CN117904186A

CN117904186A - Application of rice calcium-hydrogen antiport protein gene OsCAX a in improving rice ammonium absorption

Info

Publication number: CN117904186A
Application number: CN202410010772.7A
Authority: CN
Inventors: 徐国华; 张誉译; 张金飞; 刘鹰; 景媛媛
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2024-01-04
Filing date: 2024-01-04
Publication date: 2024-04-19
Anticipated expiration: 2044-01-04
Also published as: CN117904186B

Abstract

The invention discloses application of a rice calcium-hydrogen antiport protein gene OsCAX a in improving rice ammonium absorption. Experiments prove that OsCAX a knockout can improve the absorption capacity of rice to ammonium nutrition, and two ways of analyzing calcium ions from the transcription level and the protein level to participate in regulating and controlling the absorption of root system ammonium nutrition are provided for the first time.

Description

Application of rice calcium-hydrogen antiport protein gene OsCAX a in improving rice ammonium absorption

Technical Field

The invention belongs to the technical field of agricultural biology, and relates to application of a rice calcium-hydrogen antiport protein gene OsCAX a in improving ammonium absorption of rice.

Background

At present, 6 calhX genes CAXs, CAX1a, CAX1b, CAX1c, CAX2, CAX3 and CAX4, respectively, are found in rice. In rice, other OsCAXs genes in rice, in addition to OsCAX2, respond to low concentrations of Ca ²⁺; osCAX1a and OsCAX c can promote accumulation of Cd ²⁺, osCAX4 can reduce accumulation of Cd ²⁺ (Zou W,Chen J,Meng L,Chen D,He H,Ye G.The Rice Cation/H⁺Exchanger Family Involved in Cd Tolerance and Transport.International Journal of Molecular Sciences.2021;22(15):8186). and in addition, pH has a crucial effect on cation transport activity of OsCAXs (Pittman et al 2005), especially for Ca ²⁺. OsCAX1a can transport Ca ²⁺ into vacuoles and participate in maintaining intracellular Ca ²⁺ homeostasis under conditions of high Ca ²⁺ concentration. OsCAX1a has at least two physiological functions: one is the protection of the companion and endothelial cells, which cause calcium toxicity due to the influx of Ca ²⁺ from neighboring cells; one is to actively transport Ca ²⁺ into the vacuole, the second signal molecule (Takehiro Kamiya;Taro Akahori;Motoyuki Ashikari;Masayoshi Maeshima Expression of the Vacuolar Ca²⁺/H⁺Exchanger,OsCAX1a,in Rice:Cell and Age Specificity of Expression,and Enhancement by Ca²⁺Plant and Cell Physiology,2006,47(1):96-106)., which is a mineral nutrient element and signaling, lacks the chlorophyll content in the leaves of CAX1a to be significantly reduced, and histochemical staining and quantitative detection of ROS-related indicators indicate that there is a significant accumulation of superoxide anions at the leaf tips with concomitant occurrence of cell death; the element content detection shows that the content of various metal elements on the upper part of the leaf is obviously reduced, and the metal elements are unbalanced; the nutrient substance transportation is blocked and the small spike is degenerated due to abnormal development of flower stalks and vascular bundles; the leaves are dehydrated and curled, and the fresh weight of seedlings is obviously reduced; sensitive to high concentration Ca ²⁺, and plays a role in calcium salt stress (Wang Zhonghao, shi Yongfeng, map cloning and functional analysis of the calcium-hydrogen ion exchanger gene CAX1a of paddy rice. National academy of agricultural sciences of Shuoshi. China).

