CN111187780B - Genetic engineering application of rice potassium ion transport protein gene OsHAK18 - Google Patents
Genetic engineering application of rice potassium ion transport protein gene OsHAK18 Download PDFInfo
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Abstract
The invention discloses a gene engineering application of a rice potassium ion transport protein gene OsHAK 18. Application of rice potassium ion transporter gene OsHAK18 in creating new rice germplasm for improving effective tillering and yield of rice, improving potassium nutrient efficiency and enhancing the transport of photosynthetic products from overground part (source) to root (reservoir). The invention discovers that the rice K+The recombinant expression vector constructed by the transport protein gene OsHAK18 is transfected into wild rice Nipponbare through an agrobacterium tumefaciens-mediated rice transgenic technology, and the total tillering, effective tillering number and grain yield of the OsHAK18 transgenic rice material are obviously improved compared with wild rice. In addition, OsHAK18 enhances the transport of photosynthetic products from above ground to roots and is the only K transporter of the HAK/KT/KUP family members found to have this function. On the basis, a new rice germplasm resource is provided, which can improve the yield by improving the effective tillering of rice and improve the nutrient efficiency.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and relates to genetic engineering application of a rice potassium ion transporter gene OsHAK 18.
Background
Potassium (K) is one of the three major mineral nutrients essential to plant growth, and is also the most abundant monovalent cation in plants, accounting for approximately 4-10% of the dry weight of the plant (Leigh and Wyn,1984), and plays an extremely important role throughout the growth and development of plants, such as maintaining ionic balance, regulating osmotic pressure, regulating enzyme activity, participating in protein metabolism, promoting photosynthetic efficiency, participating in nutrient transport and redistribution, etc. (Armengaud et al, 2004; Ansch ü tz et al, 2014). Meanwhile, the high mobility of K in the plant body, the K absorbed by the root is transported to the overground part by the xylem, and then about 80% of K is transported to the underground part again by the phloem, so that the K is recycled and reused in the plant body (Deeken et al, 2002). Generally, the concentration of K in the plant cytoplasm is kept at about 100 mM; when the absorption of the plant K is insufficient, a plurality of physiological and biochemical processes are blocked, and the growth and development of the plant are also influenced; in crops, low K directly affects crop yield (Britto et al, 2008). Due to the large depletion and uneven distribution of potassium ore resources around the world (Mengel and Kirkby,1987), K deficiency in crops becomes a limiting factor for global food production. How to enhance the K absorption of crops in a low-K environment by a biotechnology means, maintain the steady-state balance of K in the crops and improve the K utilization efficiency is significant for improving the crop yield and quality, and is also an important way for reducing the agricultural production cost and maintaining the agricultural sustainable development.
In addition to mineral nutrients, their plant type is undoubtedly a decisive factor among the factors that influence crop yield. For example, the sixties green revolution is to improve the lodging resistance of wheat and rice by dwarfing breeding of the wheat and the rice and improve the yield by matching with the application of chemical fertilizers and pesticides, thereby saving billions of hungry population all over the world. The plant type of the upper part of the rice field consists of plant height, effective tiller number, tiller angle, ear grain number and the like, wherein the effective tiller number has a crucial influence on yield (Jiao et al, 2010). Therefore, the improvement of the effective tillering number of the plant is also an important factor of attention of breeders.
Disclosure of Invention
The invention aims to provide rice K+The engineering application of the transport protein gene OsHAK18 mainly has the functions of increasing tillering, improving yield and improving potassium nutrient efficiency.
The purpose of the invention is realized by the following technology:
the rice potassium ion transport protein gene OsHAK18 has a cDNA sequence shown in SEQ ID NO. 1.
A recombinant expression vector contains the rice potassium ion transport protein gene OsHAK 18.
Preferably, the recombinant expression vector is pTCK303 vector.
The recombinant expression vector is further preferably obtained by inserting the rice potassium ion transporter gene OsHAK18 into a KpnI and SpeI enzyme cutting site of a pTCK303 vector, then cutting Ubiquitin by using Hind III and KpnI double enzymes, and inserting the promoter of the OsHAK18 gene into the recombinant expression vector.
The rice potassium ion transport protein gene OsHAK18 is applied to increasing the effective tillering number of crops and improving the yield of the crops.
The application of the rice potassium ion transporter gene OsHAK18 in enhancing the transport of photosynthetic products from overground parts (sources) to roots (sinks) is provided.
