Method for improving plant phosphorus absorption capacity by wheat phosphorus transport protein TaPHT1 and 9-4B and application thereof
阅读说明：本技术 小麦磷转运蛋白TaPHT1;9-4B在提高植物磷吸收能力的方法及其应用 (Method for improving plant phosphorus absorption capacity by wheat phosphorus transport protein TaPHT1 and 9-4B and application thereof ) 是由 康国章 王鹏飞 李鸽子 葛强 韩巧霞 刘国芹 王永华 郭天财 于 2021-09-26 设计创作，主要内容包括：本发明公开了小麦磷转运蛋白技术领域的小麦磷转运蛋白TaPHT1;9-4B在提高植物磷吸收能力的方法,包括以下步骤：S1、TaPHT1.9-4B磷转运蛋白编码基因的克隆；S2、TaPHT1.9-4B过表达载体的构建；S3、水稻遗传转化与转基因水稻植株的检测；S4、TaPHT1.9-4B过表达转基因水稻在水培与土培环境下的功能验证；通过测定不同磷肥处理条件下水稻根系、籽粒以及地上部分的总磷浓度发现,转基因与野生型植株根系与地上部分的磷浓度随着磷肥的增加而增加,在四种磷肥处理条件下,转基因株系根系与地上部分的磷浓度均显著大于野生型植株,而籽粒中的磷浓度在中等磷肥与高等磷肥条件下,没有显著差异；在磷供给不足的环境下,转基因植株籽粒中的磷浓度大于野生型水稻,并且,转基因株系的地上部分生物量和单株产量显著高于野生型转基因。(The invention discloses a method for improving plant phosphorus absorption capacity of wheat phosphorus transporters TaPHT1 and 9-4B in the technical field of wheat phosphorus transporters, which comprises the following steps: cloning coding genes of S1 and TaPHT1.9-4B phosphorus transport protein; constructing an over-expression vector of S2 and TaPHT1.9-4B; s3, detecting rice genetic transformation and transgenic rice plants; s4 and TaPHT1.9-4B overexpression transgenic rice function verification in water culture and soil culture environments; the phosphorus concentrations of the root system and the overground part of the transgenic and wild plants are increased along with the increase of the phosphate fertilizer by measuring the total phosphorus concentrations of the root system, the seed and the overground part of the rice under different phosphate fertilizer treatment conditions, the phosphorus concentrations of the root system and the overground part of the transgenic plant are obviously greater than those of the wild plants under the four phosphate fertilizer treatment conditions, and the phosphorus concentrations in the seed have no obvious difference under the conditions of medium phosphate fertilizer and high phosphate fertilizer; under the environment of insufficient phosphorus supply, the phosphorus concentration in the transgenic plant grains is higher than that of wild rice, and the biomass of the overground part and the single plant yield of the transgenic line are obviously higher than that of the wild transgenic line.)
1. Wheat phosphate transporter TaPHT1;9-4B in the method for improving the phosphorus absorption capacity of plants, is characterized by comprising the following steps:
cloning coding genes of S1 and TaPHT1.9-4B phosphorus transport protein;
constructing an over-expression vector of S2 and TaPHT1.9-4B;
s3, detecting rice genetic transformation and transgenic rice plants;
and S4 and TaPHT1.9-4B overexpression transgenic rice function verification in water culture and soil culture environments.
2. The wheat phosphate transporter TaPHT1 of claim 1;9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: the step S1 includes the steps of:
s1.1, extracting total RNA of wheat:
a. extracting RNA of a root system of a Zhoumai 18 wheat seedling in a two-leaf one-heart period by adopting a Trizol method, firstly carrying out liquid nitrogen grinding on 0.1g of a freshly collected root system to form powder, putting the powder into a 1.5mL enzyme-free centrifuge tube, adding 1mL Trizol, then violently oscillating and uniformly mixing, and standing at room temperature for 30 min;
b. centrifuging at 12000rpm at low temperature (4 deg.C) for 5min, sucking supernatant into a new centrifuge tube, adding 1/3 volumes of chloroform, mixing, and standing for 15 min;
c.12000rpm low temperature (4 ℃) centrifugation for 5min, transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume, fully mixing, and standing for 30 min. Centrifuging at 12000rpm at low temperature (4 ℃) for 5min, and then removing the supernatant;
d. 1mL of 75% ethanol (made up of DEPC treated water) was added to each tube, washed thoroughly, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the process repeated twice. Drying RNA, adding 40 mu L of enzyme-free water, dissolving on ice, and measuring the concentration and quality of RNA in an ultraviolet spectrophotometer by taking 1 mu L of RNA solution;
s1.2, reverse transcription synthesis of a first strand cDNA:
e. using reverse transcription kit (PrimeScript)TMII 1st Strand cDNA Synthesis Kit) to synthesize the first chain of cDNA by reverse transcription;
f. add to a 100. mu.L enzyme-free centrifuge tube in sequence: mu.L oligo dT primer (50. mu.M), 1. mu.L dNTP mix (10mM each), 2. mu.g Total RNA, enzyme-free water to a Total volume of 10. mu.L, incubation at 65 ℃ for 5min and rapid cooling on ice;
g. mu.L of 5 XPrimeScript II Buffer, 1. mu.L of PrimeScript II RTase (200U/. mu.L), 0.5. mu.L of RNase Inhibitor (40U/. mu.L), and 4.5. mu.L of enzyme-free water were added to the above reaction solution in this order. Inactivating at 42 deg.C for 60min and 95 deg.C for 5min, and cooling on ice to obtain wheat cDNA;
s1.3, PCR amplification of TaPHT1.9-4B gene CDS: according to the wheat phosphate transporter TaPHT1;9-4B gene (GenBank No. AIZ11192.1) in the latest wheat genome database corresponding gene sequence (TramesCS 4B02G317000), design amplification primer TaPHT1; 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGCCATCGT, synthesizing primers by Henan Shanghai Biotechnology company, and performing PCR amplification on TaPHT1 by taking wheat cDNA as a template; 9-4B gene coding region (CDS), PCR amplification system including: 10 μ L of 5 XPrimeSTAR Buffer (Mg)2 +Plus)，0.5μLPrimeSTAR HSDNApolymeras(2.5U/μL)，4.0μL dNTP Mixture(2.5mM each)，TaPHT1；9-4B-CD1.0. mu.L each of S-F, R primers (10. mu.M), 1.0. mu.L of LcDNA, plus ddH2O to a total volume of 50. mu.L. The PCR amplification procedure is the first step: 5min at 94 ℃; the second step is that: 30s at 94 ℃, 30s at 56 ℃, 2min at 72 ℃ and 32 cycles; and thirdly, carrying out electrophoresis separation on the PCR product at 72 ℃ for 5min by using 1.0% agarose gel, wherein the result is shown in figure 1, the size of a strip of the PCR product is 1566bp, recovering the PCR product, carrying out enzyme ligation with a pMD19-T vector to transform escherichia coli DH5 alpha competence, screening ampicillin (100 mu g/mL) plates, carrying out colony PCR detection, sequencing the positive clone and extracting plasmid, wherein the sequencing result is the same as a reference sequence in a Chinese spring wheat genome (the homology reaches 100%), the gene does not contain an intron, a coding region contains 1566bp nucleotide and 521 coded amino acids (shown in figure 2).
