CN110922460B - Heat shock protein gene CaHSP90-1 and method for assisting in breeding heat-resistant pepper varieties by using same - Google Patents
Heat shock protein gene CaHSP90-1 and method for assisting in breeding heat-resistant pepper varieties by using same Download PDFInfo
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Abstract
The invention discloses a hot shock protein gene of hot pepperCaHSP90‑1Provides a heat shock protein-based geneCaHSP90‑1Method for assisting in breeding heat-resistant pepper varieties, in which heat shock protein genes are selectedCaHSP90‑1Designing primers for the non-conservative region, checking the relative expression quantity, screening in breeding groups, and selecting varieties with heat-resistant properties. The invention establishes heat shock protein geneCaHSP90‑1The relation between the relative expression quantity and the hot pepper heat-resistant character, the heat-resistant condition of hot pepper filial generation individuals is rapidly and accurately detected by utilizing the real-time fluorescence quantitative PCR technology, so that individuals with excellent characters are screened for further breeding.
Description
Technical Field
The invention relates to a hot pepper variety breeding method, in particular to a hot shock protein gene based hot shock proteinCaHSP90-1A method for assisting in breeding heat-resistant pepper varieties.
Background
Chili (Chili)Capsicum annuum LSolanaceae vegetable crops, native to tropical regions in China, south China and America, are the first major vegetable crops in China, and have the growth area and yield of the first vegetable crops, and moreover, hot peppers are favored by people all over the world as cooking seasonings. The pepper has wide application value, and can be used as medicaments, natural dyes and cosmetics, ornamental plants or as an effective component of an insect repellent; the pepper can provide various vitamins, minerals and nutrients necessary for human health according to statistics, the pepper is the first major vegetable crop in China, the planting area and the yield are the first of the vegetable crops, the planting area is about 2000 ten thousand mu, the total yield is 2800 ten thousand tons, and the yield is more than 1000 million yuan.
Although the pepper originates from tropical areas of China and south America, most cultivated species have the characteristic of being fond of temperature and heat intolerance, the optimum growth temperature of the pepper is 20-30 ℃, and the pepper can be subjected to heat stress when the temperature is higher than 35 ℃. After hot pepper is stressed by heat, phenomena of poor pollination and fertilization, flower and fruit drop, and serious decline of fruit setting rate, yield and quality can occur, and the development of the hot pepper industry is seriously influenced. With the rapid development of modern industry, the emission of large amount of greenhouse gases inevitably leads to global temperature rise, and the production of capsicum faces a serious challenge of heat stress. In extremely hot summer, the dry hot air in northwest and north China and the hot weather temperature in south China usually reach over 40 ℃, which leads to the dysplasia of pepper reproductive organs, the shortening of the growth period and the reduction of the yield and the quality.
Therefore, a new heat-resistant variety needs to be selected and bred in the production process of the pepper so as to improve the yield and the quality of the pepper. In the existing hot pepper variety breeding process, the heat resistance of the identified and screened filial generation mainly depends on the phenotype, but the method for breeding the heat-resistant variety depending on the phenotype generally requires at least 5 years from parent screening to later-stage variety breeding, has overlong breeding cycle and low breeding efficiency, and needs to apply a rapid and efficient breeding method. The hot pepper heat-resistant character mark is established on the gene level, auxiliary breeding is carried out, hot peppers with strong heat resistance are quickly and accurately screened out, the breeding goal and the breeding efficiency are greatly improved, and the hot pepper heat-resistant character mark has important significance for long-term development of the hot pepper industry.
Heat shock proteins are a class of proteins produced by cells in a stress response, where 90kd of the protein is rapidly produced when plants are subjected to stress, assisting the protein folding to protect it from damage. The function of the protein is more researched in animals, and research shows that the protein is widely involved in stress regulation, and the protein is less researched in plants, particularly solanaceae crops.
Disclosure of Invention
In view of the above problems, the technical problem to be solved by the present invention is to provide a heat shock protein (heat shock protein) -based geneCaHSP90-1The method for auxiliary breeding heat-resisting hot pepper variety by establishing heat shock protein geneCaHSP90-1The connection between the copy number and the heat-resistant character of the pepper realizes the purpose of gene-assisted breeding, and can greatly improve the goal and efficiency of breeding.
The present invention provides a heat shock protein geneCaHSP90-1The nucleotide sequence of the gene is shown as SEQ ID NO. 7.
