CN118063577A - Pineapple Ca2+/H+Antiport protein AcCAX, isolated nucleic acid molecule and application thereof - Google Patents
Pineapple Ca2+/H+Antiport protein AcCAX, isolated nucleic acid molecule and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant gene targets, and particularly relates to pineapple Ca 2+/H+ antiport protein AcCAX, an isolated nucleic acid molecule and application thereof. The amino acid sequence of the pineapple Ca 2+/H+ antiport protein comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence comprising a sequence identical to SEQ ID NO:2, wherein the sequence homology is more than or equal to 85%. The Ca 2+/H+ antiport protein AcCAX3 in the pineapple can play an important role in the pineapple water-borne heart disease process by regulating and controlling the calcium ion steady state in cells; the high-quality pineapple germplasm with the water-resistant heart disease can be cultivated by utilizing the molecular breeding technologies such as transgene, gene fixed-point editing and the like in the later period, so that a new thought and research direction are provided for cultivating the water-resistant heart disease variety, the important problems faced by the pineapple industry are really solved, and the economic benefit and the rapid development of the pineapple industry are promoted.
Description
Technical Field
The invention belongs to the technical field of plant gene targets, and particularly relates to pineapple Ca 2+/H+ antiport protein AcCAX, an isolated nucleic acid molecule and application thereof.
Background
Pineapple (Ananascomosus l., merr.) is a perennial evergreen herb fruit tree of the genus pineapple of the family anaceae, and is juxtaposed with banana, coconut, mango as four major tropical fruits. The pineapple in China has long planting history, the planting area and the yield are respectively in the fourth and sixth world, and the pineapple is a special and efficient tropical economic crop and is also an important economic source for peasants in hot-working areas. In recent years, the watery heart disease occurs in a large area in the major pineapple producing area. The watery heart disease is a physiological disease before harvest, the interstitial space in the diseased fruits is filled with cell fluid and presents a water stain shape, and when serious, vinasse flavor and malodor flavor are emitted, so that the edible quality and commodity value of the fruits are greatly reduced. In 2018, the quality of fruits is reduced due to the epidemic of the water-borne heart disease, so that Xuwen fresh pineapple fruits are sold at base, and a great deal of losses are brought to growers under the condition of diapause and rotten market. The statistics shows that the area of the diapause is as high as 10 mu, and the proportion of pineapple growers is over 50%. Therefore, the method for exploring the pathogenesis of the pineapple water-borne heart disease, digging key genes of the water-borne heart disease and cultivating the high-quality pineapple variety with the water-borne heart disease is a fundamental way for solving the water-borne heart disease, so that the bottleneck problem faced by the pineapple industry can be effectively solved, and the healthy and rapid development of the pineapple industry is promoted.
Pineapple water-borne heart disease is a complex physiological and biochemical process, and the cause of the pineapple water-borne heart disease is not completely analyzed, and researches show that the incidence of the pineapple water-borne heart disease is closely related to external environment factors, plant growth and development states, endogenous hormone levels, mineral nutrition in fruits, sugar content and the like. The study also shows that the pineapple water-borne heart disease can be obviously induced by carrying out high-temperature treatment at 38 ℃ and low-temperature treatment at 10 ℃ 50 days after the pineapple flowers are removed. In addition, the size of pineapple coronary buds is inversely related to the incidence of the water-borne heart disease, the incidence of the pineapple coronary buds is lower, the size of plant leaves is positively related to the incidence of the water-borne heart disease, and the incidence of the pineapple coronary buds is higher when the leaves are more exuberant. As early as 1999 Chen et al found that, starting from 6 weeks of pineapple harvest, the content of sucrose and cell wall invertase in pineapple fruit gradually increased, and the cell wall invertase can break down sucrose into glucose and fructose, doubling the osmotic pressure within the cell. The concentration of calcium ions in fruits rapidly decreases from 8 weeks before harvest, and calcium ions are known to be an important component constituting the cell wall skeleton, and as the concentration of calcium ions decreases, the permeability of cell membranes gradually increases, causing abnormal intracellular osmotic pressure and inducing a water-borne heart disease.
