CN118581105A

CN118581105A - Method for regulating heavy metal content of crops by WRKY51 gene and application thereof

Info

Publication number: CN118581105A
Application number: CN202410756006.5A
Authority: CN
Inventors: 武亮; 谭景艾; 张蓝天
Original assignee: Hainan Research Institute Of Zhejiang University
Current assignee: Hainan Research Institute Of Zhejiang University
Priority date: 2024-06-12
Filing date: 2024-06-12
Publication date: 2024-09-03

Abstract

The invention discloses a method for regulating and controlling heavy metal content of crops by a WRKY51 gene and application thereof, and belongs to the technical field of biology. The invention provides a WRKY51 gene for regulating and controlling heavy metal content of rice grains, and the nucleotide sequence of the WRKY51 gene is shown as SEQ ID No. 1. The WRKY51 gene is inhibited from being expressed after being treated by heavy metal cadmium for different time. The WRKY51 gene plays an important role in regulating and controlling the accumulation process of cadmium in rice grains, but does not influence the Mn content in the grains. The invention can reduce the influence of soil cadmium (cadmium, cd) pollution on the growth and development of rice plants on the premise of ensuring the rice yield, and relieve the grain safety problem caused by the exceeding of the cadmium content of seeds, thereby providing effective reference for the cultivation of low-cadmium rice and being beneficial to enriching the accumulation of low-cadmium rice genetic resources.

Description

Method for regulating heavy metal content of crops by WRKY51 gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for regulating and controlling heavy metal content of crops by using a WRKY51 gene and application thereof.

Background

Heavy metal pollution in paddy soil is aggravated by wastewater and waste gas emissions caused by industrial production and excessive use of pesticides and fertilizers in agricultural production. Cadmium (Cd) is one of the most toxic heavy metals, mainly flowing into the natural environment by human factors and gradually accumulating in the soil. Rice is one of the main grain crops in the world, and the rice root system is extremely easy to absorb Cd, so that Cd can enter the rice root through the root system, enter rice seeds through transfer and redistribution and accumulate, and further enter a human body through a food chain. Since the half-life of Cd is as long as 20-30 years, cd can be stored in the liver and kidney of human body continuously, and can also cause osteoporosis, renal dysfunction, cancer and other diseases, thus seriously endangering life safety. The rice with the exceeding Cd content is a main source for the accumulation of human Cd, so that the cultivation of crops with Cd stress resistance or low Cd accumulation is beneficial to guaranteeing national grain safety and life and property safety of people.

Cd is a non-essential element for rice growth, and enters the root system of rice mainly by means of transport channels of essential elements such as manganese (MANGANESE, mn), zinc (Zinc, zn), iron (Ferrum, fe) and the like, and is transported to leaves and grains. Cd transport mainly includes 4 steps, root absorption, xylem transport, vascular bundle transport, phloem migration to grain, etc. (CLEMENS ET al, 2013).

The natural resistance related macrophage protein 5 (Natural Resistance-Associated Macrophage Protein, osNramp 5) of rice is a manganese transport protein expressed by root system and positioned by plasma membrane, and is also a main transport protein for absorbing cadmium by rice root system, and is responsible for transporting Cd from soil to root cells. The nramp mutant greatly reduced the uptake of Cd by the root, thereby reducing Cd accumulation in the stalks and kernels (Sasaki et al 2012). However, deletion of the Nramp5 gene also reduces the uptake of Mn, an essential element by rice. HMA3 belongs to the family of Heavy metal atpases (Heavy-METALATPASE, HMA) and is a Cd transporter located on the vacuole membrane of rice roots. HMA3 is able to transport Cd into vacuoles and sequester, thereby significantly reducing Cd transport to the aerial parts and thus reducing Cd accumulation in the aerial parts and kernels (Ueno et al, 2010). LCT (Low-affinity cation transporter) codes Low-affinity cation transport protein, and rice LCT1 knockout does not affect xylem transport, but can obviously reduce phloem-mediated Cd transport, so that grain Cd accumulation can be effectively reduced, and the edible safety of rice is improved (Uraguchi et al., 2011).

The N-terminus of a WRKY transcription factor superfamily member contains a conserved WRKYGQK-stretch and the C-terminus contains a zinc finger motif Cx4-5Cx22-23HxH or Cx7Cx23HxC (Rushton et al, 2010). 102 and 98 WRKY transcription factors were identified in indica and japonica, respectively (Ross et al, 2007). The WRKY protein plays an important role in plant stress response, participation in plant hormone signal transduction and regulation of plant growth and development by combining with W-box regulation gene expression of target genes. However, the function of the transcription factor WRKY51 under Cd stress of rice is not reported at present, and whether the transcription factor WRKY51 can be used for low-cadmium rice breeding is unclear.

Reference is made to:

Clemens S,Aarts MG,Thomine S,Verbruggen N.(2013)Plant science:the key topreventing slow cadmiumpoisoning.TrendsPlantSci 18(2):92-99.

