Application of over-expressed TaHAK1 in improving potassium stress tolerance of rice
阅读说明：本技术 过表达TaHAK1在提高水稻钾胁迫耐性中的应用 (Application of over-expressed TaHAK1 in improving potassium stress tolerance of rice ) 是由 李鸽子 康国章 柳海涛 王鹏飞 刘金 张丹丹 于 2020-12-29 设计创作，主要内容包括：本发明属于转基因植物领域,特别是涉及过表达TaHAK1在提高水稻钾胁迫耐性中的应用。本发明提供了过表达TaHAK1在提高水稻钾胁迫耐性中的应用。本发明过表达TaHAK1基因的水稻在低钾及缺钾的状态下仍能够表现出优良的成长状态,植株的干重、根系和叶片的生物量显著高于对照植株,在低钾条件下,转基因水稻植株的K含量在根系和叶片中积累均显著上升；在缺钾条件下,过表达TaHAK1基因则影响了K在水稻根系向叶片的转移,有效提高了转基因水稻耐低钾的能力。(The invention belongs to the field of transgenic plants, and particularly relates to application of over-expressed TaHAK1 in improving potassium stress tolerance of rice. The invention provides application of over-expressed TaHAK1 in improving potassium stress tolerance of rice. The rice over-expressing the TaHAK1 gene can still show an excellent growth state under the states of low potassium and potassium deficiency, the dry weight of a plant and the biomass of a root system and leaves are obviously higher than those of a control plant, and the K content of a transgenic rice plant is obviously increased in the root system and the leaves when the K content is accumulated under the condition of low potassium; under the condition of potassium deficiency, the overexpression of the TaHAK1 gene influences the transfer of K from a rice root system to a leaf, and effectively improves the low-potassium resistance of the transgenic rice.)
1. Application of over-expressed TaHAK1 in improving potassium stress tolerance of rice.
2. Application of over-expressed TaHAK1 in improving dry weight of potassium-stressed rice plants.
3. The application of the over-expressed TaHAK1 in improving the potassium content in the root system and the leaf of the potassium-stressed rice.
4. The application of the over-expressed TaHAK1 in improving the transfer of potassium from a root system to a leaf blade in potassium-stressed rice.
5. The use according to any one of claims 1 to 4, wherein said method for over-expressing TaHAK1 comprises: the overexpression vector of the TaHAK1 protein is transferred into rice to obtain transgenic rice capable of overexpressing TaHAK 1.
6. The use according to claim 5, wherein said overexpression vector comprises a backbone vector and the coding sequence of TaHAK 1.
7. The use of claim 6, wherein the backbone vector comprises a pCUN-1301 plasmid.
8. The use according to claim 6, wherein said coding sequence of TaHAK1 is amplified by PCR using a primer pair.
9. The use of claim 8, wherein the primer pair comprises a forward primer as shown in SEDID NO. 1 and a reverse primer as shown in SED ID NO. 2.
Technical Field
The invention belongs to the field of transgenic plants, and particularly relates to application of over-expressed TaHAK1 in improving potassium stress tolerance of rice.
Background
Potassium (K) is one of the essential nutrient elements for plant growth and development. It is involved in a series of physiological and biochemical processes of plant, such as maintaining cell osmotic pressure, maintaining charge balance, regulating enzyme activity, participating in protein metabolism, influencing ion balance in plant body, influencing photosynthesis of plant and growth of cell, and has important effect on plant growth and development. In addition, K is one of the constituent elements of the matrix minerals of the soil, although the content of K in most soils is high, the content of effective K available for plants in the soil of the cultivated land is low at present due to the fixation of secondary minerals in the soil and the influence of plants on the depletion of effective potassium in the rhizosphere. The K utilization rate of winter wheat is 31.5%, and about 1/3 soil has the problem of effective K deficiency, which becomes one of the important factors troubling the sustainable development of agriculture. Therefore, the method enhances the absorption and transportation of K in soil by crops, improves the utilization efficiency of K fertilizer, and is a problem to be solved urgently in agricultural sustainable development in China.
The plants have formed a relatively perfect regulation and control mechanism in vivo, such as K ion absorption, transport, utilization and the like, in order to adapt to stress tolerance formed by the continuously changing K ion content in different soils. If the plant is in a K-deficient condition, the K can be transported from a mature tissue to a tender tissue organ to maintain the life activity of the plant due to the strong mobility of the plant in vivo; in the later period of plant growth, K can also be transported from the aged plant tissues to seed setting organs such as seeds. In addition, K ions can not be fully absorbed and utilized due to antagonism of different mineral substances absorbed by plants in soil. Therefore, the improvement of the absorption and utilization capacity of the plant K can ensure the transportation of the plant from a source to a storehouse, and is one of important measures for the sustainable development of crops. There are few promising studies related to the tolerance of plants to potassium stress in the prior art.