Calcium is a very important nutrient element in plants, and calcium in plants exists mainly in the form of free calcium and bound calcium, and is widely distributed in cell nuclei, cytoplasm, vacuoles, cell membranes and cell walls, wherein vacuoles are calcium stores in cells and are important for regulating osmotic pressure in cells. Calcium is not only a very important nutrient element in plants, but also plays an important role in stabilizing plant cell membranes and cell walls, regulating and controlling enzymes, regulating permeation and the like. For example, ca 2+ can play an important role in plants as a second messenger involved in plant response to biotic and abiotic stresses. Can be combined with exposed carboxyl of methyl galacturonate to form calcium pectate, so that the cell wall structure is more stable, and can be coupled with phospholipid and protein on cell membrane to stabilize the cell membrane structure. In the later stage of cell mitosis, the cell plate separating two daughter cells mainly consists of calcium pectate, and calcium deficiency influences the formation of the cell plate and spindle filaments, so that the daughter cells cannot be separated in the cell division process, a binuclear phenomenon occurs, and finally the cells die. In addition, ca 2+, as a divalent cation, can be transported into the cytoplasm as well as vacuoles through ion channels, involved in regulation of cytoplasmic osmotic pressure. Of these CAXs (Ca 2+/H+ exchange) is a Ca 2+/H+ antiport protein which is mainly located on the vacuole membrane in plants, and is responsible for the transport of Ca 2+ from cytoplasm to vacuole, and is important for the maintenance of cell osmotic pressure.
The research shows that 6 CAXs proteins exist in Arabidopsis, wherein the mutation of AtCAX can effectively reduce the content of Ca 2+ and the activity of Ca 2+/H+ antiporter protein in vacuoles, and enhance the tolerance of plants to freezing stress. Over-expression of AtCAX a in arabidopsis thaliana into tomato can lead to a significant increase in ion permeability of cell membranes in tomato pulp cells, a decrease in the content of Ca 2+ in apoplast and cytoplasm, an increase in the content of Ca 2+ in vacuoles, abnormal cell osmotic pressure and occurrence of plasma wall separation phenomena, and finally cell rupture and death. Apples mainly comprise 11 CAXs proteins, and can be involved in the response of plants to various abiotic stresses. For example, when apples are infested with cold stress, this results in a rapid increase in the content of calcium ions in the cytoplasm, and after calcium ion-dependent protein kinases receive Ca 2+ signals, a series of cascade responses are initiated to help plants resist the stress. However, sustained high Ca 2+ concentrations in the cytoplasm can be toxic to plant cells, where high calcium signaling can induce expression of the BHLH4 transcription factor in apples. BHLH4 can bind to the promoter region of CAXs protein to activate its expression, promote transport of cytosolic Ca 2+ to vacuoles for storage, reduce cytosolic Ca 2+ concentration, and negatively regulate cold signaling pathways. In conclusion, calcium plays an important role in the growth and development process of plants, and CAXs protein is used as Ca 2+/H+ antiport protein on cell membranes to participate in various processes such as calcium ion transportation in plants, cell osmotic pressure maintenance and the like.
CAXs protein plays an indispensable role in Arabidopsis, rice, apple, tomato, etc., while in pineapple, the function of CAXs protein has not been studied in detail.
Disclosure of Invention
Aiming at the problems, the invention aims to provide pineapple Ca 2+/H+ antiport protein AcCAX and a gene AcCAX3 for encoding the pineapple Ca 2+/H+ antiport protein, and transient expression of AcCAX3 in pineapple fruits can obviously inhibit pineapple aquatic heart disease, which indicates that the gene plays an important role in the pineapple aquatic heart disease process.
In order to achieve the above purpose, the present invention may adopt the following technical scheme:
In one aspect, the invention provides pineapple Ca 2+/H+ antiport protein AcCAX, the amino acid sequence of which comprises the amino acid sequence as shown in SEQ ID NO:2 or a sequence as set forth in seq id no:2, wherein the sequence homology is more than or equal to 85%.
In another aspect, the invention provides an isolated nucleic acid molecule encoding pineapple Ca 2+/H+ antiport protein AcCAX of the invention.
In yet another aspect, the invention provides a recombinant expression vector comprising an isolated nucleic acid molecule of the invention.
In a further aspect, the invention provides a recombinant engineering bacterium comprising an isolated nucleic acid molecule of the invention or a recombinant expression vector of the invention.
In a further aspect, the invention provides a recombinant engineered cell comprising an isolated nucleic acid molecule of the invention or a recombinant expression vector of the invention or a recombinant engineered bacterium of the invention.
In a further aspect, the invention provides the use of pineapple Ca 2+/H+ antiport protein AcCAX of the invention or an isolated nucleic acid molecule of the invention in genetic breeding of pineapple.
In a further aspect, the invention provides the use of pineapple Ca 2+/H+ antiport protein AcCAX of the invention or of an isolated nucleic acid molecule of the invention for the cultivation of transgenic plants.
In a further aspect, the invention provides an application of the pineapple Ca 2+/H+ antiport protein AcCAX3 expression promoter in inhibiting pineapple aquatic heart disease.
In yet another aspect, the invention provides a method of controlling calcium ion homeostasis in pineapple cells, comprising: by modulating pineapple Ca 2+/H+ antiport AcCAX3 content in pineapple or modulating the expression level of an isolated nucleic acid molecule in pineapple.
