Method for predicting drought resistance of tea germplasm resources using differential gene expression

Abstract

The invention belongs to the field of biotechnology, and relates to a method for rapidly predicting drought resistance of different tea tree germplasm resources by using gene differential expression, comprising the following steps: 1) harvesting budding tender branches; performing the following steps 2) to 3) on tender branches harvested from each tea tree to be tested for each germplasm resource; 2) all tender branches are first inserted into water for 1 to 2 days, and then divided into two groups in equal amounts, which are respectively used as water supply tender branches and non-water supply tender branches; collecting leaves of each water supply tender branch and each non-water supply tender branch, respectively amplifying Act gene and PMO gene, so as to obtain corresponding Cт values, and correspondingly obtaining the difference ΔΔCT of the Cт values ​​of the two genes, and the relative expression amount X of the p-hydroxyphenylacetaldehyde oxime monooxygenase-like PMO gene is X=2 ‑ΔΔCT ; Determine the drought resistance of the tested tea tree germplasm based on the relative expression level.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to a method for rapidly predicting the drought tolerance of different tea germplasm resources by using gene differential expression. Background Art

[0002] Drought is one of the most important abiotic stresses faced by tea production and occurs frequently in the main tea-producing areas of China and even tea-producing countries around the world. Drought not only causes a decline in tea yield and quality, but also seriously affects the growth vitality of tea trees, and even can lead to the death of the whole tea tree, which is extremely unfavorable for high-quality and high-yield tea.

[0003] Drought tolerance is the key content for identification and evaluation in the process of breeding new tea tree varieties. However, it is relatively difficult to evaluate this trait, mainly because there are few evaluation methods and the operations are relatively cumbersome. The existing methods are divided into two categories: indirect identification and direct identification. Among them, the indirect identification method is mainly the determination of wilting coefficient. The specific method of direct identification is to conduct a field survey on different tea tree varieties or germplasms over 5 years old after natural drought occurs. Any leaf with more than 1 / 3 dry or red-brown is considered a damaged leaf. Survey the damaged leaves of 10 tea trees, and according to the leaf damage ratio ≤ 5%, 5% - 15% (including 15%), 15% - 25% (including 25%), 25% - 50% (including 50%), > 50%, determine the drought damage levels of 0, 1, 2, 3, 4 respectively, and then calculate the drought damage index according to the formula DI = ∑(n i *x i ) / (N*4)*100% (where DI is the drought damage index, n i is the number of tea tree plants at each drought damage level, x i is the drought damage level, N is the total number of surveyed tea trees, and 4 is the highest drought damage level); if DI ≤ 10, the drought tolerance of the tea germplasm is "strong", if 10 < DI ≤ 20, the drought tolerance is "relatively strong", if 20 < DI ≤ 50, the drought tolerance is "medium", if DI > 50, the drought tolerance is "weak". Generally speaking, for the above indirect identification of drought tolerance, the pot experiment requires more than one and a half months, which is very time-consuming, and the daily management is also more complicated; while the direct identification is mainly carried out in summer and autumn every year, but the drought degree varies greatly between summer and autumn of each year, resulting in obvious differences in the identification results between years. And to reduce the influence of soil and other conditions, the drought tolerance identification of different germplasm resources needs to be limited to the same plot, so the number of germplasm resources identified each time is relatively limited.

[0004] The invention CN113403322B, "A Tea Tree Drought-Response Gene CsNAC168, Its Encoded Protein, and Applications," provides a tea tree drought-responsive gene, CsNAC168, which significantly enhances the tolerance of transgenic Arabidopsis thaliana lines to drought stress and high-concentration sodium chloride stress, significantly improving the survival rate of transgenic plants under water-deficit and drought conditions. Therefore, CsNAC168 can be used in genetic engineering and genetic improvement.