The application of the rice potassium ion transport protein gene OsHAK18 in creating a new rice germplasm for improving the effective tillering and yield of rice, improving the potassium nutrient efficiency and enhancing the transport of photosynthetic products from overground parts (sources) to roots (banks).
The recombinant expression vector is applied to increasing the effective tillering number of crops and improving the yield of the crops.
The recombinant expression vector is applied to enhancing the transport of the photosynthetic product from the overground part (source) to the root (sink).
The recombinant expression vector is applied to the establishment of a new rice germplasm for improving the effective tillering and yield of rice, improving the potassium nutrient efficiency and enhancing the transfer of a photosynthetic product from an overground part (source) to a root (reservoir).
The invention has the beneficial effects that:
our research on OsHAK18, a member of Cluster III, discovers that the plant height of a transgenic Nippon japonica rice variety guided by an OsHAK18 self promoter is reduced in field and barrel culture, the effective tillering and total tillering number are greatly increased compared with those of a wild type material, the single-plant yield of the rice is increased by about 25%, and a new way for realizing the synergistic effect of high yield of the rice and high nutrient efficiency is provided by developing and utilizing the gene. In addition, the OsHAK18 transporter is involved in the circulation of potassium from the overground part to the root in rice, and the circulation determines the transportation and distribution of photosynthetic products to the root, and the function is the latest discovery of the function of the HAK family gene at present. Therefore, the application of the gene is beneficial to improving the rice yield by improving effective tillering and simultaneously improving the nutrient efficiency of the rice.
1. In the invention, the rice K is found for the first time in the world+The recombinant expression vector constructed by the transporter gene OsHAK18 is transfected into wild type rice Nipponbare (Oryza sativa. ssp. cv. Japonica) by an agrobacterium-mediated rice transgenic technology, and the total tillering, the effective tillering number and the grain yield of the OsHAK18 transgenic rice material are found to be remarkably improved compared with those of the wild type rice.
2. The OsHAK18 gene is derived from rice, the promoter is also a sequence at the upstream of the OsHAK18 gene and is not a foreign gene, so that the OsHAK18 gene has biological safety, and the constructed rice K is+The carrier gene OsHAK18 plant expression vector can be directly used for agrobacterium tumefaciens-mediated plant genetic transformation to obtain a new germplasm of OsHAK18 gene for increasing the effective tillering of plants.
3. Up to 80% of K in plants circulates through the plant with the transport and distribution of other substances, including photosynthetic products, and there are currently limited studies on the involvement of the K transporter in this transport, while OsHAK18 is the only K transporter among members of the HAK/KT/KUP family that has been found to have this function.
4. On the basis, a new rice germplasm resource is provided, which can improve the yield by improving the effective tillering of rice and improve the nutrient efficiency.
Drawings
FIG. 1 molecular characterization of OsHAK18 transgenic rice material.
FIG. 2 Tak culture and field trial phenotypes of OsHAK18 transgenic rice.
FIG. 3OsHAK18 transgenic rice mature period K and soluble total sugar content.
FIG. 4 map of pTCK303 expression vector.
Detailed Description
Example 1 cloning of OsHAK18 Gene
1. Template: RNA of leaves of wild type rice of Nipponbare of 2 weeks size in normal water culture is extracted and reverse transcribed into cDNA, which is used as a PCR amplification template for cloning OsHAK18 gene.
PCR primer design: find the gene sequence of OsHAK18 on ARAMEMNON website (http:// arammunon. botanik. uni-koeln. de /), design the primer by using primer design software Primer5.0, add KpnI (GGTACC) and SpeI (ACTAGT) cleavage site sequence on the 5 'end and 3' end of the primer, and add a sequence on pTCK303 plasmid vector to form a 46bp homologous recombination primer (F1 and R1).
The upstream primer F1:
5'-TCGACTCTAGAGGATCCCCGGGTACCATGGAGACCAGAACAAATGA-3'(SEQ ID NO.2)；
the downstream primer R1:
5'-TCATGGTCTTTGTAGTCCATACTAGTCACGTAGAAAACCTGCCCAA-3'(SEQ ID NO.3)。
PCR amplification of OsHAK18 gene: 2.5 mul of PCR Buffer, 2 mul of dNTP Mix, 1 mul of each of the upstream primer and the downstream primer, 1 mul of template, 0.5 mul of KOD high fidelity enzyme and 17 mul of double distilled water. The PCR amplification procedure was as follows: pre-denaturation at 94 deg.C for 3min, denaturation at 94 deg.C for 30s, renaturation extension at 58 deg.C for 2min, 35 cycles, full extension at 72 deg.C for 10min, and keeping at 10 deg.C. The size of the OsHAK18 gene is 2382b of the amplified PCR product detected by 0.8% agarose gel electrophoresis, and the sequence is shown in SEQ ID NO. 1.