Disclosure of Invention
In order to solve the above problems, the present invention provides the use of over-expressed TaHAK1 for improving potassium stress tolerance in rice. The application provided by the invention improves the tolerance of rice to low potassium ions by over-expressing the TaHAK1 gene in rice.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides application of over-expressed TaHAK1 in improving potassium stress tolerance of rice.
The invention also provides application of the over-expressed TaHAK1 in improving the dry weight of potassium stress rice plants.
The invention also provides application of the over-expressed TaHAK1 in improving the potassium content in roots and leaves of potassium-stressed rice.
The invention also provides application of the over-expressed TaHAK1 in improving transfer of potassium from a root system to a leaf blade in potassium stress rice.
Preferably, the method for overexpressing TaHAK1 comprises: the overexpression vector of the TaHAK1 protein is transferred into rice to obtain transgenic rice capable of overexpressing TaHAK 1.
Preferably, the overexpression vector comprises a backbone vector and the coding sequence of TaHAK 1.
Preferably, the backbone vector comprises the pCUN-1301 plasmid.
Preferably, the coding sequence of TaHAK1 is amplified by PCR using a primer pair.
Preferably, the primer pair includes a forward primer as shown by SED ID NO. 1 and a reverse primer as shown by SED ID NO. 2.
The invention provides application of over-expressed TaHAK1 in improving potassium stress tolerance of rice. The invention can effectively over-express the TaHAK1 gene in rice. The rice over-expressing the TaHAK1 gene can still show an excellent growth state under the conditions of low potassium and potassium deficiency, the dry weight of the plant, the biomass of the root system and the leaf blade are obviously higher than those of a control plant, and simultaneously, the K content of the transgenic rice plant is obviously increased in the root system and the leaf blade under the condition of low potassium; however, under the potassium deficiency condition, the K content of the transgenic rice plant is obviously higher than that of the wild rice plant only on the overground part, and is obviously reduced in the root system, which indicates that under the potassium deficiency condition, the TaHAK1 gene influences the transfer of K to the leaf from the rice root system, and improves the low potassium resistance of the transgenic rice.
Drawings
FIG. 1 shows the sequence of the coding region of the gene TaHAK1 obtained by amplification;
FIG. 2 is a physical map of the overexpression vector pCUN1301-TaHAK 1;
FIG. 3 shows the genetic transformation process of transgenic rice;
FIG. 4 shows the identification of transgenic rice, wherein A is a transgenic plant identified by hygromycin gene (Hpt II), B is a transgenic plant identified by a vector and a target gene, and C is the protein expression level of a transgenic plant identified by a tag antibody HA;
FIG. 5 is a phenotypic observation of transgenic rice after 14 days of potassium stress;
FIG. 6 Biomass statistics after potassium stress of transgenic rice for 14 days;
FIG. 7 shows the K content in plants 14 days after potassium stress of transgenic rice.
