Primer combination for rapid screening of salt-tolerant brassica and application thereof

Abstract

The application discloses a primer combination for rapidly screening salt-tolerant and alkali-tolerant rape and application thereof. With salt-tolerant and alkali-tolerant Brassica napus as material, salt treatment and control material are subjected to transcriptome sequencing at the seedling stage, mRNA sequences corresponding to genes with an expression increase of 3.0 times or more in roots are obtained, the correlation between the expression change of the genes and the salt tolerance of the rape is analyzed, seven genes with an expression increase of 3.0 times or more in all treatment groups and a very significant positive correlation between the expression and the salt tolerance are found, and the genes are determined as salt-tolerant genes. The primers of the seven genes are used to detect the gene expression change of the rape seedlings in MS culture medium containing NaCl, the rape with an expression increase of 3.00 times or more than the control in the seven salt-tolerant genes in roots and a biomass not lower than 95% of the control is high salt-tolerant and alkali-tolerant rape. The detection method has high sensitivity and strong specificity, and can rapidly detect the salt tolerance of the rape.

Description

Technical Field

[0001] This invention relates to the field of biological breeding, specifically to a primer combination for rapid screening of salt-tolerant rapeseed and its application. Background Technology

[0002] Soil salinization has become a key factor restricting the sustainable development of grain and oil production in my country. It is one of the main factors leading to desertification and farmland degradation, causing significant negative impacts on agricultural production and the ecological environment. my country has a long coastline, and the saline-alkali land along the coast is increasing daily. Therefore, the restoration and utilization of saline-alkali land has important economic, ecological, and social benefits. Rapeseed has significant value for oil production, feed, vegetable cultivation, and tourism. As a major source of edible vegetable oil and plant protein, rapeseed occupies an important position in my country's agricultural products and is particularly important for ensuring the security of my country's oilseed supply. Previous studies have shown that some salt-tolerant rapeseed resources exist and can grow well in both coastal high-salinity tidal flats and inland saline-alkali lands in China. Furthermore, salt-tolerant rapeseed combines the "utilization" and "improvement" of saline-alkali land, making it the most advantageous field crop for improving and utilizing saline-alkali land. Rapidly selecting new salt-tolerant rapeseed germplasm, utilizing salt-tolerant rapeseed, and implementing integrated measures such as agro-pastoral integration are economical, effective, and sustainable ways to restore and utilize saline-alkali land. Summary of the Invention

[0003] To address the aforementioned issues, this solution provides a primer combination for rapid screening of salt-tolerant rapeseed and its application. Under saline-alkali conditions, the method utilizes changes in the expression of early salt uptake-related gene mRNAs in the plant to screen for rapeseed with strong salt-alkali tolerance. This method can be used for the identification of rapeseed germplasm resources with salt-alkali tolerance.

[0004] To achieve the above objectives, this invention first provides a primer combination for rapid screening of salt-tolerant rapeseed, characterized in that it includes primer combination U1, primer combination U2, primer combination U3, primer combination U4, primer combination U5, primer combination U6, and primer combination U7.

[0005] The primer combination U1 includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2;

[0006] The primer combination U2 includes an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4;

[0007] The primer combination U3 includes an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6;

[0008] The primer combination U4 includes an upstream primer as shown in SEQ ID NO.7 and a downstream primer as shown in SEQ ID NO.8;

[0009] The primer combination U5 includes an upstream primer as shown in SEQ ID NO.9 and a downstream primer as shown in SEQ ID NO.10;

[0010] The primer combination U6 includes an upstream primer as shown in SEQ ID NO.11 and a downstream primer as shown in SEQ ID NO.12;

[0011] The primer combination U7 includes an upstream primer as shown in SEQ ID NO.13 and a downstream primer as shown in SEQ ID NO.14.

[0012] Based on a general inventive concept, this solution also provides an application of primer combinations in screening salt-tolerant rapeseed, including the following steps:

[0013] S1. Using rapeseed seeds as the material, the surface was sterilized and placed in a greenhouse for inoculation on conventional MS solid medium for culture. A control group and a treatment group were set up, and the culture medium of the treatment group contained NaCl.