The beneficial effects of the invention at least comprise: the instant expression of the Ca 2+/H+ antiport protein AcCAX3 in the pineapple can obviously inhibit the pineapple aquatic heart disease, which indicates that the Ca 2+/H+ antiport protein plays an important role in the pineapple aquatic heart disease process; the high-quality pineapple germplasm with the water-resistant heart disease can be cultivated by utilizing molecular breeding technologies such as transgene, gene fixed-point editing and the like in the later period, so that a new thought and research direction are provided for cultivating the water-resistant heart disease variety, the serious problems faced by the pineapple industry are really solved, and the economic benefit and the rapid development of the pineapple industry are promoted.
Drawings
FIG. 1 is a schematic diagram of AcCAX gene cloning and functional verification techniques according to the present invention;
FIG. 2 is a gel electrophoresis diagram of PCR amplification of coding region sequence of pineapple AcCAX gene in the present invention;
FIG. 3 shows the result of NCBI prediction AcCAX CDS sequence alignment with the actual clone AcCAX CDS sequence;
FIG. 4 shows the result of NCBI predicted AcCAX protein sequence alignment with the actual clone AcCAX protein sequence;
FIG. 5 shows AcCAX transcriptional levels induced by varying concentrations of CaCl 2;
FIG. 6 shows MgCl 2、MnCl2 and NaCl induced AcCAX3 transcript levels;
FIG. 7 shows the calcium content of pineapple after various days of flower metabolism;
FIG. 8 is a plot of AcCAX transcript levels as a function of decreasing endogenous calcium ion levels;
FIG. 9 shows the transcript levels of AcCAX in pineapple fruits, stems, old leaves, young leaves and inflorescences;
FIG. 10 is a view of AcCAX subcellular localization by confocal microscopy;
FIG. 11 shows the AcCAX gene of the present invention in fruits with different degrees of pineapple's water-bearing heart disease;
FIG. 12 shows transcript levels of AcCAX genes of the invention in fruits of pineapple with different degrees of water-borne heart disease;
FIG. 13 shows that the instant pineapple fruit transformation of AcCAX of the invention can significantly inhibit the pineapple water-borne heart disease;
fig. 14 shows that the instant pineapple fruit transformation of AcCAX of the invention can significantly inhibit the area of pineapple water-borne heart disease lesions.
Detailed Description
The examples are presented for better illustration of the invention, but the invention is not limited to the examples. Those skilled in the art will appreciate that various modifications and adaptations of the embodiments described above are possible in light of the above teachings and are intended to be within the scope of the invention.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless the context clearly differs, singular forms of expression include plural forms of expression. As used herein, it is understood that terms such as "comprising," "having," "including," and the like are intended to indicate the presence of a feature, number, operation, component, part, element, material, or combination. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, elements, materials or combinations thereof may be present or added. As used herein, "/" may be interpreted as "and" or "as appropriate.
In the present invention, "AcCAX3" represents a gene name, and "AcCAX3" represents a protein name.
An embodiment of the present invention provides a pineapple Ca 2+/H+ antiport protein AcCAX, the amino acid sequence of which comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence comprising a sequence identical to SEQ ID NO:2, wherein the sequence homology is more than or equal to 85%.
Specifically, SEQ ID NO:2 is as follows:
MPLKVLSTVLLRIALLVAMFHSRGNWAYIWTILHPTARPNWPAAWVFALSLIGLAPLAERVSFLSEQIAEYAGPTVGGLLNATCGNAPELIIALLALHNGKLEVLKWSLLGSILSNLLLVLGSSLVFGGFANLGNERPFDRRQSDVCLSLLLLGALCHVMALIFRFITNTSEYKGSSITVLKLSRPCSIVMLLAYAGCLFFQLKTHHQFFEAREDESDEDDDIVSSAPVIGFTSAIVWLLGMTAVIAILSDYVVSTIEAASESWGLPVSFISVILLPIVGNAAEHAGAIIFAAKNKIDITLGVALGSSTQISMFVIPLTLVVAWIKGVPMDLNFGFLETASLVMAILITAFVLQDGNWHYMKGFILFLCYIVVAICFFILKTSPNPTNGGHLDDD.
It should be noted that pineapple's water-borne heart disease is a complex physiological and biochemical process, the cause of which is not completely resolved, and researches have shown that the incidence of the water-borne heart disease is closely related to external environmental factors, plant growth and development states, endogenous hormone levels, mineral nutrition in fruits, sugar content and the like. However, until now, all studies on pineapple's aqueous heart disease have focused on physiological and biochemical levels, and no further excavation has been made from the gene level. However, only by starting from the gene level, the key genes affecting the water-borne heart disease are deeply mined, the pathogenesis of the water-borne heart disease is explored, and the water-borne heart disease-resistant high-quality pineapple varieties are cultivated based on the gene level, so that the water-borne heart disease can be fundamentally solved, and the important problem in pineapple production is really solved.
