CN117210471A - Glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12, expression vector and application thereof - Google Patents
Glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12, expression vector and application thereof Download PDFInfo
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Abstract
The invention belongs to the fields of genetic engineering and molecular breeding, and discloses a glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12 in wheat, and an expression vector and application thereof. The cDNA sequence of the wheat TaCOBL-A12 gene is shown as SEQ ID NO.2, the DNA sequence of the coding region is shown as SEQ ID NO.1, and the coded amino acid sequence is shown as SEQ ID NO. 3. The gene is from common wheat (Triticum asetivum L.) in China spring. TaCOBL-A12 is enhanced by high temperature induced expression in China spring. The gene of the invention can obviously improve the heat resistance of the Arabidopsis through genetic transformation of the Arabidopsis. The wheat TaCOBL-A12 is expected to be used for genetic engineering breeding, and plays an important role in cultivating heat-resistant crop varieties.
Description
Technical Field
The invention belongs to the technical field of genetic engineering and molecular breeding, and particularly relates to a glycosyl phosphatidyl inositol anchoring protein (COBRA-Like) gene TaCOBL-A12, and an expression vector and application thereof.
Background
Wheat (Triticum asetivum L.) is one of the most important grain crops in China, and the yield of the wheat is directly related to national folk life, so that ensuring high and stable yield of the wheat is a primary target of wheat breeding. Wheat originates from the 'crescent soil' region of western asia, is a typical cool-loving crop and has poor high-temperature adaptability. In recent years, as global warming increases, the high temperature stress suffered by wheat during the growth period becomes an important threat restricting the stable production of wheat worldwide. The high temperature suffered during the growth period of wheat can cause growth defects of different degrees, the seedling stage suffers from high temperature to cause excessive growth of seedlings, weak growth vigor and organ dysplasia; the high temperature stress in the booting stage is easy to cause the pollen activity reduction, the flowering stage is shortened, and the fertilization effect is weakened; the high temperature in the grouting period directly influences the grouting degree of the seeds, so that the grouting period of the seeds is shortened to generate shrunken seeds. The yield loss caused by the proper growth temperature of the wheat is exceeded, and the yield of the wheat is reduced by 6% in the whole world every time the average air temperature rises by 1 ℃. Therefore, the genetic engineering is utilized to excavate the gene responding to the high temperature stress, the molecular mechanism of the high temperature stress is researched, and the cultivation of the heat-resistant new germplasm is the key for solving the damage of the high temperature stress.
The heat resistance of plants belongs to complex biological characters, and the regulatory genes involved in the heat resistance and the physiological and biochemical processes involved in the regulation of the heat resistance are very complex and various. Heat shock proteins (Heat Shock Protein, HSP) are the earliest discovered class of proteins involved in plant high temperature stress responses. When the plant is subjected to high temperature stress, the protein begins to unfold, and heat shock protein can be combined with the protein to prevent denaturation and depolymerization of the protein, maintain the bioactivity of the protein and slow down the damage of the high temperature stress to cells. In addition to heat shock proteins, heat shock transcription factors (Heat Shock Transcription Factor, HSF), dehydration response element binding proteins (DREB), polyprotein-binding factors (MBF 1 c), and the like are involved in high temperature stress responses. A large number of heat shock proteins and heat shock transcription factors were identified in Arabidopsis and their expression was demonstrated to enhance tolerance to high temperature stress.
Wheat has complex genetic characteristics in heat resistance and is greatly affected by the environment. Researchers have identified multiple heat-resistant Quantitative Trait Loci (QTLs) on multiple chromosomes of wheat, and cloning the genes behind QTL loci is difficult. But the genes, miRNAs, long-fragment non-coding RNAs, proteins and the like responding to high temperature stress can be identified by a reverse genetics method and by utilizing a comparative genomics method, a transcriptome method, a proteome method, an epigenetic group method and the like, and the important roles of candidate genes in the wheat resisting high temperature stress are explained by functional analysis. For example, wheat TaHsfs obtained by whole genome identification has obvious cycle and tissue specific expression patterns, and experiments prove that TaHsfA2-10 can improve the basal Heat resistance and the acquired Heat resistance of transgenic Arabidopsis seedlings (references: guo XL, yuan SN, zhang HN, zhang YY, zhang YJ, wang GY, li YQ, li GL (2020) Heat-response patterns of the Heat shock transcription factor family in advanced development stages of wheat (Triticum aestivum L.) and thermotolerance-regulation by TaHsfA2-10.BMC Plant Biol20 (1): 364.). 210 DREB genes were identified in wheat using reverse genetics methods, and overexpression of TaDREB3-AI in Arabidopsis increased resistance to heat stress (ref: niu X, luo T, zhao H, su Y, ji W, li H (2020) Identification of wheat DREB genes and functional characterization of TaDREB3 in response to abiotic stresses. Gene 740:144514.).
