CN116655762A - White konjak AaCaM3 gene, protein coded by same and application thereof - Google Patents

White konjak AaCaM3 gene, protein coded by same and application thereof Download PDF

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CN116655762A
CN116655762A CN202310700747.7A CN202310700747A CN116655762A CN 116655762 A CN116655762 A CN 116655762A CN 202310700747 A CN202310700747 A CN 202310700747A CN 116655762 A CN116655762 A CN 116655762A
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gene
aacam3
konjak
high temperature
seq
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牛义
王志敏
蒋学宽
张盛林
胡玲玉
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Research Institute Of Yibin Southwest University
Southwest University
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Research Institute Of Yibin Southwest University
Southwest University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

The invention relates to the field of plant molecular biology, in particular to a konjak calmodulin AaCaM3 gene, a protein coded by the same and application thereof. The full length of cDNA sequence of the gene is shown as SEQ ID No.1, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2. The AaCaM3 gene of the invention is transiently expressed in tobacco leaf cells and is positioned in cell nuclei and cytoplasm. The AaCaM3 gene has potential high temperature stress resistance, and the transcription level is obviously improved under high temperature treatment. The AaCaM3 gene expression vector is transferred into wild arabidopsis through an agrobacterium-mediated inflorescence infection method, and the result shows that an arabidopsis plant transformed with the AaCaM3 gene shows obvious high temperature stress resistance under high temperature stress, has important significance for cultivating plants with high temperature stress resistance, can be used for breeding heat-resistant new varieties of white konjak, and has excellent application prospect.

Description

White konjak AaCaM3 gene, protein coded by same and application thereof
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to a konjak AaCaM3 gene, a protein encoded by the konjak AaCaM3 gene and application of the konjak AaCaM3 gene.
Background
The high temperature stress is one of the most important environmental stresses, and can cause serious and irreversible damage to plants, the most intuitive damage to plants caused by the high temperature stress is that the morphology of crops is changed, and the external morphology change is mainly reflected in obvious inhibition effects on seed germination, plant morphology, yield, quality, reproductive development and the like of the crops, and the effects can occur at any stage of plant growth and development. High temperature stress can also cause damage to plants at the cellular level, particularly the cell membrane system can be severely affected, and leads to an increased probability of misfolding of proteins resulting in accumulation of protein aggregates, protein toxicity, and accumulation of Reactive Oxygen Species (ROS) in plant cells, causing cytoskeletal disintegration. Most current studies report that the molecular mechanism of plant regulation of high temperature stress is classified into the following: signal sensing and signal transduction pathways, transcription factor regulation pathways, ROS scavenging systems, and hormone regulation pathways.
Calcium ion (Ca) 2+ ) Is an essential element for keeping the integrity of plant cell walls, plays a role in stabilizing cell membranes and cell walls, and plays an important role in cell division, microtubule formation and post-harvest physiology of crops, ca 2+ Are typically stored in the vacuoles, mitochondria, and endoplasmic reticulum organelles of plant cells, where they are confirmed to be secondary messengers involved in signal transduction. Calmodulin CaM is a Ca that is ubiquitous in eukaryotes 2+ Susceptors, also Ca 2+ -an important member of the CaM signal transduction pathway. CaM is the most important multifunctional receptor protein in plant cells, and affects stress-tolerant functions of plants by participating in activities of various signal molecules. The current research shows that the CaM plays an important role in plant growth and development, stress resistance and other aspects, and has high research value.
White konjak (Amorphophallus albus) is a perennial monocotyledonous She Caoben plant of the genus konjak of the family Araceae, and is widely cultivated and planted in China because of its rich glucomannan content and good quality in underground enlarged corms of white konjak in the regions of origin. However, konjak is a bottom plant originated from tropical rain forest, is kept to be loved and warm and moist in long-term domestication cultivation, is prohibited from high-temperature and strong-light cultivation environment conditions, can show burn and heat injury symptoms when the cultivation environment is high-temperature for a long time, even falls down and dies, and seriously affects the growth, development and yield of konjak. Therefore, the invention clones and analyzes the sequence and the transgene function of the konjak calmodulin AaCaM3 gene, is helpful for comprehensively knowing the process of the AaCaM3 gene participating in konjak signal perception and signal transduction pathway to regulate and control high temperature stress, and provides gene resources for breeding of new konjak heat-resistant varieties and development of molecular markers.
