CN108048469A - The Ravenna grass class Calmodulin Gene ErCML30 expressed in Ravenna grass wild species by low temperature stress - Google Patents

Abstract

The invention discloses in Ravenna grass wild species by low temperature stress expression Ravenna grass class Calmodulin Gene ErCML30 and expand its primer special and detect differential expression method and its special primer of the gene in Ravenna grass wild species by low temperature stress.The gene is persistently risen in Ravenna grass wild species by expression quantity under low temperature stress, directly takes part in low temperature stress response.For the present invention to recognize the degeneration-resistant mechanism of the gene, important germ plasm resource and utilization that Ravenna grass wild species are developed as sugarcane new cold-resistant species provide technical support.

Description

The Calmodulin Gene ErCML30 of the Pseudomonas-like Calmodulin Gene Expressed by Low Temperature Stress in the Wild Species of Pseudomonas

technical field

The invention belongs to the technical field of biomolecular cloning, and in particular relates to a gene encoding a calmodulin-like (Calmodulin-like, CML) in the wild species of succulents under low temperature stress, and primers are designed based on the sequence of the gene, and detected by relative quantitative RT-PCR The method for the differential expression of the calcium-binding protein gene in the wild species of succulent grass.

Background technique

E.fulvus is a wild species of Erianthus, which is closely related to Saccharum. It is mainly distributed in Yunnan, Sichuan and Tibet in my country. It grows at an altitude of 500-2700m and has cold and drought resistance. , resistance to barrenness, strong perennial roots, anti-embroidery disease and other excellent characteristics, and easy to bloom, with a large amount of pollen. The Sugarcane Research Institute of Yunnan Agricultural University collected more than 150 clones of different ecological types from the main distribution areas of the wild species of Canegrass, and concentrated them in the sugarcane resource garden of the school (1930m above sea level). Sugarcane is native to tropical regions and is suitable for planting in tropical and subtropical climates. It is the sugar crop with the largest planting area in the world. At present, my country has basically formed three major sugarcane regions: Guangxi, Yunnan and Guangdong. In recent years, extreme weather has occurred frequently in winter, and sugarcane has been severely affected by low temperature stress during the growth and development of the seedling stage. The current sugarcane varieties often suffer from growth point necrosis and serious damage to the root system structure under low temperature at the seedling stage, which seriously restricts the growth and sugar content of sugarcane. and biological production. Therefore, how to improve the resistance of sugarcane varieties to low temperature stress and cultivate new varieties resistant to low temperature has become an important topic in the research of sugarcane genetics and breeding (Li Fusheng et al. Thoughts on the development and utilization of wild canegrass germplasm resources [J]. Biological resources ,2004,20(4):266-270; Sun Bo, Liu Guangling, Yang Litao, Li Yangrui.Response of sugarcane seedling root structure and protection system to low temperature stress[J].Journal of China Agricultural University,2014,19(06) :71-80.; Lu Xin et al. Research on the innovation and utilization of the wild sugarcane species Saccharomyces sativa germplasm [J]. Journal of Plant Genetic Resources, 2012,13(02):321-324.). Therefore, it is of great significance to stress-resistant breeding of sugarcane by discovering new stress-resistant genes in the wild species of Saccharomyces spp.

