CN111187780A - 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 can enhance the transport of photosynthetic products from the overground part to the root,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), one of the three major mineral nutrients essential for plant growth, also the most abundant monovalent cation in plants, accounts for approximately 4-10% of the dry weight of the plant (Leigh and Wyn,1984), plays an extremely important role throughout the growth and development of plants, e.g., 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 tzet al, 2014), while K is highly mobile in plants, K absorbed at roots is transported from xylem to aboveground, and then about 80% is re-transported through phloem to underground, achieving cyclic distribution and re-utilization of K in plants (deen et al, 2002), generally, K concentration in plants is kept at about 100mM, when K is insufficiently absorbed, many physiological and biochemical processes are hindered, plant growth is also influenced by K, growth is also influenced by K in plants, and global growth is a major factor that increases in crop production by global environmental and environmental losses, K is a factor that increases the steady state of crops, and crop production by grain production.
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 (Oryzasativa. 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 is transported and distributed through the circulation in the plant with the concomitant transport and distribution of other substances, including photosynthetic products, and there are currently limited studies on the involvement of K transporters in this transport, and OsHAK18 is the only K transporter among the members of the HAK/KT/KUP family 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:// aramnon. 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
The construction of OsHAK18 gene intermediate vector includes agarose electrophoresis separating OsHAK18 gene PCR product, cutting and recovering, connecting the purified segments with pEASY-Blunt intermediate vector separately, enzyme connecting system including 1 microliter pEASY-Blunt vector and 4 microliter PCR purified product, connecting at 25-28 deg.c for 25min, transferring into colibacillus DH5 α competent cell for propagation, extracting the vector for double enzyme digestion verification, further sequencing verification, adding isometric 30% glycerin into the bacteria liquid, storing at-70 deg.c to obtain recombinant plasmid containing OsHAK18 gene full-length sequence, named OsHAK 18-P.
The construction of OsHAK18 gene expression vector comprises the steps of digesting pTCK303(Eamens A L, Blancard C L, Dennis E S, et al. A bidirectional gene trap ligation competent for T-DNA and Ds-mediated expression of plasmid in rice, recovering the digested linearized expression vector, performing homologous recombination with the sequenced correct PCR linear product containing the target gene [ J ]. Plant biotechnology journal,2004,2(5): cona 380.) plasmid vector, obtaining a homologous recombination vector by adding a homologous recombination Promoter sequence to the digested plasmid DNA, cloning the digested plasmid DNA into Escherichia coli DH5 α cells, obtaining a homologous recombination vector DNA, cloning the digested plasmid DNA, cloning the homologous recombination vector DNA, cloning the digested plasmid DNA, the homologous recombination vector DNA, the digested plasmid DNA, the digested plasmid DNA, the homologous recombination vector DNA, the digested by the homologous recombination vector DNA, the digested by the homologous recombination vector DNA, the digested by the Promoter DNA, the homologous recombination vector DNA, the digested by the Promoter DNA, the digested by the Promoter DNA, the.
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 grain length, the setting rate, the grain number per grain, 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, the bucket culture and field test shows that the tillering (total tillering and effective tillering) capability of the transgenic rice is stronger than that of the wild type, thereby achieving the effect of increasing the yield of the rice.
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
Since our previous studies have found that OsHAK18 affects K transport in 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 criticalpotassium concentrations for growth to the distribution and function of thision in the plant cell.New Phytol.97,1-13.
Armengaud P,et al.(2004)The potassium-dependent transcriptome ofArabidopsis reveals a prominent role of jasmonic acid in nutrientsignaling.Plant Physiology,136:2556–2576.
Anschütz U,et al.(2014)Going beyond nutrition:regulation of potassiumhomoeostasis as a common denominator of plant adaptive responses toenvironment.Journal of Plant Physiology,171:670–687.
Deeken D,et al.(2002)Loss of the AKT2/3potassium channel affectssugar loading into the phloem of Arabidopsis.Planta,216:334–344.
Britto DT,et al.(2008)Cellular mechanisms of potassium transport inplants.Physiologia Plantarum,133:637-650.
Mengel K,Kirkby EA.(1987)Principles of plant nutition.Internationalpotash Institute:Worblaufen-Bern
Jiao Y,et al.(2010)Regulation of OsSPL14 by OsmiR156 defines idealplant 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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Claims (8)
1. A recombinant expression vector is characterized by containing a rice potassium ion transport protein gene OsHAK18, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown in SEQ ID No. 1.
2. The recombinant expression vector of claim 2, 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 OsHAK18 gene.
3. The application of the rice potassium ion transport protein gene OsHAK18 in increasing the effective tiller number of crops and/or improving the crop yield is disclosed, wherein the cDNA sequence of the rice potassium ion transport protein gene OsHAK18 is shown as SEQ ID No. 1.
4. The application of the rice potassium ion transport protein gene OsHAK18 in 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.
5. 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.
6. Use of the recombinant expression vector of any one of claims 1-2 to increase the effective tiller number and/or increase crop yield of a crop.
7. Use of the recombinant expression vector of any one of claims 1-2 to enhance transport of a photosynthetic product from above ground to roots.
8. Use of the recombinant expression vector of any one of claims 1-2 to create a new rice germplasm that improves efficient tillering and yield of rice, improves potassium nutrient efficiency, and enhances transport of photosynthetic products from above ground to roots.
Priority Applications (1)
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