained after centrifugation to a filter column, and reserving the supernatant after re-centrifugation;
s104, adding absolute ethyl alcohol with the volume 0.4 times that of the supernatant into the supernatant obtained after the operation of the step S103, transferring the mixture into an adsorption column, centrifuging, and keeping the adsorption column;
s105, adding protein removing liquid into the adsorption column operated in the step S104, centrifuging, and reserving the adsorption column;
s106, mixing the DNase I storage solution and the RDD solution, adding the mixed solution into the adsorption column treated in the step S105, standing at room temperature for 15min, adding the deproteinized solution again, centrifuging, and keeping the adsorption column;
s107, adding rinsing liquid into the adsorption column processed in the step S106, centrifuging, and reserving the adsorption column;
s108, repeating the step S107 at least once, then centrifuging again, and dropwise adding RNase-Free ddH into the centrifuged adsorption column2And O, standing at room temperature for 2min, and centrifuging to obtain a total RNA solution.
Further, in the step S101, the mass-to-volume ratio of the tree bud to the lysate is 1-2: 10mg/μ l.
Preferably, in the step S101, the mass of the tree bud is 50-100 mg, and the volume of the lysis solution is 500 μ l.
Further, the speed of centrifugation in step S102 is at least 13400 Xg, and the time is at least 2 min.
Further, in the step S2, the specific steps of reverse transcription of total RNA into cDNA template for qRT-PCR are as follows: s201, genomic DNA removal reaction: prepare 10 μ l reaction: 2.0. mu.l of 5 XgDNA Eraser Buffer, 1.0. mu.l of gDNA Eraser, 2.0. mu.l of Total RNA RNase, Free dH2Supplementing O to 10.0 μ l, and incubating at 42 deg.C for 2 min;
s202, reverse transcription reaction: preparing 20 μ L of reaction system: 4.0. mu.L of 5 XPrimeScript Buffer 2, 1.0. mu.L of PrimeScript RT Enzyme Mix I, and 10.0. mu.L of the reaction mixture from step S201, to which freedH was added2O the volume of the solution is supplemented to 20.0 mu l, the reaction is carried out for 15min at 37 ℃ in a PCR instrument, the denaturation is carried out for 5s at 85 ℃, and the storage is carried out at 4 ℃.
Further, in the step S3, the total volume of qRT-PCR is 20 μ l, and the reaction system is 10.0 μ l of SYBR Green; 1.0. mu.l of cDNA; 1.0 μ l of each of the upstream and downstream primers; 7 μ l of ddH2O。
Further, in the step S3, the qRT-PCR reaction program is pre-denatured at 95 ℃ for 30S; denaturation at 95 ℃ for 5s, annealing at 58-62 ℃ for 30s, and extension at 72 ℃ for 30s for 38 cycles; 70-95 ℃, the temperature rises by 0.5 ℃ respectively per cycle, and 2 is adopted-ΔΔCTMethod comparison CTThe relative expression ratio of CYP71a26 to CYP71B34 was calculated.
Further, the step S3 further includes a step of creating a spectrum library of relative expression ratios of different tea plant varieties, and distinguishing the tea plant varieties by comparing the detected relative expression ratios with the spectrum library of relative expression ratios.
From the above description, the beneficial effects of the present invention are: and the subsequent identification efficiency is accelerated by establishing a ratio library.
Further, the variety of the tea tree to be identified is one or more of Fuyun No. 6, golden kwan-yin, cinnamon or yellow denier.
Further, the identification standards of Fuyun No. 6, golden kwan-yin, orange osmanthus and yellow denier are as follows: and when the relative expression ratios are within the ranges of 0.16 +/-0.8, 1.80 +/-0.90, 62.20 +/-31.10 and 204.42 +/-102.21 in turn, judging that the tea plant belongs to the corresponding tea plant variety.