Calmodulin-like (CML) belongs to a class of calcium-binding proteins. Calcium ion signaling is the main regulator of plant growth and stress response. The Ca ²⁺ -CML signaling system is involved in the response of plants to multiple stress signals. Transduction, when the plant is under stress, the concentration of calcium ions increases, and the calcium ions bind to calcium-binding proteins, causing the hydrophobic regions of these calcium-binding proteins to be exposed and bind to biofilms or hydrophobic biomacromolecules, thereby regulating biofilms or these targets The function and enzymatic activity of the protein, that is, the calcium-binding protein is stimulated to sense and transmit calcium signals (Liu Wei. Analysis of the function of tomato calmodulin and calmodulin-like regulation of disease resistance [D]. Zhejiang University, 2015.; Luo Mei et al. Cloning and bioinformatics analysis of calmodulin gene in Pygonia hibiscus cotton[J]. Journal of Huazhong Agricultural University, 2012,31(03):320-324.; Function [J]. Life Science Research, 2005, (S2): 1-5.). At present, there are few calmodulin-like studies. In terms of abiotic stress, the rice CML genes OsMSR2 and CML4 cloned from rice are induced by salt and drought, and their functions are related to the signal transmission mediated by the hormone ABA in plants; CML10 found in Arabidopsis is involved in the formation of ascorbic acid and maintains the redox balance in plants; in terms of biotic stress, CML regulates plant defense responses to pathogenic bacteria, and the expression of Arabidopsis CML9 is induced by pathogenic bacteria and the hormone salicylic acid to produce NO and Hypersensitivity, the homologous gene APR134 in tomato also positively regulates plant defense responses to pathogenic bacteria (Zeng Houqing et al. Research progress on the physiological functions of plant calmodulins[J]. Chinese Science: Life Science, 2016, 46( 06):705-715.). However, there is no report on the mechanism of action of calmodulin-like genes on plants under low temperature stress, and no calmodulin-like genes have been cloned in Saccharum.

Contents of the invention

The purpose of the present invention is to improve the stress resistance of existing cultivated sugarcane varieties, so that sugarcane can produce high and stable yields in low temperature adversity so as to adapt to the situation of climate deterioration in recent years, and provide a new calcium-like calcium that is induced by low temperature stress in the wild species of sugarcane grass The modulin gene also provides a primer for amplifying the gene and a method for detecting the differential expression of the gene in the wild species of Saccharomyces chinensis under low temperature stress, thereby further laying the foundation for sugarcane cold resistance breeding and providing candidate genes.

In this study, a calmodulin-like gene was cloned from the wild species of Saccharomyces japonicus under low temperature stress by electronic cloning technology. After BLASTn comparison analysis in NCBI, it was 95% homologous to the known CML gene. The protein encoded by the gene is a calmodulin-like protein, so the gene is named the Calmodulin-like gene ErCML30.

Technical scheme of the present invention is as follows:

1. The full-length nucleotide sequence of the calmodulin gene ErCML30 expressed in the wild species of Pseudomonas under low temperature stress is shown in SEQ ID NO:1.

2. the special-purpose primer of the canegrass calmodulin gene ErCML30 expressed in the wild species of canegrass described in technical scheme 1 by low temperature stress, the special-purpose is made up of upstream primer GP-F and downstream primer GP-R, so The base sequence of the upstream primer GP-F is shown in SEQ ID NO:2, and the base sequence of the downstream primer GP-R is shown in SEQ ID NO:3.

3. A method for detecting the differential expression of the calmodulin gene ErCML30 of the succulents in the wild species of succulents under low temperature stress, including the extraction of total RNA, the synthesis of the first strand of cDNA, and the detection of calcium in the succulents by relative quantitative RT-PCR Differential expression of the modulin gene ErCML30 in the wild species of Saccharomyces cerevisiae under low temperature stress, in which:

In the extraction of total RNA, the wild species of succulata at the seedling stage were set as the control group and the experimental group. The control group grew normally without other treatments at room temperature at 25°C. The experimental groups were divided into treatment Ⅰ and treatment Ⅱ. Grow at a low temperature of 4°C for 24 hours, and grow at a low temperature of 4°C for treatment II for 72 hours. The leaves of the control, treatment I and treatment II were collected, respectively, and quickly ground with liquid nitrogen to obtain three samples, and the total RNA was extracted from the three samples respectively. ;

In the relative quantitative RT-PCR technique to detect the differential expression of the succulata calmodulin gene ErCML30 in the wild species of succulents under low temperature stress, the designed specific primers were used as RT-PCR primers to carry out gene differences between different treatments and controls Expression detection, described specific primer is made up of upstream primer QF and downstream primer QR, and described upstream primer QF nucleotide sequence is as shown in SEQ ID NO:4, and the nucleotide sequence of described downstream primer QR is as shown in SEQ ID NO:5 The full-length nucleotide sequence of the Calmodulin gene ErCML30 is shown in SEQ ID NO:1.