Sasaki A,Yamaji N,Yokosho K,Ma JF.(2012)Nramp5 is a major transporterresponsible for manganese and cadmium uptake in rice.Plant Cell

24(5):2155-2167.

Ueno D,Yamaji N,Kono I,et al.(2010)Gene limiting cadmium accumulation in rice.

ProcNatlAcadSci USA 107(38):16500-16505.

Rushton PJ,Somssich IE,Ringler P,Shen QJ.(2010)WRKY transcription factors.

TrendsPlantSci 15(5):247-258.

Ross CA.,Liu Y,Shen QXJ.(2007)The WRKY gene family in rice(Oryza sativa).JIntegrPlantBiol49(6):827-842.

Disclosure of Invention

The invention aims to provide a method for regulating and controlling heavy metal content of crops by using a WRKY51 gene and application thereof, wherein the content of Cd in seeds can be obviously reduced after the WRKY51 gene is knocked out, and the content of Mn in the seeds is not influenced. After the WRKY51 gene is excessively expressed, the Cd content in the seeds can be obviously increased without influencing the Mn content in the seeds.

The invention provides a WRKY51 gene for regulating and controlling heavy metal content of rice grains, and the nucleotide sequence of the WRKY51 gene is shown as SEQ ID No. 1.

Preferably, the heavy metal species includes cadmium.

The invention also provides application of the WRKY51 gene in cultivation of rice germplasm related to cadmium content.

Preferably, the expression of the WRKY51 gene is knocked down or the cadmium content in rice grains is reduced after the WRKY51 gene is knocked down.

The invention also provides a method for reducing the cadmium content in rice grains, which comprises the following steps: knocking down the expression of the WRKY51 gene or knocking out the WRKY51 gene.

Preferably, the knockdown or knockdown method includes gene editing.

Preferably, when the CRISPR/Cas9 gene editing technique is used for the knockdown or knockdown, the nucleotide sequence of the gene editing target is shown in SEQ ID No. 2.

The invention also provides a construction method of the rice WRKY51 gene CRISPR/Cas9 gene knockout vector, which comprises the following steps:

(1) Carrying out first-round PCR amplification by using pYLsgRNA-OsU3 vectors as templates and respectively using the first pair of primers and the second pair of primers; taking the mixture of the first round of PCR amplification products as a template, and carrying out second round of PCR amplification by using a third pair of primers; taking the PCR amplification product of the second round as a template, and carrying out third round of PCR amplification by using a fourth pair of primers to obtain U3; the first pair of primers are U-F and WRKY51-OsU T1, and the sequences are shown as SEQ ID No.3 and SEQ ID No. 4; the second pair of primers are WRKY51-gRT1 and gR-R, and the sequences are shown as SEQ ID No.5 and SEQ ID No. 6; the third pair of primers are U-F and gR-R; the fourth pair of primers are Pps-GGL and Pgs-GGR, and the sequences are shown as SEQ ID No.7 and SEQ ID No. 8.

(2) And (3) performing enzyme digestion on the U3 by using Bsa I, and then performing T4 connection to obtain the WRKY51 gene knockout binary vector.

The invention also provides a WRKY51 gene knockout binary vector obtained by the construction method.

The invention also provides a method for cultivating rice germplasm with low cadmium content, which comprises the following steps: and transforming the WRKY51 gene knockout binary vector into rice, and carrying out tissue differentiation on the obtained antibiotic resistance callus to obtain the WRKY51 gene knockout transgenic rice material which is the rice germplasm with low cadmium content.

The invention also provides a method for increasing the cadmium content in rice grains, which comprises the following steps: the WRKY51 gene is overexpressed.

Preferably, the method of overexpression comprises overexpression using the Ubiquitin promoter.

Preferably, when the overexpression technique is used, the cleavage sites for gene insertion are Sac I and Kpn I.

The invention also provides a construction method of the rice WRKY51 gene overexpression vector, which comprises the following steps:

(1) PCR amplification is carried out by using the Japanese cDNA as a template and using a primer pair; (2) cleavage pRGV of the vector with Sac I and Kpn I. The primer pairs are W51-Sac I-adapter-F and W51-Kpn I-adapter-R, and the sequences are shown as SEQ ID No.9 and SEQ ID No. 10. (3) And (3) carrying out homologous recombination connection on the PCR product and the vector subjected to enzyme digestion to construct a recombinant vector.

The invention also provides a WRKY51 gene overexpression vector obtained by the construction method.

The invention also provides a method for cultivating the high-cadmium rice germplasm, which comprises the following steps: and transforming the WRKY51 gene overexpression vector into rice, and carrying out tissue differentiation on the obtained resistant callus to obtain the WRKY51 overexpression transgenic material which is the rice germplasm with high cadmium content.