[0005] The p-hydroxyphenylacetaldehyde oxime monooxygenase-like gene PMO (TEA017496.1) was disclosed in the first version of the 'Shucha Zao' genome database (http: / / tpia.teaplant.org / index.html) and the paper "Draft genome sequence of Camellia sinensis var. sinensis provides insights into the evolution of the tea genome and tea quality" (PNAS, 2018, 115(18): E4151–E4158), and its sequence is described as SEQ ID NO: 1.

[0006] So far, there is no method to predict and evaluate the drought tolerance of tea germplasm resources using differential gene expression. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for predicting the drought resistance of tea germplasm resources by using differential gene expression; specifically, the method predicts the drought resistance of different tea germplasm resources by analyzing the differential expression of p-hydroxyphenylacetaldehyde oxime monooxygenase-like genes in leaves on detached branches under water supply and no water supply conditions.

[0008] To solve the above technical problems, the present invention provides a method for predicting the drought resistance of tea germplasm resources by using gene differential expression, comprising the following steps:

[0009] 1) When the tea trees to be tested form shoots that meet the following conditions, the shoots are harvested;

[0010] The number of budding shoots is ≥10, and each shoot has 4 complete leaves;

[0011] The following steps 2) to 3) are performed on the tender branches harvested from each tea tree to be tested;

[0012] 2) In a constant temperature and light incubator: All the tender branches harvested from each tea tree of the germplasm resources to be tested are first inserted into water for 1 - 2 days (the 3 - 4 cm at the base of the tender branches is immersed in water for adaptive adjustment), and then equally divided into two groups;

[0013] One group continues to be inserted into water to supply water to the tender branches, serving as the water - supplied tender branches; the other group is taken out of the water (i.e., without water supply), serving as the non - water - supplied tender branches;

[0014] The above - mentioned water - supplied tender branches and non - water - supplied tender branches are continuously placed in the constant temperature and light incubator for 22 - 26 h;

[0015] The temperature of the incubator is 25 ± 2 °C, the light intensity is 5000 ± 100 Lux, and the relative humidity is 65 ± 5%;

[0016] 3) After step 2), collect the leaves (one leaf at the same position) of each water - supplied tender branch and each non - water - supplied tender branch as samples. Each sample is respectively amplified for the Act gene and the PMO gene to obtain the corresponding Ct value, and the difference in the Ct values of the two genes is obtained accordingly;

[0017] The difference in the Ct values of the two genes corresponding to the water - supplied tender branches is denoted as ΔCt1;

[0018] The difference in the Ct values of the two genes corresponding to the non - water - supplied tender branches is denoted as ΔCt2;

[0019] ΔΔCT = ΔCt2 - ΔCt1;

[0020] The relative expression level X of the hydroxyphenylacetaldoxime monooxygenase - like PMO gene = 2 -ΔΔCT ;

[0021] When X ≤ 11.6, it is determined that the drought tolerance of the germplasm of the tea tree to be tested is "strong";

[0022] When 11.6 < X ≤ 37.4, it is determined that the drought tolerance of the germplasm of the tea tree to be tested is "relatively strong";

[0023] When 37.4 < X ≤ 174.4, it is determined that the drought tolerance of the germplasm of the tea tree to be tested is "medium";

[0024] When X > 174.4, it is determined that the drought tolerance of the germplasm of the tea tree to be tested is "weak".

[0025] Note: For the samples obtained by sampling, after grinding with liquid nitrogen respectively, total RNA is extracted and reverse - transcribed into cDNA, and then corresponding amplification is carried out.

[0026] As an improvement to the method for predicting the drought resistance of tea tree germplasm resources by using gene differential expression of the present invention, in step 3): Collect the second leaf under the dormant bud of each water - supplied tender branch and each non - water - supplied tender branch.

[0027] As a further improvement of the method for predicting the drought resistance of tea germplasm resources by using gene differential expression according to the present invention, in step 3):

[0028] The primers corresponding to the hydroxyphenylacetaldoxime monooxygenase-like gene PMO are:

[0029] PMO upstream primer: atgcgaaagc tctgcgtgat

[0030] PMO downstream primer: agtgatgtgt ctgatcatct tacc;

[0031] The primers corresponding to the actin ACT gene are:

[0032] Act upstream primer: cttcctcatg ctatcctccg tctt

[0033] Act downstream primer: atttcccgtt cagcagtggt g.

