Rice salt stress tolerance gene osHAK11 coding protein and application thereof

Abstract

The application discloses a salt stress tolerance gene of rice OsHAK11 The application belongs to the field of plant genetic engineering, and relates to a coding protein and application thereof. The method is to knockout a rice OsHAK11 Gene (nucleotide sequence as shown in SEQ ID NO. 1) through a CRISPR / Cas9 gene editing technology, so that a mutant with significantly enhanced salt tolerance is obtained. The gene editing vector pEGCas9Pubi-B-OsHAK11 contains sgRNA which is targeted to the region shown in SEQ ID NO. 4, and a rice receptor material is transformed through an agrobacterium mediation, so that a plant with a frame shift mutation of the gene is screened and obtained. OsHAK11 Salt stress experiment shows that the survival rate of the mutant oshak11 is significantly higher than that of a wild type. The application provides a new gene resource and an efficient technical means for salt tolerance breeding of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and specifically relates to a rice salt stress tolerance gene. OsHAK11 Encoded proteins and their applications. Background Technology

[0002] Rice is often subjected to a variety of abiotic stresses during its growth, among which salt stress is one of the main factors limiting its yield and planting area.

[0003] Rice, as a moderately salt-sensitive crop, is ideal for developing saline-alkali land resources due to its hydroponic characteristics and suitability for bioremediation and utilization. Therefore, improving the salt tolerance of rice is of great significance.

[0004] Currently, rice salt tolerance breeding mainly relies on quantitative trait loci (QTLs), such as qSKC-1 on chromosome 1 and Saltol With the development of molecular biology, gene editing technology has been used to discover salt-tolerant genes and apply them for genetic improvement, becoming an effective way to enhance the salt tolerance of rice. Potassium transporter (K⁺) families (such as HAK class) play a crucial role in maintaining K⁺ homeostasis and salt tolerance. The rice genome contains 27 such genes. HAK Genes, as research has shown... OsHAK9 Participating in seed germination under salt stress OsHAK21 K⁺ homeostasis and salt tolerance can be regulated by OsCYB5-2. OsHAK18 It participates in regulating rice plant height, tiller number, panicle length, seed setting rate, number of grains per panicle, yield, and grain-to-weed ratio. OsHAK26 It plays an important role in maintaining rice pollen development and fertility, and OsHAK11 Its function in the salt stress response has not been reported. Summary of the Invention

[0005] The core of this invention lies in the fact that, through rigorous gene editing and phenotypic identification experiments, it has been confirmed that... OsHAK11 The gene is a negative regulator of salt tolerance in rice, and a gene with a specific single-base deletion mutation has been successfully created. OsHAK11 A gene mutant. This mutant exhibits significantly enhanced salt tolerance, providing valuable germplasm resources and a clear target for salt-tolerant rice breeding.

[0006] Specifically, the present invention provides the following technical solutions:

[0007] A method to improve the salt tolerance of rice plants involves using gene editing technology to modify the endogenous components of the rice plant genome. OsHAK11 Introducing site-directed mutations into the gene makes the... OsHAK11The gene has a T base deleted at position 2368, starting from the start codon ATG, resulting in a mutant nucleotide sequence as shown in SEQ ID NO. 5. OsHAK11 Genes were used to obtain rice plants with significantly enhanced salt tolerance.

[0008] Furthermore, the gene editing technology is CRISPR / Cas9 gene editing technology.

[0009] A method for obtaining the site-directed mutation that produces the mutation in the rice genome includes the following steps:

[0010] S1. Design CRISPR / Cas9 gene editing vectors to target... OsHAK11 The nucleotide sequence of the gene coding region from base 2362 to base 2384, starting from the start codon ATG, is shown in SEQ ID NO.4;

[0011] S2. Construct a pEGCas9Pubi-B-OsHAK11 gene editing vector containing a specific targeting sequence;

[0012] S3. The pEGCas9Pubi-B-OsHAK11 gene editing vector was introduced into rice recipient material using Agrobacterium-mediated genetic transformation.

[0013] S4. Screen the transgenic plants to obtain rice carrying the site-directed mutation.

[0014] Furthermore, the gene-editing vector is constructed through the following steps:

[0015] (1) According to OsHAK11 Gene sequence design of target gRNA, and synthesis of primer pairs containing target gRNA sequences;

[0016] (2) The sgRNA expression cassette was amplified by overlap PCR and nested PCR and then ligated with the binary vector pEGCas9Pubi-B digested with Bsa I to obtain the recombinant vector pEGCas9Pubi-OsHAK11.

[0017] (3) The recombinant vector was sequenced to verify that the target sequence was correct and the pEGCas9Pubi-B-OsHAK11 gene editing vector was obtained.

[0018] Furthermore, the aforementioned OsHAK11 The gene sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.3 for the OsHAK11 protein. Beneficial effects

[0019] (1) This invention uses CRISPR / Cas9 gene editing technology to knock out OsHAK11 Genetic confirmation, OsHAK11 Genes negatively regulate rice salt tolerance.