It should be noted that AcCAX's 3 transcript levels were significantly up-regulated in mild and down-regulated in moderate as well as severe watery heart disease. The instant conversion of AcCAX < 3 > into pineapple fruit can obviously inhibit pineapple aquatic heart disease, which shows that AcCAX < 3 > protein plays an important role in the pineapple aquatic heart disease process.
Another embodiment of the invention provides an isolated nucleic acid molecule encoding pineapple Ca 2+/H+ antiport protein AcCAX3 of the invention. It should be noted that, the pineapple Ca 2+/H+ antiport protein AcCAX is an isolated nucleic acid molecule in the invention, which can be obtained by optimizing the pineapple Ca 2+/H+ antiport protein family gene AcCAX; wherein, pineapple Ca 2+/H+ antiport gene sequence is as SEQ ID NO: 1. In addition, optimization methods are well known in the art, such as promoter and codon optimization.
Specifically, SEQ ID NO: the sequence 1 is as follows:
atgccgctgaaagtattatctacagtacttcttcggatagcattacttgtcgctatgttccatagcaggggcaactgggcttatatatggacaatattgcaccctacagcacggccaaattggcctgcggcttgggtgtttgccttgagcttaataggccttgcccctcttgccgaacgcgtgagctttctcagcgagcagattgcagagtatgctggtccaactgttggtggcctactgaatgcgacatgcggtaatgcccctgagctgataatagcgctactagcactgcacaatggtaagctagaagtcctaaaatggtctctcctcggctccattctctccaaccttctgctagtccttggctcctcacttgtctttggcggatttgccaaccttggcaatgaacggccatttgatcgaaggcaatcagatgtatgcttaagcctgcttctattgggtgcgttgtgccatgtcatggctttaatttttcggtttatcacgaacactagtgagtataaaggatcatcgatcacagtactcaagctctcaagaccgtgcagcattgtgatgctcctggcctatgctggatgcctcttcttccagctgaaaacccatcaccagttctttgaagcacgagaggatgaatctgatgaggatgatgatatcgtatcatcggcacctgttattggatttactagtgcaattgtctggctgcttgggatgactgctgtaatcgcgatcttatcagattatgtcgttagtaccattgaggcagcatcagagtcatggggcttaccggttagcttcataagtgttatattacttcctattgtgggtaacgccgctgagcatgcaggtgcaatcatcttcgcagccaaaaacaaaattgatatcaccttgggtgtagccttgggctcatcgacgcaaatctccatgtttgtgattcctttgacattagttgtagcatggataaaaggtgtccctatggatctcaacttcggcttcctcgaaactgcatcactggttatggcaattctcattactgccttcgtacttcaggatggaaattggcactacatgaagggttttattctcttcctctgctacattgttgtggccatatgctttttcatcctcaaaacctctccaaatccaacaaatggcggtcacttggacgacgactaa.
a further embodiment of the invention provides a recombinant expression vector comprising an isolated nucleic acid molecule of the invention.
In a further embodiment, the invention provides a recombinant engineering bacterium comprising an isolated nucleic acid molecule of the invention or a recombinant expression vector of the invention.
In a further embodiment, the invention provides a recombinant engineered cell comprising an isolated nucleic acid molecule of the invention or a recombinant expression vector of the invention or a recombinant engineered bacterium of the invention.
The isolated nucleic acid molecules of the present invention may be prepared into recombinant expression vectors, recombinant engineering bacteria or recombinant engineering cells. The pineapple Ca 2+/H+ antiport protein gene AcCAX3 in the invention can obviously inhibit pineapple water-borne heart disease, and based on the pineapple Ca 2+/H+ antiport protein gene AcCAX, the recombinant expression vector, the recombinant engineering bacteria and the recombinant engineering cells containing the isolated nucleic acid molecules in the invention are transfected into plants, and can also inhibit the water-borne heart disease of the plants.
In a further embodiment, the invention provides the use of pineapple Ca 2+/H+ antiport protein AcCAX3 of the invention or an isolated nucleic acid molecule of the invention in genetic breeding of pineapple.
It should be noted that the traditional hybridization breeding has the advantages of time and labor consumption, large workload, low breeding efficiency, long period required for breeding new varieties of pineapple with water-resistant heart disease, high efficiency, orientation, short period and the like.
In a further embodiment, the invention provides the use of pineapple Ca 2+/H+ antiport protein AcCAX of the invention or of an isolated nucleic acid molecule of the invention for the cultivation of transgenic plants.