[0034] As a further improvement of the method for predicting the drought resistance of tea germplasm resources by using gene differential expression according to the present invention, in step 3):

[0035] The PCR system is: 5.0 μl of SYBR Green Master Mix, 0.4 μl each of the forward and reverse primers (10 μM), 0.8 μl of the first-strand cDNA, and 3.8 μl of ultrapure water;

[0036] The PCR reaction procedure is: pre-denaturation at 95 °C for 2 min, and then 40 cycles of denaturation at 95 °C for 3 s and annealing at 60 °C for 30 s.

[0037] Note: After the reaction, analyze the melting curve and Ct value according to the default program of the instrument.

[0038] As a further improvement of the method for predicting the drought resistance of tea germplasm resources by using gene differential expression according to the present invention, it further includes the following step 4):

[0039] Set the relationship equation DI = 2.3151X 0.5953 , substitute the X value obtained from the tea plant to be tested for the germplasm resource into the above relationship equation, so as to obtain the drought damage index DI corresponding to the tea plant to be tested for the germplasm resource;

[0040] When DI ≤ 10, it is determined that the drought tolerance of the germplasm of the tea plant to be tested is "strong";

[0041] When 10 < DI ≤ 20, it is determined that the drought tolerance of the germplasm of the tea plant to be tested is "relatively strong";

[0042] When 20 < DI ≤ 50, it is determined that the drought tolerance of the germplasm of the tea plant to be tested is "medium";

[0043] When DI > 50, it is determined that the drought tolerance of the germplasm of the tea plant to be tested is "weak".

[0044] The present invention provides a new method for predicting the drought tolerance of different tea germplasm resources by sampling the leaves of in vitro branches after being placed for a period of time under water supply and non - water supply conditions and analyzing the differential expression of the hydroxyphenylacetaldoxime monooxygenase - like gene therein. Specifically, the present invention provides a relationship equation DI = 2.3151X between the relative expression level X of the hydroxyphenylacetaldoxime monooxygenase - like PMO gene in the leaves of in vitro嫩枝 leaves under non - water supply to water supply conditions and the drought injury index DI of tea plants 0.5953 , and predicts the drought tolerance of different tea germplasm resources based on the analysis of the relative expression level of the hydroxyphenylacetaldoxime monooxygenase - like gene in the leaves on in vitro嫩枝.

[0045] In the present invention, primers for quantitative expression analysis of the hydroxyphenylacetaldoxime monooxygenase - like gene PMO (TEA017496.1) (up - and down - stream primers of PMO), and primers for expression analysis of the β - actin gene Act used as a control (up - and down - stream primers of Act) are designed and synthesized, and then quantitative PCR reactions are carried out using the synthesized cDNA as a template; Using 2 -ΔΔct Calculate the relative expression level of the hydroxyphenylacetaldoxime monooxygenase - like gene in the leaves of non - water - supplied嫩枝 leaves and water - supplied嫩枝 leaves, and record it as X.

[0046] According to the relationship equation DI = 2.3151X between the relative expression level X of the hydroxyphenylacetaldoxime monooxygenase - like gene in the leaves under non - water supply to water supply conditions and the drought injury index DI obtained by direct field identification 0.5953 (correlation coefficient r > 0.97), predict and evaluate the drought tolerance of the tea germplasm to be tested: If X ≤ 11.6, then DI ≤ 10, and the drought tolerance of the tea germplasm to be tested is "strong"; If 11.6 < X ≤ 37.4, then 10 < DI ≤ 20, and the drought tolerance of the tea germplasm to be tested is "relatively strong"; If 37.4 < X ≤ 174.4, then 20 < DI ≤ 50, and the drought tolerance of the tea germplasm to be tested is "medium"; If X > 174.4, then DI > 50, and the drought tolerance of the tea germplasm to be tested is "weak".