Example 2 construction of plant expression vector pTCK303-OsHAK18 and Rice transgene
Construction of OsHAK18 gene intermediate vector: separating the PCR product of the OsHAK18 gene by agarose electrophoresis, cutting the gel, recovering, connecting the purified fragments with a pEASY-Blunt intermediate vector respectively, wherein an enzyme linked system comprises 1 mul of pEASY-Blunt vector and 4 mul of PCR purified product, and connecting for 25min at 25-28 ℃; then transferring the vector into escherichia coli DH5 alpha competent cells for propagation, extracting the vector for double enzyme digestion verification, and further carrying out sequencing verification. Adding the correctly sequenced bacterial liquid into glycerol with the volume equal to the volume ratio of 30% for storage at the temperature of-70 ℃ for later use, and obtaining a recombinant plasmid containing the OsHAK18 gene full-length sequence, which is named as OsHAK 18-P.
Construction of OsHAK18 Gene expression vector: the plasmid vector pTCK303(Eamens A L, Blanchard C L, Dennis E S, et al. A bidirectional gene trap construction able for T-DNA and Ds-mediated transformation in rice (Oryza sativa L.) [ J ]. Plant biotechnology journel, 2004,2(5):367 and 380.) was digested with KpnI and SpeI, and the digested linearized expression vector was recovered by gel and subjected to homologous recombination with the correctly sequenced PCR linear product containing the target gene. And transforming the homologous recombination product into an escherichia coli DH5 alpha competent cell, screening by antibiotics, selecting a positive monoclonal, storing the positive clone, extracting an escherichia coli plasmid, sending the escherichia coli plasmid to a company for sequencing, and obtaining the Ubiquitin strong promoter overexpression vector of the pTCK303+ OsHAK18 gene after the sequencing is correct. On the basis, the vector is subjected to double digestion by Hind III and KpnI to obtain a pTCK303 linear vector which carries the OsHAK18 gene but is cut off with Ubiquitin, and the digestion product is recovered by glue. A1958 bp sequence is cut out according to the Promoter sequence of the OsHAK18 gene self Promoter, a pair of homologous recombination primers (F2 and R2) for amplifying the OsHAK18 gene self Promoter (Pro) is designed by using software Primer5.0, and KpnI (GGTACC) and Hind III (AAGCTT) cleavage site sequences are added to the 5 'end and the 3' end of the primers, and a sequence on a pTCK303 plasmid vector is added. And obtaining a target promoter linear fragment by a PCR technology, and after the target promoter linear fragment is verified to be correct, carrying out homologous recombination with the pTCK303-OsHAK18 gene linear vector from which the Ubiquitin is cut. Transforming the homologous recombination product into escherichia coli DH5 alpha competent cells, screening by antibiotics, picking positive monoclonals, storing the positive clones, extracting escherichia coli plasmids, sending the escherichia coli plasmids to a company for sequencing, obtaining Pro over-expression vectors of pTCK303+ OsHAK18 genes after the sequencing is correct, transferring the Pro over-expression vectors into EHA105 agrobacterium-infected cells by an electric shock method, screening by antibiotics, picking positive clones, extracting plasmids, turning escherichia coli, adding isometric 30% glycerol into agrobacterium liquid for storing in a refrigerator at-70 ℃ after the extracted plasmids are verified to be correct by sequencing, and reserving subsequent tests for later use.