The invention further provides an application of the pineapple Ca 2+/H+ antiport protein AcCAX3 expression promoter in inhibiting pineapple aquatic heart disease.
In some embodiments, the pineapple Ca 2+/H+ antiport AcCAX3 expression promoter is CaCl 2.
It should be noted that CaCl 2 can induce pineapple Ca 2+/H+ antiporter AcCAX3 to express, so CaCl 2 can inhibit pineapple aquatic heart disease by inducing AcCAX3 to express.
In another embodiment, the present invention provides a method for controlling calcium ion homeostasis in pineapple cells, comprising: by modulating pineapple Ca 2+/H+ antiport AcCAX3 content in pineapple or modulating the expression level of an isolated nucleic acid molecule in pineapple.
For a better understanding of the present invention, the content of the present invention is further elucidated below in connection with the specific examples, but the content of the present invention is not limited to the examples below.
In the following examples, acCAX gene cloning and functional verification were performed according to the technical route shown in FIG. 1.
1. AcCAX3 Gene amplification sequencing
Construction of pineapple cDNA library
Taking pineapple mature pulp tissue, and extracting total RNA by using an RNA extraction kit (product number: 0416-100GK type) of Beijing Hua Vietnam biological Co., ltd; the obtained total RNA was used as a template, and cDNA double strand was synthesized by using a reverse transcription kit (cat# M20105) from Beijing full gold biology Co., ltd under the action of reverse transcriptase and polymerase.
(Two) AcCAX Gene amplification
(1) 4 Specific primers were designed as follows:
AcCAX3-TF-ATGCCGCTGAAAGTATTATCTACA；
AcCAX3-TR-GTCGTCGTCCAAGTGACC；
AcCAX3-SmaI-F-CCCGGGATGCCGCTGAAAGTATTATCTACA；
AcCAX3-XbaI-R-TCTAGAGTCGTCGTCCAAGTGACC。
(2) Amplification of AcCAX Gene
The AcCAX gene is amplified according to the primer sequence, and specifically comprises the following steps: the cDNA was used as a template for amplification using the primers designed as described above and Phusion Hi-Fi enzyme, the specific amplification system is shown in Table 1 below, the amplification procedure is shown in Table 2 below, and the list of primers is the above primers (AcCAX-TF and AcCAX-TR).
TABLE 1 Phusion enzyme amplification System
| Phusion enzyme amplification system | 30μL |
| 5×HF buffer | 6μL |
| 10mM dNTPs | 0.6μL |
| 20 Mu M F primer | 0.6μL |
| 20 Mu M R primer | 0.6μL |
| Phusion enzyme | 0.3μL |
| cDNA | 1μL |
| ddH2O | 21.2μL |
TABLE 2 Phusion enzymatic amplification procedure
| Step (a) | ℃ | Time of | goto | Cycle number |
| 1 | 98 | 1min | - | - |
| 2 | 98 | 10s | - | - |
| 3 | Annealing | 30s | - | - |
| 4 | 72 | 15-30s/kb | 2 | 34 |
| 5 | 72 | 10min | - | - |
| 6 | 4 | 30min | - | - |
The annealing temperature depends on the primer and the nature of the Phusion enzyme and can be predicted at TmCalculator website (https:// www.thermofisher.cn/cn /); the extension time (Phusion enzyme amplification rate 15-30 s/kb) depends on the length of the gene. After amplification is finished, detecting the specificity and the integrity of the PCR product by agarose gel electrophoresis, specifically observing whether the band is single, the brightness of the band and the like, if the band is single, recovering the PCR product, and if the band is not single, cutting the band with the correct size and recovering the fragment by using a Shanghai biological reagent Limited glue recovery kit; the gel electrophoresis diagram of PCR amplification is shown in FIG. 2.
(III) AcCAX cloning and sequencing
The amplified fragments were sent to Jin Weizhi Bio Inc for sequencing analysis, and the fragments with correct sequencing were cloned into the pEASY-T5 Zero vector to obtain a ligation vector using the pEASY-T5 Zero Cloning Kit (cat# CT 501-01) from Beijing all-gold Bio Inc.
(IV) E.coli transformation
Adding 5 microliters of the connection product into 50 microliters of DH5 alpha competence, and mixing gently; ice-bath for 30min, 42 deg.C water bath for 60 s, ice-bath for 2min, adding 1ml LB liquid medium, culturing at 37 deg.C shaking table for 1 hr at 150rpm, applying 200 microliter bacterial liquid onto solid medium containing Kana, and culturing at 37 deg.C upside down overnight.