3. The wheat phosphate transporter TaPHT1 of claim 2; 9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: in the step d, when the value of OD260/OD280 is between 1.80 and 2.00, and OD260/OD230 is greater than 2.0, the RNA quality is better, and the RNA can be used as a reverse transcription template.
4. The wheat phosphate transporter TaPHT1 of claim 1;9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: the step S2 includes the steps of:
s2.1, designing a primer TaPHT1 according to a CDS sequence of a TaPHT1.9-4B gene and a multiple cloning site of a plant expression vector pCUN1301 (containing a Ubi promoter); 9-4B-trangle-F: TCC (transmission control center)CCCGGGATGGCGACTGAACAGCTC (underlined sequence is SacI cleavage site), R: GCGTCGACCTAAGCTTCGATGCCATCGT (the underlined sequence is the SalI cleavage site);
s2.11, using the primer pair, using the TaPHT1.9-4BCDS cloning vector as a template, carrying out PCR amplification on TaPHT1.9-4BCDS with two ends respectively connected with SacI and SalI enzyme cutting sites, recovering a target fragment, connecting with pMD19-T, transforming DH5 alpha escherichia coli to obtain a positive bacterial colony, carrying out amplification culture, and extracting a plasmid by the same method as the above description;
s2.12, double enzyme digestion is carried out on the TaPHT1.9-4B-CDS-T carrier by using restriction endonucleases SacI and SalI,the enzyme cutting system is as follows: mu.g of plasmid, 5. mu.L of 10 Xdigestion buffer, 1. mu.L each of Sac I and Sal I (10U/. mu.L), plus ddH2Supplementing the reaction system to 50 mu L by O, and carrying out enzyme digestion at 37 ℃ for 4 hours;
s2.13, separating the enzyme digestion product by using 1.0% agarose gel electrophoresis, recovering a fragment of about 1566bp by using a DNA gel recovery kit of Takara company, carrying out double enzyme digestion on the plasmid of the plant expression vector pCUN1301 by using restriction enzymes SacI and SalI, separating the enzyme digestion product by using 1.0% agarose gel electrophoresis, and recovering a linearized pCUN1301 large fragment;
s2.2, mixing the enzyme digestion product of 6 mu LTaPHT1.9-4B-CDS with the enzyme digestion large fragment of 2 mu LpCUN1301, 1 mu L (10U/. mu.L) of T4DNA ligase and 1 mu L of 10 Xligase buffer solution, connecting for 16 hours at 16 ℃, transforming escherichia coli DH5 alpha competent cells by the obtained connection product, and screening and sequencing by a resistance plate containing kanamycin (100 mu.g/mL) to obtain positive clones;
s2.21, extracting recombinant plasmids in the positive clones, and naming the recombinant plasmids as pCUN 1301-TaPHT1.9-4B. (vector map is shown in FIG. 3a), the promoter and terminator are the maize Ubiquitin promoter (Ubiquitin promoter) and the Agrobacterium nopaline synthase terminator (NosT), respectively.
5. The wheat phosphate transporter TaPHT1 of claim 4; 9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: the enzyme cutting system in the step S2.13 is as follows: 10. mu.L of plasmid, 10. mu.L of digestion buffer 5. mu. L, BamHI 1 (10U/. mu.L), 0.8. mu.L of KpnI (10U/. mu.L), and ddH2O supplemented the reaction to 50. mu.L, and cleaved at 37 ℃ for 4 hours.
6. The wheat phosphate transporter TaPHT1 of claim 1;9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: the step S3 includes the steps of:
s3.1, rice genetic transformation: transferring the pCUN1301-TaPHT1.9-4B recombinant vector into the callus of the mature embryo of the Nipponbare rice by adopting an agrobacterium-mediated dip-dyeing method;
s3.12, firstly, inducing mature embryos of rice to generate callus, transforming agrobacterium EHA105 by pCUN1301-TaPHT1.9-4B through a heat shock transformation method, co-culturing with the callus for infection transformation, culturing the infected callus in a screening culture medium containing kanamycin and hygromycin until the callus is differentiated into seedlings, and rooting in a rooting culture medium;
s3.2, identification of positive transgenic rice plants: DNA of the transgenic and wild rice plants is extracted, and first, a primer HptII-F is detected by using an HptII gene (KT 184677.1): CACGGCCTCCAGAAGAAGAT, R: CCTGCCTGAAACCGAACTGC, using the extracted DNA as a template to carry out PCR detection;
s3.21, reusing TaPHT1; carrying out PCR detection on CDS amplification primers (TaPHT 1; 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATG-CCATCGT) and a carrier primer (Ubi-F: AAAGGATCTGTATGTATGTG) of the 9-4B gene per se (the result is shown in figures 3B and c), and obtaining 6 positive plants which respectively contain HptII and TaPHT1;9-4B gene.
7. The method for improving plant phosphorus uptake capacity of wheat phosphorus transporters according to claim 6, wherein the method comprises the following steps: and in the step S3.12, picking out test-tube seedlings with relatively intact root and stem leaf growth, adding a proper amount of sterile water into a solid culture medium, hardening seedlings for about one week, and transplanting the seedlings into a rice nutrient solution.
8. The wheat phosphate transporter TaPHT1 of claim 1;9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: in the step S3, to detect TaPHT1;9-4B is normally expressed in rice plants, RT-PCR detection is carried out on target genes, firstly RNA of positive transgenosis and wild rice is extracted, cDNA first chain is synthesized by reverse transcription, and TaPHT1 is utilized by taking cDNA as a template; 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGCCATCGT and performing PCR amplification by the same method as above, wherein rice OsAction gene (AB047313) is used as an internal reference, and the OsAction amplification primer is F: TATGGTCAAGGCTGGGTTCG, R: CCTAATATCCACGTCGCACT, detected by agarose electrophoresis, TaPHT1;9-4B was successfully expressed in these 6 transgenic rice plants (as shown in FIG. 3 d). Transferring the transgenic positive seedlings to a greenhouse for cultivation, harvesting seeds according to different strains, and performing seed reproduction on the seeds to obtain homozygous T2 generation seeds of the transgenic pCUN1301-TaPHT1.9-4B rice.