Disclosure of Invention
The invention clones a Glycosyl Phosphatidyl Inositol (GPI) anchoring protein gene TaCOBL-A12 from common wheat, and discovers that the gene is up-regulated to express after being induced by high temperature stress. Further constructs a TaCOBL-A12 gene over-expression vector under the promotion of a 35S promoter of cauliflower mosaic virus (CaMV), and converts the vector into arabidopsis thaliana Col-0 by using an agrobacterium inflorescence infection method to obtain T 0 The generation single plant is separated and screened to obtain homozygous T 3 And (5) replacing single plants. Homozygous T 3 The plant of the generation shows enhanced tolerance to high temperature. TaCOBL-A12 is expected to be used for genetic engineering breeding, and introduction of the TaCOBL-A12 into wheat varieties is expected to improve the high temperature tolerance of wheat.
The aim of the invention is realized by the following technical scheme:
the glycosyl phosphatidyl inositol anchor protein gene TaCOBL-A12, the nucleotide sequence of which is shown as follows:
the cDNA sequence corresponding to the glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12 is shown as follows:
the amino acid sequence of the protein encoded by the glycosyl phosphatidyl inositol anchor protein gene TaCOBL-A12 is shown as follows:
the invention also provides an expression vector which comprises the glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12.
The expression vector is pBI121. The construction method of the expression vector comprises the following steps: the TaCOBL-A12 gene was inserted between the XbaI and SacI cleavage sites of pBI121.
The invention also provides an application of the glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12 in creating high-temperature resistant plants or high-temperature resistant microorganisms.
Further, the refractory plant comprises wheat.
The beneficial effects of the invention are as follows: according to the invention, a glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12 and a protein TaCOBL-A12 coded by the glycosyl phosphatidyl inositol anchoring protein gene are cloned from wheat by a method combining bioinformatics and molecular cloning technology, and are inserted into an expression vector pBI121, so that an over-expression vector of the gene is introduced into arabidopsis thaliana, and the high expression of the TaCOBL-A12 can obviously improve the high temperature tolerance of plants. The TaCOBL-A12 overexpression vector is used for genetic engineering breeding, and can provide important gene resources for wheat stress-resistant molecular breeding.
Drawings
FIG. 1 shows agarose gel electrophoresis of TaCOBL-A12 cloned from cDNA.
FIG. 2 is an agarose gel electrophoresis of TaCOBL-A12 amplified from a cloning vector.
FIG. 3 is an agarose gel electrophoresis of the enzyme-digested linearized expression vector pBI121.
FIG. 4 shows the relative expression level of TaCOBL-A12 gene in homozygous Arabidopsis transformants and verification of heat resistance of plants.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the following description. The experimental methods used in the following examples are conventional methods unless otherwise specified. The material reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1 cloning of the coding region sequence of the TaCOBL-A12 Gene in seedlings induced by high temperature in China spring.
The wheat local variety China spring (well known public materials, ref. International Wheat Genome Sequencing Consortium (IWSSC) (2018) Science 361,661) is a wheat reference genome sequencing variety. A glycosyl phosphatidyl inositol anchoring protein gene (TaCOBL-A12) is cloned from the wheat variety China spring. The specific process is as follows:
1) Wheat COBLs gene search. First, 40 TaCOBLs in wheat were obtained by on-line alignment of the amino acid sequences of the glycosyl phosphatidyl inositol anchoring protein TaBr1 (reference: deng Q, kong Z, wu X, ma S, yuan Y, jia H, ma Z (2019) Cloning of a COBL gene determining brittleness in diploid wheat using a MapRseq app reach. Plant Science 285:141-150.) in wheat with the amino acid sequences of the wheat genome database (WheatOmics 1.0, http:// 202.194.139.32/blast. Html). Detecting the relative expression of TaCOBLs after high-temperature induction to obtain the gene TaCOBL-A12 subjected to induced expression.