(Fifth) sequence determination of target fragment
Picking single colony, culturing in 1ml liquid culture medium containing Kana antibiotics at 37 deg.C and 150rpm for 12h, and delivering to Jin Weizhi company to sequence by using common primers M13F and M13R to obtain AcCAX gene, wherein the base sequence information is shown as SEQ ID NO: 1.
AcCAX3 Gene characteristics are shown in Table 3 below.
TABLE 3 AcCAX3 Gene characterization
As can be seen from Table 3 above, acCAX gene ID was Aco020084 (Loc 109715942), located at position 5144006-51451210 of chromosome 10 of pineapple, CDS length was 1188bp, protein size was 396 amino acids, relative molecular mass was 42642.24, isoelectric point was 5.43 (CDS length and protein size were corrected data).
(Sixth) alignment
The NCBI predicted AcCAX CDS sequence was aligned with the actual clone AcCAX CDS sequence and the results are shown in FIG. 3.
In addition, NCBI predicted AcCAX protein sequences were aligned with the actual clone AcCAX protein sequence, and the results are shown in FIG. 4.
2. AcCAX3 Gene functional verification
(One) CaCl 2 induces AcCAX3 transcriptional level changes
Collecting stem base with thickness of more than 0.5cm under sunny day, taking the normal 'Bali' bud sucking bud back to laboratory, completely stripping the aging leaf from the base, flushing the bud sucking bud with tap water, and cutting off all leaves at a position about 0.5cm above the bud sucking growth point. Pre-sterilizing the bud by 2% NaClO for 10min, washing with sterile water for 3 times, sterilizing with 0.1% HgCl for 8 min, and washing with sterile water for 3 times; each white leaf base that was grown on the bud was excised as piggybacked partial bud-sucking tissue, and the leaf base was sterilized with 0.1% HgCl for 5 minutes, rinsed 3 times with sterile water, inoculated on callus induction medium (MS+2.0 mg/LBA+2.5 mg/LNAA) for callus induction, and cultured under light.
After four weeks of culture, the callus is excised and transferred to a proliferation medium containing (Mock、100MmCaCl2、200MmCaCl2、400MmCaCl2、200MmMgCl2、200MmNaCl2、400MmMnCl2), and the callus is treated for 24 hours to obtain the material. Grinding the callus material in liquid nitrogen, and extracting total RNA with RNA extraction kit (product number: 0416-100GK type) of Beijing-Hua Vietnam biological Co., ltd; the obtained total RNA was used as a template, and cDNA double strand was synthesized by using a reverse transcription kit (cat# M20105) from Beijing full gold biology Co., ltd under the action of reverse transcriptase and polymerase.
Quantitative PCR was performed using Beijing complete gold company PerfectStartGreenQpcrSuperMix (cat# TG-AQ 601-02-V2) with AcCAX-QF-CACCTTGGGTGTAGCCTTG and AcCAX-QR-TTAGTCGTCGTCCAAGTGAC as primers and cDNA as a template, and the transcription level of AcCAX was detected.
The conditions of AcCAX transcription levels induced by exogenous CaCl 2 and endogenous CaCl 2 are shown in fig. 5, 6, 7 and 8, and the results show that AcCAX transcription levels increase with increasing CaCl 2 concentration, and AcCAX transcription levels increase (see fig. 5); acCAX3 transcript levels were not induced by MgCl 2、MnCl2 and NaCl (see FIG. 6).
In addition, pineapple fruits were sampled 20 days, 30 days, 40 days, 50 days, and 60 days after the pineapple flowers were removed. The pineapple fruit material is sent to an analysis experiment center of a southern subtropical crop institute for total calcium content measurement, and is ground in liquid nitrogen, and total RNA is extracted by using an RNA extraction kit (product number: 0416-100GK type) of Beijing-type Vietnam biological Co., ltd; the obtained total RNA was used as a template, and cDNA double strand was synthesized by using a reverse transcription kit (cat# M20105) from Beijing full gold biology Co., ltd under the action of reverse transcriptase and polymerase. By Beijing all-gold company PerfectStartGreenQpcrSuperMix (product number: TG-AQ 601-02-V2)
AcCAX3-QF-CACCTTGGGTGTAGCCTTG and AcCAX-QR-TTAGTCGTCGTCCAAGTGAC were used as primers, and quantitative PCR was performed using cDNA as a template to detect the transcription level of AcCAX 3.
The detection results are shown in fig. 7 and 8, and the results show that the calcium content in pineapple fruits gradually decreases 20 days, 30 days, 40 days, 50 days and 60 days after the pineapple flowers are removed (see fig. 7); acCAX3 transcript levels gradually decreased with decreasing endogenous calcium ion content (see figure 8).