[0020] (2) The invention created oshak11 The mutant (SEQ ID NO.5) is a specific, stable, and heritable germplasm. Under NaCl stress, oshak11 The survival rate of the mutant reached 91.66%, which was significantly higher than that of the wild-type ZH11 (8.33%), demonstrating a remarkable effect.

[0021] (3) This invention designs specific sgRNAs to target OsHAK11 The coding region is used to achieve single-base deletion frameshift mutations. The method is reliable and the mutations are stable, making it suitable for targeted improvement of salt-tolerant rice varieties.

[0022] (4) The gene editing vector and mutant materials provided by the present invention can be directly used for the breeding of new salt-tolerant rice varieties, which has important application value for expanding rice planting in saline-alkali land and increasing grain yield. Attached Figure Description

[0023] Picture 1 for OsHAK11 Schematic diagram of gene structure and target sequence elements of CRISPR / Cas-OsHAK11 vector.

[0024] Picture 2 These are peak diagrams of sequencing results; the top diagram shows the sequencing peaks of the wild-type ZH11 target, and the bottom diagram shows the peaks of the mutant. oshak11 Target sequencing peak diagram.

[0025] Picture 3 for oshak11 Salt tolerance test of mutants. A represents ZH11 and... oshak11 Plant growth status before salt treatment, bar = 5cm; B represents ZH11 and oshak11 Plant growth status after salt treatment, bar = 5 cm; C is ZH11 and oshak11 Survival rate statistics after salt treatment. Values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference (P < 0.05); ** indicates extremely significant difference (P < 0.01). Statistical analysis was performed using one-way ANOVA. Detailed Implementation

[0026] To enable those skilled in the art to better understand the technical solutions of this invention, the present application will be further described in detail below with reference to embodiments.

[0027] Unless otherwise specified, all methods described in the following embodiments can be performed using conventional methods in the art. Unless otherwise specified, all commercially available materials can be used in the experiments described below. Example 1

[0028] This embodiment provides rice. OsHAK11 The functions and applications of genes include the following:

[0029] 1. Rice OsHAK11 Functional verification of genes

[0030] To clarify OsHAK11 To investigate the function of a gene in rice, this invention employs the CRISPR / Cas9 gene editing method to site-directedly mutate the gene sequence and knock out its function in rice.

[0031] This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. This invention selects the nucleotide sequence from base 2362 to base 2384 of the gene coding region, starting from the start codon ATG (as shown in SEQ ID NO. 4), as the target region for CRISPR / Cas9 gene editing (see...). Picture 1 ).

[0032] (1) OsHAK11 Construction of CRISPR / Cas9 gene editing vector

[0033] The gene editing vector of this invention is pEGCas9Pubi-B-OsHAK11, and the base vector of this vector is pEGCas9Pubi-B. This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into the base vector using a one-step cloning method. The specific construction process is as follows:

[0034] i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsHAK11 The gene sequence was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set to NGG. The DNA sequence of the target region selected in this invention is shown in SEQ ID NO.4.

[0035] ii) Amplify the sgRNA expression cassette by overlap PCR and nested PCR. Primer pairs containing the above-mentioned sgRNA target sequences were synthesized and annealed. The primer pairs were then ligated with the binary vector pEGCas9Pubi-B (see Ma X, Zhang Q, Zhu Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant. 2015, 8(8):1274-1284, vector pEGCas9Pubi-B was kindly provided by Professor Long Tuan of Hainan University) to obtain the recombinant vector pEGCas9Pubi-OsHAK11. The recombinant vector pEGCas9Pubi-OsHAK11 was transformed into E. coli DH5α, and positive clones were selected for sequencing. The specific steps were as described in the reference "Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. BMC plant biology 14:327."

[0036] iii) Sequencing verification.

[0037] A positive clone with correct sequencing results is a successfully constructed pEGCas9Pubi-OsHAK11 gene editing vector.

[0038] (2) Agrobacterium-mediated genetic transformation of rice

[0039] The pEGCas9Pubi-OsHAK11 gene editing vector successfully constructed above was transformed into Agrobacterium EHA105 via heat shock. After identification by PCR, the bacterial culture was stored at -80 °C with glycerol.

[0040] Freshly peeled embryos of the hybrid rice variety Zhonghua 11, approximately 1.5 mm in size, were used as recipient materials. The peeled rice embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension and left for no more than 1 hour. Approximately 100 embryos were placed in each centrifuge tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount of suspension at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43°C for 2 minutes, followed by an ice bath for 1 minute. The remaining washings at the bottom of the tubes were aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then left to stand in the dark for 8 minutes.

[0041] Next, pour the embryos and infection solution from the centrifuge tubes onto the co-culture medium, shake well, and then use a pipette to remove any excess infection solution. Place all embryos with their scutes facing upwards and co-culture at 23°C in the dark for 3 days.

