A gene related to stress resistance of Wucai and its application

Abstract

The invention discloses a gene related to stress resistance of wucai and an application thereof, and relates to the technical field of genetic breeding, wherein the stress is specifically high temperature, salt and drought, the gene is specifically a bHLH57 gene, and its nucleotide sequence is shown in SEQ ID NO.1. When screening relevant stress-resistant varieties, it is only necessary to detect the expression amount of the bHLH57 gene when the screened varieties have 6 true leaves, wherein the higher the expression amount of the bHLH57 gene, the stronger the stress resistance of the variety; the invention can provide a guiding direction for cultivating transgenic wucai varieties with stress resistance.

Description

Technical Field

[0001] The invention relates to the technical field of genetic breeding, and in particular to a technology related to the stress resistance of wucai. Background Art

[0002] Wucai, also known as collapsed vegetable, collapsed vegetable, collapsed ground vegetable, black vegetable, etc., is a variant of the cabbage subspecies of the Brassica species of the genus Brassica in the family Cruciferae. It is a biennial herb of the family Cruciferae. Adversities such as high temperature, drought, and osmotic stress will cause the economic yield and quality of Wucai to decline, affecting the growth and development of the plant.

[0003] The identification of the stress resistance of Wucai can effectively determine the corresponding stress resistance, thereby facilitating protective treatment of the plants in advance to avoid economic losses; however, the traditional identification method has a long cycle, and the test results are greatly affected by the natural environment, which is not conducive to the screening of high-quality germplasm resources.

[0004] Therefore, how to quickly identify the stress resistance of Wucai in order to screen high-quality germplasm resources is an urgent problem to be solved. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a gene related to the stress resistance of Wucai and its application, so as to solve the technical problem of slow screening of high-quality seed resources in the prior art.

[0006] The present invention is achieved through the following technical solutions:

[0007] A gene related to stress resistance of Wucai, the gene is named bHLH57 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the stress resistance is specifically any combination of high temperature, high salt and drought.

[0008] The present invention also provides a method for identifying the stress resistance of wucai using the above-mentioned gene. The method comprises culturing the germplasm to be compared for stress resistance to 6 true leaves under the same conditions, measuring the expression level of the bHLH57 gene at the same position and sorting the germplasm. The higher the expression level of the bHLH57 gene, the stronger the stress resistance. The stress resistance is specifically any combination of high temperature, high salt and drought.

[0009] The present invention also provides an application of the bHLH57 gene in cultivating a transgenic wucai variety with stress resistance, wherein the stress resistance is any combination of high temperature, high salt and drought.

[0010] Compared with the prior art, the present invention has the following advantages:

[0011] By measuring the expression level of the bHLH57 gene in plant seedlings, the stress resistance of multiple varieties can be effectively judged, which improves the screening speed and can effectively provide guidance for the cultivation of transgenic varieties of Wucai with stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a comparison chart of the bHLH57 gene expression levels of different Wucai varieties at the six-leaf stage under normal conditions;

[0013] Figure 2 This is a comparison chart of the bHLH57 gene expression levels of different Wucai varieties under different conditions;

[0014] Figure 3 This is a comparison of the relative expression levels of BcbHLH57 in different transgenic Arabidopsis plants;

[0015] Figure 4 It is a comparison chart of the germination of each strain under different stresses (scale bar = 1 cm);

[0016] Figure 5 This is a comparison chart of the germination rates of each strain under different stresses;

[0017] Figure 6 It is a comparison chart of the growth of each strain under high temperature, salt and drought stress (scale bar = 1 cm). DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Example 1

[0020] Obtaining bHLH57 gene from Wucai

[0021] Specifically:

[0022] 1. Extraction of total RNA from Wucai genome and cDNA synthesis (Four Wucai varieties were selected as materials in this experiment, namely W19-6, W19-11, W20-4 and W22-3)

[0023] The total RNA of the Wucai genome was extracted according to the instructions of the Takara RNA extraction kit. After the obtained RNA was quantified, the cDNA was synthesized according to the system in Table 1, and the specific steps were carried out according to the Takara reverse transcription instructions.