(Two) tissue localization and subcellular localization of AcCAX Gene
Taking the pineapple mature fruit pulp, stems, old leaves, young leaves and inflorescences of plants before flowering, quick-freezing the materials with liquid nitrogen, grinding the materials in the liquid nitrogen, and extracting total RNA with an RNA extraction kit (product number: 0416-100GK type) of Beijing-type Vietnam biological Co., ltd; the obtained total RNA was used as a template, and cDNA double strand was synthesized by using a reverse transcription kit (cat# M20105) from Beijing full gold biology Co., ltd under the action of reverse transcriptase and polymerase. Quantitative PCR was performed using full Beijing golden company PERFECTSTART GREEN QPCR Supermix (cat# TG-AQ 601-02-V2) with AcCAX-QF-CACCTTGGGTGTAGCCTTG and AcCAX-QR-TTAGTCGTCGTCCAAGTGAC as primers and cDNA as a template, and the transcript level of AcCAX3 was detected. The detection results are shown in FIG. 9.
In addition, acCAX-SmaI-F-CCCGGGATGCCGCTGAAAGTATTATCTACA was used as a template for the ligation vector prepared in the third step; acCAX3-XbaI-R-TCTAGAGTCGTCGTCCAAGTGACC primer and Phusion high-fidelity enzyme, recovering PCR products of amplified PCR products by using a PCR product purification kit (product number: B518141-0050) of a manufacturer SanPrep column type, carrying out enzyme digestion on the recovered PCR products (2 micrograms) and pCOMBIA2300 carriers (2 micrograms) for 30 minutes by using SmaI (0.5 microliter) and XbaI (0.5 microliter) enzymes at 37 ℃, carrying out gel electrophoresis on the digested products, cutting target strips, and carrying out gel recovery by using a DNA gel recovery kit (product number: B518131-0100) of the manufacturer SanPrep column type. The product AcCAX obtained by recovery and pCOMBIA2300 vector were ligated by using the Sieimer T4DNA ligase (cat# EL 0011);
Adding 5 microliters of the connection product into 50 microliters of DH5 alpha competence, and mixing gently; ice-bath for 30min, 42 deg.C water bath for 60 s, ice-bath for 2 min, adding 1ml LB liquid medium, shaking table for 1 hr at 37deg.C at 150rpm, applying 200 microliter bacterial liquid onto solid medium containing Kana, and culturing at 37deg.C overnight. Picking single colony, culturing in 1ml liquid culture medium containing Kana antibiotic at 37deg.C and 150rpm for 12 hr, and delivering to Jin Weizhi company for use
Sequencing CFP-N-CGTCGCCGTCCAGCTCGACCAG to finally obtain a 35s-CAX3-eYFP construction;
Taking 100 nanograms of the ligature plasmid, adding the ligature plasmid into 50 microliters (high feather organism, product number: G6039) GV3101 competence, flicking and uniformly mixing; sequentially standing on ice for 5 minutes, liquid nitrogen for 5 minutes, water bath at 28 ℃ for 5 minutes and ice bath for 5 minutes. Then, 700. Mu.l of LB or YEB liquid medium without antibiotics was added thereto, and the culture was continued at 28℃for 2-3 hours with shaking. Centrifuging at 6000rpm for one minute to collect bacteria, collecting about 100 μl supernatant, gently blowing to resuspension bacteria block, coating on LB or YEB plate containing corresponding antibiotics, and culturing in 28 deg.C incubator for 2-3 days; picking single colony in 2ml liquid culture medium containing Kana antibiotics, culturing at 30 degree and 150rpm for 12h;
The cultured bacterial liquid was centrifuged at 4000 Xg for 10min, and the supernatant was discarded. Adding 1ml of transformation buffer solution to resuspend the thalli, centrifuging for 10min at 4000 Xg, and pouring out the supernatant; finally, the sediment is resuspended with 1mL of buffer, and then another 1.5mL centrifuge tube is taken, and a few thalli are taken to adjust the OD 600 value to be about 0.6-0.8. Tobacco leaves grown for about 4 weeks were selected for injection. Sucking a proper amount of agrobacterium suspension by a syringe (with a needle removed) and injecting the agrobacterium suspension into tobacco leaves to soak the leaves; subcellular localization of the protein was observed for about 36h-48 h; this is shown in fig. 10.