9. The wheat phosphate transporter TaPHT1 of claim 1;9-4B in the method for improving the phosphorus absorption capacity of plants, which is characterized in that: the step S4 includes the steps of:
s4.1, TaPHT1;9-4B transgenic rice responds to different phosphorus concentrations under the condition of water culture, T2 generation seeds and wild type rice seeds of positive transgenic rice plants (OE1, OE3) germinate in a culture chamber, then the seeds are cultured in a rice nutrient solution (IRRI) for two weeks, then the rice plants with the same size are selected and cultured in nutrient solutions containing normal (CK, 1mM Pi), low phosphorus (LP, 50 mu M Pi) and phosphorus deficiency (NP, 0 mu M Pi) for three weeks, the growth phenotypes of the transgenic rice plants and the wild type rice plants are observed, the plant height root length and the dry weight of the root system and the overground part are determined, and the nutrient solution is replaced every two days;
s4.12, under the conditions of low phosphorus and phosphorus deficiency, the plant height, the dry weight of roots and the dry weight of the overground part of the transgenic plant are all obviously higher than those of a wild type plant (as shown in figures 4a and b), under the normal phosphorus supply condition, although the difference of the plant height and the root length is not obvious, the dry weight of the root system and the overground part of the transgenic plant is obviously higher than that of the wild type plant, and the results show that: wheat phosphate transporter TaPHT1; the coding gene of 9-4B is expressed heterologously in the rice body, and can promote the growth of the transgenic rice in the low-phosphorus and phosphorus-deficient environment;
s4.13, TaPHT1;9-4B transgenic and wild rice root and leaf tissue are digested and boiled with concentrated sulfuric acid and hydrogen peroxide, and phosphorus concentration is measured by molybdenum-antimony colorimetry. The results are shown in fig. 4c, where the phosphorus concentration of the transgenic plant root system and aerial parts is significantly higher than that of the wild type plant under both normal and low-phosphorus hydroponic conditions; under the condition of phosphorus deficiency, the phosphorus concentration of the overground part of the transgenic plant is higher than that of the wild plant, but the phosphorus concentration of the root system is obviously lower than that of the root system of the wild plant, which indicates TaPHT1; the heterologous expression of the 9 gene in the rice can improve the absorption and utilization of the transgenic rice to the phosphorus and enhance the adaptability of the transgenic rice to the low-phosphorus environment;
s4.2, TaPHT1; the function of 9-4B transgenic rice in different phosphorus supply level soil culture tests is verified, and TaPHT1 is further verified; 9-4B in the rice body, TaPHT1, which influences the absorption efficiency and yield of phosphate fertilizer in the whole growth period of transgenic rice in the soil growth environment; 9-4B transgenic rice lines (OE1 and OE3) were potted with wild type rice (WT);
s4.21, taking potted soil from a farm paddy field of Maozhuang of Henan university of agriculture, respectively applying 200mg/kg of nitrogen fertilizer (urea) and 50mg/kg of potassium fertilizer (potassium sulfate), and applying phosphate fertilizer (calcium superphosphate) to set four levels: phosphorus-free (NP: 0mg/kg Ca (H)2PO4)2.H2O), low phosphorus (LP: 50mg/kg Ca (H)2PO4)2.H2O), middle phosphorus (MP: 100mg/kg Ca (H)2PO4)2.H2O) and high phosphorus (HP: 200mg/kg Ca (H)2PO4)2.H2O), respectively and uniformly mixing the soil treated by different phosphate fertilizers, and subpackaging 10kg of soil in each pot;
s4.22, seeds of wild type and transgenic rice plant lines (OE1 and OE3) are subjected to seed soaking and germination accelerating, water culture seedling is carried out in a laboratory, tillering of rice seedlings occurs after 30 days, the rice seedlings of different plant lines are respectively planted in pot plants treated by different phosphate fertilizers, each plant line is treated by 6 pots, and two plants are grown in a field environment. Observing the phenotype and taking a picture in the mature period, harvesting seeds, testing the seeds, measuring the biomass of the overground part (straw) without the seeds, and simultaneously measuring the phosphorus concentration of the seeds, the straw and the root system;
s4.23, under the condition of not applying phosphate fertilizer (NP) and low phosphate fertilizer (LP), the growth condition of the transgenic line is superior to that of a wild plant, under the condition of medium phosphate fertilizer (MP), the growth phenotype of the transgenic plant is not obviously different from that of the wild plant, and under the condition of high phosphate fertilizer (HP), the growth of the transgenic plant is even inhibited to a certain extent compared with that of the wild plant, which is probably the phosphorus poisoning phenomenon caused by the high phosphorus environment.
10. The wheat phosphate transporter TaPHT1 of claim 1; the application of 9-4B in improving the phosphorus absorption capacity of plants is characterized in that: the results of the measurement of the yield of the individual plants and the biomass of the overground part show that the yield of the individual plants of the transgenic plants and the wild plants increases along with the increase of the applied phosphate fertilizer in the soil, the yield of the individual plants does not increase due to the application of the phosphate fertilizer after the medium phosphate fertilizer application amount is reached, the yield of the individual plants of the transgenic rice reaches the maximum yield under the condition of low phosphate fertilizer, and the biomass of the overground part shows a similar rule (fig. 5a and b). Thousand kernel weight measurements showed that the transgenic lines had a thousand kernel weight greater than wild type plants under phosphorus deficient and low phosphorus conditions (as shown in FIG. 5 c), which indicates TaPHT1; the expression of 9-4B in rice can make transgenic rice reach the yield of WT under higher phosphorus supply level at lower phosphate fertilizer level.
Technical Field
The invention relates to the technical field of wheat phosphorus transporters, in particular to a method for improving plant phosphorus absorption capacity by using wheat phosphorus transporters TaPHT1 and 9-4B and application thereof.
Background
Phosphorus is one of three essential nutrients for plant growth and development, phosphorus deficiency affects plant growth and development, and deficiency of available phosphorus (Pi) in soil also affects crop yield and quality (Raghothama, 2005). In addition, the utilization efficiency of the phosphate fertilizer by crops is low, so that the phosphate fertilizer is wasted and lost, and a series of resource and environmental problems are caused (sattariet, 2012). Therefore, the method improves the utilization efficiency of the crops to the phosphorus, and has important significance for the sustainable development of agriculture and the protection of resource environment.
Plants have evolved sophisticated transport systems to perform uptake of phosphorus in soil and partitioning of phosphorus in different plant organs, including proton-coupled Phosphate Transporter (PT) family proteins and other phosphorus transporters (populus et al, 2006; Rausch & Bucher, 2002; Mlodzinska & zboiska, 2016; Wang et al, 2018). The proteins known to have phosphorus transport activity include PHT proteins involved in phosphorus absorption and transport in soil, SPX-EXS subfamily proteins involved in phosphorus transport from the root system to the aerial parts through xylem vascular bundles, and SPX-MFS subfamily proteins involved in phosphorus transport in vacuoles. The PHT phosphate transporters are the most important phosphate absorption and transport system of plants, and the phosphate in the soil mainly enters the plant body through the PHT phosphate transporters on the cell membrane of the root system and is transported in the plant body through the PHT (Smith et al, 2011). Since the first discovery of the gene encoding the high affinity phosphate transporter PHO84 in yeast cells, an increasing number of PHT phosphorus transporters have been identified and functionally validated in a variety of plants based on amino acid homology to yeast phosphorus transporters and functional complementation tests of yeast mutants lacking the endogenous phosphorus transporter gene PHO84 (Bun-Ya et al, 1991; Wang et al, 2017). PHT phosphorus transporters in plants can be classified into high-affinity and low-affinity phosphorus transporters according to the size of the kinetic constant of phosphorus uptake (Km), with Km values for high-affinity phosphorus transporters being in the μmol/L range and Km values for low-affinity phosphorus transporters being in the mmol/L range (Schachtman et al, 1998; Vance, 2001; Lopez-Arredondo et al, 2014). The high affinity phosphorus transport protein is induced and expressed by phosphorus deficiency, is mainly responsible for absorbing low-concentration phosphorus element from the external environment, and is an important plant low-phosphorus response mechanism; low affinity phosphorus transporters are constitutively expressed, are not affected by changes in phosphorus concentration in the external environment, and function primarily under normal phosphorus supply conditions (Poirier & Bucher, 2002; Bucher, 2007). Plant phosphorus transporter genes are mainly divided into four gene families according to functional differences and subcellular localization: PHT1, PHT2, PHT3 and PHT4, where the PHT1 subfamily members are the most, mainly localized to the plasma membrane, the PHT2 protein is localized to the chloroplasts, the PHT3 protein is localized to the mitochondria, and the PHT4 protein is localized to the plastids and Golgi (Rausch & Bucher, 2002; Nussaume et al, 2011; Liu et al, 2011);