Meanwhile, the invention provides a heat shock protein-based geneCaHSP90-1The method for assisting in breeding the heat-resistant pepper variety realizes efficient breeding of the heat-resistant pepper variety, and comprises the following steps:
(1) with reference to HMM model (hidden Markov model) of HSP90 and ID of PF00183, HSP90 family genes were selected from pepper genome by hmmsearch tool and named as HSP90 family genesCaHSP90-1、CaHSP90-2、 CaHSP90-3、CaHSP90-4、 CaHSP90-5、 CaHSP90-6、 CaHSP90-7；
(2) Determining the expression pattern of HSP90 family gene under high temperature stressCaHSP90-1The gene is the most obvious gene responding to high temperature stress;
(3) cloningCaHSP90-1A gene;
(4) designing gene in non-highly conserved segment of HSP90 family geneDue to the fact thatCaHSP90-1Primers for fluorescent quantitative PCR;
(5) with hot pepperβ-actinThe gene is internal reference, and qPCR primers of the gene are designed;
(6) extracting pepper total RNA for qPCR analysis, and detecting the relative expression quantity of genes;
(7) performing qPCR detection on known heat-resistant pepper varieties and control varieties by using the method, setting three groups of repeats, and respectively calculating the heat-resistant pepper varieties and the control pepper varietiesCaHSP90-1Relative expression amount of the gene, thereby establishingCaHSP90-1Correlation between relative expression level and heat-resistant character;
(8) and (3) screening in a breeding population by using the same pair of fluorescent quantitative primers, detecting the copy number of an amplified product, and selecting individuals with heat-resistant properties.
The HMM model is utilized to pass through the hmmsearch tool (E)<1 e-5), whose conserved domain was predicted by the pfamscan tool, 7 HSP90 genes without complete domain were deleted and finally determinedHSP90Genes, and names them according to gene family naming rules.
The expression pattern of the HSP90 family genes was based on published expression profiling data (pepperhub).
In the design of the non-conservative section primer, because the highly conserved homology of the HSP90 gene family reaches more than 70 percent, a specific primer needs to be designed in the non-conservative section for qPCR according to a sequence comparison result.
The primer is a specific primer designed based on a non-conservative region of a gene. Heat shock protein genesCaHSP90- 1The sequence of the forward primer of qPCR primer of (1) is shown in SEQ ID NO.3, namely: 5 '-CCTTTGCTTTCCAGGCTGAG'; the reverse primer sequence is shown as SEQ ID NO.4, namely: 5'-CGAATGAAGAGTTCCGGCTG-3' are provided. Internal reference geneβ-actinThe forward primer sequence of the qPCR primer of (1) is shown as SEQ ID NO. 5, namely: 5'-CCACCTCTTCACTCTCTGCTCT-3'; the reverse primer sequence is shown as SEQ ID NO. 6, namely: 5'-ACTAGGAAAAACAGCCCTTGGT-3' are provided.CaHSP90-1The forward primer sequence of the primer used for gene cloning is shown as SEQ ID NO. 1, namely: 5' -TGATTCAGGACGAGGAGGAG-3'; the sequence of the reverse primer is shown as SEQ ID NO. 2, namely: 5'-TGATCAACTTCTTGGAATCCTTG-3' are provided.
The extraction of the total RNA of the hot pepper can adopt a Trizol method. The specific method comprises the following steps: grinding pepper leaves in liquid nitrogen to powder, transferring to a 2ml centrifuge tube, adding 1ml Trizol, standing at room temperature for 5min, centrifuging, taking the supernatant, adding chloroform, mixing uniformly, standing for 15min, centrifuging, taking the supernatant, adding isopropanol to precipitate, centrifuging, taking the supernatant, adding 75% alcohol to the supernatant, suspending and precipitating, centrifuging, taking the supernatant, drying in the air, adding ddH2And O is reserved.
The heat shock protein geneCaHSP90-1The correlation between the relative expression level and the heat-resistant trait was performed as follows: extracting RNA of hot pepper variety with obvious heat-resisting property and common control hot pepper variety, adopting the above-mentioned designed heat shock protein geneCaHSP90-1 And (3) carrying out fluorescence quantitative specific amplification on the qPCR primers (SEQ ID NO.3 and SEQ ID NO. 4) to respectively obtain relative expression values of the heat-resistant pepper and the common pepper.CaHSP90-1The higher the expression level of (2), the stronger the heat resistance of the variety, and the relative expression level of more than 50 is the heat-resistant variety as the selection standard.