[0024]

[0025] Table 1

[0026] 2. Acquisition and recovery and purification of bHLH57 gene from Wucai

[0027] The cDNA template was obtained by reverse transcription of RNA of Wucai, and the bHLH57 gene was cloned using upstream primer F: SEQ ID NO 4 and downstream primer R: SEQ ID NO 5 (the primers were sent to Sangon Biotech Co., Ltd. for synthesis). Samples were added according to the system in Table 2 (each cycle extension time was 1 min, the annealing temperature was 56°C, and 34 cycles), and PCR products were synthesized. The obtained products were detected by 1.5% agarose electrophoresis. After the internal reference test was qualified, the obtained fragments of the expected size were cut from the agarose gel, and the target DNA fragments were recovered according to the instructions of AxyPrepTMDNA Gel Extraction Kit.

[0028]

[0029]

[0030] Table 2

[0031] 3. Construction of cloning vector and transformation of E. coli

[0032] The recovered target DNA fragment and pMD TM The 19-T vector was connected at a volume ratio of 1:4 (Table 3) and placed in a 4°C refrigerator overnight. The next day, 5 μL of the connection product was taken and added to 50 μL of thawed E. coli competent cells. After standing on ice for 30 minutes, it was heat-shocked at 40°C for 45 seconds, and then stood on ice for 3 minutes. After adding 1 mL of LB liquid culture medium, it was cultured in a shaking incubator at 37°C and 220 rpm for 1 hour. After the culture was completed, centrifuged at 4000 rpm for 3 minutes, most of the supernatant was discarded, and the remaining 150 μL was gently blown with a pipette and evenly spread on a plate containing 100 mg·L -1 Amp on LB plates. Culture at 37°C overnight, and pick 3-5 positive colonies the next day to plate on LB plates containing 100 mg·L -1 Amp were cultured in 4 mL LB centrifuge tubes at 37°C overnight.

[0033]

[0034] Table 3

[0035] 4. Sequencing and Bioinformatics Analysis

[0036] Take the bacterial solution obtained in the previous step, use the upstream primer F: SEQ ID NO 4 and the downstream primer R: SEQ ID NO 5 to amplify the bHLH57 gene according to the system in Table 4 (each cycle extension time is 1 min, the annealing temperature is 56°C, and 34 cycles). After the product is qualified by 1.5% agarose electrophoresis, take 1 mL of the bacterial solution and send it to Shanghai BGI for sequencing to obtain the bHLH57 gene with the nucleotide sequence shown in SEQID NO.1. Take another 500 μL, add 500 μL of 40% glycerol and store it in a -20°C refrigerator.

[0037]

[0038] Table 4

[0039] Example 2

[0040] Study on the Expression Pattern of bHLH57 Gene in Wucai

[0041] 1. Determination of bHLH57 gene expression under normal conditions

[0042] Four varieties of Wucai were selected as materials in this experiment, namely W19-6, W19-11, W20-4 and W22-3. The four varieties of Wucai were placed in a room with a daytime temperature of 25±1℃, a night temperature of 20±1℃, a photoperiod of 14h / 10h (light / darkness), and a light intensity of 300μmol·m -2 ·s -1 , in an artificial climate room with a relative humidity of 70%. When the seedlings grew to 6 true leaves, the bHLH57 gene expression level was determined at the center of the third leaf, avoiding the leaf veins. The specific determination of the bHLH57 gene expression level was as follows:

[0043] According to the nucleotide sequence of the bHLH57 gene shown in SEQ ID NO 1, fluorescent primers (F: SEQ ID NO 2; R: SEQ ID NO 3) for detecting the expression of the bHLH57 gene were designed using Oligo 7.0 software, and the housekeeping gene Actin was used as a reference. The primer sequence was sent to Sangon Biotech Co., Ltd. for synthesis. After semi-quantitative PCR detection, the primers had no dimers and non-specific amplification. NTC was set to detect whether the operation and primers met the requirements. After both were qualified, the samples were added on ice, and each sample was repeated 3 times and three biological replicates were performed. After the sample cover was closed, it was slightly centrifuged and the gene expression was detected in a CFX 96PCR instrument.