The beneficial effects are that: the invention provides a WRKY51 gene for regulating and controlling heavy metal content of rice grains, and the nucleotide sequence of the WRKY51 gene is shown as SEQ ID No. 1. The WRKY51 gene can be inhibited from being expressed under the stress treatment conditions of heavy metal cadmium with different concentrations.

In the embodiment of the invention, a rice material WRKY for knocking out WRKY51 is also constructed by using a gene editing method, after WRKY seeds planted in cadmium-polluted soil are harvested, the cadmium content of brown rice is measured, and the result shows that the Cd content of rice in the WRKY51 knocking-out strain is obviously reduced, but the Mn content in the seeds is not influenced, so that the WRKY51 plays an important role in regulating and controlling the accumulation process of Cd in the rice seeds. Compared with wild rice, the wrky rice plant has no obvious difference in agronomic characters. On the premise of ensuring the rice yield, the method can reduce the influence of soil Cd pollution on the growth and development of rice plants, relieve the grain safety problem possibly caused by the heavy metal content of seeds, further provide effective reference for low-cadmium rice cultivation and facilitate the accumulation of low-cadmium rice genetic resources.

In the embodiment of the invention, the over-expression method is also utilized to construct a WRKY51-OE rice material with the over-expression of WRKY51, after the WRKY51-OE seeds planted in the cadmium-polluted soil are harvested, the cadmium content of brown rice is measured, and the result shows that the Cd content of the rice of the WRKY51-OE strain is obviously increased, but the Mn content in the seeds is not influenced, so that the WRKY51 plays an important role in regulating and controlling the accumulation process of Cd in the rice seeds.

Drawings

FIG. 1 is a graph showing the results of the relative expression levels of WRKY51 in rice roots after 10. Mu.M CdCl ₂ treatment for various times;

FIG. 2 is a map of WRKY51 CRISPR/Cas9 gene knockout cloning vector PYLCRISPR/Cas9Pubi-H, pYLsgRNA-OsU3 and pYLsgRNA-OsU6 a;

FIG. 3 is a vector map of WRKY51 overexpression vector pRGV;

FIG. 4 is a diagram of the genetic transformation process of WRKY51 knockout transgenic material;

FIG. 5 is a diagram showing the genetic transformation process of WRKY51-OE transgenic rice;

FIG. 6 is a graph of the result of identifying the mutation sites of WRKY51 CRISPR/Cas9 knockout transgenic plants;

FIG. 7 is a graph showing the detection result of the overexpression multiple of WRKY51-OE transgenic rice;

FIG. 8 is a graph of Cd content measurement results of brown rice of a WRKY51 knockout transgenic plant;

FIG. 9 is a graph showing the Mn content measurement result of brown rice of WRKY51 knockout transgenic plant;

FIG. 10 is a graph of Cd content measurement results of brown rice of WRKY51-OE transgenic plants;

FIG. 11 is a graph showing the Mn content measurement result of brown rice of WRKY51-OE transgenic plant;

FIG. 12 is a graph of the result of examining agronomic traits of WRKY51 knock-out rice plants, wherein A represents plant type, B represents plant height, C represents effective ears of individual plants, D represents the number of grains per ear, and E represents thousand grain weight.

Detailed Description

The heavy metal preferably comprises cadmium, and in the embodiment, WRKY51 plays an important role in regulating and controlling the cadmium accumulation process in rice grains, and a mutant strain with a knocked-out gene is constructed through a gene editing technology, and a gene overexpression strain is also constructed through an overexpression vector. After the mutant strain and the over-expressed strain are planted with the cadmium contaminated soil, seeds are harvested and the Cd and Mn content in the brown rice is measured. As a result, it was found that the Cd content in the brown rice of the mutant strain was significantly lower than that of the wild type, but that the Mn content was not different from that of the wild type. The content of Cd in the brown rice of the over-expression strain is obviously higher than that of the wild type, but the Mn content is not different from that of the wild type.

In the invention, the expression of the WRKY51 gene is knocked down or the cadmium content in rice grains is reduced after the WRKY51 gene is knocked down. The method of the present invention is not particularly limited, and the method of knocking down or knocking out may be performed by using a conventional gene editing method in the art. In the examples, the knockdown or knockdown is preferably performed by CRISPR/Cas9 gene editing technology, so that the WRKY51 gene is expressed in a genome in a reduced amount or no longer expressed. The cadmium content in rice grains is increased after the WRKY51 gene is overexpressed. The method of the present invention is not particularly limited, and the over-expression may be performed by using an over-expression vector conventional in the art. In the examples, it is preferable to use pRGV vectors for overexpression, so that the expression level of the WRKY51 gene is significantly increased.

The knockdown or knockdown method of the present invention preferably includes gene editing, and when the knockdown or knockdown is performed using CRISPR/Cas9 gene editing technology, the nucleotide sequence of the gene editing target is preferably as shown in SEQ id No. 2.