Detailed Description
The invention provides application of over-expressed TaHAK1 in improving potassium stress tolerance of rice. In the present invention, the method for overexpressing TaHAK1 preferably comprises: transferring the overexpression vector of the TaHAK1 protein into rice to obtain transgenic rice capable of overexpressing TaHAK 1; the over-expression vector preferably comprises a backbone vector and a coding sequence for TaHAK 1; the backbone vector preferably comprises the pCUN-1301 plasmid; the coding sequence of TaHAK1 is preferably obtained by PCR amplification using a primer pair; the primer pair preferably comprises a forward primer as shown in SED ID NO: 1: 5'-GCCGGTACCATGCGGAGAAGGTCTCCGACTCG-3', SED ID NO: 1; and a reverse primer as shown in SED ID NO: 2: 5'-CGCGTCGACTATCTCGTATGTGATCCCGACTTGAGC-3', SED ID NO: 2. The sequence of the TaHAK1 gene is shown in SED ID NO: 3: [ TramesCS4D02G308200.1 ]
ATGTCGGTCCAGGCGGAGGAGCCGCGGGACACGGAGACAGCGCCTGCCCCGCTCAAGCGCCATGACTCGCTTTGGGGTGATGCAGAGAAGGTGTCCCATACCAACCACCATGGCTCCCGGGTGAGCTGGGTCCGGACGCTGAGCCTCGCCTTCCAGAGCGTCGGCATCATCTACGGCGACATCGGGACGTCGCCGCTCTACGTCTACTCCAGCACCTTCCCCGACGGCATCAAGCACAACGACGACCTCCTGGGCGTCCTGTCGCTCATCATCTACACCCTCATCATAATACCCATGCTCAAGTACGTCTTCATCGTGCTCTACGCAAACGACAACGGAGATGGTGGCACGTTTGCGCTTTACTCCCTGATATCGCGGTATGCAAAGATCAGGCTGATACCGGACCAGCAGGCCGAGGATGCTGCGGTGTCGAATTACCGGATAGAAGCGCCCAACTCGCAGCTGAGGAGGGCGCAGTGGGCCAAGCAGAAGCTCGAGTCTAGCAAGGCGGCCAAGATCGCGCTCTTCACCCTCACCATCCTCGGCACATCCATGGTGATCGGCGATGGAACCTTGACGCCCGCAATCTCTGTGCTGTCTGCAGTGAGTGGGATCAGAGAAAAAGCACCAAGCCTCACTCAAACACAAGTGGTGCTCATCTCGGTGGCGATCCTGTTCATGCTCTTCTCGGTCCAGCGTTTCGGGACCGACAAGGTCGGCTACACGTTTGCTCCTGTCATCTCGGTGTGGTTCCTTTTCATTGCGGGCATCGGCTTGTACAACCTCGTCATTCATGATGTCGGTGTCCTACGGGCCTTCAATCCGATATATATCATACAGTACTTCAAGAGGAATGGCAAGGAGGGATGGGTTTCACTTGGTGGAGTCATCTTGTGTGTCACAGGCACAGAAGGTATGTTTGCTGACCTGGGACATTTCAACATCAGGGCTGTTCAGATCAGCTTCAACGGCATCTTGTTCCCGGCTGTTGGGCTGTGTTACATCGGCCAGGCGGCTTACCTGAGGAAATTCCCAGAGAACGTGGCAAACACCTTCTATAGATCTATCCCAGCACCAATGTTCTGGCCAACCTTCATCGTTGCCATCCTTGCTGCCATCATAGCAAGCCAAGCTATGCTGTCCGGCGCATTTGCCATCCTCTCCAAGGCTCTGTCTCTGGGTTGCATGCCCAGGGTTCAAGTCATCCACACATCACACAAATACGAGGGGCAGGTGTACATTCCTGAAGTCAACTTCATAATGGGATTGGCGAGCATCGTAGTCACCGTCGCCTTCAGAACCACCACAAGCATCGGGCATGCTTATGGGATCTGTGTTGTTACTACATTCATCATCACCACCCACCTGATGACTGTCGTGATGCTCCTCATATGGAAGAAGCACGTCATCTTCATCGCGCTCTTCTACGTCGTGTTTGGCTCCATAGAGATGATCTACCTCTCTTCCATACTGTCGAAATTCATCGAGGGCGGGTACCTCCCCATCTGCTTCGCACTGGTCGTGATGAGCCTGATGGCGGCATGGCACTACGTCCAAGTCAAGAGGTACTGGTACGAGCTGGACCACATCGTGCCTACTAGTGAACTGACAGTGCTGCTCGAGAAGAACGATGTGAGGAGGATCCCAGGGGTTGGCCTCCTCTACACGGAGCTGGTCCAGGGAATCCCTCCGGTGTTCCCTCGGCTGATCGAGAGGATACCATCCGTGCACTCCATCTTCATGTTCATGTCCATCAAGCACCTGCCCATCTCACGTGTACTGCCCGCAGAGAGGTTTCTCTTCCGGCAGGTCGGCCCGAGGGAGCAGCGGATGTTCCGCTGCGTGGCGCGGTATGGATATACCGACACGCTGGAGGAGCCCAAGGAGTTTGTTGCCTTCCTCATGGATGGGCTCAAGATGTTCATTCAAGAGGAGAGCGCATTCGCACACAACGAAGTGGAGGAGATCACCGCTGGTGGTGAAGCTTCCAATGATCAGCCATCTATGGCATCGGGGCGATCCACACGCAATGCAGTGCACAGCGAGGAGATGGTCCAAGCCAGGGTGAGCAGCCACTCGTCGGGGAGGATCGGTAGCTTCCACTCCAACCGGACAGTTGAGGAGGAGAAGCAACTGATTGACAGAGAGGTGGAACACGGGATGGTGTATCTGATGGGGGAGGCCAATGTCACCGCCAAAGCCAACTCCTCGGTCTTCAAGAAGGTGGTGGTCAACTATGTTTACACATTCTTGAGGAAGAACTTGACGGAGGGGCACAAGGCACTAGCCATTCCGAAAGATCAGTTGCTCAAAGTTGGGATCACATATGAGATATAG, SED ID NO: 3; the sequence of the TaHAK1 protein is shown in SED ID NO: 4: [ TramesCS4D02G308200.1 ]