[0047] The present invention has the following technical advantages compared with the prior art:

[0048] 1. The prediction of the drought tolerance of tea germplasm by the present invention takes a short time and can be completed within 4 days;

[0049] 2. The prediction and evaluation of the drought tolerance of tea germplasm by the present invention are not restricted by external climate conditions and can be carried out during the tea - growing season;

[0050] 3. The method for predicting and evaluating the drought tolerance of tea plants provided by the present invention has a high throughput, and a large number of tea germplasms can be evaluated at one time;

[0051] 4. The method for predicting and evaluating the drought resistance of tea plants provided by the present invention is simple to operate and the evaluation results are reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0053] Figure 1 Schematic diagram of the shoot and leaf positions. DETAILED DESCRIPTION

[0054] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto: Example 1

[0055] (1) In spring, the tender shoots that have just begun to form buds on tea trees such as Anhui No. 1, Longjing Longye, Green Bud Buddha's Hand, Shubeizhong, Zhe Nong 21, and Zhe Nong 113 were harvested. Each tender shoot had 4 complete leaves, and 10 tender shoots were harvested from each tea germplasm resource to be tested. The length of each tender shoot was basically the same, about 10 cm. These tender shoots were used as tender shoots for the tested germplasm resources (tender shoots for buds of the tested tea germplasm resources).

[0056] Each shoot of the germplasm resource to be tested was subjected to the following steps (2) to (4):

[0057] (2) All the young shoots of the germplasm resources to be tested were transferred to a constant temperature and light culture room, and all were immersed in water for 1 day (3-4 cm of the base of the young shoots were submerged in water for adaptive adjustment), and then divided into two groups in equal numbers;

[0058] Five shoots were continued to be inserted in water as water-supply shoots and placed in a constant temperature and light culture room for 26 hours; the other five shoots were removed from the water and placed on ordinary bamboo sieves as non-water-supply shoots and placed in a constant temperature and light culture room for 26 hours;

[0059] The culture room temperature was 25 ± 2°C, the light intensity was 5000 ± 100 Lux, and the relative humidity was 65 ± 5%;

[0060] Water the shoots and ensure that the base of the shoots is in contact with water throughout the entire placement time.

[0061] (3) After the two groups of shoots were placed for 26 h, the second leaf below the bud of the watered and non-watered shoots of each tea plant germplasm was sampled, ground with liquid nitrogen, and total RNA was extracted using a polysaccharide and polyphenol plant RNA extraction kit (Hangzhou Easyside Biotechnology Co., Ltd.). The specific operation was referred to the kit instructions, and the first chain of cDNA was synthesized using a reverse transcription kit (Takara Biotechnology Dalian Co., Ltd.). The specific operation was referred to the kit instructions.

[0062] (4) Based on the p-hydroxyphenylacetaldehyde oxime monooxygenase-like gene PMO (gene ID TEA017496.1) in the first edition of the Shuchazao genome database (http: / / tpia.teaplant.org / index.html), primers for quantitative expression analysis (NO1 in Table 1) and primers for expression analysis of the β-actin gene Act used as a control (NO2 in Table 1) were designed. The above primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the quantitative PCR reaction was performed using the PowerUP SYBR Green Master Mix kit (ABI, USA). Each reaction tube contained SYBR Green Master Mix. 5.0 μl, 0.4 μl of forward and reverse primers (10 μM), 0.8 μl of first-strand cDNA, and 3.8 μl of ultrapure water; the quantitative PCR reaction program was 95°C pre-denaturation for 2 min, followed by 40 cycles of 95°C denaturation for 3 s and 60°C annealing for 30 s. After the reaction, the melting curve and Cт value were analyzed using the default program of the StepOnePlus real-time fluorescence quantitative PCR instrument (ABI, USA); 2 -ΔΔCT The relative expression levels X of the p-hydroxyphenylacetaldehyde oxime monooxygenase-like PMO gene in the leaves of unwatered and watered shoots of Anhui No. 1, Longjing Changye, Green Bud Foshou, Shubeizhong, Zhenong 21, and Zhenong 113 were 9.65, 3.89, 256.00, 12.64, 96.34, and 1.20, respectively, as shown in Table 2.