The present invention provides for the first time the succulata calmodulin gene ErCML30 expressed in the succulata wild species subjected to low temperature stress, as well as special primers for amplifying it and specific primers for detecting the differential expression of the gene in the succulent grass wild species subjected to low temperature stress. In the relative quantitative RT-PCR detection, the gene was almost not expressed in the absence of low temperature stress. After 24 hours of 4 ℃ low temperature treatment, the expression level of the gene was 55 times that of the control. After 72 hours of 4 ℃ low temperature treatment After treatment, the expression level in the wild species of Canegrass was 75 times that of the control, indicating that the expression of the gene increased after the wild species of Canegrass was subjected to low temperature stress, and increased with the increase of stress time. The gene ErCML30 belongs to the inducible expression gene expressed by low temperature stress, which indicates that the calmodulin gene ErCML30 of canegrass can quickly and persistently participate in the response to low temperature stress after the wild species of canegrass are subjected to low temperature stress. The stress resistance mechanism of the prime gene ErCML30 and the utilization of the wild species of sugarcane grass provide technical support.

The method of the present invention detects the differential expression of the calmodulin gene ErCML30 of the canegrass species under low temperature stress in the wild species of the canegrass, and the designed specific primers have strong specificity in relative quantitative RT-PCR, no miscellaneous bands are generated, and the melting curve shows No primer-dimers were produced. In the selection of internal reference genes, GAPDH gene and β-actin gene were not selected, but the more suitable 25SrRNA gene of sugarcane was selected as internal reference, and its expression stability was higher than that of other internal reference genes (see Figure 3). The relative quantitative RT-PCR is carried out by SYBR Green I chimeric fluorescence method, which has the characteristics of strong specificity, high sensitivity and good repeatability.

SEQ ID NO: 1 in the sequence listing is the full-length nucleotide sequence of the calmodulin gene ErCML30 of the succulent species.

Shown in the sequence listing as SEQ ID NO: 2 is the nucleotide sequence of the upstream primer GP-F for amplifying the calmodulin gene ErCML30 of the succulent species.

Shown in the sequence listing as SEQ ID NO: 3 is the base sequence of the downstream primer GP-R for amplifying the calmodulin gene ErCML30 of the succulent species.

SEQ ID NO:4 in the sequence listing is the base sequence of the upstream primer QF of the specific primer.

SEQ ID NO:5 in the sequence listing is the base sequence of the downstream primer QR of the specific primer.

Shown in SEQ ID NO:6 in the sequence listing is the base sequence of the upstream primer 25S-F of the internal reference gene 25SrRNA.

Shown in SEQ ID NO: 7 in the sequence listing is the base sequence of the downstream primer 25S-R of the internal reference gene 25SrRNA.

Description of drawings

Figure 1: Amplification results of the calmodulin gene ErCML30 from the succulent species. In Figure 1, M: maker; lane 1, lane 2, and lane 3 are amplification products at annealing temperatures of 52°C, 55°C, and 57°C, respectively.

Fig. 2: The relative fluorescence quantitative differential expression of the calmodulin gene ErCML30 in the leaves of the wild species of Saccharomyces succulata under different low temperature stress treatments. In Fig. 2, the ordinate represents the relative expression level of the gene, and the abscissa represents the samples treated at different low temperatures for different times. ER-1: Pseudomonas leaves treated at room temperature at 25°C, as a control; ER-2: Pseudomonas leaves treated at low temperature at 4°C for 24 hours; ER-3: Pseudomonas leaves treated at low temperature at 4°C for 72 hours.

Figure 3: Detection results of specific fluorescent quantitative primers. M: maker; lane 1, lane 2, and lane 3 are amplification products at annealing temperatures of 55°C, 57°C, and 60°C, respectively.

Detailed ways

The following examples are used to further illustrate the present invention, but are not intended to limit the protection scope of the present invention. In each embodiment, those without special instructions are conventional methods.

Experimental material: The experimental material is the wild species of Canegrass (Kunming Canemao 99-1), which was planted in the sugarcane resource garden of the Sugarcane Research Institute of Yunnan Agricultural University (Kunming, Yunnan, altitude 1930m).