Disclosure of Invention
In order to solve the problems, the invention provides a konjak AaCaM3 gene, a protein coded by the konjak AaCaM3 gene and application of the konjak AaCaM3 gene.
First, the present invention provides a white konjak AaCaM3 protein, which is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
The invention also provides a gene for encoding the konjak AaCaM3 protein.
Preferably, the sequence of the gene is shown as SEQ ID No. 1.
The invention also provides an over-expression vector containing the gene, a host cell and engineering bacteria.
The invention also provides application of the gene in cultivation of transgenic heat-resistant plants.
In one embodiment of the invention, the gene is transferred into the plant genome and overexpressed in the transgenic plant, so that the transgenic plant exhibits significant heat resistance under high temperature stress.
The invention obtains the calmodulin AaCaM3 gene from the white konjak leaves, which is located in the nucleus and cytoplasm. The qRT-PCR proves that the transcriptional level of the AaCaM3 gene can be obviously improved under high temperature treatment, and the expression quantity of the AaCaM3 gene can influence the high temperature stress resistance of plants and is closely related to the high temperature stress resistance of konjak. The plant expression vector of the AaCaM3 gene is constructed by utilizing a genetic engineering means and is transferred into wild type Arabidopsis thaliana, and the result shows that the transgenic plant shows obvious heat resistance under high temperature stress, the survival rate of the transgenic plant is obviously higher than that of the wild type under high temperature stress, and the method has important significance for cultivating plants with high temperature stress resistance capability and has excellent application prospect.
Drawings
FIG. 1 is a photograph of PCR electrophoresis of CDS of the calmodulin AaCaM3 gene of konjak obtained by cloning using the cDNA of konjak leaves as a template. Wherein M is 2K Plus II DNA Marker,1, and the black arrow represents the position of the amplified target gene.
FIG. 2 shows subcellular localization of the konjak calmodulin AaCaM3 gene transiently expressed in tobacco leaves. GFP: green fluorescent protein; mCherry: nuclear localization red fluorescent dye Marker; bright: bright field imaging; merge: pooled images of GFP, mCherry and Bright.
FIG. 3 shows the expression level of the calmodulin AaCaM3 gene of konjak in leaves of konjak treated with heat stress at 41℃for 0h, 1h,8h and 24 h.
FIG. 4 is a PCR identification of AaCaM3 transgenic Arabidopsis plants.
FIG. 5 shows the analysis of AaCaM3 gene expression in transgenic Arabidopsis.
FIG. 6 is a photograph showing normal growth of transgenic plants and wild type Arabidopsis under light at 25℃for 16 hours and dark conditions at 20℃for 8 hours without high temperature treatment. Wherein WT is wild type arabidopsis; l1 is transgenic Arabidopsis line 1, L4 is transgenic Arabidopsis line, and L5 is transgenic Arabidopsis line 5.
FIG. 7 is a photograph of transgenic plants and wild type Arabidopsis thaliana after heat treatment at 41℃for 1h 8h and 24h, and after recovery from growth for 3d under dark conditions of 25℃and 16h light, 20℃and 8 h. Wherein 41 ℃/1h is 41 ℃ heat treatment for 1h, and 41 ℃/8h is 41 ℃ heat treatment for 8h; 41 ℃/24h is heat treatment at 41 ℃ for 24h, the light irradiation is performed at 25 ℃/16h after the heat treatment is finished, the growth is recovered for 3d under the dark condition at 20 ℃/8h, and the WT is wild arabidopsis; l1 is transgenic Arabidopsis line 1, L4 is transgenic Arabidopsis line, and L5 is transgenic Arabidopsis line 5.
Detailed Description
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the examples are in accordance with conventional experimental conditions, such as the molecular cloning laboratory Manual of Sambrook et al (Sambrook J & Russell DW, molecular cloning: a laboratory manual, 2001), or in accordance with the manufacturer's instructions.