[0063] Table 1

[0064]

[0065] Table 2

[0066]

[0067]

[0068] Note: ΔCт1 is the CT value of the target gene PMO of the same variety under water supply minus the CT value of the internal reference gene ACT. ΔCт2 is the CT value of the target gene PMO of the same variety under no water supply minus the CT value of the internal reference gene ACT. ΔΔCт is ΔCт2 under no water supply of the same variety minus ΔCт1 under water supply, that is, ΔΔCт = ΔCт2 - ΔCт1; the relative expression level X of the hydroxyphenylacetaldoxime monooxygenase-like gene = 2 -ΔΔCт .

[0069] (5) According to the relationship equation DI = 2.3151X between the relative expression level X of the hydroxyphenylacetaldoxime monooxygenase-like PMO gene in leaves under water supply and drought injury index DI provided by the present invention 0.5953 , it can be calculated that the relative expression levels X of 'Zhenong 113', 'Longjing Changye' and 'Anhui No. 1' are 1.20, 3.89 and 9.65 respectively, all less than 11.6, then DI ≤ 10, and the drought tolerance of these three germplasms is all "strong"; the relative expression level X of 'Yabukita' is 12.64, between 11.6 and 37.4, then 10 < DI ≤ 20, and the drought tolerance of this tea germplasm is "relatively strong"; the relative expression level X of 'Zhenong 21' is 96.34, between 37.4 and 174.4, then 20 < DI ≤ 50, and the drought tolerance of this tea germplasm is "medium"; while the relative expression level X of 'Lvya Foshou' is 256.00 (greater than 174.4), then DI > 50, and the drought tolerance of this tea germplasm is "weak". See Table 3 for details.

[0070] Table 3

[0071]

[0072] Thus, it can be seen that after the spring detached tender shoots are placed at 25 ± 2 °C, 5000 ± 100 Lux, relative humidity 65 ± 5% for 26 h under water supply and no water supply conditions, according to the analysis of the relative expression level X of the hydroxyphenylacetaldoxime monooxygenase-like PMO gene in the second leaf below the dormant bud, and using the relationship equation DI = 2.3151X provided by the present invention 0.5953 The drought tolerance levels of each tea germplasm obtained are consistent with the direct field identification results.

[0073] Example 2

[0074] (1) In summer, harvest the tender shoots that have just formed dormant buds on tea trees such as 'Phoenix Dancong', 'Fuding Dabaicha', 'Lvya Foshou', 'Zhenong 113', 'Zhenong 25', 'Zhenong 901', etc. Each tender shoot has 4 complete leaves, and there are 12 tender shoots for each tea germplasm resource;

[0075] The tender shoots of each germplasm resource are respectively carried out the following steps (2) - (4):

[0076] (2) All the collected shoots of the germplasm resources to be tested were transferred to a constant temperature and light culture room, and all were first immersed in water for 2 days (the base of the shoots was 3-4 cm submerged in water for adaptation), and then divided into two groups in equal numbers;

[0077] Six shoots were continued to be inserted into the water as water-supply shoots and placed in a constant temperature and light culture room for 22 hours; the other six shoots were removed from the water and placed on ordinary bamboo sieves as non-water-supply shoots and placed in a constant temperature and light culture room for 22 hours;

[0078] The culture room temperature was 25 ± 2°C, the light intensity was 5000 ± 100 Lux, and the relative humidity was 65 ± 5%;

[0079] (3) After the two groups of shoots were placed for 22 h, the second leaf below the bud of the watered and non-watered shoots of each tea plant accession was sampled, ground with liquid nitrogen, and total RNA was extracted using a polysaccharide and polyphenol plant RNA extraction kit (Hangzhou Easyside Biotechnology Co., Ltd.). The specific operation was referred to the kit instructions, and the first chain of cDNA was synthesized using a reverse transcription kit (Takara Biotechnology Dalian Co., Ltd.). The specific operation was referred to the kit instructions.