[0014] S2. Detect the biomass of the control group and the treatment group, and extract total RNA from roots or leaves. After reverse transcription, quantitative PCR experiments were performed using primer combination U1-U7. Gene expression of primer combination U1-U7 was detected, with Actin as the reference gene.

[0015] If the U1-U7 genes detected in the treatment group are 3.00 times or more higher than those in the control group, and the biomass is greater than or equal to 95% of that in the control group, then the rapeseed is determined to be salt-tolerant rapeseed.

[0016] Preferably, the rapeseed in step S1 is Brassica napus or Brassica oleracea var. chinensis.

[0017] Preferably, the greenhouse conditions in step S1 are: a day-night length ratio of 16 / 8h, a day-night temperature of 22 / 16℃, and a light intensity of 200-250 μmol m². –2 s –1 .

[0018] Preferably, the culture time in step S1 is 10-15 days.

[0019] Preferably, in step S1, the pH of the treatment culture medium is 7.5, and the NaCl concentration is 1.25%-1.5%.

[0020] Preferably, the PCR experimental procedure in step S2 is as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 10 s, 52℃ annealing for 20 s, 72℃ extension for 10 s, cycled 30 times; 72℃ further extension for 2 min.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Based on the salt uptake gene sequence expressed under salt-alkali induction, primers were designed and a reverse transcription-quantitative PCR technology system was established. Based on this, the system was optimized and a method for rapidly selecting salt-tolerant rapeseed plants by detecting changes in gene mRNA under salt-alkali induction was established.

[0023] (2) The detection method provided by this scheme is fast, sensitive and specific, saving identification time and cost, improving the identification efficiency of salt tolerance traits of rapeseed plants, and has great application value. Detailed Implementation

[0024] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0025] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0026] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0027] Experimental Example 1

[0028] Primer design for rapeseed salt-alkali tolerance genes

[0029] S1. Using salt-tolerant rapeseed (Brassica napus) as the material, plump seeds were collected, surface-sterilized with mercuric chloride, and then the surface moisture was blotted dry with sterile absorbent paper before inoculation onto conventional MS solid medium. Control and treatment groups were set up. The treatment group had a salt concentration of 1.25% NaCl and a pH of 7.5. The day-night ratio was 16 / 8 h, the day-night temperature was 22 / 16 °C, and the light intensity was 250 μmol / m². -2 s -1 They are grown in a greenhouse for 10 days.

[0030] S2. Biomass of each group was measured, and total RNA was extracted from the roots of Brassica napus in both the treatment and control groups. Transcriptome deep sequencing was performed using a high-throughput sequencing platform. Reads with an average quality value less than 20 were removed to obtain usable reads. These usable reads were then de novo spliced ​​to assemble a mixed transcriptome library of Brassica napus. Data analysis revealed differentially expressed mRNA sequences. mRNA sequences with an expression level increasing more than 3.00-fold in roots and their corresponding genes were selected. Combined with published homologous gene sequences from Brassica spp., primers were designed in conserved gene regions using Primer Premier 5.0 software. Primers were designed using the Actin gene as an expression reference gene to prepare for the next step of quantitative PCR analysis of the correlation between changes in gene expression levels and salt tolerance.

[0031] S3. Based on the results of quantitative PCR detection of gene mRNA expression levels and salt content determination, compared with the control, seven genes were found whose expression levels increased by more than 3.00 times in the roots of all treatment groups, and the biomass of the treatment groups was more than 95% of that of the control. Therefore, these genes were identified as salt-tolerant genes, and the primer sequences for amplifying these genes are shown in Table 1 below.

[0032] Table 1 Primers for salt tolerance gene and Actin gene

[0033]

[0034]

[0035] Experiment Example 2

[0036] Investigating the feasibility of increasing expression of salt-tolerant genes in the roots of Brassica napus under saline-alkali conditions.

[0037] S1. Select salt-tolerant rapeseed varieties as materials. Take plump seeds, sterilize them with mercuric chloride, and then blot dry the seeds with sterile absorbent paper before inoculating them onto standard MS solid medium. Set up control and treatment groups. The treatment groups had a pH of 7.5, salt concentrations of 0.75, 1.00, 1.25, 1.50, and 1.75% NaCl, a day-night ratio of 16 / 8 h, a day-night temperature of 22 / 16℃, and a light intensity of 250 μmol / m². -2 s -1 They are grown in a greenhouse for 15 days.