The method for detecting whether the hot pepper variety has heat-resisting property or not utilizes the heat shock protein geneCaHSP90-1 And (3) amplifying the qPCR primer (SEQ ID NO 1), the qPCR system and the program, calculating the relative expression quantity of the heat-shock protein 90 gene in the tested pepper variety according to the amplification result, and comparing the relative expression quantity with the heat-resistant pepper variety and the control pepper variety to preliminarily judge the heat resistance of the pepper variety to be tested.
Thus, the present invention provides heat shock protein-based genesCaHSP90-1The method for assisting in breeding the heat-resistant pepper variety comprises the following steps: (1) detecting in individuals in a breeding populationCaHSP90-1The relative expression level of (3);
(2) selectingCaHSP90-1The individual having a high relative expression level of (a) is an individual having a candidate heat-resistant trait.
Wherein, detectingCaHSP90-1The relative expression of the gene was performed by fluorescence quantitative PCR. Further, the sequence of the primer of the fluorescent quantitative PCR is as follows:
the forward primer is shown as SEQ ID number 3, namely: 5'-CCTTTGCTTTCCAGGCTGAG-3', respectively;
the reverse primer is shown as SEQ ID number 4, namely: 5'-CGAATGAAGAGTTCCGGCTG-3' are provided.
Further, an internal reference gene is also set, which isβ-actinA gene ofβ-actinqPCR primers for gene detection have the following sequences:
the forward primer is shown as SEQ ID number 5, namely: 5'-CCACCTCTTCACTCTCTGCTCT-3', respectively;
the reverse primer is shown as SEQ ID NO. 6, namely: 5'-ACTAGGAAAAACAGCCCTTGGT-3' are provided.
In order to rapidly and efficiently breed hot pepper heat-resistant varieties, the invention screens HSP90 family genes from hot pepper genomes by using a hmmsearch tool by referring to an HMM (hidden markov model) of HSP90, so that a heat shock protein gene is firstly establishedCaHSP90-1The relation between the relative expression quantity and the hot pepper heat-resistant character (the relative expression quantity is more than 50, namely a heat-resistant variety) is adopted, the heat-resistant condition of hot pepper filial generation individuals is rapidly and efficiently detected by utilizing real-time fluorescent quantitative PCR, so that individuals with excellent characters are screened for further breeding, and the breeding method is assisted by a target gene, so that the breeding goal and efficiency are remarkably improved.
Drawings
FIG. 1 shows the expression pattern of a pepper HSP90 family gene under high temperature stress.
FIG. 2 shows the leaves of each pepper materialCaHSP90-1Relative expression level of gene.
Detailed Description
The invention is further illustrated by the following detailed description of specific embodiments, which are not intended to be limiting but are merely exemplary.
20 parts of pepper materials J01-7, 8506, 8505, SJ11-3, CT14-11-2-3, T0155, SJ07-23-2-3-1, A62, H1023 and W which are bred by the material used in the embodiment for the subject group23F2-4-2-1、16CT32、H1306-1-2-1、17CL30、14ML13-1、11L209F23-2-1, 16HL28, 16Y49, C68, LS22 and G16-6-2, and the heat resistance is strong or weak according to the identification result of the heat resistanceIs 17CL30>11L209F2-3-2-1>16HL28> H1306-1-2-1> LS22> SJ07-23-2-3-1>16CT32> SJ11-3> G16-6-2> A62> C68>8505> CT14-11-2-3>14ML13-1> J01-7·> W23F2-4-2-1>8506> H1023> T0155>16Y49, the most Heat-resistant Pepper varieties are 17CL30 (Wang, J.; Lv, J.; Liu Z. et al. Integration of transformations and metabolism for Pepper (Capsicum annuum L.) in Response to Heat stress. Int. J. mol. Sci. 2019, 20, 5042.) and 11L209F2-3-2-1, both of which are heat-resistant varieties.
The present invention is based on heat shock protein genesCaHSP90-1The method for assisting in breeding the heat-resistant pepper variety realizes efficient breeding of the heat-resistant pepper variety, and comprises the following steps:
(1) with reference to HMM model (hidden Markov model) of HSP90, ID is PF00183, and hmmsearch tool (E)<1 e-5) predicting the conserved structural domain by a pfamscan tool, removing HSP90 gene without complete structural domain, screening 7 HSP90 family genes from pepper genome, naming the genes according to the gene family naming rule, and finally determining 7 HSP90 family genesHSP90Genes, respectively namedCaHSP90-1、CaHSP90-2、 CaHSP90-3、 CaHSP90-4、 CaHSP90-5、 CaHSP90- 6、 CaHSP90-7。
(2) Determining the expression pattern of HSP90 family gene under high temperature stressCaHSP90-1The gene is the most obvious gene responding to high temperature stress; the expression pattern of HSP90 family gene under high temperature stress is shown in figure 1.