EXAMPLE 1 cloning of the cDNA sequence of the Amorphophallus konjac AaCaM3 Gene
1. Extraction of total RNA of konjak leaves
Fresh tender leaves of white konjak are cut, quickly placed into a centrifuge tube, quickly frozen in liquid nitrogen, and placed into an ultralow temperature refrigerator at-80 ℃ for standby. Extracting total RNA of white konjak leaves by using an RNA extraction kit: pouring white konjak leaves frozen and preserved in an ultralow temperature refrigerator at the temperature of minus 80 ℃ into a mortar, adding liquid nitrogen, rapidly grinding the white konjak leaves into powder, adding sample powder into a 1.5mL RNase-Free centrifuge tube which is prepared in advance and is mixed with 1mL cell lysate A and 300 mu L beta-mercaptoethanol, oscillating for 30s on a vortex oscillator, then standing for 15min at room temperature, and reversing for many times every 3 min; centrifuging at 12,000rpm in a low-temperature centrifuge at 4deg.C for 1min, and transferring the supernatant to a new 1.5mL RNase-Free centrifuge tube; adding 300 mu L of deproteinized solution B and 200 mu L of chloroform into a tube, oscillating for 30s on a vortex oscillator, fully and uniformly mixing the solution to be in an emulsion state, and standing for 2min at room temperature; after centrifugation at 12,000rpm for 10min in a low-temperature centrifuge at 4 ℃, the tube is obviously divided into three layers, and when the supernatant of 600-700 mu L of the upper layer is transferred to a new 1.5mL RNase-Free centrifuge tube, the suction of an intermediate layer and a lower layer organic phase containing DNA, protein and impurities is avoided; adding rinsing liquid C with the same volume as the supernatant into a centrifuge tube, adding the liquid which is mixed reversely into an adsorption column, centrifuging at 12,000rpm in a low-temperature centrifuge at 4 ℃ for 1min, pouring out the waste liquid in a collecting tube, and repeating the step once; adding 500 μl of column washing liquid D into the adsorption column, centrifuging at 12,000rpm in a low temperature centrifuge at 4deg.C for 1min, and pouring out the waste liquid in the collection tube, and repeating the steps once; centrifuging at 12,000rpm in a low-temperature centrifuge at 4deg.C for 3min, and volatilizing for 5min after uncovering; preparing a DNA digestion solution with a proper volume (each RNA sample needs 45 mu L of DNase buffer and 5 mu L of RNase-free Dnase I mixed solution), preheating the digestion solution for 1min at 37 ℃ after the preparation, adding the digestion solution into the center of a membrane of an adsorption column, and standing for 5min at room temperature; adding 500 μl of enzyme-removed solution E into the adsorption column, centrifuging at 12,000rpm in a low temperature centrifuge at 4deg.C for 1min, and pouring out the waste liquid in the collection tube, and repeating the steps once; centrifuging at 12,000rpm in a low-temperature centrifuge at 4deg.C for 3min, discarding the collecting tube, placing the adsorption column into a new 1.5mL RNase-Free centrifuge tube, and volatilizing at room temperature for 10min to thoroughly air dry the absolute ethanol in the adsorption column; adding 50 μl of RNA eluent F to the center of the adsorption column membrane, standing at room temperature for 3min, centrifuging at 12,000rpm in a low temperature centrifuge at 4deg.C for 2min, adding the first eluent to the adsorption column, and repeating. An appropriate amount of RNA sample is sucked, RNA integrity is detected by 1% ordinary agarose gel electrophoresis, and RNA concentration is detected by a Nanodrop instrument. Finally, the total RNA of the obtained white konjak leaves is stored in an ultralow temperature refrigerator at the temperature of-80 ℃ for standby.
2. Synthesis of white konjak leaf cDNA
cDNA of the white konjak leaves was synthesized using a reverse transcription kit (PrimeScript RT reagent Kit with gDNA Eraser): 2.0. Mu.L of 5X gDNA Eraser Buffer, 1.0. Mu.L of gDNA Eraser, 5. Mu.L of Total RNA and 2. Mu.L of RNase-Free ddH2O are sucked up, 10. Mu.L of genome DNA removal system is prepared on ice, the mixed system is centrifuged and placed in a PCR instrument, and the reaction procedure is 25 ℃ for 5min; after the PCR was completed, 4. Mu.L of 5X PrimeScript Buffer 2, 1. Mu. L PrimerScript RT Enzyme Mix 1, 1. Mu.L of RT Primer Mix and 4. Mu.L of RNase-Free ddH2O were added to the genome DNA removal system on ice to obtain a reverse transcription reaction system having a total volume of 20. Mu.L, and the mixed system was centrifuged and placed in a PCR apparatus at 37℃for 15 minutes; 85 ℃,5s. And (3) placing the synthesized white konjak leaf cDNA at the temperature of-20 ℃ for standby.
