Method for regulating and controlling heavy metal content of rice through MORC6s and application

Abstract

The invention provides a method for regulating and controlling the heavy metal content of rice through MORC6s and application, and particularly belongs to the technical field of rice cultivation. The invention provides a gene for regulating and controlling the heavy metal content of the root part and the overground part of rice. The gene comprises any one or more of MORC6a, MORC6b and MORC6c. The MORC6a gene, the MORC6b gene and the MORC6c gene are subjected to induced expression after being treated by the heavy metal cadmium for different times. The invention finds that the MORC6a, MORC6b and MORC6c genes play an important role in regulating and controlling the accumulation process of cadmium at the root part and the overground part of rice. By utilizing the MORC6a, MORC6b and MORC6c genes, the harm of soil cadmium pollution to the growth and development of rice plants can be relieved, the food safety problem caused by excessive cadmium content is relieved, effective reference is further provided for low-cadmium rice cultivation, and accumulation of genetic resources of the low-cadmium rice is enriched.

Description

Technical Field

[0001] The invention belongs to the technical field of rice cultivation, and specifically relates to a method and application of MORC6s for regulating the heavy metal content of rice. Background Art

[0002] Cadmium (Cd), as a non-essential element in the growth and development of plants, mainly flows into nature through human industrial activities, then accumulates in soil and water sources, and eventually enters the human body through the food chain, thus seriously endangering human life and health. Rice is the main food crop in Asia. The root system of rice has a strong ability to absorb and transport Cd, which makes rice sensitive under Cd pollution conditions. Therefore, it is one of the crops that is extremely susceptible to environmental Cd pollution. Cd, as a toxic heavy metal, will accumulate in the human body for a long time, and over time, it will chronically damage important organs such as the liver and kidneys of humans, which is not conducive to human life and health. In view of the above situation, my country's national standard "National Food Safety Standard Limit of Cadmium in Rice and Rice Products" stipulates that the Cd content in rice shall not exceed 0.2 mg / kg. Therefore, cultivating rice germplasm with low Cd absorption and low Cd accumulation is an important task at present. This is not only conducive to protecting people's life and health, but also helps to ensure national food security. Summary of the invention

[0003] The purpose of the present invention is to provide a method and application of MORC6s to regulate the heavy metal content of rice. Knocking out MORC6a, MORC6b and MORC6c genes simultaneously can significantly reduce the Cd content in the root and aboveground part.

[0004] The present invention provides genes for regulating the heavy metal content in rice roots and aerial parts, wherein the genes include any one or more of MORC6a, MORC6b and MORC6c, and the nucleotide sequences of the MORC6a, MORC6b and MORC6c genes are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.

[0005] Preferably, the heavy metal includes cadmium.

[0006] The present invention also provides the use of the gene described in the above technical solution in regulating the heavy metal content in rice roots and aerial parts or in cultivating rice germplasm related to cadmium content.

[0007] The present invention also provides the use of knocking down or knocking out MORC6a, MORC6b and MORC6c genes in reducing the cadmium content in rice roots and aerial parts or in cultivating rice germplasm with low cadmium content.

[0008] The present invention also provides a method for reducing the cadmium content in the root and aboveground part of rice or cultivating rice germplasm with low cadmium content, comprising the following steps:

[0009] Knock down the expression of the MORC6a, MORC6b and MORC6c genes described in the above technical solution in rice or knock out the MORC6a, MORC6b and MORC6c genes.

[0010] Preferably, the knockdown or knockout method comprises gene editing; and the gene editing method comprises CRISPR / Cas9 gene editing method.

[0011] Preferably, when the knockdown or knockout is performed using the CRISPR / Cas9 gene editing method, the nucleotide sequences of the gene editing target are shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.

[0012] The present invention also provides a method for constructing a vector for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on the CRISPR / Cas9 gene editing method, comprising the following steps:

[0013] (1) Using the pYLsgRNA-OsU3 vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, the third pair of primers were used for the second round of PCR amplification; using the second round of PCR amplification products as a template, the fourth pair of primers were used for the third round of PCR amplification to obtain the U3 promoter-driven sgRNA expression cassette; the first pair of primers were UF and MORC6a-OsU3T1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.8; the second pair of primers were MORC6a-gRT1 and gR-R, and the nucleotide sequences were shown in SEQ ID No.9 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GGL and Pgs-GG2, and the nucleotide sequences were shown in SEQ ID No.11 and SEQ ID No.12;

[0014] (2) Using the pYLsgRNA-OsU6c vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, the third pair of primers were used for the second round of PCR amplification; using the second round of PCR amplification products as a template, the fourth pair of primers were used for the third round of PCR amplification to obtain the U6c promoter-driven sgRNA expression cassette; the first pair of primers were UF and MORC6b-OsU6cT1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.13; the second pair of primers were MORC6b-gRT2 and gR-R, and the nucleotide sequences were shown in SEQ ID No.14 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GG2 and Pgs-GG3, and the nucleotide sequences were shown in SEQ ID No.15 and SEQ ID No.16;