Transcript levels of AcCAX Gene in pineapple fruit with different degrees of Water heart disease
Selecting normal pineapple pulp (the fruit has no disease spots), mild water-borne pineapple pulp (the water core part is dispersed in fruit eyes or fruit cores, the disease spot area is less than or equal to 30% of the whole fruit area ratio), moderate water-borne pineapple pulp (the water core part is arranged in fruit eyes and the periphery of the fruit eyes, the water core part is connected with fruit cores, the disease spot area is less than or equal to 30% and less than or equal to 60% of the whole fruit area ratio) and severe water-borne pineapple pulp (the water core part is distributed in large-area connected with pieces, the water core symptoms are extended to fruit peel and fruit cores, the disease spot area is more than 60%) to be quick-frozen in liquid nitrogen, grinding pineapple fruit materials in the liquid nitrogen, and extracting total RNA by using an RNA extraction kit (product number: 0416-100 GK) of Beijing-China transoceanic biological limited company; the obtained total RNA was used as a template, and cDNA double strand was synthesized by using a reverse transcription kit (cat# M20105) from Beijing full gold biology Co., ltd under the action of reverse transcriptase and polymerase. Quantitative PCR was performed using full Beijing golden company PERFECTSTART GREEN QPCR Supermix (cat# TG-AQ 601-02-V2) with AcCAX-QF-CACCTTGGGTGTAGCCTTG and AcCAX-QR-TTAGTCGTCGTCCAAGTGAC as primers and cDNA as a template, and the transcript level of AcCAX3 was detected.
Transcript levels of AcCAX genes in fruits of pineapple with different degrees of water-borne heart disease are shown in fig. 11 and 12, and the transcript level of AcCAX genes is obviously down-regulated in mild water-borne heart disease and obviously up-regulated in moderate and moderate water-borne heart disease.
(IV) AcCAX) instant conversion of pineapple fruits, which can obviously inhibit the situation of pineapple water-borne heart disease
Picking up single colony of 35s-CAX3-eYFP agrobacterium transformed with pCOMBIA to 2300 in 10ml liquid culture medium containing Kana antibiotic, culturing at 30 deg.C and 150rpm for 12-24 hr; the cultured bacterial liquid was centrifuged at 4000 Xg for 10min, and the supernatant was discarded. 2ml of transformation buffer was added to resuspend the cells, centrifuged at 4000 Xg for 10min and the supernatant was discarded. Finally, the sediment is resuspended by using 2mL of conversion buffer solution, then another 15mL centrifuge tube is taken, 10mL of conversion buffer solution is added into the tube, a few thalli are taken into the 15mL tube, and the OD 600 value is regulated to be 0.6-0.8. 200 microliters of the prepared bacterial liquid is taken, a 1ml syringe is used for injecting in the gap between the fruit eyes of the mature pineapple, the pineapple is cut 60 hours after injection, a photo is taken, and the area of the disease spots is counted by using imageJ software.
TABLE 4 conversion buffer
| 1M MgCl2 | 0.5mL |
| 0.1M MESpH5.6 | 5mL |
| 0.1M AS | 7.5μL |
| Ultrapure water | Constant volume finger 50mL |
The detection results are shown in fig. 13 and 14, and the results show that the AcCAX-3 transient transformation of pineapple fruits can significantly inhibit pineapple aquatic heart disease compared with a transient expression empty vector control (EV).
Finally, it is noted that the above embodiments are only for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications and equivalents may be made thereto without departing from the spirit and scope of the technical solution of the present invention, which is intended to be covered by the scope of the claims of the present invention.
Claims (10)
1. Pineapple Ca 2+/H+ antiport protein AcCAX3, which is characterized in that the amino acid sequence thereof comprises the amino acid sequence shown in SEQ ID NO:2 or a sequence comprising a sequence identical to SEQ ID NO:2, wherein the sequence homology is more than or equal to 85%.
2. An isolated nucleic acid molecule encoding pineapple Ca 2+/H+ antiport protein AcCAX according to claim 1.
3. A recombinant expression vector comprising the isolated nucleic acid molecule of claim 2.
4. A recombinant engineering bacterium comprising the isolated nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3.
5. A recombinant engineered cell comprising the isolated nucleic acid molecule of claim 2 or the recombinant expression vector of claim 3 or the recombinant engineered bacterium of claim 4.
6. Use of pineapple Ca 2+/H+ antiport protein AcCAX3 of claim 1 or the isolated nucleic acid molecule of claim 2 in genetic breeding of pineapple.
7. Use of pineapple Ca 2+/H+ antiport protein AcCAX3 of claim 1 or the isolated nucleic acid molecule of claim 2 for the cultivation of transgenic plants.
8. Application of pineapple Ca 2+/H+ antiport protein AcCAX3 expression promoter in inhibiting pineapple aquatic heart disease.
9. The use according to claim 8, wherein AcCAX's 3 expression promoter is CaCl 2.
10. A method of controlling calcium ion homeostasis in pineapple cells, comprising: by modulating the pineapple Ca 2+/H+ antiport AcCAX content of claim 1 in pineapple or modulating the expression level of the isolated nucleic acid molecule of claim 2 in pineapple.
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