Example 1 Acquisition of the Calmodulin Gene ErCML30 Expressed by Low Temperature Stress in the Wild Species of Canegrass

1. Design and amplify the special primers for amplifying the full-length sequence of the calmodulin gene ErCML30 expressed in the wild species of Pseudomonas under low temperature stress.

In the early stage, the differently treated wild species of Canegrass were sent for high-throughput transcriptome sequencing, and the returned transcriptome sequencing results were analyzed and screened by bioinformatics to obtain the spliced Unigene sequence of the transcriptome data of the gene. According to the spliced Unigene Sequence Utilize biological software Primer5.0, Oligo 7 to design and amplify the special primer of succulent class calmodulin gene ErCML30, this special primer is made up of upstream primer GP-F and downstream primer GP-R, described upstream primer GP-F The base sequence is shown in SEQ ID NO: 2, the base sequence of the downstream primer GP-R is shown in SEQ ID NO: 3, and the special primer was sent to Shanghai Sangong Company for synthesis.

2. Extraction of total RNA from the leaves of the wild species of succulata treated at low temperature.

(1) Sample treatment: Take 6 pots of the wild species of Canegrass with basically the same growth state at the seedling stage and use the light incubator for experimental treatment. Set up a control group and an experimental group, and 2 pots grow at room temperature (25°C) and the others are not treated as a control. (ER-1), 2 pots of low temperature treatment for 24h (Er-LT2), 2 pots of low temperature treatment for 72h (Er-LT3), the low temperature treatment temperature is 4°C.

(2) Place the treated leaves in a mortar and add liquid nitrogen to grind quickly, add 1ml TRIzol extract per 100mg sample, and vortex for 15s. The sampling in each period is a treated sample, a total of 6 treated samples. Place each sample at 15-30°C for 5 minutes; add chloroform, the amount of chloroform is 200 μl of chloroform for every 1ml of TRIzol used, cover the tube, vortex for 15 seconds, and place at 15-30°C for 3 minutes.

(3) Centrifuge at 12000rpm for 15min at 4°C. At this time, the solution is divided into three layers: the yellow one is the organic phase, the middle and upper layers are the water phase, and the RNA mainly exists in the water phase. Transfer the water phase to another new phase. in an RNase-free centrifuge tube.

(4) Add an equal volume of isopropanol to the obtained water phase, mix well, and place at 15-30°C for 20-30min.

(5) Centrifuge at 12,000 rpm for 10 min at 4°C, and discard the supernatant.

(6) Add a 75% ethanol solution prepared with DEPC water to wash the precipitate. The amount used is 75% ethanol solution prepared with 1 ml DEPC water for every 1 ml TRIzol used.

(7) Centrifuge at 5000 rpm for 3 minutes at 4°C, carefully suck out the upper layer liquid with a pipette tip, and keep the precipitate.

(8) Dry the precipitate at 15-30°C for 2-3min, add 30-100μl RNase-free double-distilled water, fully dissolve the RNA, and mix the two control sample RNAs as a total control sample and store them at -70°C. The processed samples during the period were stored at -70°C;

3. Synthesis of the first strand of cDNA

(1) Thaw template RNA on ice; thaw 5×gDNA Buffer, FQ-RT Primer Mix, 10×Fast RTBuffer, RNase-Free ddH ₂ O at room temperature (15-25°C), and place on ice immediately after thawing . Each solution was vortexed before use, and briefly centrifuged (6000rpm, 30s) to collect the liquid remaining on the tube wall. In order to ensure the accuracy of the preparation of the reaction solution, when performing various reactions, the Mix was prepared first, and then distributed into each reaction tube.

(2) According to the following system, the gDNA removal system in RNA is used to prepare the mixed solution and mix thoroughly. Briefly centrifuge (6000rpm, 30s) and place at 42°C for 3min. Then place on ice.

gDNA removal reaction system:

5×gDNA Buffer 2μl

Total RNA 2μl

Make up to 10 μl with RNase-Free ddH ₂ O.