(3) CloningCaHSP90-1A gene.
Design with Primer3.0CaHSP90-1Primers and pairs for gene cloningCaHSP90-1Cloning was performed.CaHSP90- 1The forward primer sequence of the primer used for gene cloning is shown as SEQ ID NO. 1, namely: 5'-TGATTCAGGACGAGGAGGAG-3', respectively; the reverse primer sequence is shown as SEQ ID NO. 2, namely: 5'-TGATCAACTTCTTGGAATCCTTG-3' are provided. Cloning from Capsicum annuum leaves by RT-PCRCaHSP90-1A gene. The method specifically comprises the following steps: grinding Capsici fructus leaf in liquid nitrogen, and usingTotal RNA was extracted using an RNA extraction Kit (TransZol Up Plus RNA Kit, Transgen, Beijing). The First Strand of cDNA obtained by reverse transcription using a reverse transcription kit (TransScript All-in-One First-Strand cDNA Synthesis Supermix for PCR, Transgen, Beijing) was subjected to PCR as follows: 5min at 95 ℃, 30s at 95 ℃, 2.5min at 72 ℃, 40 cycles, 10min at 72 ℃ and constant temperature at 16 ℃. The PCR product was then subjected to agarose gel electrophoresis, cut and recovered, and sequenced. The coding sequence is a DNA sequence shown in SEQ ID NO. 7.
(4) Designing genes in non-highly conserved segments of HSP90 family genesCaHSP90-1Primers for fluorescent quantitative PCR.
(5) With hot pepperβ-actinThe gene is an internal reference, and a qPCR primer of the gene is designed.
Sequence alignment followed by Primer3.0 design in non-highly conserved segments of this familyCaHSP90-1The specific primer is used for qPCR and simultaneously designs the internal reference gene of the hot pepperβ-actinThe qPCR primers of (1), used for qPCR analysis.
Heat shock protein geneCaHSP90-1The forward primer sequence of the qPCR primer of (1) is shown as SEQ ID NO.3, namely: 5 '-CCTTTGCTTTCCAGGCTGAG'; the sequence of the reverse primer is shown as SEQ ID NO.4, namely: 5'-CGAATGAAGAGTTCCGGCTG-3' are provided. Internal reference geneβ-actinThe forward primer sequence of the qPCR primer of (1) is shown as SEQ ID NO. 5, namely: 5'-CCACCTCTTCACTCTCTGCTCT-3', respectively; the reverse primer sequence is shown as SEQ ID NO. 6, namely: 5'-ACTAGGAAAAACAGCCCTTGGT-3' are provided.
(6) Extracting pepper total RNA to carry out qPCR analysis, and detecting the relative expression quantity of the gene.
The method for extracting the total RNA of the pepper leaves by adopting a Trizol method comprises the following specific operation steps: grinding Capsici fructus leaves in liquid nitrogen to powder, transferring into centrifuge tube, adding Trizol, standing for 5min, centrifuging, collecting supernatant, adding chloroform, standing for 15min, extracting, centrifuging, collecting supernatant, adding isopropanol, standing for 10min, centrifuging, removing supernatant, adding 75% ethanol, suspending, precipitating, centrifuging, removing supernatant, drying, precipitating, and adding ddH2And O is reserved.
(7) qPCR detection is carried out on known heat-resistant pepper varieties and reference varieties, and three groups are arrangedRepeating, respectively calculating heat-resisting pepper variety and control pepper varietyCaHSP90-1Relative expression amount of the gene, thereby establishingCaHSP90-1The correlation between the relative expression level and the heat-resistant trait.
(8) And (3) screening in a breeding population by using the same pair of fluorescent quantitative primers, detecting the copy number of an amplified product, and selecting individuals with heat-resistant properties.
To pairCaHSP90-1And reference geneβ-actinThe (Mir-X miRNA qRT-PCR SYBR kit; Takara, Dalian, China) kit is used for amplification, and the amplification system (25 mul) is as follows: ddH2O 9.5.5. mu.l, SYBR Advantage Premix (2X) 12.5. mu.l, primer-F (10. mu.M) 0.5. mu.l, primer-R0.5. mu.l, cDNA 2. mu.l. The amplification procedure was: 95 ℃ for 10 s, 35 cycles were: 95 ℃ for 5 s and 60 ℃ for 20 s.