The first embodiment of the invention is as follows: a method for identifying tea plant varieties by using tea plant cytochrome P450 comprises the following steps:
1. tea sample treatment and sampling
Respectively taking 4 varieties of 1-year-old and uniformly-grown Fuyun No. 6, Jinguanyin, Dangui and Huangdan in a tea garden, respectively taking one bud and one leaf (each sample is repeated for 3 times), freezing by using liquid nitrogen, and storing at-80 ℃ for later use.
2. Extraction of total RNA and construction of qRT-PCR template
2.1 Total RNA extraction
Extracting the total RNA of the tea trees from the samples prepared in the steps according to the operation steps of the instruction of a plant total RNA extraction kit (RNAprep pure plant kit, dp441), and specifically comprising the following steps:
(1) 50mg of the sample is rapidly ground into powder in liquid nitrogen, 500. mu.l of lysate is added, and the mixture is vortexed and vigorously shaken and uniformly mixed.
(2) Centrifuge at 12,000rpm (. about.13,400 Xg) for 2 min.
(3) The supernatant was transferred to the filtration column CS and centrifuged at 12,000rpm (. about.13,400 Xg) for 2min, and the supernatant in the collection tube was carefully pipetted into a new RNase-Free centrifuge tube, with the tip being kept from touching the cell debris pellet in the collection tube as much as possible.
(4) Adding 0.4 times of the volume of the supernatant of absolute ethyl alcohol slowly, mixing uniformly, transferring the obtained solution and the precipitate into an adsorption column CR3, centrifuging for 15s at 12,000rpm (13,400 Xg), pouring off the waste liquid in the collecting tube, and putting the adsorption column CR3 back into the collecting tube.
(5) 350 μ l of deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm (. about.13,400 Xg) for 15s, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.
(6) Preparing DNase I working solution: add 10. mu.l DNase I stock into a new RNase-Free centrifuge tube, add 70. mu.l RDD solution, mix gently.
(7) 80. mu.l of DNase I working solution was added to the center of the adsorption column CR3, and the mixture was left at room temperature for 15 min.
(8) 350 μ l of deproteinizing solution RW1 was added to the adsorption column CR3, centrifuged at 12,000rpm (. about.13,400 Xg) for 15s, the waste liquid in the collection tube was discarded, and the adsorption column CR3 was returned to the collection tube.
(9) 500. mu.l of the rinsing solution RW was added to the adsorption column CR3, and the tube was centrifuged at 12,000rpm (. about.13,400 Xg) for 15 seconds to remove the waste liquid from the collection tube, and the adsorption column CR3 was returned to the collection tube.
(10) Step 9 is repeated.
(11) Centrifuging at 12,000rpm (13,400 Xg) for 2min, placing adsorption column CR3 into a new RNase-Free centrifuge tube, and adding 30-50 μ l RNase-Free ddH dropwise into the middle part of the adsorption membrane2O, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution.
2.2 construction of qRT-PCR templates
The total RNA is reversely transcribed into a cDNA template of qRT-PCR according to the instruction of the PrimeScript RT reagent Kit with gDNA Eraser Kit of TaKaRa, and the specific steps are as follows:
(1) removal reaction of genomic DNA: prepare 10 μ l reaction: 2.0. mu.l of 5 XgDNA Eraser Buffer, 1.0. mu.l of gDNA Eraser, 2.0. mu.l of Total RNA RNase, Free dH2O is supplemented to 10.0 mu l, and the mixture is incubated for 2min at the normal temperature of 42 ℃.
(2) Reverse transcription reaction: preparing 20 μ L of reaction system: 4.0. mu.l of 5 XPrimeScript Buffer 2, 1.0. mu.l of PrimeScript RT Enzyme Mix I, 10.0. mu.l of the reaction mixture from step (1), Free dH2And O is supplemented to 20.0 mu L. Reacting at 37 deg.C for 15min in PCR instrument, denaturing at 85 deg.C for 5s, and storing at 4 deg.C.