[0015] (3) Using the pYLsgRNA-OsU6a vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, the third pair of primers were used for the second round of PCR amplification; using the second round of PCR amplification products as a template, the fourth pair of primers were used for the third round of PCR amplification to obtain the U6a promoter-driven sgRNA expression cassette; the first pair of primers were UF and Morc6c-OsU6aT1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.17; the second pair of primers were Morc6c-gRT3 and gR-R, and the nucleotide sequences were shown in SEQ ID No.18 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GG3 and Pgs-GGR, and the nucleotide sequences were shown in SEQ ID No.19 and SEQ ID No.20;

[0016] (4) The U3 promoter-driven sgRNA expression cassette, the U6c promoter-driven sgRNA expression cassette, and the U6a promoter-driven sgRNA expression cassette were digested with BsaⅠ and then ligated to the pYLCRISPR / Cas9Pubi-H vector by T4 to obtain a binary vector for simultaneously knocking out the three genes MORC6a, MORC6b, and MORC6c.

[0017] The present invention also provides a vector for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on the CRISPR / Cas9 gene editing method, which is constructed by the construction method described in the above technical scheme.

[0018] The present invention provides genes for regulating the heavy metal content in the root and aboveground parts of rice. MORC6a, MORC6b and MORC6c genes can be induced to express under the conditions of heavy metal cadmium stress treatment at different times. By regulating the genes, the regulation of the heavy metal cadmium content in rice can be achieved. Specifically, knocking out or knocking down the MORC6a, MORC6b and MORC6c genes can reduce the heavy metal cadmium content in the root and aboveground parts of rice, reduce the harm of soil cadmium pollution to the growth and development of rice plants, alleviate the food security problems caused by excessive cadmium content, and thus provide an effective reference for the cultivation of low-cadmium rice, and help to enrich the accumulation of low-cadmium rice genetic resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0020] Figure 1 The 10 μM CdCl provided by the present invention 2 The relative expression level of MORC6a in rice roots after treatment for different time periods;

[0021] Figure 2 The 10 μM CdCl provided by the present invention 2 The relative expression level of MORC6b in rice roots after treatment for different time periods;

[0022] Figure 3 The 10 μM CdCl provided by the present invention 2 The relative expression level of Morc6c in rice roots after treatment for different time periods;

[0023] Figure 4 Vector maps of the vector backbone pYLCRISPR / Cas9Pubi-H of the CRISPR / Cas9 gene knockout vectors of MORC6a, MORC6b and MORC6c provided by the present invention, and the amplification plasmid templates pYLsgRNA-OsU3, pYLsgRNA-OsU6a and pYLsgRNA-OsU6c of the three expression cassettes;

[0024] Figure 5The vector map of pCambia1390 provided by the present invention;

[0025] Figure 6 A diagram showing the genetic transformation process of MORC6a, MORC6b and MORC6c knockout transgenic materials and MORC6b-OE transgenic rice provided by the present invention;

[0026] Figure 7 A diagram showing the results of identifying mutation sites of MORC6a, MORC6b and MORC6c knockout transgenic plants provided by the present invention;

[0027] Figure 8 A graph showing the results of overexpression multiple detection of MORC6b-OE transgenic rice provided by the present invention;

[0028] Fig. 9 This is a graph showing the results of measuring the Cd content in the roots of MORC6a, MORC6b and MORC6c knockout transgenic plants provided by the present invention;

[0029] Fig.10 A graph showing the results of measuring the Cd content in the aerial parts of MORC6a, MORC6b and MORC6c knockout transgenic plants provided by the present invention;

[0030] Fig.11 This is a graph showing the results of measuring the Cd content in the roots of the MORC6b-OE transgenic plants provided by the present invention;

[0031] Fig.12 This is a graph showing the results of measuring the Cd content in the aerial part of the MORC6b-OE transgenic plant provided by the present invention. DETAILED DESCRIPTION

[0032] The present invention provides a gene for regulating the heavy metal content in the root and aboveground part of rice, the gene comprising any one or more of MORC6a, MORC6b and MORC6c, and the nucleotide sequences of the MORC6a, MORC6b and MORC6c genes are shown in SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively. In a specific embodiment, the heavy metal comprises cadmium. In the embodiment, it is confirmed that MORC6a, MORC6b and MORC6c play an important role in regulating the accumulation of cadmium in the root and aboveground part of rice, and a gene knockout mutant strain is constructed by gene editing technology, and a gene overexpression strain is also constructed by an overexpression vector. After being treated with 2 μM Cd for 10 days, the roots and aboveground parts of the treated rice seedlings were taken for Cd content determination. The results showed that the Cd content in the roots and aboveground parts of the gene knockout mutant strain was significantly lower than that of the wild type. The Cd content in the roots and aboveground parts of the overexpression strain was significantly higher than that of the wild type.