(3) Prepare the mixed solution according to the reverse transcription reaction system of the following system:

Reverse transcription reaction system:

10×Fast RT Buffer 2μl

RT Enzyme Mix 1μl

FQ-RT Primer Mix 2μl

Make up to 10 μl with RNase-Free ddH ₂ O.

(4) Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step, and mix well. Incubate at 42°C for 15 minutes, at 95°C for 3 minutes and place on ice to obtain cDNA for subsequent experiments, or store at -20°C.

4. Amplification of the calmodulin gene ErCML30 of the succulent succulata wild species

The cDNA obtained by reverse transcription in step 3 (4) is used as the template of the PCR reaction, and the special primer designed in the step 1 is used as the primer of the PCR reaction to amplify the calmodulin expressed by low temperature stress in the leaves of the succulent grass wild species Gene ErCML30 full-length, PCR reaction system: Template 1 μl; upstream primer GP-F 1 μl; downstream primer GP-R 1 μl; Buffer 2 μl; dNTP ₂ μl; Pfu enzyme 0.5 μl; The PCR reaction program was: pre-denaturation at 95°C for 2 min; denaturation at 95°C for 30 s; annealing at 57°C for 30 s; extension at 72°C for 2 min, 35 cycles; 6 min at 72°C, and storage at 4°C.

5. Recovery of the target gene

The PCR product was subjected to 1% agarose gel electrophoresis, and the target band in the gel was gel-recovered, and the steps were as follows:

(1) Cut off the gel block of the target band under ultraviolet light, remove as much as possible the blank gel that does not contain DNA fragments, and put it into a 1.5ml centrifuge tube.

(2) Add 500 μl of sol solution to the gel block at a ratio of 500 μl of sol solution per 100 μl of gel block, and place the sol at 60° C. for about 10 minutes, shaking continuously during this period.

(3) After the gel blocks are completely dissolved, transfer the sol solution to an adsorption column, centrifuge at 12000 rpm for 30 s at 18-22 ° C, and remove the waste liquid.

(4) Add 500 μl of washing solution to the adsorption column, centrifuge at 12000 rpm for 30 s at 18-22 ° C, and discard the waste liquid.

(5) Repeat step (4), centrifuge the centrifuge tube at 12,000 rpm for 2 minutes after the waste liquid is discarded, completely remove the waste liquid, and keep the adsorption column.

(6) Move the adsorption column to a clean 1.5ml centrifuge tube, and place it at 18-22°C for 2 minutes to allow the remaining ethanol in the rinse solution to evaporate to dryness.

(7) Add 30μl-50μl of elution buffer to the center of the adsorption column membrane, place it at 18-22°C for 1-2min, and centrifuge at 12000rpm for 2min to elute DNA. The solution eluted from the adsorption column membrane collected in the centrifuge tube is the target Fragments of purified product recoveries.

The amplification result is shown in Fig. 1, when the annealing temperature is 57°C, the amplification effect is the best, and no impurity bands are generated. After the amplified fragment was recovered by gel cutting, the sequence of the recovered fragment was consistent with the Unigen sequence obtained from the transcriptome, that is, the recovered product was the calmodulin gene ErCML30 expressed in the leaves of the wild species of Canegrass species under low temperature stress, The sequencing result of the recovered product is shown as SEQ ID NO: 1 in the sequence listing.

Embodiment 2 A method for detecting the differential expression of the calmodulin gene ErCML30 of the succulata class provided by the present invention in the wild species of the succulent grass under low temperature stress, comprising the following steps:

1. Sample processing:

Take 6 pots of wild species of Canegrass with basically the same growth state at the seedling stage, and use the light incubator for experimental treatment. Set up the control group and the experimental group. (ER-2), 2 pots of low temperature treatment for 72h (ER-3), low temperature treatment temperature is 4 ℃.