(1) Carrying out first-round PCR amplification by using pYLsgRNA-OsU3 vectors as templates and respectively using the first pair of primers and the second pair of primers; taking the mixture of the first round of PCR amplification products as a template, and carrying out second round of PCR amplification by using a third pair of primers; and carrying out third round of PCR amplification by using the second round of PCR amplification product as a template and utilizing a fourth pair of primers to obtain U3. The first pair of primers are U-F and WRKY51-OsU T1, and the sequences are shown as SEQ ID No.3 and SEQ ID No. 4; the second pair of primers are WRKY51-gRT1 and gR-R, and the sequences are shown as SEQ ID No.5 and SEQ ID No. 6; the third pair of primers are U-F and gR-R; the fourth pair of primers are Pps-GGL and Pgs-GGR, and the sequences are shown as SEQ ID No.7 and SEQ ID No. 8.

The coding region DNA sequence of the rice WRKY51 is preferably edited by using a CRISPR/Cas9 gene editing technology: firstly, an editing target point of WRKY51 is designed on line by utilizing CRISPR-GE (http:// skl. Scau. Edu. Cn /), and an optimal target sequence is selected for primer design; secondly, constructing a PYLsgRNA-OsU3 expression cassette containing the double targets of the gene through Overlapping PCR; and then cloning the sgRNA expression cassette to a Cas9 vector by utilizing BsaI endonuclease and T4 DNA ligase in a side-by-side edge connection mode, and finally constructing the WRKY51 gene knockout binary vector.

The invention also provides a method for increasing the cadmium content in rice grains, which comprises the following steps: overexpression was performed using the Ubiquitin promoter.

The over-expression method comprises the steps of utilizing a Ubiquitin promoter, and inserting genes with enzyme cutting sites of Sac I and Kpn I.

The WRKY51 gene knockout binary vector is preferably transformed into an agrobacterium AGL1 strain, rice is transformed by using an agrobacterium-mediated method, and PCR sequencing is performed on transgenic positive seedlings. And (3) propagating the homozygous plant, and further carrying out PCR sequencing on the newly harvested plant to obtain the homozygous mutant plant.

The invention preferably converts the WRKY51 gene over-expression vector into agrobacterium AGL1, converts rice by using an agrobacterium-mediated method, and carries out qRT-PCR detection on transgenic positive seedlings. And (3) propagating the over-expressed strain, and further carrying out resistance screening on newly harvested plants to gradually obtain the homozygous over-expression material.

In order to further illustrate the present invention, the following detailed description of the method and application of the WRKY51 gene provided by the present invention to regulate heavy metal content in crops is provided in connection with examples, but should not be construed as limiting the scope of the present invention.

The test methods used in the embodiment of the invention are all conventional methods unless specified otherwise; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Wherein:

(1) The method for measuring the Cd and Mn content of brown rice of the WRKY51 knockout transgenic rice plant and the WRKY51-OE transgenic rice plant under low-cadmium cultivation comprises the following steps:

Firstly, culturing wild type, WRKY mutant and WRKY51-OE transgenic rice plants in common soil to a five-leaf state, and then simultaneously planting the wild type, WRKY mutant and WRKY51-OE transgenic rice plants with consistent growth vigor in the soil with Cd content of 2 mg/kg. Harvesting rice kernels, drying and shelling. After weighing the dry weight of each group of samples, placing the samples in a 20mL test tube, adding 1-2 mL of nitric acid, placing the samples in a constant temperature metal bath, and carrying out nitrolysis for 4 hours at 120 ℃. Then evaporating and discharging acid at 140 ℃ for about 2 hours until the residual liquid is less than 1mL, and adding ultrapure water to dilute to 20mL after cooling. 10mL of the sample is sucked by a gun head and is filled in a centrifuge tube with 15mL of a sharp bottom for measurement. The content of Cd and Mn elements in the sample was determined by means of an ICP/MS electrically coupled plasma mass spectrometer (PERKINELMER, USA).

(2) The field agronomic trait investigation method comprises the following steps:

The WRKY51 gene knocked rice plants with stable inheritance and wild rice are planted in the same paddy field, the same field management is carried out in the whole growth period, and important agronomic yield traits such as plant height, effective spike of single plant, thousand grain weight, grain number per spike and the like are examined.

Example 1

Expression level of WRKY51 in wild type rice after 10. Mu.M CdCl ₂ treatment for various times

1. CdCl ₂ treatment of rice seedlings

Culturing germinated wild rice in 1/2 nutrient solution for 7 days, selecting seedlings with consistent growth vigor, respectively culturing in 1/2 nutrient solution and 1/2 nutrient solution+10mu M CdCl ₂ water culture solution, and sampling roots of the materials for extracting RNA at 12h, 24h, 48h and 10d after treatment.