Blast alignment was performed in the NCBI database using the mRNA sequence of Amorphophallus konjac AaCaM3 in the transcriptome tested. Primer3 plus was used to design Primer AaCaM3-F (SEQ ID NO. 3): 5'-TACAGTTTTTACGTTTCACAATGGT-3' and AaCaM3-R (SEQ ID No. 4): 5'-GAATCGGAACTCACATGGTACT-3' PCR was performed using Phanta Max Super-Fidelity DNA Polymerase as a DNA polymerase for PCR reaction, the reaction system was as follows:
the reaction procedure was as follows:
after completion of the PCR reaction, the product was mixed with 5. Mu.L of 10X DNA Loading buffer and subjected to 1.5% agarose gel electrophoresis (electrophoresis conditions: 1 XTAE electrophoresis buffer; 110V,25 min) to examine the mixture (FIG. 1).
And (3) carrying out PCR product recovery by adopting a universal DNA purification recovery kit: adding the adsorption column CB2 into a collecting pipe, adding 500 μl Buffer BL into the collecting pipe, centrifuging at 12,000rpm for 1min, pouring out waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again; cutting an electrophoresis product containing a single target strip under an ultraviolet lamp, putting the cut electrophoresis product into a 1.5mL centrifuge tube, adding Buffer PC with equal volume into the centrifuge tube, and putting the centrifuge tube on a sol meter with the speed of 300rpm at 55 ℃ to fully dissolve gel blocks; adding the solution obtained after dissolution into a column-balanced adsorption column CB2, centrifuging at 12,000rpm for 1min, pouring out the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again; adding 600 μl Buffer PW into the adsorption column CB2, standing for 5min, centrifuging at 12,000rpm for 1min, pouring out the waste liquid in the collecting pipe, and placing the adsorption column CB2 into the collecting pipe, wherein the steps are repeated once; centrifuging at 12,000rpm for 3min, discarding the collecting tube, transferring the adsorption column CB2 to a new 1.5mL centrifuge tube, and volatilizing at room temperature for 10min to thoroughly dry the absolute ethyl alcohol in the adsorption column; 30 μl of Buffer EB was added to the center of the adsorption column membrane, and the mixture was left at room temperature for 2min, centrifuged at 12,000rpm for 2min at room temperature, and the first eluate was added to the adsorption column and repeated once more.
After product recovery, the product was ligated into a pEASY-Blunt Simple vector by pEASY-Blunt Simple Cloning Kit kit: to the centrifuge tube, 0.8. Mu.L pEASY-Blunt Simple Cloning Kit and 4.2. Mu.L gum were added to recover the product, and the mixed system was centrifuged and placed in a linker for 20min at 25 ℃.
The mixed system is transformed into Trans1-T1 E.coli competent cells by a heat shock transformation method. After the transformed competence was shake-activated at 37℃and 220rpm for 1 hour, 300. Mu.L of the supernatant was discarded after 5min at 4000rpm at room temperature, and after pipetting the bottom of the well-mixed tube with a gun, the cells were uniformly spread on LB solid resistant medium containing 50mg/L kanamycin solution (Kana), and cultured upside down overnight in a constant temperature incubator at 37 ℃. And (3) picking a single colony into a PCR detection system by using a sterilized toothpick, carrying out positive monoclonal screening, picking a correct monoclonal detected by electrophoresis by using the sterilized toothpick, placing the monoclonal on a culture medium into an LB/Kana liquid resistance culture medium, and sequencing, wherein the cDNA sequence is shown as SEQ ID No.1, and the coded amino acid sequence is shown as SEQ ID No. 2.
EXAMPLE 2 subcellular localization analysis of the Amorphophallus konjac AaCaM3 Gene
The CDS sequence of the AaCaM3 gene was subjected to cleavage site analysis using software BioXM 2.7, and primers containing the cleavage site were designed, aaCaM3-1300-F-Xba I (SEQ ID No. 5): 5'-GCTCTAGAATGCTGTGTCCACGTA-3'; aaCaM3-1300-R-Kpn I (SEQ ID No. 6): 5'-CGGGGTACCCTTGGCCATCATAACT-3'.