[0038] S2. Biomass of the control and experimental groups was measured. Total RNA was extracted from rapeseed roots, reverse transcribed, and then subjected to quantitative PCR. Primer sequences are shown in Table 1. Actin gene was used as a reference gene, and the experiment was repeated three times. Based on the results of quantitative PCR detection of gene mRNA expression levels, the fold increase in expression level is shown in Table 2, and the increase in biomass is shown in Table 3.

[0039] Table 2. Effects of salt-alkali treatment on the expression levels of salt tolerance genes in salt-tolerant rapeseed.

[0040]

[0041] Table 3. Effects of salt-alkali treatment on biomass of salt-tolerant rapeseed.

[0042]

[0043] Compared with the control group, seven genes were identified whose expression levels in roots increased significantly by more than 3.00-fold in all treatment groups. The correlation coefficient between the expression levels of these genes and the salt treatment concentration was above 0.95 and the two showed a significant positive correlation. Therefore, these genes were identified as salt-tolerant genes. Thus, the salt-tolerant ability of rapeseed can be evaluated by whether the expression of these genes is upregulated by more than 3.00-fold and the biomass is not less than 95% of the control.

[0044] Comparative Example 1

[0045] This study investigated the expression of salt-tolerant genes and changes in biomass in salt-alkali intolerant Brassica napus under saline-alkali conditions.

[0046] S1. Select the salt- and alkali-intolerant Brassica napus type as material. Take plump seeds, sterilize them with mercuric chloride, and then blot the surface moisture with sterile absorbent paper before inoculating them onto conventional MS solid medium. Set up control and treatment groups. The treatment group has a pH of 7.5, salt concentrations of 0.75, 1.00, 1.25, 1.50, and 1.75% NaCl, a day-night ratio of 16 / 8h, a day-night temperature of 22 / 16℃, and a light intensity of 250 μmol / m². -2 s -1 They are grown in a greenhouse for 15 days.

[0047] S2. Biomass of the control and experimental groups was measured. Total RNA was extracted from rapeseed roots, reverse transcribed, and then subjected to quantitative PCR. Primer sequences are shown in Table 1. Actin gene was used as a reference gene, and the experiment was repeated three times. Based on the results of quantitative PCR detection of gene mRNA expression levels, the fold increase in expression level is shown in Table 4, and the increase in biomass is shown in Table 5.

[0048] Table 4. Effects of salt-alkali treatment on the expression levels of salt-tolerant genes in salt-intolerant Brassica napus.

[0049]

[0050] Table 5. Effects of salt-alkali treatment on biomass of salt-alkali tolerant rapeseed.

[0051]

[0052] Compared with the control group, the expression levels of the seven genes in the roots of all salt-tolerant rapeseed treatment groups increased by a relatively small fold, less than 3 times. The biomass decreased with increasing treatment concentration, further demonstrating the high feasibility of the primer genes selected in this scheme for screening salt-tolerant rapeseed. If the genes detected by primers U1, U2, U3, U4, U5, U6, and U7 are significantly increased by more than 3.00 times in the roots of the treatment groups compared with the control group, and the biomass is not less than 95% of the control, then the rapeseed material can be determined to have a strong salt-tolerant ability and can be used as a salt-tolerant rapeseed.

[0053] Experimental Example 3

[0054] Investigating the salt and alkali tolerance of rapeseed (Brassica napus) growing normally in coastal tidal flats

[0055] S1. Seeds of Brassica napus, a type of rapeseed that grows normally in coastal tidal flats, were used as the material. After surface sterilization, the seeds were dried with sterile absorbent paper and inoculated onto conventional MS solid medium. A control group and a treatment group were set up. The treatment group had a salt concentration of 1.25% NaCl and a pH of 7.5. The growing conditions included a day-night ratio of 16 / 8 h, a day-night temperature of 22 / 16 °C, and a light intensity of 200 μmol / m². –2 s –1 Cultured for 15 days under the specified conditions.

[0056] S2. Total RNA was extracted from the roots of the above-mentioned Brassica napus, reverse transcribed, and then subjected to quantitative PCR. The Actin gene was used as a reference gene, and the experiment was repeated 3 times, and the average was taken.