Extraction of RNA

① Taking a rice seedling root sample, rapidly placing the rice seedling root sample into a 2mL RNA-free centrifuge tube with steel balls, and putting the rice seedling root sample into liquid nitrogen;

② Freezing a metal block of a grinder in liquid nitrogen to constant temperature, symmetrically placing a sample tube in a metal block hole, screwing up a screw, and grinding at 60Hz for 60s;

③ Quick adding RNAiso Plus (Trizol) 1mL into the sample tube, immediately shaking and mixing, and standing at room temperature for 5min;

④ Continuously adding 200 mu L of chloroform into the sample tube, uniformly mixing, and standing at room temperature for 5min;

⑤ Centrifuging at 12000rpm at 4deg.C for 15min, collecting 400 μl of supernatant, adding into a new RNAfree mL centrifuge tube, adding 400 μl isopropanol, slightly turning upside down, standing at-20deg.C overnight or standing at room temperature for 20min;

⑥ Centrifuging at 12000rpm for 10-15min at 4deg.C, and discarding supernatant;

⑦ Adding 1mL of 75% ethanol, cleaning the precipitate, centrifuging at 12000rpm and 4 ℃ for 5min, and discarding the supernatant;

⑧ Repeating the step 7;

⑨ Centrifuging the centrifuge tube again at 12000rpm and 4deg.C for 1min, removing residual liquid in the tube, opening the tube cover, air drying at room temperature until white flaky RNA is transparent, adding 30mu LRNase-FREE WATER, dissolving on ice, and immediately performing reverse transcription or long-term storage at-80deg.C.

2. Reverse transcribed RNA

Reaction one: 5 μl system: RNA was not more than 5ng, oligo d (T) 1. Mu.L, DEPC H ₂ O was made up.

The reaction system is evenly mixed and then reacts for 5 minutes at 70 ℃, and then is immediately placed on ice for at least 5 minutes. Then, 15. Mu.L of the system ：GoScript^TM 5×ReactionBuffer4μL、MgCl₂2μL、PCR Nucleotide Mix 1μL、Recombinant RNase Ribonuclease Inhibitor 0.5μL、GoScript^TM Reverse Transcriptase 1μL、Nuclease-Free Water 6.5μL and 5. Mu.L of the reaction system were added to the system.

After the reaction system is uniformly mixed, the following reaction is carried out: 25℃for 5min,42℃for 90min,70℃for 5min and 4℃for 60min.

The product after reverse transcription is cDNA, and can be stored at-20 ℃ for a long time.

3. Fluorescent quantitative PCR

Fluorescent quantitative PCR experiments were performed using PowerUp ^TM SYBP Green Mix (appliedbiosystems) kit.

The cDNAs obtained by reverse transcription were diluted 4-fold to prepare a quantitative PCR system (15. Mu.L): SYBP Green Mix 7.5.5. Mu.L, forward primer 0.3. Mu.L, REVERSE PRIMER 0.3. Mu. L, cDNA 1.5.5. Mu.L and RNase-free H ₂ O5.4. Mu.L.

The cDNA obtained by reverse transcription is used as a template, qGAPDH-F (shown as SEQ ID No. 9) is used as a forward primer, qGAPDH-R (shown as SEQ ID No. 10) is used as a reverse primer, and the reverse primer is used as an internal reference expression quantity.

Quantitative PCR was performed using reverse transcribed cDNA as a template (the amount of template was determined by the amount adjusted), qWRKY-F (as shown in SEQ ID No. 11) as a forward primer, and qWRKY-R (as shown in SEQ ID No. 12) as a reverse primer.

As a result, as shown in FIG. 1, 10. Mu.M Cd treatment strongly inhibited the expression of the WRKY51 gene as compared with no treatment. Therefore, WRKY51 may play a role in regulating Cd uptake in rice, but the specific regulation mode is not clear yet.

Example 2

Construction of rice WRKY51 CRISPR/Cas9 gene knockout vector

The carrier construction mainly comprises three rounds of PCR amplification and edge trimming connection, and the PCR procedure and the system are as follows:

First round PCR amplification: PCR amplification was performed simultaneously with pYLsgRNA-OsU vector (upper panel in FIG. 2) as template, U-F (shown as SEQ ID No. 3) and WRKY51-OsU T1 (shown as SEQ ID No. 4) as a first pair of primers and WRKY51-gRT1 (shown as SEQ ID No. 5) and gR-R as a second pair of primers (shown as SEQ ID No. 6), respectively, as follows;

Second round PCR amplification: PCR amplification is carried out by mixing PCR products of the two pairs of primers in the first round of amplification products by taking pYLsgRNA-OsU as a template, taking U-F (shown as SEQ ID No. 2) as a forward primer and gR-R (shown as SEQ ID No. 6) as a reverse primer as a template for the second round of PCR, wherein the amplification procedure is as follows.

Third round of PCR amplification: and (3) taking the last round of PCR product glue recovery as a template, respectively taking Pps-GGL (shown as SEQ ID No. 7) and Pgs-GGR (shown as SEQ ID No. 8) as primers for PCR amplification, and recording the recovered product glue as a U3 amplification final product after the amplification procedure is shown as follows.