Amplifying by using the AaCaM3-Blunt Simple plasmid with correct sequence as a template to obtain the AaCaM3 gene CDS sequence containing Xba I and Kpn I enzyme cutting sites and removed the stop codon. The target gene and subcellular localization vector pCAMBIA1300 plasmid were extracted, double digestion reactions were performed with restriction enzymes Xba I and Kpn I, respectively, and the target gene and subcellular localization vector pCAMBIA1300 plasmid were recovered by 1.5% agarose gel electrophoresis. And (3) connecting the target gene AaCaM3 subjected to double enzyme digestion with a subcellular localization vector pCAMBIA1300 by using T4 DNA ligase, transferring the recombinant vector into a competent cell of Trans1-T1 escherichia coli, and then carrying out bacterial liquid PCR and sequencing after double enzyme digestion verification to ensure that the target gene sequence is successfully connected to the vector. The constructed vector plasmid is extracted and transferred into competent cells of agrobacterium LBA4404 by a chemical transformation method.
The stock solutions of AaCaM3-pCAMBIA1300-GFP and pCAMBIA1300-GFP, which had been competent with the transformed Agrobacterium tumefaciens LBA4404, were respectively prepared at 1:100 was added to YEB liquid medium containing 50. Mu.g/mL kan and 20. Mu.g/mL Rif at 28℃and propagated at 220rpm to OD 600 0.8-1.6. The cells were collected by centrifugation at 4000rpm for 10min at 4℃in a low temperature centrifuge, and the supernatant medium was discarded and the permeation buffer (10 mM MgCl) was injected with Agrobacterium 2 10mM MES-KOH, ph=5.6, 150 μm acetosyringone) was resuspended to adjust OD 600 The value is 0.6-0.8, and the mixture is kept stand for 2 hours at room temperature. Selecting robust leaf blades of Nicotiana benthamiana growing for 3-4 weeks, sucking the agrobacterium injection penetrating bacterial suspension by using a 1mL sterile syringe without a needle, avoiding veins at the back of the leaf, injecting bacterial liquid into the leaf blades, and repeating the injection operation until the leaf blades are completely injected. After injection, carrying out wet dark culture for 36-48 h, and then taking 1cm near the injection site 2 The left and right tobacco leaf tissues were slide-prepared, and GFP fluorescence was observed under a confocal microscope at 488nm (FIG. 2). The results showed that the expression product of the konjak calmodulin AaCaM3 gene was localized in the nucleus and cytoplasm.
EXAMPLE 3 analysis of expression level of the Amorphophallus konjac AaCaM3 Gene
Total RNA in the white konjak leaves subjected to heat stress treatment at 41℃for 1h,8h and 24h was extracted, respectively, and reverse transcribed into cDNA as a template (for a specific method, see example 1). According to the cDNA sequence of the konjak AaCaM3 gene, real-time fluorescent quantitative primers AaCaM3-RT-F (SEQ ID No. 7): 5'-TCTTCGACAAGGACCAGAACG-3' and AaCaM3-RT-R (SEQ ID No. 8): 5'-AACCTCCTCATCCGTCAACTTC-3' are designed by using a Primer3 plus on-line website. The specificity of the PCR is detected by common PCR, and the PCR specific amplification is ensuredOn the premise of increasing, the real-time fluorescence quantitative PCR can be carried out. The konjak EIF4A gene is taken as an internal reference gene, and the primer is AaEIF4A-RT-F (SEQ ID No. 9): 5'-ACAAGATGAGGAGCAGGG-3' and AaEIF4A-RT-R (SEQ ID No. 10): 5'-GGTGATAAGGACACGAGA-3'. Each reaction was repeated 3 times, and each biological repetition was repeated three more times. The PCR reaction procedure is that the PCR reaction is performed for 5min at 95 ℃;95 ℃ for 30s,56.5 ℃ for 30s,40 cycles; the dissolution profile was collected. Based on the Ct value obtained, 2 was used -ΔΔCT The expression levels of the AaCaM3 gene in the heat stress treatment of the white konjak leaves at 41℃for 1h,8h and 24h were calculated, respectively (FIG. 3). The results show that: the expression level of the AaCaM3 gene in the white konjak leaves increases with the increase of the heat treatment time, which shows that the change of the expression pattern of the AaCaM3 gene is closely related to heat stress.