[0044] The test results are as follows Figure 1 As shown, the expression levels of bHLH57 gene in different Wucai varieties at the six-leaf stage were significantly different. W19-6 had the lowest expression level, and the expression levels of bHLH57 gene in W20-4, W19-11, and W22-3 were approximately 2, 3, and 4 times that of W19-6, respectively.

[0045] 2. Analysis of the expression pattern of bHLH57 gene in Wucai under different stress conditions

[0046] When the seedlings of the four varieties grew to 6 true leaves, Wucai with the same size and growth were selected and subjected to different stress treatments, including:

[0047] High temperature stress: The conditions of the high temperature artificial climate chamber were set as follows: 40±1℃ / 30±1℃ (day / night), photoperiod of 14h / 10h (day / night), light intensity of 300μmol·m-2s-1, and relative humidity of 70%; the conditions of the control artificial climate chamber were set as follows: 25±1℃ / 20±1℃ (day / night), photoperiod of 14h / 10h (day / night), light intensity of 300μmol·m-2s-1, and relative humidity of 70%. -2 ·s -1 , relative humidity is 70%.

[0048] Salt stress: The artificial climate chamber conditions were set at 25 ± 1 °C / 20 ± 1 °C (day / night), the photoperiod was 14 h / 10 h (day / night), and the light intensity was 300 μmol·m -2 ·s -1 , relative humidity was 70%. The salt stress treatment group was irrigated with 150 mM NaCl, and the control group was irrigated with the same amount of water.

[0049] Drought stress: The artificial climate chamber was set at 25±1℃ / 20±1℃ (day / night), the photoperiod was 14h / 10h (day / night), and the light intensity was 300μmol·m -2 ·s -1 , relative humidity was 70%. The drought stress treatment group was irrigated with 250mmol·L-1 mannitol, and the control group was irrigated with the same amount of water.

[0050] After 3 days of treatment under the respective control, high temperature, salt and drought stress conditions, the seedlings were sampled (the sampling position was the center of the third leaf, avoiding the veins, three biological replicates), and frozen for subsequent experiments.

[0051] The obtained samples were respectively measured for bHLH57 gene expression, superoxide anion, hydrogen peroxide and relative conductivity.

[0052] The measured results are as follows:

[0053] Effects of different stress treatments on O2 .- The influence of content

[0054]

[0055] Table 5

[0056] Effects of different stress treatments on H2O2 content in different varieties of Wucai

[0057]

[0058] Table 6

[0059] Effects of different stress treatments on relative electrical conductivity of different varieties of Wucai

[0060]

[0061] Table 7

[0062] The results of the detection of bHLH57 gene expression under control, high temperature, salt and drought stress are as follows Figure 2 shown.

[0063] From the above, it can be seen that under normal conditions, compared with W19-6, the O2.- content of W19-11, W20-4 and W22-3 decreased by 36.64%, 17.58% and 42.75%, respectively; which is opposite to the expression trend of bHLH57 gene in each variety. The O2.- content under high temperature, salt stress and drought stress increased by 48.09%, 104.58% and 11.45% compared with normal conditions, and the change trend of O2.- content in each variety was consistent with that under normal conditions, which was W19-6>W20-4>W19-11>W22-3 (Table 5). Under normal conditions, compared with W19-6, the H2O2 content of W19-11, W20-4 and W22-3 decreased by 34.94%, 31.50% and 43.90%, respectively; which is opposite to the expression trend of bHLH57 gene in each variety. The H2O2 content under high temperature, salt stress and drought stress increased by 713.37%, 276.74% and 490.70% compared with that under normal conditions, and the change trend of H2O2 content in each variety was consistent with that under normal conditions, which was W19-6>W20-4>W19-11>W22-3 (Table 6). Under normal conditions, compared with W19-6, the relative conductivity of W19-11, W20-4 and W22-3 decreased by 29.10%, 20.68% and 36.53%, respectively; which was opposite to the expression trend of bHLH57 gene in each variety. The relative conductivity under high temperature, salt stress and drought stress increased by 57.27%, 65.80% and 29.39% compared with that under normal conditions, and the change trend of relative conductivity of each variety was consistent with that under normal conditions, which was W19-6>W20-4>W19-11>W22-3 (Table 7).