MSVARDTTAAKRHDSWGDAKVSHTNHHGSRVSWVRTSASVGIIYGDIGTSYVYSSTDGIKHNDDGVSIIYTIIIMKYVIVYANDNGDGGTAYSISRYAKIRIDADAAVSNYRIANSRRAWAKKSSKAAKIATTIGTSMVIGDGTTAISVSAVSGIRKASTTVVISVAIMSVRGTDKVGYTAVISVWIAGIGYNVIHDVGVRANIYIIYKRNGKGWVSGGVICVTGTGMADGHNIRAVISNGIAVGCYIGAAYRKNVANTYRSIAMWTIVAIAAIIASAMSGAAISKASGCMRVVIHTSHKYGVYIVNIMGASIVVTVARTTTSIGHAYGICVVTTIITTHMTVVMIWKKHVIIAYVVGSIMIYSSISKIGGYICAVVMSMAAWHYVVKRYWYDHIVTSTVKNDVRRIGVGYTVGIVRIRISVHSIMMSIKHISRVARRVGRRMRCVARYGYTDTKVAMDGKMISAAHNVITAGGASNDSMASGRSTRNAVHSMVARVSSHSSGRIGSHSNRTVKIDRVHGMVYMGANVTAKANSSVKKVVVNYVYTRKNTGHKAAIKDKVGITYI，SED ID NO:4。
In the present invention, the potassium stress preferably includes a low potassium stress in which the potassium content is preferably 3mM K/L or less and more than 0mM K/L, and in the present embodiment, the potassium content is 0.3mM K/L in the low potassium stress, and a potassium deficiency stress in which the potassium content is preferably 0mM K/L (in practice, the potassium ion is replaced by another element). The rice over-expressing the TaHAK1 gene can still show an excellent growth state under the states of low potassium and potassium deficiency, the dry weight of a plant and the biomass of a root system and leaves are obviously higher than those of a control plant, and simultaneously, the K content of a transgenic rice plant is obviously increased in the root system and the leaves; however, under the potassium deficiency condition, the K content of the transgenic rice plant is obviously higher than that of the wild rice plant only on the overground part, and is obviously reduced in the root system, which indicates that under the potassium deficiency condition, the TaHAK1 gene influences the transfer of K to the leaf from the rice root system, and improves the low potassium resistance of the transgenic rice.
The invention also provides application of the over-expressed TaHAK1 in improving the dry weight of potassium stress rice plants. In the invention, under the conditions of low potassium and potassium deficiency, the rice plant over expressing TaHAK1 can effectively improve the dry weight of root systems and leaves.
The invention also provides application of the over-expressed TaHAK1 in improving the potassium content in roots and leaves of potassium-stressed rice. In the invention, under the low potassium state, the dry weight of the root system of the rice plant over expressing TaHAK1 is increased by 1.33 times compared with that of the wild rice, and the dry weight of the leaves is increased by 1.19 times; under the potassium deficiency state, the dry weight of the leaves of the rice plants over expressing TaHAK1 is increased by 1.19 times.
The invention also provides application of the over-expressed TaHAK1 in improving transfer of potassium from a root system to a leaf blade in potassium stress rice. In the invention, under the low potassium state, the K content of the rice plant over expressing TaHAK1 is obviously increased, and the maximum K content is as high as 34.2%; under the potassium deficiency condition, the K content of the rice plant over-expressing TaHAK1 is increased in the overground part and is reduced in the underground part, which shows that under the potassium deficiency condition, the over-expression vector in the rice plant over-expressing TaHAK1 influences the transfer of K from the rice root system to the leaf.