[0080] (4) Using the same primers and quantitative PCR as in Example 1, 2 -ΔΔCT The relative expression levels X of the p-hydroxyphenylacetaldehyde oxime monooxygenase-like PMO gene in the leaves of unwatered and watered shoots of 'Phoenix Narcissus', 'Fuding Dabaicha', 'Green Bud Buddha's Hand', 'Zhe Nong 113', 'Zhe Nong 25' and 'Zhe Nong 901' were 237.21, 34.30, 274.37, 1.36, 40.50 and 27.86, respectively, as shown in Table 4.

[0081] Table 4

[0082]

[0083] (5) According to the present invention, the relative expression level X of the p-hydroxyphenylacetaldehyde oxime monooxygenase-like gene in leaves under the conditions of no water supply and water supply is related to the drought damage index DI: DI = 2.3151X 0.5953, it can be calculated that: the relative expression level X of 'Zhenong 113' is less than 11.6, then DI ≤ 10, and the drought tolerance of this germplasm is all'strong'; the relative expression levels X of 'Zhenong 901' and 'Fuding Dabaicha' are both between 11.6 and 37.4, then 10 < DI ≤ 20, and the drought tolerance of these two tea germplasms is '较强' (relatively strong); the relative expression level X of 'Zhenong 25' is between 37.4 and 174.4, then 20 < DI ≤ 50, and the drought tolerance of this tea germplasm is'medium'; while the relative expression levels X of 'Lvyaboshu' and 'Fenghuang Shuixian' are both greater than 174.4, then DI > 50, and the drought tolerance of these two tea germplasms is 'weak'. See Table 5 for details.

[0084] Table 5

[0085]

[0086] It can be seen that after the summer detached young branches are placed at 25 ± 2 °C, 5000 ± 100 Lux, relative humidity 65 ± 5% for 22 h under water supply and non - water supply conditions, according to the analysis of the relative expression level X of p - hydroxyphenylacetaldoxime monooxygenase - like PMO gene in the second leaf below the resting bud, and using the relational equation DI = 2.3151X provided by the present invention 0.5953 The drought tolerance levels of each tea germplasm obtained are consistent with the results of direct field identification.

[0087] Example 3

[0088] (1) In autumn, harvest the young branches that have just formed resting buds on tea trees such as 'Hanlv', 'Lvyaboshu', 'Pingyun', 'Yingshuang', 'Zhenghe Dabaicha', 'Zhenong 21', etc. Each young branch has 4 complete leaves, and there are 10 young branches for each tea germplasm resource;

[0089] The young branches of each germplasm resource are respectively carried out the following steps (2) - (4):

[0090] (2) Transfer all the collected young branches of the tested germplasm resources to a constant - temperature light culture chamber. First, insert them all into water for 1 day (the 3 - 4 cm at the base of the young branch is submerged in water for adaptive adjustment), and then divide them into two equal groups;

[0091] 5 young branches are continued to be inserted into water as water - supplied young branches and placed in the constant - temperature light culture chamber for 24 h; another 5 young branches are taken out of the water and placed on a common bamboo sieve as non - water - supplied young branches and placed in the constant - temperature light culture chamber for 24 h;

[0092] The temperature of the culture chamber is 25 ± 2 °C, the light intensity is 5000 ± 100 Lux, and the relative humidity is 65 ± 5%;