most of the currently identified plant PHT1 phosphorus transporters belong to the group of high affinity phosphorus transporters that share a high similarity in structure, with all plant PHT1 proteins having a conserved protein tag (GGDYPLASTIXSE) and 12 transmembrane domains (Raghothama, 1999; Vance, 2001; Karandashov & Bucher, 2005; Nussaume et al, 2011). The first plant PHT1 gene was cloned from Arabidopsis thaliana, similar to the Pi transporter gene (PHO 84) in Saccharomyces cerevisiae (Muchhal et al, 1996; Bun-Ya et al, 1991). Based on protein sequence homology and conserved protein tag analysis, multiple PHT1 members have been identified and cloned from multiple plants, most PHT1 genes are preferentially expressed in roots and are subject to phosphorus deficiency-induced expression, and are widely involved in phosphorus uptake in soil and phosphorus transport in plants (Wang et al, 2017; Victor et al, 2019);
the Arabidopsis genome contains 9 PHT1 family members, 8 of which are expressed induced by phosphorus starvation (Muchhal et al, 1996; Mudge et al, 2002; Aung et al, 2006). AtPHT1;1 and AtPHT1;4 are two high expression PHT1 proteins, responsible for the absorption of most of the phosphorus, whether under low Pi or high Pi conditions (Misson et al, 2004; Shin et al, 2004); AtPHT1;1, AtPHT1;2 and AtPHT1;3 is primarily involved in Pi absorption, AtPHT1;1 is involved in Pi transfer from root to leaf (Ayadi et al, 2015); AtPHT1, 8 and AtPHT1, 9 are involved in the absorption of Pi and the transport of root systems to the aerial parts, and show genetic interactions with other AtPHT1 (Remy et al, 2012; Lapis-Gaza et al, 2014); AtPHT1;5 has higher transcription levels in leaves than in roots and plays an important role in Pi mobilization in sink-source organs (Nagarajan et al, 2011). In the rice genome, there are 13 PHT1 transporters, 9 of which have been functionally validated, most of which are involved in Pi uptake and transport. OsPHT1;2 has been shown to be associated with the transfer of Pi in the root cap, while OsPHT1;6 is associated with both Pi uptake and root cap transfer (Ai et al, 2009); OsPHT1, 1 and OsPHT1, 8 still maintain high expression level under sufficient phosphorus, participate in the absorption of Pi and the transfer from root to overground part, and OsPHT1, 8 also participate in the growth and development of plants (Jia et al, 2011; Sun et al, 2012); OsPHT1, 9 and OsPHT1, 10 have redundancy to the function of Pi absorption (Wang et al, 2014); OsPHT1, 4 not only shadow(iv) response to the uptake and transport of Pi, and also influence embryo development (Zhang et al, 2015); OsPHT1, 11a and OsPHT1, 13 are induced to express by arbuscular bacteria (AM), are necessary for symbiotic development of AM fungi and rice roots, are similar to MtPHT1 in alfalfa, 4 in function, but only OsPHT1 and 11 play a main role in the Pi absorption process in which AM participates (Javot et al, 2007; Yang et al, 2012). In addition, the phosphorus transporter gene of rapeseedBnPht1；4Heterologous expression in arabidopsis plants can alter root morphology and response to phosphorus starvation in transgenic arabidopsis (Feng et al, 2014). The tobacco phosphorus transport protein NtPT1 is expressed heterologously in rice, so that the utilization efficiency of phosphorus of transgenic rice and the accumulation of phosphorus in plants can be obviously improved (Myoung et al, 2007);
wheat has 14 PHT1 phosphorus transporters, and most of TaPHT1 genes are induced to express by phosphorus deficiency stress. However, compared with rice and Arabidopsis, the research on wheat PHT1 gene is still deficient, the function of PHT1 protein is still little known, and proteomics analysis finds that a phosphorus transport protein TaPHT1 and 9-4B which are obviously up-regulated and expressed in the root system of phosphorus-deficient wheat can obviously express the phosphorus absorption capacity and utilization efficiency of transgenic rice through heterologous expression in rice bodies, so that the method for improving the plant phosphorus absorption capacity by wheat phosphorus transport protein TaPHT1 and 9-4B and the application thereof are provided.
Disclosure of Invention
The invention aims to provide a method for improving plant phosphorus absorption capacity by wheat phosphorus transport proteins TaPHT1 and 9-4B and application thereof, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
a method for improving the phosphorus absorption capacity of plants by using wheat phosphorus transportprotein TaPHT1 and 9-4B comprises the following steps:
cloning coding genes of S1 and TaPHT1.9-4B phosphorus transport protein;
constructing an over-expression vector of S2 and TaPHT1.9-4B;
s3, detecting rice genetic transformation and transgenic rice plants;
and S4 and TaPHT1.9-4B overexpression transgenic rice function verification in water culture and soil culture environments.
1. Preferably, the step S1 includes the steps of:
s1.1, extracting total RNA of wheat:
a. extracting RNA of a root system of a Zhoumai 18 wheat seedling in a two-leaf one-heart period by adopting a Trizol method, firstly carrying out liquid nitrogen grinding on 0.1g of a freshly collected root system to form powder, putting the powder into a 1.5mL enzyme-free centrifuge tube, adding 1mL Trizol, then violently oscillating and uniformly mixing, and standing at room temperature for 30 min;
b. centrifuging at 12000rpm at low temperature (4 deg.C) for 5min, sucking supernatant into a new centrifuge tube, adding 1/3 volumes of chloroform, mixing, and standing for 15 min;
c.12000rpm low temperature (4 ℃) centrifugation for 5min, transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume, fully mixing, and standing for 30 min. Centrifuging at 12000rpm at low temperature (4 ℃) for 5min, and then removing the supernatant;
d. 1mL of 75% ethanol (made up of DEPC treated water) was added to each tube, washed thoroughly, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the process repeated twice. Drying RNA, adding 40 mu L of enzyme-free water, dissolving on ice, and measuring the concentration and quality of RNA in an ultraviolet spectrophotometer by taking 1 mu L of RNA solution;
s1.2, reverse transcription synthesis of a first strand cDNA:
e. using a reverse transcription kit (PrimeScript. sup. II 1)stStrand cDNA Synthesis Kit) to synthesize the first chain of cDNA;
f. add to a 100. mu.L enzyme-free centrifuge tube in sequence: mu.L oligodT primer (50. mu.M), 1. mu.L dNTP mix (10mM each), 2. mu.g Total RNA, enzyme-free water to a Total volume of 10. mu.L, incubation at 65 ℃ for 5min and rapid cooling on ice;
g. mu.L of 5 XPrimeScript II Buffer, 1. mu.L of PrimeScript II RTase (200U/. mu.L), 0.5. mu.L of RNase Inhibitor (40U/. mu.L), and 4.5. mu.L of enzyme-free water were added to the above reaction solution in this order. Inactivating at 42 deg.C for 60min and 95 deg.C for 5min, and cooling on ice to obtain wheat cDNA;
s1.3, PCR amplification of TaPHT1.9-4B gene CDS: according to the wheat phosphorus transport protein TaPHT1 and the 9-4B gene (GenBank No. AIZ11192.1)The corresponding gene sequence (TramesCS 4B02G317000) in the wheat genome database is used for designing an amplification primer TaPHT1, 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGCCATCGT, synthesizing primers by Henan Shanghai Biotechnology company, and performing PCR amplification on TaPHT1 by taking wheat cDNA as a template; 9-4B gene coding region (CDS), PCR amplification system including: 10 μ L of 5 XPrimeSTAR Buffer (Mg)2+Plus), 0.5. mu.L of PrimeSTAR HS DNApolymeras (2.5U/. mu.L), 4.0. mu.L of dNTP mix (2.5 mM each), TaPHT1, 9-4B-CDS-F, R primers (10. mu.M), 1.0. mu.L of each cDNA, Plus ddH2O to a total volume of 50. mu.L. The PCR amplification procedure is the first step: 5min at 94 ℃; the second step is that: 30s at 94 ℃, 30s at 56 ℃, 2min at 72 ℃ and 32 cycles; and thirdly, carrying out electrophoresis separation on the PCR product at 72 ℃ for 5min by using 1.0% agarose gel, wherein the result is shown in figure 1, the size of a strip of the PCR product is 1566bp, recovering the PCR product, carrying out enzyme ligation with a pMD19-T vector to transform escherichia coli DH5 alpha competence, screening ampicillin (100 mu g/mL) plates, carrying out colony PCR detection, sequencing the positive clone and extracting plasmid, wherein the sequencing result is the same as a reference sequence in a Chinese spring wheat genome (the homology reaches 100%), the gene does not contain an intron, a coding region contains 1566bp nucleotide and 521 coded amino acids (shown in figure 2).