For further illustration of the present invention, the following detailed description will be made of the application of the overexpressed TaHAK1 provided by the present invention in improving potassium stress tolerance of rice, with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
TaHAK1 protein and obtaining of coding gene thereof
1.1 extraction of Total RNA from wheat and preparation of cDNA
1) Extracting total RNA of wheat: taking 0.1g of wheat seedling root system as a material, grinding the wheat seedling root system in liquid nitrogen to obtain freeze-dried powder, transferring the freeze-dried powder into a 1.5mL centrifuge tube containing 1mL of a trizol reagent (purchased from Invitrogen), and fully and uniformly mixing the freeze-dried powder; standing at 25 deg.C for 5 min; adding 0.2mL of precooled chloroform into a centrifuge tube, shaking for 15s, and standing for 2-3 min at 25 ℃; centrifugation (4 ℃, 12000rpm, 15 min); transferring the supernatant (about 0.5mL) to a new 1.5mL centrifuge tube, adding 0.5mL of pre-cooled isopropanol, and standing at 25 ℃ for 10 min; centrifugation (4 ℃, 12000rpm, 15 min); taking the precipitate, washing the precipitate for 2 times by using 75% ethanol (1mL), and blowing the washed precipitate on a super clean bench for 3-5 min; adding 50 mu L of LDEPC-ddH2O for heavy suspension precipitation to obtain an extracted total RNA solution, and storing the total RNA solution at-70 ℃ for later use after quality detection. A template for reverse transcription is prepared.
2) RT-PCR: the total RNA solution obtained by extraction was diluted with reference to the RT-PCR kit (purchased from Taraka) instructions; according to the quantitative result of RNA, 0.5. mu.g Oligo dT primer and DEPC-ddH were added to 1. mu.g RNA2Supplementing O to 7.5 μ L, mixing, denaturing at 65 deg.C for 5min, and standing on ice for 5 min; after transient centrifugation, 12.5. mu.L of Reverse transcription mix (including 5 × ReactionBuffer, 2.5. mu.L; dNTPMixture (2.5mM), 3.0. mu.L; Reverse Transcriptase Enzymes, 0.5. mu.L; RNase Inhibitor, 0.5. mu.L; DEPC-ddH) was added2O, 6.0. mu.L). After mixing, PCR conditions: 42 ℃ for 1 h; at 95 ℃ for 10 min; and (4) completing the reverse transcription of the cDNA to obtain cDNA, and determining the concentration of the cDNA for later use.
1.2 amplification of TaHAK1
Primers were designed based on the sequence of TaHAK1, shown as SED ID NO. 3 for TaHAK 1.
The forward primer in the designed primer is shown as SED ID NO: 5: 5'-ATGTCGCTCCAGGTCGAGG-3', SED ID NO: 5;
the reverse primer is shown as SED ID NO: 6: 5'-CTATATCTCGTATGTGATCCCGA-3', SED ID NO: 6;
adding cDNA prepared in 1.1 as a template, and configuring a reaction system as follows: prime Star (10 μ L); forward and reverse primers SED ID NO 5 and SED ID NO 6 (1. mu.L each); cDNA (1. mu.L); distilled water (8. mu.L). Then using PCR amplification, the amplification program: pre-denaturation (94 ℃, 1 min); after 32 cycles of PCR amplification (denaturation at 94 ℃, 10 min; renaturation at 56 ℃, 15 s; extension at 72 ℃, 1min), continuing extension at 72 ℃ for 10min to obtain a PCR product, separating the PCR product by agarose gel electrophoresis, and obtaining a PCR product band with about 2337bp by electrophoresis separation as shown in figure 1.
The PCR product was recovered and sequenced. The sequencing result showed that the nucleotide sequence of the PCR product was identical to the SED ID NO 3 sequence. Indicating that the sequence of TaHAK1 was obtained by amplification.
Example 2
TaHAK1 protein and application of coding gene thereof
2.1 construction of TaHAK1 overexpression vector
Designing a pair of primers, wherein the sequence of the forward primer is shown as SED ID NO. 1; the reverse primer is shown as SED ID NO: 2. The coding sequence of TaHAK1 gene is obtained by PCR amplification with SED ID NO. 1 and SED ID NO. 2, and the fragments are recovered, namely PCR products.
The plasmid of the backbone vector pCUN-1301 is subjected to double enzyme digestion by using restriction enzymes KpnI and BamHI, wherein the enzyme digestion system is as follows: mu.L of plasmid (10. mu.L (1mg), 5. mu. L, BamHI 1. mu.L of 10 Xdigestion buffer (10U/. mu.L), 0.8. mu.L of KpnI (10U/. mu.L), and ddH2O replenishes the reaction system to 50. mu.L, and the enzyme is cleaved at 37 ℃ for 3 h. And separating the enzyme digestion product by agarose gel electrophoresis, and recovering the linearized pUN1301 large fragment, namely the vector fragment.