2) And (3) obtaining cDNA of the Chinese spring wheat after high-temperature induction. The Chinese spring seedlings which are grown for 7 days in a plant growth box at 25 ℃/20 ℃ and in the light/dark for 14h/10h are transferred into a 42 ℃ growth box. Whole plants were harvested after 0.5h and 2h, respectively, rapidly placed in liquid nitrogen for freezing and stored at-70 ℃ for RNA extraction. Total RNA was extracted using TRIzol (SIGMA, MO, USA) according to the instructions and reverse transcribed using RevertAidTM Master Mix kit (Invitrogen, CA, USA) according to the instructions to obtain cDNA templates.
3) And (3) carrying out RT-PCR amplification to obtain the coding region fragment of TaCOBL-A12. Primers for amplifying TaCOBL-A12 were designed based on the gene sequence of TaCOBL-A12 in IWSSC RefSeq v1.1 (gene ID: traesCS6A02G 379800) as follows:
p1: ATGGCGGCGCTTTCTGGC (shown as SEQ ID NO. 4)
P2: TCAGACATAGTAGGCCAGCAGG (shown as SEQ ID NO. 5)
The full length of the coding region of TaCOBL-A12 in China spring is cloned by using the seedling cDNA which is induced by high temperature for 0.5h and 2h in China spring as a template and by using an RT-PCR technology. The specific amplification process is as follows: mu.l of cDNA template (50 ng/. Mu.l), 0.8. Mu.l of primer P1 and 0.8. Mu.l of primer P2, 6. Mu.l of 2mM dNTPs, 15. Mu.l of 2X PCR Buffer for KOD FX Neo, 0.3. Mu.l of KOD FX Neo (TOYOBO, japan) were added with water to 30. Mu.l. PCR amplification conditions: pre-denaturation at 94℃for 2min;98℃10s,60℃30s,68℃1min30s,36 cycles; extending at 68℃for 5min. The PCR products were electrophoresed on a 1.5% agarose gel at 100V for 20min to detect the size and specificity of the amplified band (FIG. 1), and 1344bp specific amplified band (Tiangen, china) was recovered.
4) Cloning and sequencing of the TaCOBL-A12 gene. The amplified and recovered specific bands were cloned into pEASY-Blunt Zero vector (full gold, china) and transformed into DH 5. Alpha. Competent cells. Primers P1 and P2 for amplifying TaCOBL-A12 are used for detecting the monoclonal, and the monoclonal containing the target gene TaCOBL-A12 is selected for sequencing. And analyzing the sequencing result to obtain the 1344bp TaCOBL-A12 coding region sequence.
EXAMPLE 2 amplification of the TaCOBL-A12 insert for constructing the pBI121 TaCOBL-A12 vector
The primers were designed based on the coding region sequence of the TaCOBL-A12 gene as follows:
p3: CACGGGGGACTCTAGAATGGCGGCGCTTTCTGGC (shown as SEQ ID NO. 6)
P4: GATCGGGGAAATTCGAGCTCTCAGACATAGTAGGCCAGCAGG (SEQ ID NO. 7).
The 5' ends of P3 and P4 introduce 15-20bp complementary sequences (underlined sequences) to the ends of the linearized vector, facilitating insertion of the amplified fragment into the expression vector pBI121 by a recombinase. Specific amplifications were as follows: a full-length fragment of the coding region 1344bp of TaCOBL-A12 was amplified using pEASY-Blunt Zero vector plasmid dilutions containing TaCOBL-A12 as templates. Amplification procedure: mu.l of plasmid template (20 ng/. Mu.l), 0.8. Mu.l of primer P3 and 0.8. Mu.l of primer P4, 6. Mu.l of 2mM dNTPs, 15. Mu.l of 2X PCR Buffer for KOD FX Neo, 0.3. Mu.l of KOD FX Neo (TOYOBO, japan) were added with water to 30. Mu.l. PCR amplification conditions: pre-denaturation at 94℃for 2min;98℃10s,60℃30s,68℃1min30s,36 cycles; extending at 68℃for 5min. The PCR products were subjected to 1.5% agarose gel electrophoresis at 100V for 20min, the size and specificity of the amplified bands were detected (FIG. 2), and the specific amplified bands were recovered (Tiangen, china).