PCR amplification procedure: pre-denaturation at 98℃for 2min; denaturation at 98℃for 20s, annealing at 58℃for 30s, elongation at 72℃for 1min,38 cycles; extending at 72 ℃ for 10min; preserving at 8 ℃.

Edge trimming and connecting: bsaI cleavage was first performed

And (3) enzyme cutting: 37 ℃ for 15min

And (3) enzyme cutting system: 10X Cutsmart Buffer 1.5. Mu. L, U3 amplification final product 2. Mu. L, pYLCRISPR/Cas9 Pubi-H2. Mu. L, bsa I1. Mu.L and ddH ₂ O4.5. Mu.L.

After cleavage, 1. Mu.LT 4 ligase was added to the above system and edge ligation was performed with 1.5. Mu.L of 10 XT 4 DNALIGASE BUFFER and 2.5. Mu. LddH ₂ O as follows:

PCR reaction procedure: 37 ℃ 5min,10 ℃ 5min,20 cycling at 5min and 15 deg.C; preserving at 4 ℃.

Example 3

Construction of Rice WRKY51-OE vector

The vector construction mainly comprises vector enzyme digestion, fragment amplification and homologous recombination, and an enzyme digestion system and a program, a PCR system and a program and a homologous recombination system and a program are shown as follows:

(1) And (3) carrier enzyme cutting:

pRGV vector (FIG. 3) was digested with SacI and KpnI fast-cutting enzymes.

And (3) enzyme cutting system: 10 XBuffer 4. Mu. L, pRGV empty 30. Mu.L, sac I1. Mu.L, kpn I1. Mu.L and ddH ₂ O4. Mu.L.

And (3) enzyme cutting: and (3) performing water bath enzyme digestion for 3 hours at 37 ℃.

(2) WRKY51 CDS sequence amplification:

The Japanese cDNA is used as a template, and W51-Sac I-adapter-F (shown as SEQ ID No. 9) and W51-Kpn I-adapter-R (shown as SEQ ID No. 10) are respectively used as primers for amplification.

And (3) enzyme cutting system: 2 XPAmix 10. Mu. L, cDNA template 2. Mu. L, W51-Sac I-adapter-F0.75. Mu. L, W51-Kpn I-adapter-R0.75. Mu.L and ddH ₂ O6.5. Mu.L.

PCR amplification procedure: pre-denaturation at 98℃for 2min; denaturation at 98℃for 20s, annealing at 56℃for 30s, elongation at 72℃for 1min,38 cycles; extending at 72 ℃ for 10min; preserving at 8 ℃.

Example 4

WRKY51 gene knock-out rice material and genetic transformation of WRKY51 over-expressed rice material

The WRKY51 gene knockout binary vector constructed in the example 2 and the WRKY51 overexpression vector constructed in the example 3 are respectively transformed into agrobacterium tumefaciens AGL1, rice is transformed by using an agrobacterium-mediated method to obtain hygromycin resistant callus, and tissue differentiation is carried out to respectively obtain a WRKY51 gene knockout transgenic rice material and a WRKY51 overexpression rice material.

The steps of agrobacterium-mediated transformation of rice are shown in fig. 4 and 5, respectively:

(1) Preparation of Agrobacterium solution

Single colonies of Agrobacterium were picked up on an ultra clean bench and inoculated into 3mLLB liquid medium (containing 50mg/L rifampicin and 50mg/L kanamycin) and shaken overnight on a shaker at 28℃at 200r/min until the inoculum became cloudy. 1mL of the bacterial liquid is coated on LB solid medium (containing 50mg/L rifampicin and 50mg/L kanamycin) and placed in a 28 ℃ incubator for 1-2 d. After the agrobacteria grow on LB solid medium, scraping the thalli by a sterilizing gun head, activating by an activating medium, and adjusting OD ₆₀₀ to 0.4-0.6 to be used for infection.

The activation medium (AAM) formulation is as follows:

Chu's N6 Basal Medium withVitamins 4.10.10 g/L+glycine 2 mg/L+tyrosin 500 mg/L+glucose 36 g/L+sucrose 68.5 g/L+acetosyringone 200. Mu. Mol/L, pH=5.2

(2) Induction and transformation of rice callus

Firstly, husking rice seeds, and selecting seeds with full seeds and no disease spots from the husks. Placing seeds in a sterilizing tube in an ultra-clean bench, adding a proper amount of 75% alcohol for soaking for 1-2 min, and continuously shaking during the period; then, removing alcohol, adding sterile water for washing, soaking and sterilizing for 20-30 min by using 1% NaClO solution, and fully shaking during the soaking and sterilizing; then washing 3-4 times with sterile water, and placing the sterilized seeds on sterilized paper for airing for standby; uniformly placing the sterilized seeds on an N6 culture medium, culturing for 7-10 days at 26-28 ℃ by light until golden yellow granular callus is induced by rice, stripping the rice to a new culture medium by forceps, and using the callus after subculturing for one week for infection of agrobacterium.