[0057] The reaction conditions and result analysis of quantitative PCR amplification were performed according to conventional methods. Gene expression was detected using the U1-U7 primers provided in Experiment 1 using quantitative PCR, and the results are shown in Table 6 below:

[0058] Table 6. Effects of saline-alkali treatment on the expression levels of salt tolerance genes in normally growing Brassica napus in coastal tidal flats.

[0059]

[0060] According to the results in Table 6, when the salt concentration in MS medium was 1.25% NaCl and the pH was 7.5, the expression level of these 7 salt tolerance genes in the roots of the selected Brassica napus increased by more than 3.00 times compared with the control, and the biomass was 103.7% of the control, which meets the standard of salt-tolerant plants.

[0061] Experiment Example 4

[0062] Investigating the salt and alkali tolerance of Brassica napus grown normally in the saline-alkali lands of Northwest China

[0063] S1. Seeds of Brassica napus, a type of rapeseed that grows normally in the saline-alkali land of Northwest China, were used as the material. After sterilization of the seed surface, the moisture on the seed surface was absorbed with sterile absorbent paper, and the seeds were inoculated onto conventional MS solid medium. A control group and a treatment group were set up. The treatment group had a salt concentration of 1.25% NaCl and a pH of 7.5. The day-night ratio was 16 / 8 h, the day-night temperature was 22 / 16℃, and the light intensity was 200 μmol / m². –2 s –1 Cultivate for 15 days under the specified conditions;

[0064] S2. Total RNA was extracted from the leaves of the above-mentioned Brassica napus, reverse transcribed, and then subjected to quantitative PCR. The Actin gene was used as a reference gene, and the experiment was repeated 3 times, and the average was taken.

[0065] The reaction conditions and result analysis of quantitative PCR amplification were performed according to conventional methods. Gene expression was detected using the U1-U7 primers provided in Experiment 1 using quantitative PCR, and the results are shown in Table 7 below:

[0066] Table 7. Effects of saline-alkali treatment on the expression levels of salt tolerance genes in normally growing Brassica napus in the saline-alkali lands of Northwest China.

[0067]

[0068] According to the results in Table 7, when the salt concentration in MS medium was 1.25% NaCl and the pH was 7.5, the expression levels of these 7 salt tolerance genes in the selected rapeseed leaves increased by more than 3.00 times compared with the control, and the biomass was 100.3% of the control, which meets the standard for salt-tolerant plants.

[0069] Experimental Example 5

[0070] Investigating the salt and alkali tolerance of mustard-type rapeseed that grows normally in coastal mudflats

[0071] S1. Seeds of mustard-type rapeseed, normally grown in coastal tidal flats, were used as the material. After surface sterilization, the seeds were dried with sterile absorbent paper and inoculated onto conventional MS solid medium. A control group and a treatment group were set up. The treatment group had a salt concentration of 1.50% NaCl and a pH of 7.5. The growing conditions included a day-night ratio of 16 / 8 h, a day-night temperature of 22 / 16 °C, and a light intensity of 200 μmol / m². –2 s –1 Cultivate for 15 days under the specified conditions;

[0072] S2. Total RNA was extracted from the leaves of the above-mentioned mustard-type rapeseed, reverse transcribed, and then subjected to quantitative PCR. The Actin gene was used as a reference gene, and the experiment was repeated 3 times, and the average was taken.

[0073] The reaction conditions and result analysis for quantitative PCR amplification were performed according to standard methods. Gene expression was detected using the U1-U7 primers provided in Experiment 1 using quantitative PCR, and the results are shown in Table 8 below:

[0074] Table 8. Effects of saline-alkali treatment on the expression levels of salt tolerance genes in normally growing mustard-type rapeseed in coastal tidal flats.

[0075]

[0076] According to the results in Table 8, when the salt concentration in MS medium was 1.50% NaCl and the pH was 7.5, the expression level of these 7 salt tolerance genes in the leaves of the selected mustard-type rapeseed increased by more than 3.00 times compared with the control, and the biomass was 99.1% of the control, which meets the standard of salt-tolerant plants.

[0077] Experimental Example 6

[0078] Investigating the salt and alkali tolerance of mustard-type rapeseed that grows normally in saline-alkali land in Northwest China.