And connecting the PCR product with the vector fragment by using T4 ligase, wherein the ratio of the vector fragment to the PCR product is 1:3, diluting and adding 2.0 mu L of the vector fragment and 6.0 mu L of the PCR product according to the concentration, adding 1.0 mu L of T4DNA ligase and 1.0 mu L of 10 Xligase buffer solution, mixing uniformly, centrifuging, and connecting for 10h at 16 ℃ to obtain a connecting product, namely the over-expression pCAMBIA1300-TaHAK1 vector. And transforming the ligation product into escherichia coli DH5 alpha competent cells, screening and culturing for 16h by using a resistance plate containing kanamycin to obtain a monoclonal, detecting the positive of the monoclonal by using PCR, and sequencing to verify the detection result of the PCR. The physical map of the pCAMBIA1300-TaHAK1 vector is shown in FIG. 2, which comprises Ubi (maize ubiquitin gene) promoter, TaHAK1 gene and Nos-T (Agrobacterium nopaline synthase terminator), respectively.
2.2 obtaining and identifying transgenic Rice with TaHAK1
(1) Acquisition of TaHAK1 transgenic Rice
The overexpression vector constructed by 2.1 is transferred into the agrobacterium EHA105 strain by an electric excitation instrument, positive clones are screened on a kanamycin resistant plate, and whether TaHAK1 is transferred or not is detected. Selecting and detecting correct positive clones to prepare engineering bacteria, and introducing the engineering bacteria into callus of Nipponbare paddy rice (Oryza sativa L.); after the introduction, the callus is washed for 4-5 times by using sterile water containing 300mg/L of cefuroxime, and the water is sucked dry by using sterile filter paper; transferring the callus to a medium containing hygromycin and cephalosporinN of mycin6D2Screening on a culture medium; the culture medium is changed once every 2 weeks, 3 generations of culture are continuously carried out, callus with good growth state is selected and transferred to a differentiation culture medium for culture, the photoperiod is set as day light (12h, 28 ℃), and dark (12h, 24 ℃) at night; replacing the culture medium once a week until seedlings are differentiated; and (3) selecting seedlings which grow vigorously, transferring the seedlings to a rooting culture medium for rooting culture, opening a container sealing film when the seedlings grow to about 10cm, hardening the seedlings for 2-3 days, and then transferring the seedlings to an artificial climate chamber for cultivation, namely T0 generation transgenic seedlings. The specific operation process is shown in fig. 3.
(2) Identification of TaHAK1 transgenic rice
DNA of the T0 transgenic seedlings (Nos. #1 to #8) was extracted, and hygromycin primers were used to detect whether the transgenic rice plants contained hygromycin genes, as shown in A in FIG. 4, which indicates that the plants tested contained hygromycin genes except for Wild Type (WT). Indicating that these strains carry the selection marker gene of the overexpression vector.
On the basis, a primer LBP (forward primer F shown by SED ID NO: 7: 5'-AAAAGGAAGGTGGCTCCTAC-3', reverse primer R shown by SED ID NO: 8: 5'-TCCCTCGGCCCGACCTG-3') is designed near the upstream of the insertion site of the vector gene, the LBP and the downstream fragment (RP) of the inserted gene are combined, the amplification result is shown as B in FIG. 4, and as can be seen from B in FIG. 4, the amplification result of the combination of the vector primer and the downstream primer of the gene is larger than that of the gene, which indicates that the TaHAK1 gene is over-expressed in the transgenic rice. And (3) carrying out generation-adding culture on the plants carrying the over-expression vector, and respectively extracting the total protein of the transgenic plants containing the target gene and the marker gene from a large number of planted T2 generation plants. The expression level of these strains at the protein level was determined using the HA-tag antibody. The detection results are shown in fig. 4 as C: the expression level of the transgenic strains TaHAK1-OE1 and TaHAK1-OE3 is much higher than that of other strains. These 2 lines were used for subsequent experiments.
2.3 detection of the role of transgenic Rice in K stress tolerance
The seeds of the screened homozygous strains TaHAK1-OE1 and TaHAK1-OE3 germinate independently, seedlings with consistent growth and development are selected and divided into 3 parts when the seeds grow to 3 leaf stages, and normal (+ K, 6.0mM K/L), low-potassium (LK, 0.3mM K/L) and potassium-deficiency (DK, 0mM K/L) treatment is carried out on different types of plants (a control group is wild type plants not carrying over-expression vectors and marked as WT).