2. Extraction of total RNA

(1) Place the treated leaves in a mortar and add liquid nitrogen to grind quickly, add 1ml TRIzol extract per 100mg sample, and vortex for 15s. The sampling in each period is a treated sample, a total of 6 treated samples. Place each sample at 15-30°C for 5 minutes; add chloroform, the amount of chloroform is 200 μl of chloroform for every 1ml of TRIzol used, cover the tube, vortex for 15 seconds, place at 15-30°C for 3 minutes; centrifuge at 12,000 rpm for 15 minutes at 4°C, At this time, the solution is divided into three layers: the yellow one is the organic phase, the middle layer and the upper layer are the water phase, and the RNA mainly exists in the water phase. Transfer the water phase to another new RNase-free centrifuge tube;

(2) Add an equal volume of isopropanol to the obtained aqueous phase, mix well, and place at 15-30°C for 20-30min; centrifuge at 12,000rpm at 4°C for 10min, and discard the supernatant;

(3) Add 75% ethanol solution prepared with DEPC water to wash the precipitate. The dosage is 75% ethanol solution prepared with 1ml DEPC water for every 1ml TRIzol used; centrifuge at 5000rpm for 3min at 4°C, and carefully suck out with the tip of a pipette The upper liquid, retain the precipitate;

(4) Dry the precipitate at 15-30°C for 2-3 minutes, add 30-100 μl RNase-free double-distilled water to fully dissolve the RNA, and store the RNA of the control and two treated samples at -70°C.

3. Synthesis of the first strand of cDNA

(1) Thaw template RNA on ice; thaw 5×gDNA Buffer, FQ-RT Primer Mix, 10×Fast RTBuffer, RNase-Free ddH2O at room temperature (15-25°C), and place on ice immediately after thawing. Each solution was vortexed before use, and briefly centrifuged (6000rpm, 30s) to collect the liquid remaining on the tube wall. In order to ensure the accuracy of the preparation of the reaction solution, when performing various reactions, the Mix was prepared first, and then distributed into each reaction tube.

(2) According to the following system, the gDNA removal system in RNA is used to prepare the mixed solution and mix thoroughly. Briefly centrifuge (6000rpm, 30s) and place at 42°C for 3min. Then place on ice.

gDNA removal reaction system:

5×gDNA Buffer 2μl

Total RNA 2μl

Make up to 10 μl with RNase-Free ddH ₂ O.

(3) Prepare the mixed solution according to the reverse transcription reaction system of the following system:

Reverse transcription reaction system:

10×Fast RT Buffer 2μl

RT Enzyme Mix 1μl

FQ-RT Primer Mix 2μl

Make up to 10 μl with RNase-Free ddH ₂ O.

(4) Add the Mix in the reverse transcription reaction to the reaction solution in the gDNA removal step, and mix well. Incubate at 42°C for 15 minutes, and at 95°C for 3 minutes, then place on ice to obtain cDNA for subsequent experiments, or store at low temperature.

4. The relative quantitative RT-PCR technique was used to detect the differential expression of the calmodulin gene ErCML30 expressed in the leaves of wild species of succulents under low temperature stress. The experiment was carried out on the 7500 fluorescence PCR instrument of ABI company. Gene expression was analyzed (see Figure 2).

(1) Relative quantitative RT-PCR reaction system:

The configuration of the relative quantitative RT-PCR reaction system of the calmodulin-like gene ErCML30:

The base sequence of the upstream primer QF is shown in SEQ ID NO: 4, and the base sequence of the downstream primer QR is shown in SEQ ID NO: 5.

The configuration of the internal reference gene 25SrRNA relative quantitative RT-PCR reaction system:

The base sequence of the upstream primer 25S-F is shown in SEQ ID NO:6, and the base sequence of the downstream primer 25S-R is shown in SEQ ID NO:7.

(2) Relative quantitative RT-PCR reaction procedure:

Pre-denaturation at 95°C for 15 minutes,

Denaturation at 95°C for 10s,

Anneal at 60°C for 32s, 40 cycles; test data were analyzed on ABI 7500 fluorescent quantitative PCR instrument.