[0079] S1. Using seeds of mustard-type rapeseed that grows normally in the saline-alkali land of Northwest China as material, after surface sterilization, the seeds were dried with sterile absorbent paper and inoculated onto conventional MS solid medium. A control group and a treatment group were set up. The treatment group had a salt concentration of 1.25% NaCl and a pH of 7.5. The day-night ratio was 16 / 8 h, the day-night temperature was 22 / 16℃, and the light intensity was 200 μmol / m². –2 s –1 Cultivate for 15 days under the specified conditions;

[0080] S2. Total RNA was extracted from the roots of the above-mentioned mustard-type rapeseed, reverse transcribed, and then subjected to quantitative PCR. The Actin gene was used as a reference gene, and the experiment was repeated 3 times, and the average was taken.

[0081] The reaction conditions and result analysis for quantitative PCR amplification were performed according to standard methods. Gene expression was detected using the U1-U7 primers provided in Experiment 1 using quantitative PCR, and the results are shown in Table 9 below:

[0082] Table 9. Effects of saline-alkali treatment on the expression levels of salt tolerance genes in normally growing mustard-type rapeseed in saline-alkali land in Northwest China.

[0083]

[0084] According to the results in Table 9, when the salt concentration in MS medium was 1.50% NaCl and the pH was 7.5, the expression level of these 7 salt tolerance genes in the roots of the selected mustard-type rapeseed increased by more than 3.00 times compared with the control, and the biomass was 107.2% of the control, which meets the standard of salt-tolerant plants.

Claims

1. A primer combination for rapid screening of salt-tolerant rapeseed, characterized in that, Including primer combination U1, primer combination U2, primer combination U3, primer combination U4, primer combination U5, primer combination U6 and primer combination U7; The primer combination U1 includes an upstream primer as shown in SEQ ID NO.1 and a downstream primer as shown in SEQ ID NO.2; The primer combination U2 includes an upstream primer as shown in SEQ ID NO.3 and a downstream primer as shown in SEQ ID NO.4; The primer combination U3 includes an upstream primer as shown in SEQ ID NO.5 and a downstream primer as shown in SEQ ID NO.6; The primer combination U4 includes an upstream primer as shown in SEQ ID NO.7 and a downstream primer as shown in SEQ ID NO.8; The primer combination U5 includes an upstream primer as shown in SEQ ID NO.9 and a downstream primer as shown in SEQ ID NO.10; The primer combination U6 includes an upstream primer as shown in SEQ ID NO.11 and a downstream primer as shown in SEQ ID NO.12; The primer combination U7 includes an upstream primer as shown in SEQ ID NO.13 and a downstream primer as shown in SEQ ID NO.

14.

2. The application of the primer combination as described in claim 1 in screening salt-tolerant rapeseed, characterized in that, Includes the following steps: S1. Using rapeseed seeds as the material, the surface was sterilized and placed in a greenhouse for inoculation on conventional MS solid medium for culture. A control group and a treatment group were set up, and the culture medium of the treatment group contained NaCl. S2. Biomass of the control and treatment groups was detected, and total RNA was extracted from roots or leaves. After reverse transcription, quantitative PCR was performed using primer combination U1-U7 to detect gene expression of primer combination U1-U7, with Actin as the reference gene.

3. The application according to claim 2, characterized in that, In step S1, the rapeseed is either Brassica napus or Brassica oleracea var. chinensis.

4. The application according to claim 2, characterized in that, The greenhouse conditions in step S1 are: a day-night length ratio of 16 / 8h, a day-night temperature of 22 / 16℃, and a light intensity of 200-250 μmol / m². –2 s –1 .

5. The application according to claim 2, characterized in that, The culture time in step S1 is 10-15 days.

6. The application according to claim 2, characterized in that, In step S1, the pH of the treatment group culture medium is 7.5, and the NaCl concentration is 1.25%-1.5%.

7. The application according to claim 2, characterized in that, The PCR experimental procedure in step S2 is as follows: pre-denaturation at 94℃ for 3 min; denaturation at 94℃ for 10 s, annealing at 52℃ for 20 s, extension at 72℃ for 10 s, for 30 cycles; and further extension at 72℃ for 2 min.

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