The upstream primer F2:
5'-GTAAAACGACGGCCAGTGCCAAGCTTTGTCCCACAGATCTTATTGT-3'(SEQ ID NO.4)；
the downstream primer R2:
5'-TCATTTGTTCTGGTCTCCATGGTACCGGGTTCAGACTTCAGATCAA-3'(SEQ ID NO.5)。
3. obtaining of transgenic rice: infecting the rice callus with the obtained agrobacterium tumefaciens transferred with the pTCK303+ OsHAK18 Pro overexpression vector, performing co-culture (dark culture) for 2.5 days, washing the bacteria, transferring the aired callus to a selection culture medium containing 500mg/L carbenicillin (Car) and 50mg/L hygromycin (Hyg) for first round of selection culture, performing illumination culture at 28 ℃ for 2 weeks, transferring the callus with resistance to a selection culture medium containing 500mg/L carbenicillin and 80mg/L hygromycin for second round of selection culture, and performing illumination culture at 28 ℃ until granular resistant callus grows out. Selecting the yellow resistant callus from the same callus, transferring into a plastic wide-mouth bottle filled with a differentiation culture medium for differentiation culture, waiting for differentiation into seedlings (25-30d), and placing into a rooting culture medium for strengthening the seedlings when the seedlings grow to about 2-3 cm. Picking out the differentiated seedling from the rooting tube, adding a proper amount of sterile water, and hardening the seedling for one week. And washing off the root agar culture medium, transplanting the culture medium into a rice nutrient solution to grow, identifying positive seedlings, and transferring the positive seedlings into a field to harvest to obtain T1 generation transgenic seeds.
The culture medium used therein is prior art.
Identifying overexpression effect and screening plant types of OsHAK18 transgenic rice materials: the OsHAK18 Pro overexpression strain and Nipponbare wild type rice seeds (T1 generation) are germinated by a water germination method. Selecting healthy seeds, soaking the seeds in 30% sodium hypochlorite solution for 30 minutes, washing the seeds for 5 times by using clear water, placing the seeds in a paper cup paved with a 20-mesh nylon net, immersing half of the seeds by using the clear water, and putting the seeds into an oven at 37 ℃ for 24-36 hours. And (5) 10 days after seedling emergence, performing GUS (glucuronidase) staining identification on all over-expressed seedlings, and selecting positive seedlings to be transplanted into a turnover box for water culture. After 2 weeks of culture in the total IRRI nutrient solution, total RNA in the OsHAK18 gene Pro overexpression strains and wild type Japanese rice tissues is extracted by Trizol reagent (Invitrogen), cDNA is obtained by reverse transcription, and RT-PCR (semi-quantitative) is carried out by taking the cDNA as a template to identify the expression level of the OsHAK18 gene on the transcription level. The semiquantitative primers for the semiquantitative internal reference and OsHAK18 genes are shown in Table 1.
TABLE 1 primer sequences for OsActin and OsHAK18 for RT-PCR
In order to detect the overexpression effect of the OsHAK18 gene in the transgenic rice and screen the plant type, the overground parts of wild type and transgenic rice samples cultured in water for 2 weeks are collected for semi-quantitative PCR, and the expression quantity is analyzed (figure 1). As a result, the OsHAK18 gene expression in the aerial part of the transgenic rice is found to be obviously higher than that in the wild type. Meanwhile, by combining the expression quantity and the plant type of the OsHAK18 gene, 3 representative lines are selected from 12 transgenic materials to carry out subsequent physiological experiments, and are numbered as Pro1, Pro2 and Pro3 again.
Example 3 Bitterculture and field phenotype and Main agronomic trait indices statistics for wild-type and transgenic Rice
To study the role of OsHAK18 in the phenotype and yield of rice in the maturity stage, bucket culture and field experiments were performed on wild-type and transgenic rice material, respectively at Nanjing university Tokyo Pachii test base and the eight Diagram test base. The soil for the barrel culture test is acid yellow brown soil from Nanjing urban area, the pH value is about 5.20, the quick-acting potassium concentration of the soil is 133mg/kg (the normal K content and the other nutrients are normal) by using an ammonium acetate leaching method, 10kg of soil is filled in each barrel, seedlings with consistent growth vigor of each strain are selected and transplanted into the barrel, and one strain is planted in each barrel for 6 times until the seedlings are cultured to be mature and harvested. The field trial was a plot of 49 replicates per plant line at 7x 7. Watering, fertilizing and pesticide spraying are carried out regularly until the rice is completely mature. By counting the differences of agronomic characters such as plant height, ear length, total tillering, effective tillering, grain number per ear, seed setting rate, thousand kernel weight, single plant yield and the like (figure 2 and table 2), we find that the overexpression of the OsHAK18 self promoter (Pro) improves the yield of rice by about 25%. Compared with the wild type, the plant height of the transgenic rice is reduced to about 90cm from 104cm on average, the total tillering number and the effective tillering number are both obviously increased, and 3-5 effective tillering numbers are increased on average per plant. The spike length, the setting rate, the grain number per spike, the thousand grain weight and the grain-straw ratio of the OsHAK18 overexpression rice have no significant difference compared with the wild type, so that the comprehensive single-plant yield is significantly increased compared with the wild type. Therefore, barrel culture and field experiments show that the tillering (total tillering and effective tillering) capability of the transgenic rice is stronger than that of the wild rice, so that the effect of increasing the yield of the rice is achieved.