N6 (NBD) was formulated as follows:

chu's N6 Basal Medium withVitamins 4.10.10 g/L, proline 500mg/L, glutamine 500mg/L, tyrosin 300mg/L, sucrose 30g/L+2, 4-D2.5 mg/L, pH=5.8, phytagel 3g/L

3) Rice callus transformed by agrobacterium and co-culture process

Selecting yellow hard callus, placing into an activated culture medium with activated bacterial liquid, continuously shaking for 5 minutes, and standing for 30 minutes. Pouring out the bacterial liquid, placing the callus on sterilized paper, draining the bacterial liquid, transferring to a solid co-culture medium, and culturing for 2-3 days at 28 ℃ in a dark place.

The co-culture medium (NBDC) was formulated as follows:

Chu's N' 6 Basal Medium WITH VITAMINS 4.10.10 g/L+glycine 2 mg/L+2.4-D2.5 mg/L+sucrose 20 g/L+mannitol 36.43 g/L+glucose 10 g/L+tyrosin 0.5 g/L+acetosyringone 200. Mu. Mol/L, pH=5.8, phytagel 3g/L

4) Screening and subculturing of resistant callus

The callus with clean surface and no agrobacterium pollution is transferred into a screening culture medium for first screening, and then screening is carried out once every week until the resistant callus (the bright yellow callus newly grown around brown callus) grows. Resistant calli were stripped onto new selection medium and calli stripped from one calli were a strain. Two more weeks after which screening can be used for callus regeneration.

The screening media (NBDS) formulation is as follows:

Chu's N6 Basal Medium withVitamins 4.10.10 g/L+0.3 g/L of tyrosin+0.5 g/L of proline+2 mg/L of glycine+2, 4-D2.5 mg/L+300 mg/L of timentin+50 mg/L of Hyg+30 g/L of sucrose, pH=5.8, phytagel 3g/L

5) Regeneration of resistant calli

Transferring the selected callus into a pre-differentiation culture medium for continuous culture, transferring the callus into a differentiation culture medium for illumination culture after compact callus grows out after one month, and transferring the callus into a rooting culture medium after the callus turns green and differentiating seedlings. After the transgenic seedlings grow up, leaf DNA is extracted, PCR is performed by using WRKY51-test-F (shown as SEQ ID No. 11) and WRKY51-test-R (shown as SEQ ID No. 12) primers, sequencing is performed on PCR products, and sequencing results are analyzed.

The prescaled medium (MS-PG) was formulated as follows:

MS 4.43g/L+6-BA2 mg/L+ABA5 mg/L+NAA1 mg/L+sucrose 30 g/L+sorbitol 20g/L+Hyg 50mg/L, pH=5.8, phytagel 3g/L

Differentiation medium (MS-RG)

MS 4.43g/L+6-BA2 mg/L+NAA 0.2 mg/L+sorbitol 20 g/L+sucrose 30g/L+Hyg 50mg/L, pH=5.8, phytagel 3g/L

Rooting medium (MS-RT) was formulated as follows:

1/2MS2.215 g/L+sucrose 20g/L+Hyg 50mg/L+Phytagel 2.5g/L, pH=5.8

Example 5

Identification of mutation site of WRKY51 gene knockout transgenic plant

And (5) identifying mutation sites of the screened positive transgenic plants. The PCR system and the procedure are as follows, taking positive plant genome DNA as a template, taking WRKY51-test-F (shown as SEQ ID No. 11) as a forward primer and taking WRKY51-test-R (shown as SEQ ID No. 12) as a reverse primer:

(1) PCR system: 2X TaqBuffer. Mu. L, DNA. Mu. L, WRKY 51-test-F0.75. Mu. L, WRKY 51-test-R0.75. Mu.L and ddH ₂ O6.5. Mu.L.

(2) PCR amplification procedure: pre-denaturation at 95℃for 2min; denaturation at 94℃for 20s, annealing at 58℃for 30s, elongation at 72℃for 15s,38 cycles; extending at 72 ℃ for 10min; preserving at 8 ℃.

And finally, performing rubber tapping sequencing on the PCR product, analyzing a sequencing peak diagram, and counting mutation conditions.

As a result, FIG. 4 shows that the WRKY cas9#1 mutant deletion 5bp,wrky51 cas9#2 mutant deletion 4bp,wrky51 cas9#3 mutant lacks 2bp as compared with the DNA sequence of the wild-type WRKY 51.

Example 6

Detection of overexpression fold of WRKY51 overexpression plant

And (5) carrying out over-expression multiple detection on the screened positive transgenic plants. qRT-PCR was performed using similar plant vigour as templates, and qGAPDH-F (shown as SEQ ID No. 13) and qGAPDH-R (shown as SEQ ID No. 14), qWRKY-51-F (shown as SEQ ID No. 15) and qWRKY-R (shown as SEQ ID No. 16) as primer pairs, respectively. As a result, the WRKY51-OE#1 and WRKY51-OE#2 lines had a multiple of about 10-15 times over-expression, as shown in FIG. 7.