The growth results of rice after 14 days of potassium stress are shown in fig. 5, and it can be seen from fig. 5 that the growth and development states of transgenic rice plants under LK and DK conditions are obviously better than those of wild plants.
The biomass of rice was measured as shown in FIG. 6 and Table 1. Root in fig. 6 refers to Root and Leaf.
TABLE 1 Rice biomass assay data
As can be seen from FIG. 6 and Table 1, the dry weight of the transgenic rice plants was increased in comparison with the control plants under LK and DK conditions, respectively, in which transgenic rice plants TaHAK1-OE1 and TaHAK1-OE3 were increased 1.32 and 1.33 times in the root system and 1.10 and 1.19 times in the leaves, respectively, under LK conditions; under DK conditions, 2 lines of TaHAK 1-transgenic rice plants were significantly increased in leaf blades by 1.18 and 1.19 times, respectively, while the differences in root systems were not significant.
The measurement of K content in rice is shown in FIG. 7 and Table 2. Root in FIG. 7 refers to Root and Leaf in Leaf.
TABLE 2 determination of K content in Rice
As can be seen from fig. 7 and table 2, under LK condition, the K content of the transgenic rice plants was significantly increased in both root and leaf (increased by 34.2%, 35.4% and 19.2%, 33.1% respectively compared to the control); under the DK condition, the K content of the TaHAK1 transgenic rice plant is obviously higher than that of a wild type (1.93 and 1.81 times) only in the overground part and is obviously reduced (0.47 and 0.38 times) in the root system, which indicates that the overexpression vector in the transgenic rice influences the transfer of K from the root system of the rice to leaves under the potassium deficiency condition.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<120> application of over-expressed TaHAK1 in improving potassium stress tolerance of rice
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gtcatccaca catcacacaa atacgagggg caggtgtaca ttcctgaagt caacttcata 1260
atgggattgg cgagcatcgt agtcaccgtc gccttcagaa ccaccacaag catcgggcat 1320
gcttatggga tctgtgttgt tactacattc atcatcacca cccacctgat gactgtcgtg 1380
atgctcctca tatggaagaa gcacgtcatc ttcatcgcgc tcttctacgt cgtgtttggc 1440
tccatagaga tgatctacct ctcttccata ctgtcgaaat tcatcgaggg cgggtacctc 1500
cccatctgct tcgcactggt cgtgatgagc ctgatggcgg catggcacta cgtccaagtc 1560
aagaggtact ggtacgagct ggaccacatc gtgcctacta gtgaactgac agtgctgctc 1620
gagaagaacg atgtgaggag gatcccaggg gttggcctcc tctacacgga gctggtccag 1680
ggaatccctc cggtgttccc tcggctgatc gagaggatac catccgtgca ctccatcttc 1740
atgttcatgt ccatcaagca cctgcccatc tcacgtgtac tgcccgcaga gaggtttctc 1800
ttccggcagg tcggcccgag ggagcagcgg atgttccgct gcgtggcgcg gtatggatat 1860
accgacacgc tggaggagcc caaggagttt gttgccttcc tcatggatgg gctcaagatg 1920
ttcattcaag aggagagcgc attcgcacac aacgaagtgg aggagatcac cgctggtggt 1980
gaagcttcca atgatcagcc atctatggca tcggggcgat ccacacgcaa tgcagtgcac 2040
agcgaggaga tggtccaagc cagggtgagc agccactcgt cggggaggat cggtagcttc 2100
cactccaacc ggacagttga ggaggagaag caactgattg acagagaggt ggaacacggg 2160
atggtgtatc tgatggggga ggccaatgtc accgccaaag ccaactcctc ggtcttcaag 2220