Preferably, in the step d, when the value of OD260/OD280 is between 1.80 and 2.00, the OD260/OD230 is greater than 2.0, which indicates that the quality of RNA is better, and the RNA can be used as a template for reverse transcription.
Preferably, the step S2 includes the steps of:
s2.1, designing a primer TaPHT1 according to the CDS sequence of the TaPHT1.9-4B gene and the multiple cloning site of a plant expression vector pCUN1301 (containing a Ubi promoter), 9-4B-trangene-F: TCC (transmission control center)CCCGGGATGGCGACTGAACAGCTC (underlined sequenceSacI enzyme site), R: GCGTCGACCTAAGCTTCGATGCCATCGT (underlined sequenceSalI enzyme cutting site);
s2.11, using the primer pair and the TaPHT1.9-4BCDS cloning vector as a template, respectively connecting the two ends of the PCR amplificationSacI andSalTaPHT1.9-4B CDS at the I enzyme cutting site, recovering a target fragment, connecting pMD19-T, transforming DH5 alpha largeEnterobacter, obtaining positive bacterial colony, enlarging culture, extracting plasmid, the method is the same as the above description;
s2.12 restriction enzymesSacI andSali, carrying out double enzyme digestion on a TaPHT1.9-4B CDS-T vector, wherein the enzyme digestion system is as follows: plasmid 1 ug, 10 Xdigestion buffer 5 ul,SacI andSali1. mu.L each (10U/. mu.L), plus ddH2Supplementing the reaction system to 50 mu L by O, and carrying out enzyme digestion at 37 ℃ for 4 h;
s2.13, the digested product was separated by 1.0% agarose gel electrophoresis, and the 1566bp fragment was recovered by using a DNA gel recovery kit from Takara, and restriction endonuclease was usedSacI andSali, carrying out double enzyme digestion on a plant expression vector pCUN1301 plasmid, separating the enzyme digestion product by using 1.0% agarose gel electrophoresis, and recovering a linearized pCUN1301 large fragment;
s2.2, mixing 6 mu L of TaPHT1.9-4B CDS enzyme digestion product with 2 mu L of pCUN1301 enzyme digestion large fragment, 1 mu L (10U/mu L) of T4DNA ligase and 1 mu L of 10 Xligase buffer solution, connecting for 16 h at 16 ℃, transforming Escherichia coli DH5 alpha competent cells by the obtained connection product, and screening and sequencing by a resistance plate containing kanamycin (100 mu g/mL) to obtain positive clone;
s2.21, extracting recombinant plasmids in the positive clones, and naming the recombinant plasmids as pCUN 1301-TaPHT1.9-4B. (vector map is shown in FIG. 3a), the promoter and terminator are the maize Ubiquitin promoter (Ubiquitin promoter) and the Agrobacterium nopaline synthase terminator (NosT), respectively.
Preferably, the enzyme cutting system in step S2.13 is: 10. mu.L of plasmid, 10 Xenzyme digestion buffer solution 5. mu.L,BamHI 1 μL（10 U/μL）、KpnI0.8. mu.L (10U/. mu.L), plus ddH2O supplemented the reaction to 50. mu.L, and cleaved at 37 ℃ for 4 hours.
Preferably, the step S3 includes the steps of:
s3.1, rice genetic transformation: transferring the pCUN1301-TaPHT1.9-4B recombinant vector into the callus of the mature embryo of the Nipponbare rice by adopting an agrobacterium-mediated dip-dyeing method;
s3.12, firstly, inducing mature embryos of rice to generate callus, transforming agrobacterium EHA105 by pCUN1301-TaPHT1.9-4B through a heat shock transformation method, co-culturing with the callus for infection transformation, culturing the infected callus in a screening culture medium containing kanamycin and hygromycin until the callus is differentiated into seedlings, and rooting in a rooting culture medium;
s3.2, identification of positive transgenic rice plants: DNA of the transgenic and wild rice plants is extracted, and first, a primer HptII-F is detected by using an HptII gene (KT 184677.1): CACGGCCTCCAGAAGAAGAT, R: CCTGCCTGAAACCGAACTGC, using the extracted DNA as a template to carry out PCR detection;
s3.21, reusing TaPHT1; carrying out PCR detection on CDS amplification primers (TaPHT 1; 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGCCATCGT) and a carrier primer (Ubi-F: AAAGGATCTGTATGTATGTG) of the 9-4B gene per se (the result is shown in figures 3B and c), and obtaining 6 positive plants which respectively contain HptII and TaPHT1;9-4B gene.
Preferably, in the step S3.12, test-tube plantlets with intact root and stem leaf growth are picked out, a proper amount of sterile water is added into the solid culture medium, the plantlets are hardened for about one week, and the plantlets are transplanted into the rice nutrient solution.
Preferably, in the step S3, in order to detect TaPHT1;9-4B is normally expressed in rice plants, RT-PCR detection is carried out on target genes, firstly RNA of positive transgenosis and wild rice is extracted, cDNA first chain is synthesized by reverse transcription, and TaPHT1 is utilized by taking cDNA as a template; 9-4B-CDS-F: ATGGCGACTGAACAGCTC/R: CTAAGCTTCGATGCCATCGT PCR amplification, the method is the same as above, and the rice is usedOsActionGene (AB047313) was an internal control, and the OsAction amplification primers were F: TATGGTCAAGGCTGGGTTCG, R: CCTAATATCCACGTCGCACT, and the agarose electrophoresis detection shows that TaPHT1, 9-4B is successfully expressed in the 6 transgenic rice plants (as shown in FIG. 3 d). Transferring the transgenic positive seedlings to a greenhouse for cultivation, harvesting seeds according to different strains, and performing seed reproduction on the seeds to obtain homozygous T2 generation seeds of the transgenic pCUN1301-TaPHT1.9-4B rice.