Example 4 transformation of Amorphophallus konjac AaCaM3 Gene into Arabidopsis thaliana
1. Construction of plant transgenic vector AaCaM3-pBin35SRed3 of white konjak AaCaM3 gene
The CDS sequence of AaCaM3 gene was subjected to cleavage site analysis by using software BioXM 2.7, and cleavage site primers at both ends were designed, aaCaM3-pBin35SRed3-F (SEQ ID NO. 11): 5'-GCTCTAGAATGCTGTGTCCACGTATT-3'; aaCaM3-pBin35SRed3-R (SEQ ID NO. 12): 5'-CCGCTCGAGTCACTTGGCCATCATAAC-3'. Amplifying by using the AaCaM3-Blunt Simple plasmid with correct sequence as a template to obtain the AaCaM3 gene CDS sequence containing Xba I and Xho I enzyme cutting sites. The target gene and the overexpression vector pBin35SRed3 plasmid are respectively extracted, double digestion reactions are respectively carried out by using restriction enzymes Xba I and Xho I, and the target gene and the overexpression vector pBin35SRed3 plasmid are recovered after 1.5% agarose gel electrophoresis. And (3) connecting the target gene AaCaM3 subjected to double enzyme digestion with an overexpression vector pBin35SRed3 by using T4 DNA ligase, transferring the recombinant vector into a competent cell of the Trans1-T1 escherichia coli, and then carrying out bacterial liquid PCR and sequencing after double enzyme digestion verification to ensure that the target gene sequence is successfully connected to the vector. The constructed vector plasmid is extracted and transferred into competent cells of agrobacterium GV3101 by a chemical transformation method.
2. Transgenic expression vector AaCaM3-pBin35SRed3 transferred into wild type Arabidopsis thaliana
The transformed Agrobacterium tumefaciens GV3101 was sensedThe preservation bacterial liquid of AaCaM3-pBin35SRed3 in a state is 1:100 was added to 50mL YEB liquid medium containing 50. Mu.g/mL kan and 60. Mu.g/mL Rif, and propagated to OD in a shaker at 28℃and 220rpm 600 About 1.2 to 1.6; cutting the wild arabidopsis with the inflorescence growing for 4-5 weeks, and after the pod is formed, watering the chassis with water and waiting for infection; centrifuging at 4deg.C and 6000rpm for 10min with a low temperature centrifuge, collecting cell at the bottom of tube, re-suspending cell with 1/2 MS liquid medium (100 ml containing 5g sucrose) heavy suspension, and adjusting OD 600 The value is about 0.6 to 0.8; pouring the heavy suspension into a culture dish, adding 20 mu L of Silwet L-77 into the heavy suspension, uniformly mixing, and immersing the arabidopsis inflorescence into the heavy suspension for 60s; the infected arabidopsis is subjected to moisture preservation and dark culture for 24 hours, then is subjected to normal light culture, and is repeatedly infected according to inflorescence growth conditions every 5-7d, and the total of three times is performed.
3. Transgenic arabidopsis screening and phenotype identification of konjak AaCaM3 gene
After the Arabidopsis thaliana transformed with the AaCaM3 gene is naturally dried, collecting fruit pods, and selecting out seeds which emit red light under a handheld LUYOR-3415RG excitation light source from the collected mature seeds; planting the selected transgenic arabidopsis seeds into a culture pot mixed with peat soil, vermiculite and perlite according to the proportion of 4:1:1, culturing in a climatic chamber.
Cutting leaves after bolting the transgenic plants, and extracting DNA: extracting plant DNA by using a DNA rapid release method, adding a proper amount of tender leaves into a 1.5mL centrifuge tube containing 30 mu L TPS buffer, mashing the leaves and then placing the mashed leaves on ice; boiling water for 15min, and standing on ice for 2min; 270. Mu.L of double distilled water is added, and after shaking, the mixture is centrifuged at 12000rpm for 2min; storing in a refrigerator at-20deg.C for use.
The positive plant DNA of transgenic Arabidopsis thaliana was PCR-screened using vector primers pBin35SRed3-F and vector construction AaCaM3-pBin35SRed3-R as controls. PCR amplification procedure: 94 ℃ for 1min30s; 34 cycles were performed at 94℃for 20s,58℃for 20s, and 72℃for 45 s; 72 ℃ for 5min; preserving at 4 ℃. After the completion of the reaction, 1% agarose gel electrophoresis was performed, and detection in a gel imaging system (FIG. 4) revealed that 8 positive transgenic Arabidopsis plants containing the AaCaM3 gene were obtained in total.