[0093] (3) After the two groups of tender branches were grouped and placed for 24 h respectively, the second leaves under the stationary buds of the water-supplied and non-water-supplied tender branches of each tea germplasm were sampled. After grinding with liquid nitrogen respectively, total RNA was extracted using a polysaccharide and polyphenol plant RNA extraction kit (Hangzhou Eastep Biotech Co., Ltd.), and the specific operation was referred to the kit instruction manual. Then, the first strand of cDNA was synthesized using a reverse transcription kit (TaKaRa Bio Inc., Dalian), and the specific operation was referred to the kit instruction;

[0094] (4) Using the same primers and quantitative PCR as in Example 1, 2 -ΔΔCT Calculate the relative expression levels X of the p-hydroxyphenylacetaldoxime monooxygenase-like PMO gene in the leaves of non-water-supplied tender branches and water-supplied tender branches of 'Hanlü', 'Lüyab佛手', 'Pingyun', 'Yingshuang', 'Zhenghe Dabaicha' and 'Zhenong 21' to be 21.56, 268.73, 2.08, 6.15, 6.32 and 94.35 respectively, as shown in Table 6 specifically.

[0095] Table 6

[0096]

[0097] (5) According to the relationship equation DI = 2.3151X between the relative expression level X of the p-hydroxyphenylacetaldoxime monooxygenase-like PMO gene in the leaves under non-water-supplied and water-supplied conditions provided by the present invention 0.5953 , it can be calculated that: the relative expression levels X of 'Pingyun', 'Yingshuang' and 'Zhenghe Dabaicha' are all less than 11.6, then DI ≤ 10, and the drought tolerance of these three germplasms is all'strong'; the relative expression level X of 'Hanlü' is between 11.6 and 37.4, then 10 < DI ≤ 20, and the drought tolerance of this tea germplasm is '较强'; the relative expression level X of 'Zhenong 21' is between 37.4 and 174.4, then 20 < DI ≤ 50, and the drought tolerance of this tea germplasm is'medium'; while the relative expression level X of 'Lüyab佛手' is greater than 174.4, then DI > 50, and the drought tolerance of this tea germplasm is 'weak'. See Table 7 specifically.

[0098] Table 7

[0099]

[0100] Thus, it can be seen that for autumn excised tender branches at 25 ± 2 °C, 5000 ± 100 Lux, relative humidity 65 ± 5%, after being placed for 24 h under water-supplied and non-water-supplied conditions, by analyzing the relative expression level X of the p-hydroxyphenylacetaldoxime monooxygenase-like PMO gene in the second leaf under the stationary bud, and using the relationship equation DI = 2.3151X provided by the present invention 0.5953 The drought tolerance levels of each tea germplasm obtained are consistent with the results of direct field identification.

[0101] Comparative Example 1-1: The illumination intensity in step 2) of Example 1 was changed from "5000±100Lux" to "6000±100Lux", and the rest was the same as Example 1.

[0102] Only the five germplasms described in Table 8 were selected for the experiment, and the results were compared with those of Example 1 as shown in Table 8 below.

[0103] Table 8

[0104]

[0105] Table 8 shows that the drought tolerance level of 'Anhui No. 1' is wrong.

[0106] Comparative Example 1-2: The illumination intensity in step 2) of Example 1 was changed from "5000±100 Lux" to "4000±100 Lux", and the rest was the same as Example 1.

[0107] Only the five germplasms described in Table 9 were selected for the experiment, and the results were compared with those of Example 1 as shown in Table 9 below.

[0108] Table 9

[0109]

[0110] Table 9 shows that the drought tolerance level of 'Shubei' is wrong.

[0111] Comparative experiment 1-1, using the CsNAC168 gene described in patent CN113403322B "A tea tree drought response gene CsNAC168 and its encoded protein and application" as a template, expression primers were designed (see NO3 in Table 1), and the corresponding calculation rules were set with reference to the PCR system and program of Example 1 of the present invention. The results were as follows: the drought resistance of all varieties in Table 3 was concluded to be "strong", and the differences in drought resistance between varieties could not be distinguished, which was inconsistent with the "traditional field direct identification results".