Example 7

Determination of content of Cd element and Mn element in brown rice of WRKY51 knockout transgenic plant

And (3) planting the stably inherited WRKY51 knockout transgenic plant and wild rice in soil with Cd content of 2mg/kg, harvesting the plant in the mature period, drying, unshelling, and measuring the Cd and Mn contents of the brown rice.

The digestion process is as follows:

1) Weighing 0.2 g-0.4 g brown rice into a digestion tube, adding 1-2mL of concentrated nitric acid, and standing overnight in a fume hood;

2) The temperature of the digestion furnace is adjusted to 90-100 ℃, and the digestion furnace is slightly swayed until the brown rice disappears into a fluid state. Heating to about 120deg.C, and heating for 2 hr to obtain a complete liquid;

3) Raising the temperature to 140 ℃ again for acid discharge, and keeping the residual liquid in the digestion tube to be about 1 mL;

4) And cooling, then carrying out constant volume, dilution and measurement.

As a result, as shown in fig. 8 and 9, the Cd content in wrky mutant brown rice was significantly reduced (fig. 8) but the Mn content was not significantly changed (fig. 9) compared to the wild type.

Example 8

Determination of content of Cd element and Mn element in brown rice of WRKY51-OE transgenic plant

And (3) planting the WRKY51-OE transgenic plant with stable inheritance and wild rice in soil with Cd content of 2mg/kg, harvesting the plant in the mature period, drying, unshelling, and measuring the Cd and Mn contents of the brown rice.

The digestion process is as follows:

4) And cooling, then carrying out constant volume, dilution and measurement.

The results are shown in fig. 10 and 11, where Cd content in brown rice of WRKY51-OE transgenic plants was significantly increased compared to wild type (fig. 10), while Mn content was not significantly changed (fig. 11).

Example 9

Agronomic trait investigation result of WRKY51 knockout transgenic plant

The WRKY51 gene knocked-out rice plants with stable inheritance and wild rice are planted in the same paddy field, the same field management is carried out in the whole growth period, and important agronomic yield traits such as plant height, thousand seed weight, effective tillering number of each plant, spike number and the like are examined and counted.

The results are shown in fig. 12, in which the wrky mutant showed no significant change in plant height (a), thousand kernel weight (B), effective tillering per plant (C) and kernel per ear number (D) compared to the wild type.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. The WRKY51 gene for regulating and controlling the heavy metal content of rice grains is characterized in that the nucleotide sequence of the WRKY51 gene is shown as SEQ ID No. 1.

2. The WRKY51 gene according to claim 1, wherein the heavy metal species comprises cadmium.

3. Use of the WRKY51 gene according to claim 1 or 2 for the cultivation of rice germplasm related to cadmium content.

4. The use according to claim 3, wherein the cadmium content of the rice grain is reduced after knocking down or knocking out the expression of the WRKY51 gene.

5. A method for reducing cadmium content in rice grains, comprising the steps of: knocking down the expression of the WRKY51 gene of claim 1 or 2 or knocking out the WRKY51 gene.

6. The method of claim 5, wherein the knockdown or knockdown method comprises gene editing.

7. The method of claim 6, wherein when the knockdown or knockdown is performed using CRISPR/Cas9 gene editing methods, the nucleotide sequence of the gene editing target is as shown in SEQ ID No. 2.

8. The construction method of the WRKY51 gene CRISPR/Cas9 gene knockout vector according to claim 1 or 2, comprising the steps of: (1) Carrying out first-round PCR amplification by using pYLsgRNA-OsU3 vectors as templates and respectively using the first pair of primers and the second pair of primers; taking the mixture of the first round of PCR amplification products as a template, and carrying out second round of PCR amplification by using a third pair of primers; taking the second round PCR amplification product as a template, and carrying out third PCR amplification by using a fourth pair of primers to obtain U3; the first pair of primers are U-F and WRKY51-OsU T1, and the nucleotide sequences are shown as SEQ ID No.3 and SEQ ID No. 4; the second pair of primers are WRKY51-gRT1 and gR-R, and the nucleotide sequences are shown as SEQ ID No.5 and SEQ ID No. 6; the third pair of primers are U-F and gR-R; the fourth pair of primers are Pps-GGL and Pgs-GGR, and the nucleotide sequences are shown as SEQ ID No.7 and SEQ ID No. 8;

9. The WRKY51 gene knockout binary vector obtained by the construction method of claim 8.

10. A method for cultivating rice germplasm with low cadmium content, which is characterized by comprising the following steps: transforming the WRKY51 gene knockout binary vector of claim 9 into rice, and carrying out tissue differentiation on the obtained antibiotic resistance callus to obtain the WRKY51 gene knockout transgenic rice material which is the rice germplasm with low cadmium content.