Preferably, the step S4 includes the steps of:
s4.1, TaPHT1;9-4B transgenic rice responds to different phosphorus concentrations under the condition of water culture, T2 generation seeds and wild type rice seeds of positive transgenic rice plants (OE1, OE3) germinate in a culture chamber, then the seeds are cultured in a rice nutrient solution (IRRI) for two weeks, then the rice plants with the same size are selected and cultured in nutrient solutions containing normal (CK, 1mM Pi), low phosphorus (LP, 50 mu M Pi) and phosphorus deficiency (NP, 0 mu M Pi) for three weeks, the growth phenotypes of the transgenic rice plants and the wild type rice plants are observed, the plant height root length and the dry weight of the root system and the overground part are determined, and the nutrient solution is replaced every two days;
s4.12, under the conditions of low phosphorus and phosphorus deficiency, the plant height, the dry weight of roots and the dry weight of the overground part of the transgenic plant are all obviously higher than those of a wild type plant (as shown in figures 4a and b), under the normal phosphorus supply condition, although the difference of the plant height and the root length is not obvious, the dry weight of the root system and the overground part of the transgenic plant is obviously higher than that of the wild type plant, and the results show that: the coding gene of the wheat phosphorus transport protein TaPHT1 and 9-4B is expressed heterologously in the rice body, and can promote the growth of transgenic rice in the low-phosphorus and phosphorus-deficiency environment;
s4.13, TaPHT1, 9-4B transgenic rice and wild rice root and leaf tissue are digested by concentrated sulfuric acid and hydrogen peroxide, and phosphorus concentration is measured by molybdenum-antimony colorimetry. The results are shown in fig. 4c, where the phosphorus content of the transgenic plant root system and aerial parts was significantly higher than that of the wild type plants both under normal and low-phosphorus hydroponic conditions; under the condition of phosphorus deficiency, the phosphorus content of the overground part of the transgenic plant is higher than that of a wild plant, but the phosphorus content of the root system is obviously lower than that of the root system of the wild plant, which shows that the TaPHT1, 9 gene heterologously expresses in the rice body, can improve the absorption and utilization of the transgenic rice to phosphorus, and enhances the adaptability of the transgenic rice to a low-phosphorus environment;
s4.2, TaPHT1, and 9-4B transgenic rice in different phosphorus supply level soil culture tests, in order to further verify TaPHT1;9-4B in the rice, the influence of overexpression of the transgenic rice in the rice on the absorption efficiency and yield of phosphate fertilizer in the whole growth period in the soil growth environment is realized, and a pot experiment is carried out on TaPHT1, 9-4B transgenic rice strains (OE1 and OE3) and wild rice (WT);
s4.21, taking potted soil from farm rice field of Maozhuang of Henan university of agriculture, and applying nitrogen fertilizer respectively200mg/kg of (urea), 50mg/kg of potash fertilizer (potassium sulfate), four levels of applied phosphate fertilizer (superphosphate): phosphorus-free (NP: 0mg/kg Ca (H)2PO4)2.H2O), low phosphorus (LP: 50mg/kg Ca (H)2PO4)2.H2O), middle phosphorus (MP: 100mg/kg Ca (H)2PO4)2.H2O) and high phosphorus (HP: 200mg/kg Ca (H)2PO4)2.H2O), respectively and uniformly mixing the soil treated by different phosphate fertilizers, and subpackaging 10kg of soil in each pot;
s4.22, seeds of wild type and transgenic rice plant lines (OE1 and OE3) are subjected to seed soaking and germination accelerating, water culture seedling is carried out in a laboratory, tillering of rice seedlings occurs after 30 days, the rice seedlings of different plant lines are respectively planted in pot plants treated by different phosphate fertilizers, each plant line is treated by 6 pots, and two plants are grown in a field environment. Observing the phenotype and taking a picture in the mature period, harvesting seeds, testing the seeds, measuring the biomass of the overground part (straw) without the seeds, and simultaneously measuring the phosphorus concentration of the seeds, the straw and the root system;
s4.23, under the condition of not applying phosphate fertilizer (NP) and low phosphate fertilizer (LP), the growth condition of the transgenic line is superior to that of a wild plant, under the condition of medium phosphate fertilizer (MP), the growth phenotype of the transgenic plant is not obviously different from that of the wild plant, and under the condition of high phosphate fertilizer (HP), the growth of the transgenic plant is even inhibited to a certain extent compared with that of the wild plant, which is probably the phosphorus poisoning phenomenon caused by the high phosphorus environment.
Preferably, the yield per plant of the transgenic plants and wild plants is increased along with the increase of the applied phosphate fertilizer in the soil by measuring the yield per plant and the biomass of the overground part, when the application amount of the phosphate fertilizer reaches a medium application amount, the yield per plant of the transgenic rice reaches the maximum yield under the condition of low phosphate fertilizer, and the biomass of the overground part shows a similar rule (fig. 5a and b). Thousand kernel weight measurements showed that the transgenic lines had a thousand kernel weight greater than wild type plants under phosphorus deficient and low phosphorus conditions (as shown in FIG. 5 c), which indicates TaPHT1; the expression of 9-4B in rice can make transgenic rice reach the yield of WT under higher phosphorus supply level at lower phosphate fertilizer level.
Compared with the prior art, the invention has the beneficial effects that: the invention discovers that the phosphorus concentrations of roots, seeds and overground parts of transgenic and wild plants are increased along with the increase of phosphate fertilizers by measuring the total phosphorus concentrations of the roots, the seeds and the overground parts of rice under different phosphate fertilizer treatment conditions, the phosphorus concentrations of the roots and the overground parts of transgenic lines are obviously greater than those of the wild plants under four phosphorus supply conditions (as shown in figures 5d and 5 e), the phosphorus concentration in the seeds is not obviously different under the conditions of medium phosphate fertilizers and high phosphate fertilizers, and the phosphorus concentration in wild rice seeds is less than that of the transgenic plants under the environment with insufficient phosphorus supply (NP and LP) (as shown in figure 5 f), so that TaPHT1 is considered to be in the soil environment with insufficient phosphorus supply; the overexpression of 9-4B in the rice body can improve the absorption of the transgenic plant to phosphorus in soil, promote the increase of plant biomass and thousand seed weight, increase yield and improve the utilization efficiency of phosphate fertilizer.
Drawings
FIG. 1 is a schematic diagram showing the amplification result of coding region (CDS) of TaPHT1, 9-4B gene in the present invention;
FIG. 2 is a schematic representation of the nucleic acid sequence of the TaPHT1, 9-4B coding region and the amino acid sequence of the translated protein of the present invention;
FIG. 3 is a schematic diagram showing the identification of positive plants of TaPHT1, 9-4B heterologous expression transgenic rice in the present invention;
FIG. 4 is a schematic diagram showing the functional verification of TaPHT1, 9-4B transgenic rice and wild rice plants under different phosphorus concentration water culture conditions;
FIG. 5 is a schematic diagram showing the functional verification of TaPHT1, 9-4B transgenic rice and wild rice plants under different phosphate fertilizer level soil culture conditions.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1, fig. 2, fig. 3, fig. 4 and fig. 5, the present invention provides a technical solution:
a method for improving the phosphorus absorption capacity of plants by using wheat phosphorus transportprotein TaPHT1 and 9-4B comprises the following steps:
cloning coding genes of S1, TaPHT1 and 9-4B phosphorus transport protein;
constructing an over-expression vector of S2, TaPHT1 and 9-4B;
s3, detecting rice genetic transformation and transgenic rice plants;
s4, TaPHT1 and 9-4B function verification of the overexpression transgenic rice in water culture and soil culture environments.