[0033] The present invention also provides the use of the gene described in the above technical solution in regulating the heavy metal content in rice roots and aerial parts or in cultivating rice germplasm related to cadmium content.

[0034] The specific embodiment of the present invention verifies that overexpression of the MORC6b gene can increase the cadmium content in the root and aerial part of rice, and can cultivate rice germplasm with high cadmium content. The present invention does not specifically limit the method of overexpression, and overexpression can be performed using a conventional overexpression vector in the art. Overexpression of the MORC6b gene can increase the cadmium content in the root and aerial part of rice. In a specific embodiment, the overexpression includes overexpression using the Ubiquitin promoter. In a specific embodiment, the reagent for overexpressing the MORC6b gene includes a vector for overexpressing the MORC6b gene. In a specific embodiment, overexpression is performed using the pCambia1390 vector, so that the expression amount of the MORC6b gene is significantly increased. When the overexpression technology is used, the restriction sites for gene insertion are Kpn I and BamHI. In a specific embodiment, a method for constructing a rice MORC6b gene overexpression vector comprises the following steps: (1) using Nipponbare cDNA as a template, performing PCR amplification using a primer pair to obtain a PCR product; the primer pair is MORC6b-Kpn IF and MORC6b-BamH IR, and the sequences are shown in SEQ ID No.21 and SEQID No.22; (2) Kpn I and BamHI are used to digest the pCambia1390 vector to obtain a digested vector; (3) the PCR product and the digested vector are homologously recombined to construct a recombinant vector. In a specific embodiment, a method for cultivating high-cadmium rice germplasm comprises the following steps: transforming the above-mentioned MORC6b gene overexpression vector into rice, performing tissue differentiation on the obtained resistant callus tissue, and obtaining the MORC6b overexpression transgenic material as the high-cadmium rice germplasm (MORC6b-OE). In a specific embodiment, the MORC6b gene overexpression vector is transformed into Agrobacterium AGL1, and the rice is transformed by Agrobacterium-mediated method, and the transgenic positive seedlings are detected by qRT-PCR. The overexpression strains are propagated, and the newly harvested plants are further screened for resistance, and homozygous overexpression materials are gradually obtained. The test results show that the MORC6b-OE transgenic rice is first hydroponically cultured under CK conditions for 4 days, and then rice seedlings with similar growth states are selected. After 10 days of 2μM Cd treatment, the rice seedlings are divided into two parts, the root and the aboveground part, which are used for the determination of Cd content in the seedling stage. The Cd content in the root and aboveground part of MORC6b-OE transgenic rice is significantly higher than that of the wild type, which shows that MORC6b plays an important role in regulating the accumulation of Cd in the root and aboveground part of rice.

[0035] The present invention also provides the use of knocking down or knocking out MORC6a, MORC6b and MORC6c genes in reducing the cadmium content in the root and aerial part of rice or in cultivating rice germplasm with low cadmium content. The present invention does not specifically limit the method of knocking down or knocking out, and the knocking down or knocking out can be performed using conventional gene editing methods in the art. In a specific embodiment, the knocking down or knocking out is performed using CRISPR / Cas9 gene editing technology, so that the expression amount of the MORC6a, MORC6b and MORC6c genes in the genome is reduced or no longer expressed. After the expression of the MORC6a, MORC6b and MORC6c genes, the cadmium content in the root and aerial part of rice is reduced.

[0036] The present invention also provides a method for reducing the cadmium content in the root and aboveground part of rice or cultivating rice germplasm with low cadmium content, comprising the following steps:

[0037] Knock down the expression of the MORC6a, MORC6b and MORC6c genes described in the above technical solution in rice or knock out the MORC6a, MORC6b and MORC6c genes.