Using cDNA of wild type Arabidopsis thaliana as a template, real-time fluorescent quantitative primers AaCaM3-RT-F (SEQ ID NO. 7): 5' -TCTTCGACAAGGACCAGAACG-3 and AaCaM3-RT-R (SEQ ID NO. 8): 5'-AACCTCCTCATCCGTCAACTTC-3', and reference gene Arabidopsis thaliana AtTUB2-qPCR-F (SEQ ID NO. 13) were used: 5'-ATCCGTGAAGAGTACCCAGAT-3'; atTUB2-qPCR-R (SEQ ID NO. 14): 5'-AAGAACCATGCACTCATCA GC-3' as a control. The expression level of AaCaM3 gene was analyzed on positive plants of transgenic arabidopsis thaliana, and a total of 4 transgenic lines were obtained (fig. 5).
Among the 3 transgenic lines L1, L4 and L5, heat stress-resistant phenotypes were identified, and the heat-treated survival rate phenotypes of these Arabidopsis were observed and photographed: adding 1ml of 70% absolute ethyl alcohol into a centrifuge tube of the collected seeds of the transgenic arabidopsis plants and wild arabidopsis seeds, reversing the centrifuge tube for 5-8 times every 20s, and discarding the absolute ethyl alcohol after 1 min; adding 1ml of 10% sodium hypochlorite solution into the centrifuge tube, reversing 5-8 times every 3min, and discarding absolute ethyl alcohol after 10 min; with ddH 2 After O is washed for 5 times, the transgenic seeds and the wild seeds are sown in the same square culture dish in different areas, the sealed culture dish is placed in a climatic incubator after being sealed by a sealing film and placed at the temperature of 4 ℃ for Wen Chunhua d, and the culture condition is that the light is applied at 23 ℃/16h and the dark is applied at 20 ℃/8 h. After 14d, according to the growth condition of the arabidopsis, changing the temperature of the artificial climate incubator to 41 ℃, taking out corresponding culture dishes after 1h,6h and 12h of treatment according to a pre-schedule, and after 3d of growth recovery under the dark conditions of 23 ℃/16h illumination and 20 ℃/8h, observing the survival rate of the arabidopsis. The results show that: the transgenic and wild arabidopsis seedlings are hardly affected after being treated for 1h at 41 ℃, and the plant survival rate is not greatly different; differences between transgenic and wild type seedlings began to appear after 6h treatment at 41 ℃, both began to appear a certain amount of wilting and death, but the survival rate of transgenic plants was significantly higher than that of wild type; after 12h of treatment at 41 ℃, the transgenic seedlings and the wild seedlings die in a large amount, but the survival rate of the transgenic plants is more than 60 percent and is obviously higher than that of the wild seedlings, and the survival rate of the transgenic plants is obviously higher than that of the non-transgenic plantsWild-type Arabidopsis thaliana of the gene (FIGS. 6-7, table 1).
TABLE 1 statistical table of survival after Heat treatment of wild type and transgenic Arabidopsis thaliana
The results show that: the AaCaM3 gene can enhance the heat resistance of the arabidopsis, and the transgenic arabidopsis material of the AaCaM3 gene can be used for improving the heat resistance of plants, so that the heat resistance of the plants is improved, and the variety breeding with the capability of resisting high temperature stress is facilitated.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The konjak AaCaM3 protein is:
1) A protein consisting of the amino acids shown in SEQ ID No. 2; or (b)
2) A protein derived from 1) which has equivalent activity and is obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in SEQ ID No. 2.
2. A gene encoding the konjak AaCaM3 protein of claim 1.
3. The gene of claim 2, wherein the sequence is set forth in SEQ ID No. 1.
4. A vector comprising the gene of claim 2 or 3.
5. A host cell comprising the vector of claim 4.
6. An engineered bacterium comprising the gene of claim 2 or 3.
7. Use of a gene according to claim 2 or 3 for the cultivation of transgenic heat-resistant plants.
8. The use according to claim 7, wherein the gene according to claim 2 or 3 is transferred into the genome of a plant and overexpressed in a transgenic plant, such that the transgenic plant exhibits pronounced heat tolerance under high temperature stress.
9. A method for constructing a transgenic plant, wherein the transgenic plant is obtained by transferring the gene of claim 2 or 3 into a plant genome and screening.
10. The method of claim 9, wherein the transgenic plant has a higher survival rate under high temperature stress and exhibits significant heat resistance compared to the wild type.
CN202310700747.7A 2023-06-13 2023-06-13 White konjak AaCaM3 gene, protein coded by same and application thereof Pending CN116655762A (en)

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