Referring to fig. 2, the step S1 includes the following steps:
s1.1, extracting total RNA of wheat:
a. extracting RNA of a root system of a Zhoumai 18 wheat seedling in a two-leaf one-heart period by adopting a Trizol method, firstly carrying out liquid nitrogen grinding on 0.1g of a freshly collected root system to form powder, putting the powder into a 1.5mL enzyme-free centrifuge tube, adding 1mL Trizol, then violently oscillating and uniformly mixing, and standing at room temperature for 30 min;
b. centrifuging at 12000rpm at low temperature (4 deg.C) for 5min, sucking supernatant into a new centrifuge tube, adding 1/3 volumes of chloroform, mixing, and standing for 15 min;
c.12000rpm low temperature (4 ℃) centrifugation for 5min, transferring the supernatant into a new centrifuge tube, adding isopropanol with the same volume, fully mixing, and standing for 30 min. Centrifuging at 12000rpm at low temperature (4 ℃) for 5min, and then removing the supernatant;
d. 1mL of 75% ethanol (made up of DEPC treated water) was added to each tube, washed thoroughly, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the process repeated twice. Drying RNA, adding 40 mu L of enzyme-free water, dissolving on ice, and measuring the concentration and quality of RNA in an ultraviolet spectrophotometer by taking 1 mu of LRNA solution;
s1.2, reverse transcription synthesis of a first strand cDNA:
e. by usingReverse transcription kit (PrimeScript. sup. II 1)stStrand cDNA Synthesis Kit) to synthesize the first chain of cDNA by reverse transcription;
f. add to a 100. mu.L enzyme-free centrifuge tube in sequence: mu.L oligodT primer (50. mu.M), 1. mu.L dNTP mix (10mM each), 2. mu.g Total RNA, enzyme-free water to a Total volume of 10. mu.L, incubation at 65 ℃ for 5min and rapid cooling on ice;
g. mu.L of 5 XPrimeScript II Buffer, 1. mu.L of PrimeScript II RTase (200U/. mu.L), 0.5. mu.L of RNase Inhibitor (40U/. mu.L), and 4.5. mu.L of enzyme-free water were added to the above reaction solution in this order. Inactivating at 42 deg.C for 60min and 95 deg.C for 5min, and cooling on ice to obtain wheat cDNA;
s1.3, PCR amplification of TaPHT1.9-4B gene CDS: designing an amplification primer TaPHT1 according to a gene sequence (TraseCS 4B02G317000) corresponding to a wheat phosphorus transporter TaPHT1, a 9-4B gene (GenBank No. AIZ11192.1) in a latest wheat genome database; 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGCCATCGT, synthesizing primers by Henan Shanghai Biotechnology company, and performing PCR amplification on TaPHT1 by taking wheat cDNA as a template; 9-4B gene coding region (CDS), PCR amplification system including: 10 μ L of 5 XPrimeSTAR Buffer (Mg)2+Plus), 0.5. mu.L PrimeSTAR HS DNApolymeras (2.5U/. mu.L), 4.0. mu.L dNTP mix (2.5 mM each), TaPHT1;9-4B-CDS-F, R primers (10. mu.M) 1.0. mu.L each, 1.0. mu.L of cDNA, plus ddH2O to a total volume of 50. mu.L. The PCR amplification procedure is the first step: 5min at 94 ℃; the second step is that: 30s at 94 ℃, 30s at 56 ℃, 2min at 72 ℃ and 32 cycles; thirdly, 5min at 72 ℃, separating the PCR product by 1.0% agarose gel electrophoresis, wherein the result is shown in figure 1, the size of the strip of the PCR product is 1566bp, recovering the PCR product, converting escherichia coli DH5 alpha competence after enzyme-linking with pMD19-T vector, screening ampicillin (100 mug/mL) plates, detecting colony PCR, sequencing the positive clone and extracting plasmid, the sequencing result is the same as the reference sequence in the genome of spring wheat of China (the homology reaches 100%), the gene does not contain introns, the coding region contains 1566bp nucleotides, and 521 amino acids are coded (shown in figure 2);
referring to FIG. 2, in step d, when the OD260/OD280 is between 1.80 and 2.00, and OD260/OD230 is greater than 2.0, it indicates that the RNA has good quality and can be used as a template for reverse transcription;
referring to fig. 3, the step S2 includes the following steps:
s2.1, designing a primer TaPHT1 according to the CDS sequence of the TaPHT1.9-4B gene and the multiple cloning site of a plant expression vector pCUN1301 (containing a Ubi promoter), 9-4B-trangene-F: TCC (transmission control center)CCCGGGATGGCGACTGAACAGCTC (underlined sequenceSacI enzyme site), R: GCGTCGACCTAAGCTTCGATGCCATCGT (underlined sequenceSalI enzyme cutting site);
s2.11, using the primer pair and the TaPHT1.9-4BCDS cloning vector as a template, respectively connecting the two ends of the PCR amplificationSacI andSalrecovering target fragments from TaPHT1.9-4B CDS at the I enzyme cutting site, connecting pMD19-T, transforming DH5 alpha escherichia coli to obtain positive colonies, carrying out amplification culture, and extracting plasmids by the same method as the above description;
s2.12 restriction enzymesSacI andSali, carrying out double enzyme digestion on a TaPHT1.9-4B CDS-T vector, wherein the enzyme digestion system is as follows: plasmid 1 ug, 10 Xdigestion buffer 5 ul,SacI andSali1. mu.L each (10U/. mu.L), plus ddH2Supplementing the reaction system to 50 mu L by O, and carrying out enzyme digestion at 37 ℃ for 4 hours;
s2.13, the digested product was separated by 1.0% agarose gel electrophoresis, and the 1566bp fragment was recovered by using a DNA gel recovery kit from Takara, and restriction endonuclease was usedSacI andSali, carrying out double enzyme digestion on a plant expression vector pCUN1301 plasmid, separating the enzyme digestion product by using 1.0% agarose gel electrophoresis, and recovering a linearized pCUN1301 large fragment;
s2.2, mixing 6 mu L of TaPHT1.9-4B CDS enzyme digestion product with 2 mu L of pCUN1301 enzyme digestion large fragment, 1 mu L (10U/mu L) of T4DNA ligase and 1 mu L of 10 Xligase buffer solution, connecting for 16 hours at 16 ℃, transforming escherichia coli DH5 alpha competent cells by the obtained connection product, and screening and sequencing by a resistance plate containing kanamycin (100 mu g/mL) to obtain positive clone;
s2.21, extracting recombinant plasmids in the positive clones, and naming the recombinant plasmids as pCUN 1301-TaPHT1.9-4B. (vector map is shown in FIG. 3a), the promoter and terminator are the maize Ubiquitin promoter (Ubiquitin promoter) and the Agrobacterium nopaline synthase terminator (NosT), respectively;
referring to fig. 3, the enzyme cutting system in step S2.13 is: 10. mu.l of plasmid, 10 Xenzyme digestion buffer solution 5. mu.L,BamHI 1 μL（10 U/μL）、KpnI1. mu.L (10U/. mu.L), plus ddH2Supplementing the reaction system to 50 mu L by O, and carrying out enzyme digestion at 37 ℃ for 4 hours;
referring to fig. 3, the step S3 includes the following steps:
s3.1, rice genetic transformation: transferring the pCUN1301-TaPHT1.9-4B recombinant vector into the callus of the mature embryo of the Nipponbare rice by adopting an agrobacterium-mediated dip-dyeing method;
s3.12, firstly, inducing mature embryos of rice to generate callus, transforming agrobacterium EHA105 by pCUN1301-TaPHT1.9-4B through a heat shock transformation method, co-culturing with the callus for infection transformation, culturing the infected callus in a screening culture medium containing kanamycin and hygromycin until the callus is differentiated into seedlings, and rooting in a rooting culture medium;