Design of qRT-PCR primers
Gene sequences of CYP71A26(GenBank accession No.: GARM01003853.1) and CYP71B34(GenBank accession No.: KA281555.1) were screened from the tea plant transcriptome database (Taxolomy ID:4442) into which NCBI has logged. According to the existing sequence, the sequence is introduced into primer5.0 software to design the optimal quantitative primer. Primer design must satisfy:
(1) the length of the primers is between 18bp and 23bp, and the length difference between the two primers is small;
(2) the length of the fluorescent quantitative product is between 100bp and 150 bp;
(3) the primer annealing temperature TM value is between 58 ℃ and 62 ℃;
(4) the base distribution of the primer is uniform, the T/C and A/G continuous structure is avoided, and the G + C is between 45 and 55 percent;
(5) the primer itself and the two primers can not generate continuous 4 complementary bases, and the quantitative products of the two genes with 105bp and 134bp and the sequences of the primers are shown in Table 1.
TABLE 1 quantitative products of tea plant CYP71A26 and CYP71B34 genes and sequences of primers
In addition, GAPDH gene can be selected as an internal reference gene (refer to the cloning and expression analysis of geranylgeranyl pyrophosphate synthetase gene CsGGDPS of tea plant, published in tea science by Yuan Qiaoqian equal to 2017), and the primer sequence of the internal reference gene is as follows: upstream F: TTGGCATCGTTGAGGGTCT (SEQ ID No:7), downstream R: CAGTGGGAACACGGAAAGC (SEQ ID No: 8).
qRT-PCR expression detection
Using the template and primers obtained above, 20. mu.L of total reaction system was prepared for each tube: 10.0 μ L of SYBR Green; 1.0. mu.L of cDNA; 1.0. mu.L of each of the upstream and downstream primers; ddH of 7. mu.L2And O. Pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 5s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 38 cycles; at 70 deg.C, respectively increasing by 0.5 deg.C per cycle, and adopting 2-ΔΔCTMethod comparison CTThe method of (4) calculates the relative expression level of CYP71A26 and CYP71B 34.
5. Results
According to the steps, the relative expression ratios of CYP71A26 and CYP71B34 of 4 varieties of Fuyun No. 6, Jinguanyin, Dangui and Huangdan are obtained and are respectively shown in tables 2, 3,4 and 5.
TABLE 2 relative expression ratios of Fuyun No. 6 variety
Note: the amount of CYP71B34 gene expression was used as a control (mapping 1).
TABLE 3 relative expression ratios of golden Guanyin varieties
Note: the amount of CYP71B34 gene expression was used as a control (mapping 1).
TABLE 4 relative expression ratios of Huangdan varieties
Note: the amount of CYP71B34 gene expression was used as a control (mapping 1).
TABLE 5 relative expression ratios of the species Osmanthus
Note: the amount of CYP71B34 gene expression was used as a control (mapping 1).
As can be seen from tables 2-5, the ratio difference is remarkable due to the fact that 4 varieties have different resistance capacities, and the Fuyun No. 6, the golden kwan-yin, the orange osmanthus and the yellow denier can be quickly and accurately combined by the method.
6. Establishing a relative expression ratio spectrum library
Although the molecular biology technology has the characteristics of simple, rapid and accurate method, and accurate identification of the authenticity of the tea tree variety. However, due to the different sources of samples and the inevitable differences in sample handling, there is a range of small errors in qRT-PCR results. Therefore, on the basis of the obtained data, a relative expression ratio spectrum library is established by selecting the value of +/-50%, and the spectrum is identified correspondingly. The relative expression ratio spectrum library spectrum information of Fuyun No. 6, golden kwan-yin, orange osmanthus and Huangdan varieties is shown in Table 6.
TABLE 6 relative expression ratio spectrum library spectra of Fuyun No. 6, Jinguanyin, Dangui and Huangdan varieties
By adopting the information of the spectral library in the table 6, the identification is carried out on 4 tea garden varieties of Fuyun No. 6, golden kwan-yin, red cinnamon and Huangdan with the widest popularization area in Fujian, particularly in the east Fujian region, and the accuracy of the identification result is found to be 100%.