[0064] Figure 2 It can be seen that the expression level of bHLH57 gene is also significantly different after the plants experience stress. Figure 1 , 2, Tables 5, 6, and 7, we can see that the higher the expression level of the bHLH57 gene, the lower the content of superoxide anion, hydrogen peroxide, and relative conductivity of the plant, indicating that plants with high expression levels of the bHLH57 gene can better cope with the damage caused by stress.

[0065] Example 3

[0066] The PCR gel recovery product of the bacterial solution in Example 1 was double-digested with the 1305 vector plasmid (Table 8, gently pipetting and mixing, and heated in a 37°C water bath for 2 hours), and homologous recombination and ligation were performed to transform Escherichia coli (Table 9) after gel recovery. After reacting at 50°C for 20 minutes, the centrifuge tube was immediately placed on ice for 2 minutes.

[0067]

[0068] Table 8

[0069]

[0070]

[0071] Table 9

[0072] Add 4μl of reaction solution to competent cells, incubate on ice for 30min, heat shock at 42℃ for 90s, quickly ice bath for 5min, add 500μl SOC liquid medium, incubate at 37℃, 180rpm for 45-60min. Collect the bacteria after centrifugation at 4000rpm for 3min, and evenly spread the bacteria on the antibiotic kana plate. After overnight culture in a 37℃ incubator, pick the bacteria, shake the bacteria for sequencing and store at -80℃. Take 5ul of plasmid product and add 100ul of Agrobacterium competent cells, mix well by blowing, stand on ice for 5min, freeze in liquid nitrogen for 5min, heat bath at 37℃ for 5min, and stand on ice for 5min. Add 1ml of liquid LB to the reaction solution, incubate at 28℃, 220rpm for 3h, and centrifuge at 5000rpm at 4℃ for 3min. Spread the plate containing Kan+rif (200ml LB+200ul Kan+400ul rif), seal the plate with sealing film, invert and culture at 28 degrees Celsius for 2 days, pick bacteria, shake bacteria, wash bacteria, blow and evenly mix the plaque with buffer (200ml 1 / 2MS+10g 5% sucrose+50ul Silwet-77, PH=5.8), centrifuge again, and repeat adding 20ml (buffer) to blow and evenly mix to obtain a milky white liquid, and measure OD560=0.8-1 (calibrate to zero with buffer).

[0073] The Arabidopsis inflorescence was soaked in buffer for 2 minutes and then washed with clean water. After keeping moist and dark for 24 hours, it was cultured normally. The first batch of seeds were collected at 30 days, and the second batch of seeds were collected at 40 days. They were dried in the sun for 7 to 10 days. After the seeds were disinfected and washed, they were sown on kanamycin and rifampicin selective medium for screening. After seven days, the Arabidopsis seedlings with longer roots were transplanted into nutrient pots (the conditions of the artificial climate chamber were set at 25±1℃ / 20±1℃ (day / night), the photoperiod was 14h / 10h (day / night), the light intensity was 300μmol·m-2·s-1, and the relative humidity was 70%). After the plants grew up, each Arabidopsis was sampled separately and bHLH57 fluorescence expression was verified to be a positive plant. The seeds of the positive plants were collected for single plant line propagation. After screening generation by generation, three T3 transgenic lines with relatively high expression levels (OE#4, OE#15 and OE#23) were finally obtained. Figure 3 The results of real-time quantitative PCR showed that the relative expression levels of OE#4, OE#15 and OE#23 were higher than those of the wild type (WT). These results indicate that the genetic transformation of transgenic Arabidopsis thaliana was successfully carried out, and the three transgenic lines obtained can be used for subsequent experiments. The seeds of the three T3 generation lines of transgenic Arabidopsis thaliana and the wild type were subjected to high temperature (40°C), osmotic (250mmol·L-1D-mannitol) and salt (150mmol·L-1NaCl) stress treatments, and their germination was observed, and the germination rate within 7 days ( Figure 4 ,5). The germination rates in the high temperature stress and salt stress treatment groups were in the following order: OE#4>OE#15>OE#23>WT; while in the osmotic stress treatment group, the germination rates were in the following order: OE#4>OE#23>OE#15>WT.