EXAMPLE 3 construction of pBI121:TaCOBL-A12 vector
The pBI121 vector was digested with restriction enzymes XbaI and SacI, and the digested product was subjected to 1% agarose gel electrophoresis at 100V for 30min, and the digested fragment was detected (FIG. 3), whereby the digested product of pBI121 (Tiangen, china) was recovered. The recovered product of TaCOBL-A12 was combined with linearized pBI121 vector fragment using a recombinaseSnap Assembly Master Mix (TAKARA, japan) ligation products, ligation reaction was as follows: 1. Mu.l recovery of TaCOBL-A12 (47.5 ng/. Mu.l), 1. Mu.l restriction linearized pBI121 vector fragment (45 ng/. Mu.l), 2. Mu.l 5 XIn-Fusion Snap Assembly Master Mix, and water to 10. Mu.l. Connection reaction conditions: and rapidly placing on ice at 50 ℃ for 15 min. Ligation products were blotted with 2.5. Mu.l of transformed DH 5. Alpha. Competent cells, plated on LB solid plate medium containing 100mg/L kanamycin resistance, and monoclonal selected. After the monoclonal shaking culture, the bacterial liquid is used as a template, the P3 and P4 primers are used for PCR screening, the clone with the insert fragment is selected for sequencing, and the sequence of the insert fragment is verified, so that the recombinant vector pBI121 with the complete correct insert fragment is obtained, namely TaCOBL-A12.
Example 4 Agrobacterium GV3101 competent cell preparation and transformation
The Agrobacterium on the plates was picked and inoculated into 2ml LB (25 mg/L rifampicin) liquid medium and shake-cultured at 28℃and 220rpm overnight. 2ml of the overnight culture broth was transferred to 200ml of liquid LB medium containing the same antibiotic and cultured under the same conditions until od600=0.5-0.7. The bacterial solution was transferred to a 50ml sterile centrifuge tube, centrifuged at 4000rpm at 4℃for 10min and the supernatant was discarded. The thalli are washed 3 times by pre-cooled 10% sterile glycerol, resuspended by 2ml pre-cooled 10% sterile glycerol, packaged and then placed in liquid nitrogen for quick freezing, and stored at-70 ℃ for standby. Agrobacterium competent cells were removed and frozen and thawed on ice, and 2. Mu.l of pBI121: taCOBL-A12 expression vector plasmid (100 ng total) was added and mixed in 100. Mu.l of competent cells. The agrobacterial cell and plasmid mixture was aspirated and transferred into a pre-chilled cuvette (1 mm) and shocked at 2000V high pressure in an electroporator (BioRad, USA). Taking out the electric shock cup, adding 500 μl of precooled LB (containing 25mg/L rifampicin) culture medium, sucking, mixing, transferring the sucked bacterial liquid into a 1.5ml centrifuge tube, and shake culturing at 28deg.C at 200rpm for 5h. The cells were collected by centrifugation at 4000rpm for 3min, plated on LB plates containing 25mg/L rifampicin and 100mg/L kanamycin, and cultured in inversion at 28℃for 1-2 days. The PCR detection of positive clones facilitates the genetic transformation of Arabidopsis thaliana.
EXAMPLE 5TaCOBL-A12 transformation of Arabidopsis thaliana
The arabidopsis plants were watered enough one day before infection, and pod bearing fruits were cut off. Positive Agrobacterium strains were selected and cultured overnight in LB liquid medium (containing 25mg/L rifampicin and 100mg/L kana). The overnight culture broth 1:100 was inoculated into 200ml of the same antibiotic medium, and cultured at 28℃and 220rpm until the broth OD600 = 1.8-2.0. Fresh dip, 1/2ms,5.0% sucrose, 0.02% Silwet L-77 (soltreasure, china), ph=5.7 was prepared. Agrobacterium cells were collected at 4000rpm for 15min and resuspended with infestation liquid such that od600=0.8. The upper part of the tissue of Arabidopsis thaliana was immersed in the invasion dye for about 1 minute and gently shaken. The humidity was kept with a preservative film sleeve and protected from light overnight. After one day, the preservative film is uncovered, the arabidopsis thaliana is transferred into a normal growth incubator until the seeds are ripe, and all the seeds are harvested.