Result analysis: by relative quantitative RT-PCR, the differential expression of the succulata calmodulin gene ErCML30 in the leaves of the succulata wild species at different low temperature stress times was detected (see Figure 2), and it was found that after low temperature treatment There was a significant difference in the expression of the sucrose calmodulin gene ErCML30 in the leaves of the succulent grass wild species. The sucrose calmodulin gene ErCML30 was almost not expressed in the absence of low temperature stress. After 24 hours of treatment, the succulent calmodulin gene ErCML30 The expression level in the wild species of succulata was 50 times that of the control, and the expression level of the calmodulin gene ErCML30 in the wild species of succulata was 75 times that of the control after 72 hours of treatment, indicating that the calmodulin gene ErCML30 of the succulent The expression level increased after the wild species of S. succulata was subjected to low temperature stress, and it increased with the increase of stress time. Therefore, the succulent grass calmodulin gene ErCML30 belongs to the inducible expression gene expressed by low temperature stress, indicating that the succulent grass calmodulin gene ErCML30 can quickly and persistently participate in the low temperature stress response after being subjected to low temperature stress.

sequence listing

<110> Yunnan Agricultural University

<120> Calmodulin gene ErCML30 of succulents expressed in wild species of succulents under low temperature stress

<160> 7

<170> SIP Sequence Listing 1.0

<210> 1

<211> 795

<212>DNA

<213> Canegrass wild species (E.fulvus)

<220>

<221> CDS

<222> (82)..(645)

<223> coding sequence

<220>

<221> 5'UTR

<222> (1)..(81)

<223> The 5' terminal region of the mature transcript that is not translated into protein

<220>

<221> 3'UTR

<222> (646)..(795)

<223> The 3' terminal region of the mature transcript that is not translated into protein

<220>

<221> protein_bind

<222> (163)..(636)

<223> Conserved domain EF domain

<220>

<221> primer_bind

<222> (1)..(20)

<223> Sites for PCR primer elements

<220>

<221> primer_bind

<222> (775)..(795)