aaggtggtgg tcaactatgt ttacacattc ttgaggaaga acttgacgga ggggcacaag 2280
gcactagcca ttccgaaaga tcagttgctc aaagttggga tcacatatga gatatag 2337
<210> 4
<211> 565
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Ser Val Ala Arg Asp Thr Thr Ala Ala Lys Arg His Asp Ser Trp
1 5 10 15
Gly Asp Ala Lys Val Ser His Thr Asn His His Gly Ser Arg Val Ser
20 25 30
Trp Val Arg Thr Ser Ala Ser Val Gly Ile Ile Tyr Gly Asp Ile Gly
35 40 45
Thr Ser Tyr Val Tyr Ser Ser Thr Asp Gly Ile Lys His Asn Asp Asp
50 55 60
Gly Val Ser Ile Ile Tyr Thr Ile Ile Ile Met Lys Tyr Val Ile Val
65 70 75 80
Tyr Ala Asn Asp Asn Gly Asp Gly Gly Thr Ala Tyr Ser Ile Ser Arg
85 90 95
Tyr Ala Lys Ile Arg Ile Asp Ala Asp Ala Ala Val Ser Asn Tyr Arg
100 105 110
Ile Ala Asn Ser Arg Arg Ala Trp Ala Lys Lys Ser Ser Lys Ala Ala
115 120 125
Lys Ile Ala Thr Thr Ile Gly Thr Ser Met Val Ile Gly Asp Gly Thr
130 135 140
Thr Ala Ile Ser Val Ser Ala Val Ser Gly Ile Arg Lys Ala Ser Thr
145 150 155 160
Thr Val Val Ile Ser Val Ala Ile Met Ser Val Arg Gly Thr Asp Lys
165 170 175
Val Gly Tyr Thr Ala Val Ile Ser Val Trp Ile Ala Gly Ile Gly Tyr
180 185 190
Asn Val Ile His Asp Val Gly Val Arg Ala Asn Ile Tyr Ile Ile Tyr
195 200 205
Lys Arg Asn Gly Lys Gly Trp Val Ser Gly Gly Val Ile Cys Val Thr
210 215 220
Gly Thr Gly Met Ala Asp Gly His Asn Ile Arg Ala Val Ile Ser Asn
225 230 235 240
Gly Ile Ala Val Gly Cys Tyr Ile Gly Ala Ala Tyr Arg Lys Asn Val
245 250 255
Ala Asn Thr Tyr Arg Ser Ile Ala Met Trp Thr Ile Val Ala Ile Ala
260 265 270
Ala Ile Ile Ala Ser Ala Met Ser Gly Ala Ala Ile Ser Lys Ala Ser
275 280 285
Gly Cys Met Arg Val Val Ile His Thr Ser His Lys Tyr Gly Val Tyr
290 295 300
Ile Val Asn Ile Met Gly Ala Ser Ile Val Val Thr Val Ala Arg Thr
305 310 315 320
Thr Thr Ser Ile Gly His Ala Tyr Gly Ile Cys Val Val Thr Thr Ile
325 330 335
Ile Thr Thr His Met Thr Val Val Met Ile Trp Lys Lys His Val Ile
340 345 350
Ile Ala Tyr Val Val Gly Ser Ile Met Ile Tyr Ser Ser Ile Ser Lys
355 360 365
Ile Gly Gly Tyr Ile Cys Ala Val Val Met Ser Met Ala Ala Trp His
370 375 380
Tyr Val Val Lys Arg Tyr Trp Tyr Asp His Ile Val Thr Ser Thr Val
385 390 395 400
Lys Asn Asp Val Arg Arg Ile Gly Val Gly Tyr Thr Val Gly Ile Val
405 410 415
Arg Ile Arg Ile Ser Val His Ser Ile Met Met Ser Ile Lys His Ile
420 425 430
Ser Arg Val Ala Arg Arg Val Gly Arg Arg Met Arg Cys Val Ala Arg
435 440 445
Tyr Gly Tyr Thr Asp Thr Lys Val Ala Met Asp Gly Lys Met Ile Ser
450 455 460
Ala Ala His Asn Val Ile Thr Ala Gly Gly Ala Ser Asn Asp Ser Met
465 470 475 480
Ala Ser Gly Arg Ser Thr Arg Asn Ala Val His Ser Met Val Ala Arg
485 490 495
Val Ser Ser His Ser Ser Gly Arg Ile Gly Ser His Ser Asn Arg Thr
500 505 510
Val Lys Ile Asp Arg Val His Gly Met Val Tyr Met Gly Ala Asn Val
515 520 525
Thr Ala Lys Ala Asn Ser Ser Val Lys Lys Val Val Val Asn Tyr Val
530 535 540
Tyr Thr Arg Lys Asn Thr Gly His Lys Ala Ala Ile Lys Asp Lys Val
545 550 555 560
Gly Ile Thr Tyr Ile
565
<210> 5
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atgtcgctcc aggtcgagg 19
<210> 6
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ctatatctcg tatgtgatcc cga 23
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
aaaaggaagg tggctcctac 20
<210> 8
<211> 17
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tccctcggcc cgacctg 17