The second embodiment of the invention is a method for identifying tea plant varieties by using tea plant cytochrome P450, which comprises the following steps:
s1, picking one bud and one leaf of the tea tree to be identified, and extracting total RNA;
s2, reverse transcribing the total RNA into a cDNA template for qRT-PCR, and designing a primer for the qRT-PCR according to the nucleotide sequences of CYP71A26 and CYP71B34, wherein the nucleotide sequences of the CYP71A26 and CYP71B34 are shown as SEQ ID No. 1 and SEQ ID No. 2;
s3, carrying out qRT-PCR by using the cDNA template and the primers, detecting and calculating the relative expression ratio of cytochrome P450 genes CYP71A26 and CYP71B34 in the same tea plant variety, and distinguishing the tea plant variety according to the relative expression ratio.
Wherein, the sequence of the upstream primer of CYP71A26 is as follows: 5'-TAGGCAACGGCAAGCAATAC-3', the sequence of the downstream primer is: 5'-GCAATGGAATAGGAGGATGTAGG-3', and the sequences of the upstream primer and the downstream primer of the CYP71A26 are shown as SEQ ID No. 3 and SEQ ID No. 4 respectively. The sequence of the upstream primer of CYP71B34 is as follows: 5'-GCTTCCTGCTCTCATCATCCT-3', the sequence of the downstream primer is: 5'-TGCCGAGTTGGTGGAGGTT-3', and the sequences of the upstream primer and the downstream primer of the CYP71B34 are shown as SEQ ID No. 5 and SEQ ID No. 6 respectively.
In step S1, the operation steps for extracting total RNA are as follows:
s101, quickly grinding 100mg of sample into powder in liquid nitrogen, adding 500 mu l of lysate, and violently shaking and uniformly mixing by vortex;
s102, centrifuging the tea tree bud solution subjected to the operation at 12,000rpm (13,400 Xg) for 2 min;
s103, transferring the supernatant to a filter column CS, centrifuging for 2min at 12,000rpm (13,400 Xg), carefully sucking the supernatant in a collecting tube to a new RNase-Free centrifugal tube, and avoiding the contact of a suction head with cell debris in the collecting tube to be precipitated as far as possible;
s104, slowly adding absolute ethyl alcohol with the volume 0.4 times of the volume of the supernatant into the supernatant obtained after the operation of the step S103, uniformly mixing, transferring the obtained solutions into an adsorption column CR3 together, centrifuging for 15S at 12,000rpm (13,400 Xg), pouring waste liquid in a collecting pipe, and putting the adsorption column CR3 back into the collecting pipe;
s105, adding 350 mu l of deproteinizing solution RW1 into the adsorption column CR3 after the operation of the step S104, centrifuging for 15S at 12,000rpm (13 to 400 Xg), pouring off waste liquid in the collection tube, putting the adsorption column CR3 back into the collection tube, centrifuging, and reserving the adsorption column;
s106, preparation of DNase I working solution: putting 10 mul DNase I stock solution into a new RNase-Free centrifuge tube, adding 70 mul RDD solution, and gently mixing; mixing DNase I storage solution and RDD solution, adding the mixed solution into the adsorption column CR3 treated in the step S105, standing at room temperature for 15min, adding 350 mu l of deproteinizing solution RW1 into the adsorption column CR3 again, centrifuging at 12,000rpm (13,400 Xg) for 15S, pouring waste liquid in the collection tube, and putting the adsorption column CR3 back into the collection tube;
s107, adding 500 mu l of rinsing liquid RW into the adsorption column CR3 processed in the step S106, centrifuging for 15S at 12,000rpm (13,400 Xg), pouring off waste liquid in the collection tube, putting the adsorption column CR3 back into the collection tube, adding the rinsing liquid into the collection tube, centrifuging, and keeping the adsorption column;
s108, repeating the step S107 once, centrifuging the adsorption column for 2min at 12,000rpm (13,400 Xg), placing the adsorption column CR3 into a new RNase-Free centrifuge tube, and suspending and dropwise adding 30-50 μ l of RNase-Free ddH to the middle part of the adsorption film2O, left at room temperature for 2min, and centrifuged at 12,000rpm (. about.13,400 Xg) for 1min to obtain an RNA solution.