<223> Sites for PCR primer elements

<400> 1

agaaataacc agccctcgca tccgcacgcc cacaaaacca cgaagaaaag aagaggagga 60

acgccgcaag ttcacgcgac g atg gag aac gcc act ccc gcc atc gcc aag 111

Met Glu Asn Ala Thr Pro Ala Ile Ala Lys

1 5 10

ccg tcg ctg tcc aag aag ccc tcg ccg tcg ttc cgc ctc cgg aac ggc 159

Pro Ser Leu Ser Lys Lys Pro Ser Pro Ser Phe Arg Leu Arg Asn Gly

15 20 25

agc ctc aac gct ctg cgc ctg cgc cgc gtg ttc gac ctc ttc gac cgc 207

Ser Leu Asn Ala Leu Arg Leu Arg Arg Val Phe Asp Leu Phe Asp Arg

30 35 40

aac ggc gac ggc gag atc acc ctc gac gag atg gcc tcg gcg ctc gac 255

Asn Gly Asp Gly Glu Ile Thr Leu Asp Glu Met Ala Ser Ala Leu Asp

45 50 55

tcg ctc ggc ctc ggc gcc gac cgc tcc ggc ctg gag gcc gcg gtg ggc 303

Ser Leu Gly Leu Gly Ala Asp Arg Ser Gly Leu Glu Ala Ala Val Gly

60 65 70

ggg tac atc ccg gcc ggc gcc gcg ggg ctc cgc ttc gac gac ttc gag 351

Gly Tyr Ile Pro Ala Gly Ala Ala Gly Leu Arg Phe Asp Asp Phe Glu

75 80 85 90

tcc ctc cac cgc gcg ctc ggg gac gcg ctg ttc ggc ccc atc ccg gag 399

Ser Leu His Arg Ala Leu Gly Asp Ala Leu Phe Gly Pro Ile Pro Glu

95 100 105

gag gtg cct gag gac gac gac gag ggg gac atg aag gag gcg ttc cgg 447

Glu Val Pro Glu Asp Asp Asp Glu Gly Asp Met Lys Glu Ala Phe Arg

110 115 120

gtg ttc gac gag gac ggt gac ggc ttc atc tcg gcc gcc gag ctg cag 495

Val Phe Asp Glu Asp Gly Asp Gly Phe Ile Ser Ala Ala Glu Leu Gln

125 130 135

gcc gtg ctc aag aag ctc ggt ctg ccg gag gct cgg aac ctg gcc act 543

Ala Val Leu Lys Lys Leu Gly Leu Pro Glu Ala Arg Asn Leu Ala Thr

140 145 150

gtg cag gag atg atc tgc aac gtc gac cgc aac tgc gac ggc cgc gtc 591

Val Gln Glu Met Ile Cys Asn Val Asp Arg Asn Cys Asp Gly Arg Val

155 160 165 170

gac ttc ggc gaa ttc aag aac atg atg cag ggg atc acc gtg tgg ggc 639

Asp Phe Gly Glu Phe Lys Asn Met Met Gln Gly Ile Thr Val Trp Gly

175 180 185

gct tag ctgcagagcg cctgcgttgt cagcccctct actgcgtggt ttcatgtatg 695

Ala

tatctaggca ccgtagaatg cgcttttcta cccggcgctt cttcctgttc ttgatcgcgc 755

agtggattct tgctgttctt ggctttgctc tctgactcga 795

<210> 2

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_

<222> (1)..(20)

<223> Upstream primer GP-F

<400> 2

agaaataacc agccctcgca 20

<210> 3

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_

<222> (1)..(20)

<223> downstream primer GP-R

<400> 3

tcgagtcaga gagcaaagcc 20

<210> 4

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_

<222> (1)..(20)

<223> Upstream Primer QF

<400> 4

ccctctactg cgtggtttca 20

<210> 5

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<221> misc_

<222> (1)..(20)

<223> downstream primer QR

<400> 5

tcgagtcaga gagcaaagcc 20

<210> 6

<211> 22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_

<222> (1)..(22)

<223> Upstream primer 25S-F

<400> 6

ataaccgcat caggtctcca ag 22

<210> 7

<211> 21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> misc_

<222> (1)..(21)

<223> downstream primer 25S-R

<400> 7

cctcagagcc aatccttttc c 21

Claims (3)

1. The calmodulin gene ErCML30 of the canegrass class expressed by low temperature stress in the wild species of canegrass, characterized in that: its full-length nucleotide sequence is shown in SEQ ID NO:1. 2. the special primer of the cane grass class calmodulin gene ErCML30 expressed by low temperature stress in the wild species of cane grass described in claim 1, it is characterized in that: described special primer is by upstream primer GP-F and downstream primer GP - composed of R, the base sequence of the upstream primer GP-F is shown in SEQ ID NO: 2, and the base sequence of the downstream primer GP-R is shown in SEQ ID NO: 3. 3. A method for detecting the differential expression of the calmodulin gene ErCML30 of the succulents in the wild species of succulents under low temperature stress, including the extraction of total RNA, the synthesis of the first strand of cDNA, and the detection of calcium in the succulents by relative quantitative RT-PCR The differential expression of the modulin gene ErCML30 in the wild species of Saccharomyces japonicus under low temperature stress, which is characterized by: In the extraction of total RNA, the seedling-stage wild species of Canegrass were set as the control group and the experimental group. The control group was grown at room temperature at 25°C. The experimental group was divided into treatment Ⅰ and treatment Ⅱ. Treatment 1 was grown at a low temperature of 4°C. Grow for 24 hours, and grow for 72 hours at a low temperature of 4°C for treatment II, collect the leaves of the control, treatment I and treatment II respectively, add liquid nitrogen and grind to obtain three samples, and extract total RNA from the three samples respectively; In the relative quantitative RT-PCR technique to detect the differential expression of the succulata calmodulin gene ErCML30 in the wild species of succulents under low temperature stress, the designed specific primers were used as RT-PCR primers to carry out gene differences between different treatments and controls Expression detection, described specific primer is made up of upstream primer QF and downstream primer QR, and described upstream primer QF nucleotide sequence is as shown in SEQID NO:4, and the nucleotide sequence of described downstream primer QR is as shown in SEQ ID NO:5 ; The full-length nucleotide sequence of the calmodulin gene ErCML30 of the canegrass class is shown in SEQ ID NO:1.