[0038] In a specific embodiment, the knockdown or knockout method includes gene editing; the gene editing method includes CRISPR / Cas9 gene editing method. In a specific embodiment, when the knockdown or knockout is performed using the CRISPR / Cas9 gene editing method, the nucleotide sequences of the gene editing target are shown as SEQ ID No.4, SEQ ID No.5 and SEQ ID No.6, respectively. In a specific embodiment, the present invention uses a vector based on the CRISPR / Cas9 gene editing method to knock down or knock out MORC6a, MORC6b and MORC6c genes in rice to knock down or knock out MORC6a, MORC6b and MORC6c genes. After constructing a vector for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on the CRISPR / Cas9 gene editing method, the present invention transforms the vector into rice, and the obtained antibiotic-resistant callus is subjected to tissue differentiation. The obtained MORC6a, MORC6b and MORC6c three genes are simultaneously knocked out. The transgenic rice material (morc6abc) is the low-cadmium rice germplasm, which reduces the cadmium content in the root and aboveground part of the rice. In a specific embodiment, the MORC6a, MORC6b and MORC6c gene knockout binary vector is transformed into the Agrobacterium AGL1 strain, and the rice is transformed using the Agrobacterium-mediated method, and the transgenic positive seedlings are PCR sequenced. The homozygous plants are propagated, and the newly harvested plants are further PCR sequenced to obtain homozygous mutant plants. The results showed that the morc6abc rice mutant was first hydroponically cultured under CK conditions for 4 days, and then rice seedlings with similar growth status were selected. After 10 days of 2μM Cd treatment, the rice seedlings were divided into two parts, the root and the aboveground part, for the determination of Cd content in the seedling stage. The Cd content in the roots and aboveground parts of the morc6abc rice mutant was significantly lower than that of the wild type, indicating that MORC6a, MORC6b and MORC6c play an important role in regulating the accumulation of Cd in the roots and aboveground parts of rice.

[0039] The present invention also provides a method for constructing a vector for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on the CRISPR / Cas9 gene editing method, comprising the following steps:

[0040] (1) Using the pYLsgRNA-OsU3 vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, the third pair of primers were used for the second round of PCR amplification; using the second round of PCR amplification products as a template, the fourth pair of primers were used for the third round of PCR amplification to obtain the U3 promoter-driven sgRNA expression cassette; the first pair of primers were UF and MORC6a-OsU3T1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.8; the second pair of primers were MORC6a-gRT1 and gR-R, and the nucleotide sequences were shown in SEQ ID No.9 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GGL and Pgs-GG2, and the nucleotide sequences were shown in SEQ ID No.11 and SEQ ID No.12;

[0041] (2) Using the pYLsgRNA-OsU6c vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, the third pair of primers were used for the second round of PCR amplification; using the second round of PCR amplification products as a template, the fourth pair of primers were used for the third round of PCR amplification to obtain the U6c promoter-driven sgRNA expression cassette; the first pair of primers were UF and MORC6b-OsU6cT1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.13; the second pair of primers were MORC6b-gRT2 and gR-R, and the nucleotide sequences were shown in SEQ ID No.14 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GG2 and Pgs-GG3, and the nucleotide sequences were shown in SEQ ID No.15 and SEQ ID No.16;

[0042] (3) Using the pYLsgRNA-OsU6a vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, the third pair of primers were used for the second round of PCR amplification; using the second round of PCR amplification products as a template, the fourth pair of primers were used for the third round of PCR amplification to obtain the U6a promoter-driven sgRNA expression cassette; the first pair of primers were UF and Morc6c-OsU6aT1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.17; the second pair of primers were Morc6c-gRT3 and gR-R, and the nucleotide sequences were shown in SEQ ID No.18 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GG3 and Pgs-GGR, and the nucleotide sequences were shown in SEQ ID No.19 and SEQ ID No.20;

[0043] (4) The U3 promoter-driven sgRNA expression cassette (PYLsgRNA-OsU3 expression cassette), the U6c promoter-driven sgRNA expression cassette (PYLsgRNA-OsU6c expression cassette), and the U6a promoter-driven sgRNA expression cassette (PYLsgRNA-OsU6a expression cassette) were digested with BsaⅠ and then ligated to the pYLCRISPR / Cas9Pubi-H vector by T4 to obtain a binary vector for simultaneous knockout of the three genes MORC6a, MORC6b, and MORC6c.

[0044] The present invention preferably uses CRISPR / Cas9 gene editing technology to edit the coding region DNA sequences of rice MORC6a, MORC6b and MORC6c: First, use CRISPR-GE (http: / / skl.scau.edu.cn / ) to design the editing targets of MORC6a, MORC6b and MORC6c online, respectively, and select the optimal target sequence for primer design; secondly, construct the PYLsgRNA-OsU3 expression cassette, PYLsgRNA-OsU6c expression cassette and PYLsgRNA-OsU6a expression cassette containing MORC6a, MORC6b and MORC6c genes by Overlapping PCR; then, use BsaⅠ endonuclease and T4 DNA ligase to clone the above sgRNA expression cassette into the Cas9 vector by cutting and ligating at the same time, and finally construct the MORC6a, MORC6b and MORC6c gene knockout binary vectors.

[0045] The present invention also provides a vector for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on the CRISPR / Cas9 gene editing method, which is constructed by the construction method described in the above technical scheme.