In the step S2, the specific steps of cDNA template operation of reverse transcription of total RNA into qRT-PCR are as follows: s201, genomic DNA removal reaction: prepare 10 μ l reaction: 2.0. mu.l of 5 XgDNA Eraser Buffer, 1.0. mu.l of gDNA Eraser, 2.0. mu.l of Total RNA RNase, Free dH2Supplementing O to 10.0 μ l, and incubating at 42 deg.C for 2 min;
s202, reverse transcription reaction: preparing 20 μ L of reaction system: 4.0. mu.l of 5 XPrimeScript Buffer 2, 1.0. mu.l of PrimeScript RT Enzyme Mix I, 10.0. mu.l of the reaction mixture from step S201, Free dH was added2O the volume of the solution is supplemented to 20.0 mu l, the reaction is carried out for 15min at 37 ℃ in a PCR instrument, the denaturation is carried out for 5s at 85 ℃, and the storage is carried out at 4 ℃.
In the step S3, the total volume of qRT-PCRqRT-PCR is 20 mul, and the reaction system is 10.0 mul of SYBR Green; 1.0. mu.l of cDNA; 1.0 μ L of each of the upstream and downstream primers; 7 μ l of ddH2O。
In the step S3, the qRT-PCR reaction program is pre-denatured at 95 ℃ for 30S; denaturation at 95 ℃ for 5s, annealing at 62 ℃ for 30s, and extension at 72 ℃ for 30s for 38 cycles; 95 ℃ and a rise of 0.5 ℃ per cycle respectively, using 2-ΔΔCTMethod comparison CTThe method calculates the relative expression ratio of CYP71A26 and CYP71B34, calculates the relative expression ratio of CYP71A26 and CYP71B34 of the tea plant to be 0.16, and compares the relative expression ratio with the information in the relative expression ratio library to know that the tea plant variety is Fuyun No. 6.
In conclusion, the method for identifying the tea tree varieties by using the tea tree cytochrome P450 has the advantages of simplicity and convenience in operation, high identification efficiency, accurate identification result and the like.
It should be noted that, the case in the patent specification mainly relates to the identification of four varieties of Fuyun No. 6, Jinguanyin, Dangui and Huangdan, but the invention is also applicable to the identification of other tea tree varieties.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by the present specification, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
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<213> tea plant (Camellia sinensis)
<400> 2
gcttcctgct ctcatcatcc tcctcctttc ttccatctcc ttcctctttt tcaccaaaaa 60
gtccaaaaac ccaaccaacc ttcctccaac cccaccaacc cttcccatca tcggcaacct 120
ccaccaactc ggca 138
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
taggcaacgg caagcaatac 20
<210> 4
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcaatggaat aggaggatgt agg 23
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcttcctgct ctcatcatcc t 21
<210> 6
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
tgccgagttg gtggaggtt 19
<210> 7
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ttggcatcgt tgagggtct 19
<210> 8
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cagtgggaac acggaaagc 19
Claims (3)
1. A method for identifying tea plant varieties by using tea plant cytochrome P450 is characterized by comprising the following steps: the method comprises the following steps:
the variety of the tea tree to be identified is one or more of Fuyun No. 6, golden kwan-yin, orange osmanthus or yellow denier;
the identification standards of Fuyun No. 6, golden kwan-yin, orange osmanthus and Huangdan are as follows: when the relative expression ratio is within the ranges of 0.16, 1.80, 62.20 and 204.42 in sequence, judging that the tea plant belongs to a corresponding tea plant variety;
s1, picking one bud and one leaf of the tea tree to be identified, and extracting total RNA;
s2, reverse transcribing the total RNA into a cDNA template for qRT-PCR, and designing a primer for the qRT-PCR according to the nucleotide sequences of CYP71A265 and CYP71B34, wherein the nucleotide sequences of CYP71A26 and CYP71B34 are respectively shown as SEQ ID No. 1 and SEQ ID No. 2;
the sequence of the upstream primer of CYP71A26 is as follows: 5'-TAGGCAACGGCAAGCAATAC-3', the sequence of the downstream primer is: 5'-GCAATGGAATAGGAGGATGTAGG-3', wherein the sequences of the upstream primer and the downstream primer of CYP71A26 are respectively shown as SEQ ID No. 3 and SEQ ID No. 4;
the sequence of the upstream primer of CYP71B34 is as follows: 5'-GCTTCCTGCTCTCATCATCCT-3', the sequence of the downstream primer is: 5'-TGCCGAGTTGGTGGAGGTT-3', wherein the sequences of the upstream primer and the downstream primer of CYP71B34 are respectively shown as SEQ ID No. 5 and SEQ ID No. 6;
s3, carrying out qRT-PCR by using the cDNA template and the primers, detecting and calculating the relative expression ratio of cytochrome P450 genes CYP71A26 and CYP71B34 in the same tea plant variety, and distinguishing the tea plant variety according to the relative expression ratio.