EXAMPLE 6 selection of transgenic plants and acquisition of homozygotes
T harvested after transformation 0 The seeds of the generation are sterilized and sown on MS culture medium containing 50mg/L kanamycin, vernalized for 3 days at 4 ℃, and then cultivated under normal illumination. After one week, the kanamycin-screened seedlings were identified, and the transformants were normally grown but not the transformants were not resistant to the chlorosis albino state due to the kanamycin resistance site on the expression vector. Resistance to transplant 1 Seedling generation, continuous culture and harvesting of T 1 Seed generation. Will T 1 Sowing the seeds of the generation on MS culture medium containing kanamycin for continuous screening, and obtaining the seed according to T 2 The plant separation ratio is used for judging whether the plant is inserted at a single point. Selecting T inserted by single point 2 Transplanting single plant of transgene, harvesting T by single plant 2 Seed generation, sowing on MS culture medium containing kanamycin, further screening homozygote, and obtaining homozygote without separation.
EXAMPLE 7 detection of expression level of TaCOBL-A12 Gene of transgenic Arabidopsis thaliana homozygote
Wild arabidopsis thaliana Col-0 and transgenic homozygote seeds are sterilized and spread on an MS culture medium, the whole plant is sampled after 14d of illumination culture, and the whole plant is quickly frozen in liquid nitrogen and stored at the temperature of minus 70 ℃ for RNA extraction. Total RNA was extracted using TRIzol (SIGMA, MO, USA) according to the instructions and reverse transcribed using RevertAidTM Master Mix kit (Invitrogen, CA, USA) according to the instructions to obtain cDNA templates. Real-time fluorescent quantitative PCR primers were designed based on the cDNA sequence of TaCOBL-A12 as follows:
p5: TACTTCAACGGCGACAACTG (shown as SEQ ID NO. 8)
P6: TGTGGAACCGAATGAACAGA (shown as SEQ ID NO. 9)
The relative expression of TaCOBL-A12 gene is detected by taking Arabidopsis ACTIN gene as an internal reference gene. Specific amplification by fluorescent quantitative PCR was as follows: mu.l of cDNA template (20 ng/. Mu.l), 0.5. Mu.l of primer P5 and 0.5. Mu.l of primer P6, 10. Mu.l of 2X SYBR Green Master Mix (Applied Biosystems, CA, USA) and water were added to 20. Mu.l. Using Quant Studio TM 6 Flex Real-time PCR systems (Applied Biosystems, CA, USA) were subjected to Real-time fluorescent quantitative PCR amplification according to the protocol of the reagent instructions. By 2 -ΔΔCT The relative expression level of the TaCOBL-A12 gene was calculated by the method. As shown in FIG. 4, the expression of the TaCOBL-A12 gene was detected in the transgenic homozygous plants of Arabidopsis thaliana.
Example 8 identification of Heat resistance of transgenic plants
The wild arabidopsis Col-0 is used as a control, about 300 seeds of the transgenic arabidopsis homozygote obtained by transformation screening are sterilized and then treated in a water bath kettle at 50 ℃ for 1 hour, and the seeds are uniformly mixed in an upside down manner every 20 minutes. The treated seeds are planted on an MS culture medium, the germination rate of the seeds is counted after the seeds are normally cultured for 14 days in an illumination incubator, and the plants which normally grow two cotyledons are counted as survival plants. The experiment was set up for at least 3 biological replicates and significance analysis was performed using t-test. Meanwhile, as shown in FIG. 4, the survival rate of the wild type Arabidopsis thaliana Col-0 is 25.60%, the survival rate of the homozygous overexpressed strain OE1 is 39.52%, the survival rate of the homozygous overexpressed strain OE2 is 28.21%, the survival rate of the homozygous overexpressed strain OE3 is 35.60%, the survival rate of the transgenic homozygote after heat treatment is improved by 2.61% -13.92% compared with that of the wild type Arabidopsis thaliana Col-0, the average improvement is 8.84%, and the expression quantity of the transgenic homozygote is positively correlated with that of the TaCOBL-A12 gene.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (7)
1. The glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-A12 is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.
2. The glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-a12 according to claim 1, wherein the cDNA sequence corresponding to the glycosyl phosphatidyl inositol anchoring protein gene TaCOBL-a12 is shown in SEQ ID No. 2.
3. A protein encoded by the glycosyl phosphatidyl inositol anchor protein gene TaCOBL-a12 of claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID No. 3.
4. An expression vector comprising the glycosyl phosphatidyl inositol anchor protein gene TaCOBL-a12 of claim 1.
5. An expression vector according to claim 3, wherein the expression vector comprises pBI121.
6. The use of the glycosyl phosphatidyl inositol anchor protein gene TaCOBL-a12 of claim 1 to create a high temperature resistant plant or high temperature resistant microorganism.
7. The use of claim 5, wherein the refractory plant comprises wheat.
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