TABLE 2 evaluation of agronomic performance indexes of wild-type and transgenic rice barrel culture and field test
Example 4 differences in partitioning of Potassium and soluble Total sugar in mature periods of wild-type and transgenic Rice
Given that our previous studies have found that OsHAK18 affects K transport in the phloem, we further analyzed whether this transporter affects sugar distribution in rice. From statistical analysis of the K and soluble sugar content of various parts of wild-type and transgenic rice plants at maturity in the field, we found that both the K and soluble total sugar content in leaves and leaf sheaths were significantly lower than that of the wild-type, and both were significantly higher than that of the wild-type in typical sink organs (fig. 3). The experimental result shows that OsHAK18 is helpful for the transportation of K in rice leaves and leaf sheaths to roots, brown rice and glumes and is also helpful for the transportation of sugar in the rice leaves and leaf sheaths to the roots in the rice mature period. Therefore, we believe that OsHAK18 can be involved in regulating photosynthetic product transport.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Reference to the literature
Leigh,R.A.,and Wyn Jones,R.G.(1984)A hypothesis relating critical potassium concentrations for growth to the distribution and function of this ion in the plant cell.New Phytol.97,1-13.
Armengaud P,et al.(2004)The potassium-dependent transcriptome of Arabidopsis reveals a prominent role of jasmonic acid in nutrient signaling.Plant Physiology,136:2556–2576.
Anschütz U,et al.(2014)Going beyond nutrition:regulation of potassium homoeostasis as a common denominator of plant adaptive responses to environment.Journal of Plant Physiology,171:670–687.
Deeken D,et al.(2002)Loss of the AKT2/3potassium channel affects sugar loading into the phloem of Arabidopsis.Planta,216:334–344.
Britto DT,et al.(2008)Cellular mechanisms of potassium transport in plants.Physiologia Plantarum,133:637-650.
Mengel K,Kirkby EA.(1987)Principles of plant nutition.International potash Institute:Worblaufen-Bern
Jiao Y,et al.(2010)Regulation of OsSPL14 by OsmiR156 defines ideal plant architecture in rice.Nature Genetics,42(6):541-U36.
Sequence listing
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<120> genetic engineering application of rice potassium ion transporter gene OsHAK18
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tcatttgttc tggtctccat ggtaccgggt tcagacttca gatcaa 46
Claims (7)
1. The application of the rice potassium ion transport protein gene OsHAK18 in increasing the effective tiller number of rice and/or improving the rice yield is disclosed, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown in SEQ ID No. 1.
2. The application of the rice potassium ion transport protein gene OsHAK18 in enhancing the transport of rice photosynthetic products from overground parts to roots is disclosed, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown as SEQ ID No. 1.
3. The application of the rice potassium ion transport protein gene OsHAK18 in creating a new rice germplasm for improving the effective tillering and yield of rice, improving the potassium nutrient efficiency and enhancing the transport of photosynthetic products from overground parts to roots is disclosed, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown in SEQ ID No. 1.
4. The application of a recombinant expression vector containing a rice potassium ion transport protein gene OsHAK18 in increasing the effective tiller number of rice and/or improving the rice yield is disclosed, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown in SEQ ID No. 1.
5. The use according to claim 4, wherein the recombinant expression vector is obtained by inserting the rice potassium ion transporter gene OsHAK18 into the pTCK303 vector KpnI and SpeI cleavage sites, then excising Ubiquitin with HindIII and KpnI double enzymes, and inserting the promoter of the OsHAK18 gene.
6. The application of a recombinant expression vector containing a rice potassium ion transport protein gene OsHAK18 in creating a new rice germplasm for improving the effective tillering and yield of rice, improving the potassium nutrient efficiency and enhancing the transport of photosynthetic products from the overground part to the roots is disclosed, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown in SEQ ID No. 1.
7. The use according to claim 6, wherein the recombinant expression vector is obtained by inserting the rice potassium ion transporter gene OsHAK18 into the pTCK303 vector KpnI and SpeI cleavage sites, then excising Ubiquitin with HindIII and KpnI double enzymes, and inserting the promoter of the OsHAK18 gene.
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