[0046] To further illustrate the present invention, the method and application of MORC6s for regulating heavy metal content in rice provided by the present invention are described in detail below in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods; the materials and reagents used are all reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0048] in:

[0049] The method steps for determining the Cd content of brown rice of MORC6a, MORC6b and MORC6c knockout transgenic rice plants (morc6abc) and MORC6b-OE transgenic rice plants under low-cadmium hydroponics are as follows:

[0050] The wild-type, morc6abc mutant, and MORC6b-OE transgenic rice plants were first cultured in CK conditions (1 / 2 Yoshida nutrient solution (Coolaber, Yoshida nutrient solution, catalog number: NSP1040, prepared by dissolving dry powder)) for 4 days, and then the wild-type, morc6 mutant, and MORC6b-OE transgenic rice plants with the same growth potential were simultaneously transferred to 2 μM CdCl 2 In hydroponic solution. After 10 days of treatment, the roots and aerial parts of wild-type, morc6 mutant and MORC6b-OE transgenic rice plants were dried separately. After weighing the dry weight of each group of samples, they were placed in a 20 mL test tube, added with 1-2 mL nitric acid and placed in a constant temperature metal bath, and nitrolyzed at 120°C for 4 h. Then, the acid was evaporated at 140°C for about 2 h until the remaining liquid was less than 1 mL. After cooling, ultrapure water was added to dilute to 20 mL. 10 mL was drawn from it with a pipette tip and placed in a 15 mL conical centrifuge tube for measurement. The Cd and Mn element contents in the samples were determined using an ICP / MS coupled plasma mass spectrometer (PerkinElmer, USA).

[0051] Example 1

[0052] MORC6a, MORC6b and MORC6c were induced by 10 μM CdCl 2 Expression levels in wild-type rice after treatment for different time periods

[0053] 1. CdCl in Rice Seedlings 2 deal with

[0054] The germinated wild-type rice was cultured in 1 / 2 Yoshida nutrient solution for 7 days, and then the seedlings with the same growth were selected and continued to be cultured in 1 / 2 Yoshida nutrient solution and 1 / 2 Yoshida nutrient solution + 10 μM CdCl 2 In the hydroponic solution, the roots of the above materials were sampled for RNA extraction at 12 h, 24 h, and 48 h after treatment.

[0055] 2. RNA Extraction

[0056] ① Take root samples of rice seedlings, quickly place them in a 2 mL RNA-free centrifuge tube filled with steel beads, and immerse them in liquid nitrogen;

[0057] ② Place the metal block of the grinder in liquid nitrogen and freeze it to a constant temperature. Then place the sample tube symmetrically in the hole of the metal block, tighten the screw, and grind at 60 Hz for 60 s.

[0058] ③ Quickly add 1 mL of RNAiso Plus (Trizol) to each sample tube, shake and mix immediately, and place at room temperature for 5 min;

[0059] ④ Add 200 μL of chloroform to the sample tube, mix well and place at room temperature for 5 min;

[0060] ⑤ Centrifuge at 12,000 rpm for 15 min in a 4°C low-temperature centrifuge, take 400 μL of the supernatant and add it to a new RNA-free 1.5 mL centrifuge tube, then add 400 μL of isopropanol, gently turn it upside down and place it at -20°C overnight or at room temperature for 20 min;

[0061] ⑥ Centrifuge at 12,000 rpm for 10 to 15 min at 4°C and discard the supernatant;

[0062] ⑦ Add 1 mL of 75% ethanol to wash the precipitate, centrifuge at 12,000 rpm and 4°C for 5 min, and discard the supernatant;

[0063] ⑧Repeat step ⑦;

[0064] ⑨ Centrifuge the tube again at 12,000 rpm and 4°C for 1 min, then aspirate the remaining liquid in the tube. Open the tube cap and let it dry at room temperature until the white flaky RNA turns transparent. Immediately add 30 μL RNase-free water, place on ice to dissolve, and then use it immediately for reverse transcription or store it at -80°C for a long time.

[0065] 2. Reverse transcription of RNA

[0066] Reaction 1: 5 μL system: RNA no more than 5 ng, oligo d(T) 1 μL, DEPC H 2 O complement.

[0067] Mix the above reaction system and react at 70℃ for 5 minutes, then immediately place on ice for at least 5 minutes. Then add 15μL of the system: GoScript TM 5×Reaction Buffer 4μL, MgCl 2 2μL, PCR Nucleotide Mix 1μL, Recombinant RNase Ribonuclease Inhibitor 0.5μL, GoScript TM ReverseTranscriptase 1μL, Nuclease-Free Water 6.5μL and 5μL reaction system.

[0068] After mixing the above reaction system, carry out the following reaction: 25℃ for 5 min, 42℃ for 90 min, 70℃ for 5 min, and 4℃ for 60 min.

[0069] The product of reverse transcription is cDNA, which can be stored at -20°C for a long time.

[0070] 3. Fluorescence quantitative PCR

[0071] The PowerUpTM SYBP Green Mix (applied biosystems) kit was used for quantitative PCR experiments.