s3.2, identification of positive transgenic rice plants: DNA of the transgenic and wild rice plants is extracted, and first, a primer HptII-F is detected by using an HptII gene (KT 184677.1): CACGGCCTCCAGAAGAAGAT, R: CCTGCCTGAAACCGAACTGC, using the extracted DNA as a template to carry out PCR detection;
s3.21, reusing TaPHT1; carrying out PCR detection on CDS amplification primers (TaPHT 1; 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGCCATCGT) and a carrier primer (Ubi-F: AAAGGATCTGTATGTATGTG) of the 9-4B gene per se (the result is shown in figures 3B and c), and obtaining 6 positive plants which respectively contain HptII and TaPHT1;9-4B gene;
referring to fig. 3, in step S3.12, picking out test-tube plantlets with intact roots and stems and leaves, adding a proper amount of sterile water into a solid culture medium, hardening seedlings for about one week, and transplanting the seedlings into a rice nutrient solution;
referring to FIG. 3, in step S3, to detect TaPHT1;9-4B is expressed normally in rice plant, RT-PCR detection is carried out to target gene, positive transgene and wild rice are extractedAnd (3) DNA is subjected to reverse transcription to synthesize a first strand of cDNA, the cDNA is taken as a template, and a primer pair TaPHT1, 9-4B-CDS-F: ATGGCGACTGAACAGCTC, R: CTAAGCTTCGATGC-CATCGT was used for PCR amplification in the same manner as described above with riceOsActionGene (AB047313) was an internal control, and the OsAction amplification primers were F: TATGGTCAAGGCTGGGTTCG, R: CCTAATATCCACGTCGCACT, detected by agarose electrophoresis, TaPHT1;9-4B was successfully expressed in these 6 transgenic rice plants (as shown in FIG. 3 d). Transferring the transgenic positive seedlings to a greenhouse for cultivation, harvesting seeds according to different strains, and performing seed reproduction on the seeds to obtain homozygous T2 generation seeds of the transgenic pCUN1301-TaPHT1.9-4B rice;
referring to fig. 5, the step S4 includes the following steps:
s4.1, TaPHT1; responding to different phosphorus concentrations of 9-4B transgenic rice under a water culture condition, germinating T2 generation seeds and wild type rice seeds of positive transgenic rice plants (OE1, OE3) in a culture chamber, culturing in a rice nutrient solution (IRRI) for two weeks, selecting rice plants with the same size, culturing in a nutrient solution containing normal (CK, 1mM Pi), low phosphorus (LP, 50 mu M Pi) and phosphorus deficiency (NP, 0 mu M Pi) for three weeks, observing the growth phenotype of the transgenic rice plants and the wild type rice plants, measuring the height and root length of the plants and the dry weight of the root system and the overground part, and replacing the nutrient solution every two days, wherein the growth phenotype of the TaPHT1 and 9-4B transgenic rice plants is superior to that of a WT plant (as shown in a picture 4 a);
s4.12, under the conditions of low phosphorus and phosphorus deficiency, the plant height, the dry weight of roots and the dry weight of the overground part of the transgenic plant are all obviously higher than those of a wild type plant (as shown in figures 4a and b), under the normal phosphorus supply condition, although the difference of the plant height and the root length is not obvious, the dry weight of the root system and the overground part of the transgenic plant is obviously higher than that of the wild type plant, and the results show that: the coding gene of the wheat phosphorus transport protein TaPHT1 and 9-4B is expressed heterologously in the rice body, and can promote the growth of transgenic rice in the low-phosphorus and phosphorus-deficiency environment;
s4.13, TaPHT1, 9-4B transgenic rice and wild rice root and leaf tissue are digested by concentrated sulfuric acid and hydrogen peroxide, and phosphorus concentration is measured by molybdenum-antimony colorimetry. The results are shown in fig. 4c, where the phosphorus content of the transgenic plant root system and aerial parts was significantly higher than that of the wild type plants both under normal and low-phosphorus hydroponic conditions; under the condition of phosphorus deficiency, the phosphorus content of the overground part of the transgenic plant is higher than that of a wild plant, but the phosphorus content of the root system is obviously lower than that of the root system of the wild plant, which shows that the TaPHT1, 9 gene heterologously expresses in the rice body, can improve the absorption and utilization of the transgenic rice to phosphorus, and enhances the adaptability of the transgenic rice to a low-phosphorus environment;
s4.2, TaPHT1, function verification of 9-4B transgenic rice in soil culture tests with different phosphorus supply levels, namely, in order to further verify the influence of TaPHT1 and 9-4B overexpression in rice bodies on the absorption efficiency and yield of phosphate fertilizer in the whole growth period of the transgenic rice in a soil growth environment, carrying out pot culture test on TaPHT1, 9-4B transgenic rice strains (OE1 and OE3) and wild rice (WT);
s4.21, taking potted soil from a farm paddy field of Maozhuang of Henan university of agriculture, respectively applying 200mg/kg of nitrogen fertilizer (urea) and 50mg/kg of potassium fertilizer (potassium sulfate), and applying phosphate fertilizer (calcium superphosphate) to set four levels: phosphorus-free (NP: 0mg/kg Ca (H)2PO4)2.H2O), low phosphorus (LP: 50mg/kg Ca (H)2PO4)2.H2O), middle phosphorus (MP: 100mg/kg Ca (H)2PO4)2.H2O) and high phosphorus (HP: 200mg/kg Ca (H)2PO4)2.H2O), respectively and uniformly mixing the soil treated by different phosphate fertilizers, and subpackaging 10kg of soil in each pot;
s4.22, seeds of wild type and transgenic rice plant lines (OE1 and OE3) are subjected to seed soaking and germination accelerating, water culture seedling is carried out in a laboratory, tillering of rice seedlings occurs after 30 days, the rice seedlings of different plant lines are respectively planted in pot plants treated by different phosphate fertilizers, each plant line is treated by 6 pots, and two plants are grown in a field environment. Observing the phenotype and taking a picture in the mature period, harvesting seeds, testing the seeds, measuring the biomass of the overground part (straw) without the seeds, and simultaneously measuring the phosphorus concentration of the seeds, the straw and the root system;
referring to fig. 5, it was found that the yield per plant of the transgenic plants and wild-type plants increased with the increase of applied phosphate fertilizer in the soil by measuring the yield per plant and the biomass of the above-ground parts, and when the applied phosphate fertilizer reaches the medium amount, the yield per plant of the transgenic rice reaches the maximum yield under the condition of low phosphate fertilizer, and the biomass of the above-ground parts shows similar rules (fig. 5a, b). Thousand kernel weight determination results show that the thousand kernel weight of a transgenic line is greater than that of a wild type plant (shown in figure 5 c) under the conditions of phosphorus deficiency and low phosphorus, and the results show that the expression of TaPHT1, 9-4B in rice bodies can enable the transgenic rice to achieve the yield of WT under a higher phosphorus supply level under a lower phosphorus fertilizer level;
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.