The nitrogen nutrition in the soil mainly exists in the forms of inorganic nitrate, ammonium salt, organic amino acid, polypeptide and the like, and is absorbed and utilized by plants. For this reason, plants evolved a powerful inorganic nitrogen absorption system. For rice, the root system of the rice is in a flooded state for a long time. In flooded soil, ammonium nitrogen (NH ₄ ⁺) is the predominant form of nitrogen, so NH ₄ ⁺ is the predominant form of nitrogen available to rice in anaerobic paddy soil, and is also the predominant form of inorganic nitrogen for absorption by rice roots (SASAKAWA AND yamamoto,1978;Tabuchi et al, 2005). In rice, NH ₄ ⁺ is absorbed and transported mainly by the ammonium transporter (AMTs), and can be divided into four subfamilies (OsAMT 1-OsAMT 4) for a total of 12 AMTs. In agricultural production, NH ₄ ⁺ concentration in soil varies drastically, even to a degree of up to 3-4 orders of magnitude, due to field management, climate and fertilization etc. (Wolt and others, 1994). In order to accommodate the dramatic concentration changes in NH ₄ ⁺ caused by environmental changes, rice evolved a corresponding, complex ammonium absorption mechanism. The NH ₄ ⁺ absorption of the rice root system can be mainly divided into two systems: high affinity ammonium absorption system (HATS) and low affinity ammonium absorption system (LATS) (glass and a.,2002;Miller and Smith,1996). In addition to the ammonium transporter responsible for NH ₄ ⁺ uptake in rice, some other ion channels or proteins have been reported to possess NH ₄ ⁺ transport activity in recent years. For example, the K ⁺ ion channel protein, because of the similar charge and hydration radius of the K ⁺ ion and NH ₄ ⁺, can also transport NH ₄ ⁺ to some extent in part by the low selectivity of the K ⁺ ion channel protein (Adam et al, 1997).

Regulation of ammonium absorption in rice mainly involves two aspects: firstly, ammonium absorption is controlled by regulating AMTs gene expression level; secondly, ammonium absorption is regulated by altering the activity of AMTs proteins. As described above, the expression patterns of the different AMTs genes are different and are affected by a variety of environmental factors. For example, osAMT a1 under low ammonium concentration conditions; 1 and OsAMT1;2 is NH ₄ ⁺ induced to express, but OsAMT; 3 are inhibited by ammonium, thus forming a feedback mechanism for ammonium transport (Yutaka et al., 2003). This suggests that rice can autonomously regulate the ammonium absorption process by sensing the external NH ₄ ⁺ concentration. In addition to being regulated at the transcriptional level by NH ₄ ⁺ concentrations, genes encoding ammonium transporters also have a transport activity regulated by transactivation allosteric effects (Benjamin Neuhuser,2007; loqu et al, 2007). In addition, ammonium uptake is also affected by a variety of factors such as phytohormones, stress and gene editing.

In summary, regulation of ammonium uptake in rice is a complex process involving the interaction of multiple genes and environmental factors. There are few studies on regulation of ammonium absorption at present, but most focus on analysis of individual genes or factors, and lack of further mechanism studies.

Wang Zhonghao et al identified the CAX1a gene by chemically inducing a mutant strain with a degenerated phenotype at the top of the ear using EMS. Experiments such as photosynthetic pigment identification, slicing and electron microscope observation, ROS and chemical staining, element detection and the like confirm that CAX1a gene positioning re-cell membrane and vacuole membrane are related to chlorophyll content, cell death, absorption and transportation of various metal elements, flower development, calcium salt stress and the like. (Wang Zhonghao, shi Yongfeng) map cloning and functional analysis of the calcium-hydrogen ion-exchange protein gene CAX1a of paddy rice. Shuoshi national academy of agricultural scion of China) the current report of the CAX1a gene in paddy rice is limited to the transportation of calcium ions and influence on spike development, and root systems, ammonium absorption and the like are not mentioned; the same family of genes in rice only reported transport functions for part of divalent cations; arabidopsis thaliana equivalent genes also only report their transport function for metal cations of different valences and protons. No report about OsCAX a gene related to the function of root system to absorb ammonium nutrition is yet seen.

Disclosure of Invention

The invention aims at overcoming the defects in the prior art and provides application of the rice calcium hydrogen antiport protein gene OsCAX a in improving the ammonium absorption of rice.

The aim of the invention can be achieved by the following technical scheme:

The application of the rice OsCAX a gene in improving the ammonium absorption capacity of root systems is that the sequence of the rice OsCAX a gene is shown as SEQ ID NO. 1.

As a preferable mode of the invention, knocking out the rice OsCAX a gene or inhibiting the expression of the gene can improve the ammonium absorption capacity of the root system.

The application of substances for knocking out or silencing the rice OsCAX a gene in improving the ammonium absorption capacity of root systems is that the rice OsCAX a gene sequence is shown as SEQ ID NO.1, and the coded protein amino acid sequence is shown as SEQ ID NO. 2.