The heat shock protein gene in each material leaf of the pepper is obtained by taking RNA of each material as a template and adopting the qPCR systemCaHSP90-1Relative expression amount of (2).CaHSP90-1Relative gene expression level = CaHSP90-1Calculating the expression level of the gene/the expression level of the reference gene to obtain the leaf of each material CaHSP90-1The relative expression level of the genes (FIG. 2) was determined for the pepper varieties 17CL30 and 11L209F2In (e) -3-2-1CaHSP90-1The relative expression level of the gene is the highest, and the pepper variety with heat-resistant character (consistent with the previous screening result, namelyCaHSP90-1The relative expression amount of the gene is more than 50, and the gene can be determined as a heat-resistant variety).
The method for breeding the hot pepper heat-resistant variety has the characteristics of strong target, short breeding period and remarkable heat-resistant character. In addition, the variety breeding thought adopted by the invention can be applied to the screening of the complex quantitative characters of the hot pepper and other crops.
<110> institute for vegetables in Hunan province; hunan university of agriculture
<120> heat shock protein gene CaHSP90-1 and method for assisting in breeding heat-resistant pepper variety
<160> 7
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TGATTCAGGACGAGGAGGAG 20
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TGATCAACTTCTTGGAATCCTTG 23
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CCTTTGCTTTCCAGGCTGAG 20
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CGAATGAAGAGTTCCGGCTG 20
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<212> DNA
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CCACCTCTTCACTCTCTGCTCT 22
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ACTAGGAAAAACAGCCCTTGGT 22
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<211>2549
<212> DNA
<400> 7
ATGGATCAAGTTCTGTTTTTTGAAGTTGATTCAGGACGAGGAGGAGGACGAGGAGTAGATTATACGAAAAATAATCAAGAAATTAGGGAAGTTGTTTTTGTTATGGACCTTAGAAGGGGTCCATGGACTGTTGAAGAAGACTTTACACTTATCAATTTTATTGCTCATCATGGTGTTGGTCGTTGGAATTCCCTTGCACGTTGTGCTGGTAATTACCTCTATTTTCTTATTGTCATCTTCGTCTGATATTCGTGTTTCTCTGACTAAACTAATTTAGATTTGCGCCCGTGTAAGGACCCATTAAGGTAACTAAACACTTATTTTTTAATATTTTTTTGTTTTAAGGACGAAATGATTTGATCCATATAACCCATCGTTGTGTACTTTTACATATTCATTGACTAAGAAAACCTTTCTTGTTCAGCTACTAAAAAGTTGAACGACTCTTTAGTAATCCATAATTTTCGTATTCTTAGAACTCAAACTTTAGTTTTCTTGTGAAAAGGTTCAAGAAAGTTTATTATATTATCGTAAGGAGATGAAATCAAGATTTAAAGCTTATATACTTTGTTTTTTTTTTTAATAATTATATGACAATAAATCATGTTCATGTAGTTAGTTAAATCAATCATGTTCATGTAGTTAGTTAATTCTTTCTTTCTTGAGAAACCAGCTTTTCTTTATTCTAATAGATATTTCTTCTACTTTTCTTTTGTATAAATCAATTTCTTCTCGTTTCTTAACGTAGATAAATATTCGTATGGATATGCTTTTGTATAAATCTTTTCCAAAAAATAATATAAAATAAAATGTTTTCTTTATTTCCGTACAAGGTAATTTCTTAAATCCTAAACACTAGTCCAATCTGATAAGGATAAAGGAAACAAAGTGCTTTTTTGTGGTGGGGTGAGTAATTGAGTATACAAGTAGTAGTAATATTAAAGTAGTATTTATTCAATATTTCACTTTTGTTGTTGTTTCTACGATCAGAAATCAGAATTTTCATTAAAAAATTTAAAAATATAAGAAAATAAATATATGAAAAAATTGTGTTGTGTTTCTAATGAGTTTGTAATATATATATATATATATATATTATTTCTACTTAGCTCCGGCCCTATCTGCAATCGTTTTTCTTCTTAGCTTCACCCCTATCTGCAATTGTATATTTAACATAGAATATTTTATGCACCATAATATCTTTTATTGATTTTTTTGTTCAACATTAATTGTGTTCTTGAAACATGCTTTTTGAACTTAATTTATCGTTAATTAGTTATGAAATTAAATCTTTGTTTGTATTGCTAATTCAAATTTAATTAATTAAACAGGTTTGAAGAGAACAGGAAAAAGCTGCAGATTAAGATGGCTTTTTTTTCTTCGACCAGATGTTCGACGTGGGAATATTACTCTTGAAGAACAACTCTTGATTCTTGAATTGCATTCACGTTGGGGCTTTCGGTACGTTCGTTATACGGATTAATTTTACGTATGATCATTAAAATGTGTTTTCTTTATTATTAACTATAATCTTCTTTTTACTTGTCAAACAAGTAATCGAACTTATATCACATGAAAATAACTAAACTATGTGTTAATTGCTAATCAAATTTGTTTTTTAAAATGACCAATAGGTCAAGTTTCTGGCTTTGTCATTCTGTAAATTTCATGTCAGAAGAAAAAAGAAATTTTTTGCGTTTAAGTTTTAATCTAAAATATTTGAGAAAAGACATAAGTTTGATTATAGTACAAACATGCTTTAAAAGTTATCCTTTTCCTTTTCTTTTCTATATATACTTTAGAATTATCTAATAGTGCAACAAGTGTTTGTAAACTTTATTCTAATATACTTCCTTCTTTTGTATTTAATATGACTTTTCATTTCTTTTGTAGTTGGTCTAAGATTGCTCAACATCTTCCTGGAAGAACAGTCTTCGTGTTAAAAAATTACTGGCGAACTCGAGTGCAAAAGCATGCAAAACAGCTCAAATGTGATGTGAACAGCAAGCATTTCTTTGTTACCATGAAGTATCTTTGGATGCCAAGGTTAGCCGAAAGAATTCAAGCCGCTGCCGCTTCTAACCCGGCGACTGCTTCTTCCTCTGGCCCCCCCACCACCACTTACATCCAAAACCAAGAAATTCAACATCCATTACCAAACATGAATCATCTTTTGTCTGACTATACACCTATTCCATTTCATGAATCATCTTTTGTCTGACTATACACCTATTCCATTTATCGAGAAAACAAACAGTCTGAATTATTCAGCTACTTCAGCTTCGTCAGACTTTTTCTCGTCCGACCTCACCGATGGTTGCTACAATTTCCCAATTAACCAAAGCAATAATCAGGATTATTCTCAAGTTATTCTTTGTTCTAATCAAATGTGCTATGGAGAATCCACAATTAACCCAACAAATTACGATTTTCACCATGGATTTCAAGGATTCCTTGTTGTTGATCAACAAAACAATACTCAATGGATGGACAATCCATGGAATATTGAAGATGTGTGTTTCTTACAACATTTGTTCTTTGACATGTAA
Claims (6)
1. Heat shock protein geneCaHSP90-1The nucleotide sequence is shown as SEQ ID NO. 7 or degenerate sequence thereof.
2. A heat shock protein gene of claim 1CaHSP90-1Application in breeding heat-resistant pepper varieties.
3. Heat shock protein based geneCaHSP90-1The method for assisted breeding of the heat-resistant pepper variety is characterized by comprising the following steps:
(1) detecting in individuals in a breeding populationCaHSP90-1The relative expression level of (3); the above-mentionedCaHSP90-1Relative gene expression level = CaHSP90-1The expression level of the gene/the expression level of the reference geneβ-actinA gene;
(2) selectingCaHSP90-1The individual with the relative expression amount of more than 50 is an individual with a candidate heat-resistant trait;
wherein, the heat shock protein geneCaHSP90-1The nucleotide sequence of (A) is shown in SEQ ID NO. 7.
4. The method of claim 3, wherein: detection ofCaHSP90-1The relative expression of the gene was performed by fluorescence quantitative PCR.
5. The method of claim 4, wherein: the sequence of the primer for the fluorescent quantitative PCR is as follows:
the forward primer is shown as SEQ ID number 3, namely: 5'-CCTTTGCTTTCCAGGCTGAG-3', respectively;
the reverse primer is shown as SEQ ID number 4, namely: 5'-CGAATGAAGAGTTCCGGCTG-3' are provided.
6. The method according to any one of claims 3 to 5, wherein: the above-mentionedβ-actinqPCR primers for gene detection have the following sequences:
the forward primer is shown as SEQ ID number 5, namely: 5'-CCACCTCTTCACTCTCTGCTCT-3', respectively;
the reverse primer is shown as SEQ ID NO. 6, namely: 5'-ACTAGGAAAAACAGCCCTTGGT-3' is added.
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