[0042] After co-culture, use sterile forceps to transfer the embryos to recovery medium and culture at 28°C for 7-14 days. During this process, be careful to remove any sprouts that grow on the embryos.

[0043] After the recovery culture was completed, the immature embryos were placed on a selection medium containing 1.5 mg / L Bialaphos for 3 rounds of selection culture, with each round lasting 2 weeks. Then, they were transferred to a selection medium containing 2 mg / L Bialaphos for 2 rounds of selection culture, with each round lasting 2 weeks.

[0044] The resistant callus was transferred to propagation medium and cultured in the dark at 28°C for 2 weeks. The propagated resistant callus was then transferred to induction medium and cultured in the dark at 28°C for 2 weeks. Finally, it was transferred to differentiation medium and cultured under light at 25°C and 5000 lx for 2 weeks.

[0045] After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25 ℃, 5000 lx, and under light until they root. The seedlings are then transferred to small nutrient pots for growth. After they have survived, they are transplanted into a greenhouse. The offspring seeds are harvested after 3-4 months.

[0046] (3) Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0047] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection:

[0048] This invention first employs the CTAB method to extract DNA from rice leaves. The specific method is as follows: DNA extraction is performed according to the traditional CTAB method (Rogers and Bendich, 1985). A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, which is then set aside. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (A260 / A280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0049] PCR was performed using 5 μL of Biomiga 2×PCR premix (containing Mg2+, Taq DNA Polymerase, 2.5 mM dNTPs, and 10×PCR Buffer), 1 μL of primers (containing 0.5 μL each of forward and reverse primers), 1 μL of template DNA, and ddH2O to a final volume of 10 μL. The PCR amplification program was a standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min). The amplified products were analyzed by 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after color development with formaldehyde and NaOH.

[0050] For mutants oshak11 Nucleotide sequence alignment analysis revealed that ( Picture 2 Compared to the unedited wild type (WT), the mutated OsHAK11 The mutant nucleotide sequence is shown in SEQ ID NO. 5, which describes the deletion of a T base at position 2368, starting from the start codon ATG. This deletion causes a frameshift in the amino acid sequence, leading to premature termination of translation. Example 2

[0051] This embodiment describes the mutant obtained in Example 1. oshak11 Phenotypic analysis was performed, as follows:

[0052] 1. Mutant oshak11 Salt tolerance assessment

[0053] The parameters for alternating light and dark culture are as follows: light intensity is 120 μmol·m⁻¹. -2 ·s -1 The temperature is 28℃ / 25℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0054] The rice seeds to be tested are oshak11 - L1 Homozygous seeds of mutant T1 generation, along with its background material ZH11 and empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0055] (1) For each material, take 12 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48h.

[0056] (2) After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0057] (3) After completing step 2, take a 96-well plate, cut off part of the lower edge of each well, and then put one germinated seed into each well (embryo facing up, radicle facing down).

[0058] (4) After completing step 3, place the 96-well plate (with germinated seeds on it) on a plastic box containing Yoshida rice culture solution and immerse the germinated seeds in the culture solution. Culture in alternating light and dark for 3 weeks to obtain rice seedlings that have grown to the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture solution needs to be replaced every 7 days.

[0059] (5) After completing step 4, place the 96-well plate (on which rice seedlings that have grown to the three-leaf stage) on a plastic box containing 150 mM NaCl Yoshida rice culture solution and completely immerse the roots in the culture solution. After 3 days of treatment, replace the Yoshida rice culture solution with 200 mM NaCl and then replace it with 150 mM NaCl Yoshida rice culture solution. The high salt stress was carried out for 11 days under alternating light and dark culture (during the high salt stress period, the Yoshida rice culture solution was replaced every 2 days).

[0060] (6) After completing step 5, place the 96-well plate (with rice seedlings on it) on a plastic box containing Yoshida rice culture medium and recover for 7 days under alternating light and dark culture.

[0061] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0062] See the growth status of rice seedlings before treatment. Picture 3 The growth status after treatment A is shown in Figure 1. Picture 3 B. Survival rate statistics are shown in [the original text]. Picture 3 C in the middle.

[0063] The results showed that before salt treatment, oshak11 Slightly shorter than ZH11; freshwater control ZH11 and mutant oshak11 The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while that of the mutant... oshak11 The survival rate was 91.66%, and statistical analysis results showed that... oshak11 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... oshak11 The salt tolerance of the mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically consistent with those of the background material ZH11, with no statistical difference.

Claims

1. A method for improving the salt tolerance of rice plants, characterized in that, By introducing a site-directed mutation in an endogenous OsHAK11 gene of a rice plant genome through a gene editing technology, the OsHAK11 gene is deleted with one T base at the 2368th position from the start codon ATG, thereby obtaining a mutant OsHAK11 gene with a nucleotide sequence as shown in SEQ ID NO. 5, and further obtaining a rice plant with significantly enhanced salt tolerance.

2. The method according to claim 1, characterized in that, The gene editing technology mentioned is CRISPR / Cas9 gene editing technology.

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