As a preferable aspect of the invention, the substance for knocking out or silencing the rice OsCAX a gene is a gene editing system or siRNA aiming at the rice OsCAX a gene.

As a further preferred aspect of the present invention, the gene editing system is selected from the group consisting of CRISPR-CAS9 vector or CRISPR-CAS9 vector composition.

The beneficial effects are that:

Cax1a random knockout mutant material is constructed by using CRISPR-Cas9 technology, and the material identification result is shown in figure 1. Transient treatment of Japanese sunny and cax a with ¹⁵ N-labeled ammonium salt nitrogen source nutrient solution resulted in a certain (10%) increase in ¹⁵ N abundance in cax a (FIG. 2), which demonstrates that OsCAX a knockout did increase the uptake capacity of ammonium nutrition in rice. To exclude changes in ammonium uptake caused by defects in mutant growth, we monitored in real time the ammonium ion flow of the wild type of Nippon and cax a mutant root surfaces using a non-invasive detection (NMT) system. The results are shown in FIG. 3, and the mutant ammonium absorption capacity is significantly higher than that of the wild type Japanese sunny type under the same treatment conditions at the same time, which again proves that CAX1a can actually influence the absorption of ammonium nutrition by rice.

To explore how calcium hydrogen antiporters affect rice ammonium uptake, we examined the expression changes of major AMTs (AMT 1.1, 1.2, 1.3, 2.1) in mutant and wild type. The results indicate that the expression of ammonium transporter family AMTs in cax a mutant was all up-regulated to a small extent, especially the greatest extent of AMT1.3 variation (fig. 4). Therefore, it is considered that the transcriptional level is mainly to control the ammonium uptake capacity by enhancing the expression of AMT1.3 and the like.

Calcium is known as a "total regulator of plant cell metabolism" and as a second messenger in plants, calcium binds to calmodulin as an intracellular messenger and transmits various signals, regulating many physiological metabolic processes in plants, and calcium deficiency is highly likely to affect the transmission of certain ammonium absorption-related signals. CPK, CIPK, is a series of kinases regulated by calcium signaling and is widely believed to play an important role in signal transduction of plant responses, not to be excluded in regulating ammonium uptake: the ammonium absorption channel can be closed/opened by phosphorylating the rice ammonium transporter AMTs. Therefore, we have reason to speculate that following the OsCAX a deletion, there is insufficient signal from calmodulin to turn off the ammonium transporter, resulting in continued operation of the portion of the ammonium transporter that should have been stopped, and increased rice ammonium uptake. Based on this we extracted membrane proteins from both mutant and wild-type medium root line cells and identified membrane proteins that separated phosphorylation changes by mass spectrometry (fig. 5). The results indicate that the level of phosphorylation of ammonium transporter family AMTs in cax a mutant was significantly altered, especially with the greatest extent of AMT1.2 (fig. 6), and therefore we believe that the protein modification level is mainly affected by altering the protein activity of AMT1.2 etc. to affect ammonium uptake capacity.

In conclusion, the calcium-hydrogen antiport protein OsCAX a is hopefully a new regulatory ammonium absorption factor, so that the environmental and toxic effects caused by nitrogenous fertilizer application are reduced; can improve the ammonium absorption capacity of about 10% of the root system of the rice, and provides potential possibility for breeding the rice with strong ammonium absorption capacity in the future.

Drawings

Fig. 1: mutant mutation site schematic diagram and genome DNA identification result after mutation

Fig. 2: compared with the transient absorption of ammonium salt marked by the root ¹⁵ N of the mutant,

Fig. 3: the non-damage detection system detects the change of the root table NH ₄ ⁺ in real time

Fig. 4: comparison of the relative expression levels of the root systems of the wild type and mutant AMTs

Fig. 5: AMTs protein phosphorylation site (AMT 1.1-p388; AMT1.2-p453/p468/p283; AMT1.3-p 488)

Fig. 6: comparison of the phosphorylation degree of wild-type and mutant AMTs proteins in root Membrane proteins

Fig. 7: nondestructive microassay technology measurement schematic diagram

Detailed Description

Example 1 construction and characterization of Gene knockout Material

(1) And (3) constructing a carrier:

The OsCAX a gene has an accession number LOC_Os01g37690 in Rice Genome Annotation Project (RGAP 7, MSU) (http:// price. Plant biology. MSU. Edu/index. Shtml), searches for the corresponding gene specificity Spacers by using a CRISPR-P (http:// CRISPR. Hzau. Edu. Cn/CRISPR /) website according to the accession number of the gene number, randomly selects a plurality of spaners with smaller off-target rates (green) and is positioned on the exon near the 5' end; respectively adding enzyme cutting sites at two ends of the screened Spacer, and respectively synthesizing positive and negative chains by using Nanjing Jinsri biotechnology limited company; the two synthesized chains are diluted to 100 mu M and then mixed in equal quantity, and double-chain synthesis is performed by gradient annealing, wherein the annealing process is as follows;

95 ℃ for 2 minutes;

72 ℃ for 2 minutes;

55 ℃ for 2 minutes;

2 minutes at 37 ℃;

25 ℃ for 2 minutes;

Keeping the temperature at 10 ℃.

Ligation of the above product with T4 ligase to the corresponding intermediate vector previously linearized with BsaI

PYLgRNA-OsU a/LacZ; amplifying by using two pairs of B1'/BL primers after the connection is completed and recovering amplified products;

The recovered product was ligated into expression vector PYLCRISPR/Cas9-MH using the efficient "side-trim ligation" method and sequencing verified. Methods of use are disclosed in ：Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y(2015).A Robust CRISPR/Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Molecular Plant,8:1274–1284

(2) Transgenic:

100 mu L of Agrobacterium tumefaciens (EHA 105) competent cells and 2 mu L of verified expression vector were gently mixed in a pre-chilled sterile Ep tube. The mixed solution is slowly transferred into a pre-cooled electric shock cup at the temperature of minus 20 ℃ to avoid generating bubbles. Carefully dry the outer wall of the cuvette, set the electrode voltage of the Berle (bio) electric shocker 2500V, and transform the high voltage. After electric shock, pre-culturing by using pre-chilled YEP culture solution in sequence, and screening the YEP flat plate culture containing corresponding antibiotics to transfer into successful monoclonal. After 1mL of the above-mentioned bacterial liquid having an OD600 of 0.8-1.0 was cultured and subjected to centrifugal precipitation, a suspension was prepared using an inductive liquid (200. Mu. Mol/L As in 30mL of AAM). Proper amount of yellowish, round and solid high-quality rice callus is taken, poured into suspension for 5 minutes to be infected, drained or air-dried on sterile filter paper, and transferred to a co-culture medium for dark culture for 3 days.

(3) Plant tissue culture:

Selecting white and full rice seeds, peeling, sterilizing, soaking in clear water for 30 minutes, air-drying on sterile filter paper, placing in an induction culture medium, and culturing in a tissue culture chamber for 4 weeks every 12-14 grains of culture dish. And then placing the rice on a secondary culture medium for continuous culture for 2 weeks to obtain healthy rice callus. Transferring the infected callus into a sterile tube, washing for multiple times with sterile water, soaking for 30-60 min with sterile water containing 500mg/L carbenicillin (Car), blow-drying, transferring to a selective medium, screening, transferring the screened resistant callus into a differentiation tank filled with a differentiation medium, culturing at constant temperature, waiting for differentiation into seedlings (30-60 days), and then placing into a rooting medium for rooting.

(4) And (3) knocking out effect identification:

The transgenic rice genome was sequenced in the interval 1:21071497-21076720 and compared to the wild type. As shown in FIG. 1, the OsCAX a gene in the mutant was successfully knocked out.

Example 2

(1) Mutant root system ammonium absorption capacity detection (isotope tracing method)

Seedlings of rice "Nippon-Qing" and OsCAX a mutant (hereinafter referred to as mutant) were cultivated in normal nutrient solution until 1 leaf and 1 core period, starved in nitrogen-deficient rice culture solution for 3 days, soaked in CaSO ₄ solution of 0.1m M for 1 minute, dried and weighed, surface water was immediately removed by a water absorbing paper, placed in ¹⁵ N nutrient solution (0.25 Mn or 2.5nM N, with an abundance of 49.16% in terms of N) (¹⁵NH₄)₂SO₄, purchased from Shanghai isotope chemical institute), and taken out after accurate timing for 5 minutes, soaked in CaSO ₄ solution of 0.1mM again, and washed out of ¹⁵ N remaining in a root table immediately after washing and placed in a dry blow box preheated at 105 ℃, the root system of rice was completely cut off after water-removing, dried and weighed, and crushed by a ball mill.