[0072] The cDNAs system obtained by reverse transcription was diluted 4 times and then the quantitative PCR system (15 μL) was prepared: SYBP Green Mix 7.5 μL, forward primer 0.3 μL, reverse primer 0.3 μL, cDNA 1.5 μL and RNase-free H 2 O 5.4 μL.

[0073] The reverse transcribed cDNA was used as a template, qGAPDH-F (as shown in SEQ ID No. 29) was used as a forward primer, and qGAPDH-R (as shown in SEQ ID No. 30) was used as a reverse primer, which were used as internal reference expression levels.

[0074] Quantitative PCR was performed using the reverse transcribed cDNA as a template (the template amount was determined by the adjusted amount), qMORC6b-F (as shown in SEQ ID No. 31) as a forward primer, and qMORC6b-R (as shown in SEQ ID No. 32) as a reverse primer.

[0075] The results are as follows Figure 1 , Figure 2 and Figure 3 As shown in the figure, compared with no treatment, 10 μM Cd treatment strongly induced the expression of MORC6a, MORC6b, and MORC6c genes. Therefore, MORC6a, MORC6b, and MORC6c may play a role in regulating the Cd absorption of rice, but the specific regulation method is still unclear.

[0076] Example 2

[0077] Construction of CRISPR / Cas9 gene knockout vectors for rice MORC6a, MORC6b and MORC6c

[0078] The construction of this vector mainly consists of three rounds of PCR amplification and cutting and ligation. The PCR procedure and system are as follows:

[0079] (1) Using pYLsgRNA-OsU3 vector (Plasmid#66193 in Addgene) as template ( Figure 4 A), respectively using UF (as shown in SEQ ID No.7) and MORC6a-OsU3T1 (as shown in SEQ ID No.8), and MORC6a-gRT1 (as shown in SEQ ID No.9) and gR-R (as shown in SEQ ID No.10) for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, using UF (as shown in SEQ ID No.7) and gR-R (as shown in SEQ ID No.10) for the second round of PCR amplification; using the second round of PCR amplification products as a template, using Pps-GGL (as shown in SEQ ID No.11) and Pgs-GG2 (as shown in SEQ ID No.12) for the third round of PCR amplification, to obtain a U3 promoter-driven sgRNA expression cassette, which can be used to finally construct a 3-target tandem sgRNA expression cassette;

[0080] (2) Using pYLsgRNA-OsU6c vector (Plasmid#66197inAddgene) as template ( Figure 4A), using UF (as shown in SEQ ID No.7) and MORC6b-OsU6cT1 (as shown in SEQ ID No.13), MORC6b-gRT2 (as shown in SEQ ID No.14) and gR-R (as shown in SEQ ID No.10) for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, using UF (as shown in SEQ ID No.7) and gR-R (as shown in SEQ ID No.10) for the second round of PCR amplification; using the second round of PCR amplification products as a template, using Pps-GG2 (as shown in SEQ ID No.15) and Pgs-GG3 (as shown in SEQ ID No.16) for the third round of PCR amplification, a U6c promoter-driven sgRNA expression cassette is obtained, which can be used to finally construct a 3-target tandem sgRNA expression cassette;

[0081] (3) Using pYLsgRNA-OsU6a vector (Plasmid#66194 in Addgene) as template ( Figure 4 A), using UF (as shown in SEQ ID No.7) and Morc6c-OsU6aT1 (as shown in SEQ ID No.17), MORC6c-gRT3 (as shown in SEQ ID No.18) and gR-R (as shown in SEQ ID No.10) for the first round of PCR amplification; using the mixture of the first round of PCR amplification products as a template, using UF (as shown in SEQ ID No.7) and gR-R (as shown in SEQ ID No.10) for the second round of PCR amplification; using the second round of PCR amplification products as a template, using Pps-GG3 (as shown in SEQ ID No.19) and Pgs-GGR (as shown in SEQ ID No.20) for the third round of PCR amplification, a U6a promoter-driven sgRNA expression cassette is obtained, which can be used to finally construct a 3-target tandem sgRNA expression cassette;

[0082] (4) The U3 promoter-driven sgRNA expression cassette, the U6c promoter-driven sgRNA expression cassette, and the U6a promoter-driven sgRNA expression cassette were digested with BsaⅠ and then ligated to the pYLCRISPR / Cas9Pubi-H vector (Plasmid#66187inAddgene) by T4. Figure 4 In B), the MORC6a, MORC6b and MORC6c gene knockout binary vectors were obtained.

[0083] PCR amplification program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 38 cycles; extension at 72°C for another 10 min; storage at 8°C.

[0084] Cutting and ligating: First perform BsaⅠ enzyme digestion.

[0085] Enzyme digestion program: 37°C, 15 min.