[0112] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. A method for predicting drought resistance of tea germplasm resources using gene differential expression, characterized in that The following steps are involved: 1) When the tea trees to be tested form shoots that meet the following conditions, the shoots are harvested; The number of budding shoots is ≥10, and each shoot has 4 complete leaves; The following steps 2) to 3) are performed on the tender branches harvested from each tea tree to be tested for each germplasm resource. 2) In a constant temperature and light culture room: all the tender shoots of each tea tree to be tested were first inserted into water for 1-2 days, and then divided into two groups in equal amounts; One group continues to be inserted into the water to supply water to the tender branches, serving as water supply tender branches; Another group was taken out of the water and used as unwatered shoots; The water-supplied shoots and the non-water-supplied shoots are placed in a constant temperature and light culture room for 22 to 26 hours; The temperature of the culture room is 25±2℃, the light intensity is 5000±100Lux, and the relative humidity is 65±5%; 3) After step 2), leaves of each watered shoot and each unwatered shoot are collected as samples, and each sample is amplified for the Act gene and the PMO gene, so as to obtain the corresponding Cт value, and the difference between the Cт values ​​of the two genes is obtained accordingly; The difference between the Cт values ​​of the two genes corresponding to the water-supplied shoots was calculated as ΔCт1; The difference between the Cт values ​​of the two genes corresponding to the shoots without water supply was calculated as ΔCт2; ΔCт1 is the CT value of the target gene PMO of the same variety under water supply conditions minus the CT value of the internal reference gene ACT, and ΔCт2 is the CT value of the target gene PMO of the same variety under no water supply conditions minus the CT value of the internal reference gene ACT; ∆∆CT=∆Cт2-∆Cт1; Relative expression of p-hydroxyphenylacetaldehyde oxime monooxygenase-like PMO genes X= 2 -∆∆CT ; when X ≤11.6, the drought tolerance of the tested tea germplasm is judged to be "strong"; When 11.6< X ≤37.4, the drought tolerance of the tested tea germplasm is judged to be "relatively strong"; When 37.4< X ≤174.4, the drought tolerance of the tested tea germplasm is judged to be "medium"; when X >174.4, the drought tolerance of the tested tea germplasm is judged to be "weak"; The primers corresponding to the p-hydroxyphenylacetaldehyde oxime monooxygenase-like gene PMO are: PMO upstream primer: atgcgaaagc tctgcgtgat PMO downstream primer: agtgatgtgt ctgatcatct tacc; The primers corresponding to the actin ACT gene are: Act upstream primer: cttcctcatg ctatcctccg tctt Act downstream primer: atttcccgtt cagcagtggt g.

2. The method for predicting drought resistance of tea germplasm resources using gene differential expression according to claim 1, characterized in that In the step 3), the second leaf below the bud of each water-supplied shoot and each non-water-supplied shoot is collected.

3. The method for predicting drought resistance of tea germplasm resources using differential gene expression according to claim 2, characterized in that In the step 3): The PCR system was: SYBR Green Master Mix 5.0 μl, forward and reverse primers 0.4 μl each, cDNA first strand 0.8 μl, and ultrapure water 3.8 μl; The PCR reaction program was pre-denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 3 s and annealing at 60°C for 30 s.

4. The method for predicting drought resistance of tea germplasm resources by utilizing gene differential expression according to any one of claims 1 to 3, characterized in that Also includes the following step 4): Set the relational equation DI = 2.3151 X 0.5953 , the germplasm resources obtained from the tea trees to be tested X Substitute the value into the above equation to obtain the drought damage index corresponding to the tea tree to be tested in the germplasm resource. DI ; when DI ≤10, the drought tolerance of the tested tea germplasm is judged to be "strong"; When 10< DI ≤20, the drought tolerance of the tested tea germplasm is judged to be "relatively strong"; When 20< DI ≤50, the drought tolerance of the tested tea germplasm is judged to be "medium"; when DI >50, the drought tolerance of the tested tea plant germplasm is judged to be "weak".

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