2. The method of using tea tree cytochrome P450 to identify a tea tree species as claimed in claim 1, wherein: in step S1, the operation steps for extracting total RNA are as follows:
s101, grinding tea tree buds to be identified in liquid nitrogen into powder, adding a lysis solution, and vibrating and uniformly mixing after vortexing;
s102, centrifuging the tea tree bud solution after the operation;
s103, transferring the supernatant obtained after centrifugation to a filter column, and reserving the supernatant after re-centrifugation;
s104, adding absolute ethyl alcohol with the volume 0.4 times that of the supernatant into the supernatant obtained after the operation of the step S103, transferring the mixture into an adsorption column, centrifuging, and keeping the adsorption column;
s105, adding protein removing liquid into the adsorption column operated in the step S104, centrifuging, and reserving the adsorption column;
s106, mixing the DNase I storage solution and the RDD solution, adding the mixed solution into the adsorption column treated in the step S105, standing at room temperature for 15min, adding the deproteinized solution again, centrifuging, and keeping the adsorption column;
s107, adding rinsing liquid into the adsorption column processed in the step S106, centrifuging, and reserving the adsorption column;
s108, repeating the step S107 at least once, then centrifuging again, and dropwise adding RNase-Free ddH into the centrifuged adsorption column2And O, standing at room temperature for 2min, and centrifuging to obtain a total RNA solution.
3. The method of using tea tree cytochrome P450 to identify a tea tree species as claimed in claim 1, wherein: in the step S101, the mass-to-volume ratio of the tree buds to the lysis solution is 1-2: 10 mg/mu l.
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| CN101643775A (en) * | 2008-08-06 | 2010-02-10 | 中国农业大学 | Rice cytochrome P450 gene primer for assisting in identifying herbicide residue in plant culture matrix |
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| CN101643775A (en) * | 2008-08-06 | 2010-02-10 | 中国农业大学 | Rice cytochrome P450 gene primer for assisting in identifying herbicide residue in plant culture matrix |
Non-Patent Citations (2)
| Title |
|---|
| De Novo Characterization of Leaf Transcriptome Using 454 Sequencing and Development of EST-SSR Markers in Tea (Camellia sinensis);Wu Hualing等;《PLANT MOLECULAR BIOLOGY REPORTER》;20130630;第31卷(第3期);第524-538页 * |
| 番茄CYP71基因的克隆及其功能的初步分析;郝彦伟等;《热带作物学报》;20091231;第30卷(第12期);第1813-1817页 * |
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Address after: No. 104, puLong Road, Xindian Town, Jin'an District, Fuzhou City, Fujian Province Applicant after: Tea Inst., Fujian Academy of Agricultural Science Address before: 355015, Ningde City, Fujian province Fu'an city social town Applicant before: Tea Inst., Fujian Academy of Agricultural Science |
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