[0086] Enzyme digestion system: 10×Cutsmart Buffer 1.5μL, U3 promoter-driven sgRNA expression cassette 2μL, U6c promoter-driven sgRNA expression cassette 2μL, U6a promoter-driven sgRNA expression cassette 2μL, pYLCRISPR / Cas9 Pubi-H 2μL, BsaⅠ 1μL and ddH 2 O 0.5 μL.

[0087] After enzyme digestion, add 1 μL T4 ligase, 1.5 μL 10×T4 DNA Ligase Buffer and 1.5 μL ddHO to the above system. 2 O performs cutting and connecting at the same time, the procedure is as follows:

[0088] PCR reaction program: 37°C for 5 min, 10°C for 5 min, 20°C for 5 min, 15 cycles; stored at 4°C.

[0089] Example 3

[0090] Construction of rice MORC6b-OE vector

[0091] The vector construction mainly includes vector restriction enzyme digestion, fragment amplification and homologous recombination. The restriction enzyme digestion system and procedure, PCR system and procedure, and homologous recombination system and procedure are as follows:

[0092] (1) Vector digestion:

[0093] pCambia1390 vector ( Figure 5 , purchased from Cambia) was digested with Kpn I and BamHI enzymes.

[0094] Enzyme digestion system: 10×Buffer 4μL, pCambia1390 empty load 30μL, BamHI 1μL, Kpn I

[0095] 1 μL ddHO 2 2. 4 μL of HO.

[0096] Enzyme digestion procedure: 37℃ water bath digestion for 3h.

[0097] (2) MORC6b CDS sequence amplification:

[0098] Nipponbare cDNA was used as a template and MORC6b-Kpn IF (as shown in SEQ ID No. 21) and MORC6b-BamH IR (as shown in SEQ ID No. 22) were used as primers for amplification.

[0099] Amplification system: 2×Premix 10μL, cDNA template 2μL, MORC6b-Kpn IF 0.75μL, MORC6b-BamHI-R 0.75μL and ddH 2 O 6.5 μL.

[0100] PCR amplification program: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 20 s, annealing at 56°C for 30 s, extension at 72°C for 1 min, 38 cycles; extension at 72°C for another 10 min; storage at 8°C.

[0101] (3) Homologous recombination

[0102] The pCambia1390 vector after restriction digestion and the amplified MORC6b CDS sequence were homologously recombined.

[0103] Recombination system: 1.5 μL of pCambia1390 vector after restriction digestion, 1 μL of MORC6b CDS amplification product and 2.5 μL of 2×HieffClone Buffer.

[0104] The above recombinant vector was transformed into DH5α, and single clones were selected for sequencing verification to obtain the correctly connected pCambia1390+MORC 6b CDS recombinant vector.

[0105] Example 4

[0106] Genetic transformation of MORC6a, MORC6b and MORC6c knockout rice materials and MORC6b overexpression rice materials

[0107] The MORC6a, MORC6b and MORC6c gene knockout binary vectors constructed in Example 2 and the MORC6b overexpression vector constructed in Example 3 were respectively transformed into Agrobacterium tumefaciens AGL1, and rice was transformed using the Agrobacterium-mediated method to obtain hygromycin-resistant callus tissue, and tissue differentiation was performed to obtain transgenic rice materials with simultaneous knockout of MORC6a, MORC6b and MORC6c genes and MORC6b overexpression rice materials, respectively.

[0108] The steps of Agrobacterium-mediated transformation of rice are as follows: Figure 6 As shown:

[0109] (1) Preparation of Agrobacterium culture solution

[0110] Pick a single colony of Agrobacterium on the clean bench and inoculate it into 3mL LB liquid medium (containing 50mg / L rifampicin and 50mg / L kanamycin). Shake the culture overnight at 28℃ and 200r / min on a shaker until the culture liquid becomes turbid. Take 1mL of the culture liquid and spread it on LB solid medium (containing 50mg / L rifampicin and 50mg / L kanamycin), and place it in a 28℃ incubator for 1 to 2 days. After the Agrobacterium has grown all over the LB solid medium, scrape the bacteria with a sterilized pipette tip, activate it with activation medium, and then adjust the OD 600 Adjust it to 0.4-0.6 and it can be used for infection.

[0111] The activation medium (AAM) formula is as follows:

[0112] Chu's N6 Basal Medium with Vitamins 4.10g / L+Glycine 2mg / L+Tyrosine 500mg / L+Glucose 36g / L+Sucrose 68.5g / L+Acetosyringone 200μmol / L, pH=5.2.