(2) Mutant root system ammonium absorption capacity detection (in-situ non-damage real-time monitoring method)

Rice "Nippon" and OsCAX a mutant seedlings were incubated in appropriate medium at 28℃for 2 weeks, and the flow of the corresponding cations at the root tip of rice plants was determined using Non-invasive Micro-test Technology (NMT) (FIG. 7). The joint of the root system and the rhizome of the rice is soaked in test liquid and fixed, the test electrode is calibrated before measurement, the measurement time is 10 minutes continuously, and the sampling rate is once every 5 seconds.

The results are shown in FIG. 3, and the mutant ammonium absorption capacity is significantly higher than that of the wild type Japanese sunny type under the same treatment conditions at the same time, which again proves that CAX1a can actually influence the absorption of ammonium nutrition by rice. (because ammonium ions are positive cations, the potential recorded is negative, i.e., the greater the absolute value the more strongly the ion changes).

(3) Transcript level detection of ammonium transport capacity of ammonium transport proteins

Total RNA was extracted from rice root tissue using Trizol (Semerle technology, USA) reagent. RNA was reverse transcribed using reverse transcription kit HISCRIPT III 1.1. 1st Strand cDNA Synthesis Kit (Norwegian bioscience, nanjing, china) to obtain high purity cDNA from which DNA was removed. Gene expression was measured using a real-time fluorescent quantitation system ABIQuabtStudio Flex real-time PCR (Siemens technology, USA). As an internal reference, osActin1 gene (LOC_Os03g 50885) was used. The relative expression level of the gene was calculated according to methods 2 ^-ΔCT and 2 ^-ΔΔCT (Livak KJ,Schmittgen TD(2001)Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the2-ΔΔCT Method.Methods 25:402–408).

We examined the expression changes of major AMTs (AMT 1.1, 1.2, 1.3, 2.1) in mutant and wild type. The results indicate that the expression of ammonium transporter family AMTs in cax a mutant was all up-regulated to a small extent, especially the greatest extent of AMT1.3 variation (fig. 4).

(4) Protein level detection of ammonium transport capacity of ammonium transport proteins

And (3) placing the quick-frozen plant root tissues in an ultralow temperature refrigerator for 2 days to volatilize liquid nitrogen, taking out, breaking the plant root tissues by using an iron hammer, and completely transferring the plant root tissues into a grinding tool. 30ml of the extract was added and the mixture was ground on ice 2000 times. Ultracentrifugation, separation and solubilization of membrane proteins. Protein concentration was adjusted and ultrasound disruption was performed, membrane proteins were identified using Thermo Scientific Orbitrap Exploris 480,480 mass spectrometer, and protein phosphorylation states of ammonium transporter family AMTs were compared.

The formula of the extracting solution is as follows:

The results indicate that the level of phosphorylation of ammonium transporter family AMTs in cax a mutant was significantly altered, especially with the greatest extent of AMT1.2 (fig. 6), and therefore we believe that the protein modification level is mainly affected by altering the protein activity of AMT1.2 etc. to affect ammonium uptake capacity.

Claims

1. The application of the rice OsCAX a gene in improving the ammonium absorption capacity of root systems is characterized in that the sequence of the rice OsCAX a gene is shown as SEQ ID NO. 1.

2. The use according to claim 1, wherein knocking out the rice OsCAX a gene or inhibiting the expression of the gene increases the ammonium uptake capacity of the root system.

3. The application of the substance for knocking out or silencing the rice OsCAX a gene in improving the ammonium absorption capacity of root systems is characterized in that the sequence of the rice OsCAX a gene is shown as SEQ ID NO. 1.

4. The use according to claim 3, wherein the substance for knocking out or silencing rice OsCAX a gene is a gene editing system, siRNA against rice OsCAX a gene.

5. The use according to claim 3, wherein the gene editing system is selected from the group consisting of CRISPR-CAS9 vectors and CRISPR-CAS9 vector compositions.