[0074] The seedlings of wild-type Arabidopsis and three overexpressing transgenic lines were cultured in 1 / 2MS medium for 5 days and then transplanted into nutrient pots. At 3 weeks of age, the wild-type and transgenic lines were subjected to high temperature and salt stress. At this time, the survival rate of transgenic seedlings was significantly higher than that of wild-type ( Figure 6 ),in:

[0075] High temperature stress: The conditions of the high temperature artificial climate chamber were set as follows: 40±1℃ / 30±1℃ (day / night), photoperiod of 14h / 10h (day / night), light intensity of 300μmol·m-2s-1, and relative humidity of 70%; the conditions of the control artificial climate chamber were set as follows: 25±1℃ / 20±1℃ (day / night), photoperiod of 14h / 10h (day / night), light intensity of 300μmol·m-2s-1, and relative humidity of 70%. -2 ·s -1 , relative humidity is 70%.

[0076] Salt stress: The artificial climate chamber conditions were set at 25 ± 1 °C / 20 ± 1 °C (day / night), the photoperiod was 14 h / 10 h (day / night), and the light intensity was 300 μmol·m -2 ·s -1 , relative humidity was 70%. The salt stress treatment group was irrigated with 150 mM NaCl, and the control group was irrigated with the same amount of water.

[0077] The seedlings of wild-type Arabidopsis and three overexpressing transgenic lines were cultured in 1 / 2MS medium for 5 days and then transplanted into nutrient pots. At 3 weeks of age, the wild-type and transgenic lines were subjected to a 14-day drought treatment (i.e., no watering for 14 days), and then re-watered for 3 days to observe the survival of each line ( Figure 6 , Table 10). When the wild-type and transgenic Arabidopsis seedlings were treated with drought stress for 7 days, it was found that the wild-type plants had obvious leaf curling, plant drying and wilting, that is, there was a more significant dehydration phenomenon, and even a small number of plants died; while the transgenic plants had no obvious or slight dehydration, which was similar to the control group. When the drought stress was treated for 14 days, it was found that the wild-type plants had very significant leaf curling, plant drying and wilting, and a large number of plants died; while the transgenic plants also had obvious dehydration, and even a small number of plants died, but their survival rates were higher than those of the wild type. After 3 days of rehydration, the leaves of the transgenic plants turned green significantly, and the survival rate was significantly higher than that of the wild type. The OE#4 and OE#23 transgenic plants had a survival rate of more than 80% after 3 days of rehydration, while the wild-type plants had a survival rate of less than 10%, which was significantly lower than that of the transgenic plants.

[0078]

[0079] Table 10

[0080] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for identifying the stress resistance of Wucai, characterized in that: The method comprises culturing the Wucai germplasm that needs to be compared for stress resistance to 6 true leaves under the same conditions, measuring and sorting the expression amount of the bHLH57 gene at the same position, and the higher the expression amount of the bHLH57 gene, the stronger its stress resistance, and the stress resistance is specifically any combination of high temperature, high salt and drought. The nucleotide sequence of the bHLH57 gene is shown in SEQ ID NO.

1.

2. Application of the bHLH57 gene in breeding transgenic wucai varieties with stress resistance, wherein the stress resistance is any combination of high temperature, high salt and drought, and the application is overexpression of the bHLH57 gene, and the nucleotide sequence of the bHLH57 gene is shown in SEQ ID NO.1.

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