[0113] (2) Induction and transformation of rice callus

[0114] First, the rice seeds were shelled and the seeds with full grains and no disease spots were selected. The seeds were placed in a sterilized tube in a clean bench, and soaked in 75% alcohol for 2 minutes, with constant shaking; then, the alcohol was discarded, one side was washed with sterile water, and then soaked in 1% NaClO solution for 30 minutes, with sufficient shaking; then rinsed with sterile water 4 times, and the sterilized seeds were placed on sterilized paper to dry for later use; the sterilized seeds were evenly placed on N6 culture medium, and cultured in light at 28°C for 10 days until the rice induced golden granular callus, and then peeled off with tweezers to a new culture medium, and the callus after one week of subculture could be used for Agrobacterium infection.

[0115] The formula of N6 (NBD) is as follows:

[0116] Chu's N6 Basal Medium with Vitamins 4.10g / L+Proline 500mg / L+Glutamine 500mg / L+Tyrosine 300mg / L+Sucrose 30g / L+2,4-D 2.5mg / L, pH=5.8, Phytagel 3g / L.

[0117] 3) Agrobacterium transformation of rice callus and co-cultivation process

[0118] Select yellow and hard callus, place it in the activated culture medium with bacterial liquid, shake it continuously for 5 minutes, and then let it stand for 30 minutes. Pour off the bacterial liquid, place the callus on sterilized paper to drain the bacterial liquid, and then transfer it to the solid co-culture medium and culture it at 28℃ in the dark for 2 days.

[0119] The formula of co-culture medium (NBDC) is as follows:

[0120] Chu's N6 Basal Medium with Vitamins 4.10g / L+Glycine 2mg / L+2.4-D 2.5mg / L+Sucrose 20g / L+Mannitol 36.43g / L+Glucose 10g / L+Tyrosine 0.5g / L+Acetosyringone 200μmol / L, pH=5.8, Phytagel 3g / L.

[0121] 4) Screening and subculture of resistant callus

[0122] The callus with clean surface and no large area of ​​Agrobacterium contamination is transferred to the screening medium for the first screening, and then screened once a week until resistant callus grows (newly grown bright yellow callus around brown callus tissue). The resistant callus is peeled off to the new screening medium, and the callus peeled off from one callus is a strain. After another two weeks of screening, it can be used for callus regeneration.

[0123] The formulation of screening medium (NBDS) is as follows:

[0124] Chu's N6 Basal Medium with Vitamins 4.10g / L+Tyrosine 0.3g / L+Proline 0.5g / L+Glycine 2mg / L+2,4-D 2.5mg / L+Timentin 300mg / L+Hyg 50mg / L+Sucrose 30g / L, pH=5.8, Phytagel 3g / L.

[0125] 5) Regeneration of resistant callus

[0126] The screened callus tissue is transferred to a pre-differentiation medium for further culture. One month later, after dense callus grows, it is transferred to a differentiation medium for light culture. When the callus turns green and differentiates into seedlings, it is moved to a rooting medium.

[0127] The formula of pre-differentiation medium (MS-PG) is as follows:

[0128] MS 4.43g / L+6-BA 2mg / L+ABA 5mg / L+NAA 1mg / L+sucrose 30g / L+sorbitol 20g / L+Hyg 50mg / L, pH=5.8, Phytagel 3g / L.

[0129] Differentiation medium (MS-RG)

[0130] MS 4.43g / L+6-BA2mg / L+NAA 0.2mg / L+sorbitol 20g / L+sucrose 30g / L+Hyg 50mg / L, pH=5.8, Phytagel 3g / L.

[0131] The rooting medium (MS-RT) formula is as follows:

[0132] 1 / 2MS 2.215g / L+sucrose 20g / L+Hyg 50mg / L+Phytagel 2.5g / L, pH=5.8.

[0133] There are five main stages in the Agrobacterium-mediated transformation of rice, namely, the induction of rice callus ( Figure 6 A) Screening of rice resistant callus ( Figure 6 B) Pre-differentiation of rice resistant callus ( Figure 6 C) Differentiation of rice resistant callus ( Figure 6 D) and rooting ( Figure 6 E) The desired transgenic rice seedlings are obtained through the above genetic transformation process.

[0134] Example 5

[0135] Identification of mutation sites in transgenic plants with simultaneous knockout of MORC6a, MORC6b and MORC6c genes

[0136] The mutation sites of the screened positive transgenic plants were identified. Using the genomic DNA of the positive plants as templates, MORC6a-test-F (as shown in SEQ ID No. 23) and MORC6a-test-R (as shown in SEQ ID No. 24), MORC6b-test-F (as shown in SEQ ID No. 25) and MORC6b-test-R (as shown in SEQ ID No. 26), MORC6c-test-F (as shown in SEQ ID No. 27) and Morc6c-test-R (as shown in SEQ ID No. 28), the PCR system and procedure were as follows:

[0137] (1) PCR system: 2×Taq Buffer 10μL, DNA 2μL, MORC6a-test-F (or MORC6b-test-F or Morc6c-test-F) 0.75μL, MORC6a-test-R (or MORC6b-test-R or Morc6c-test-R) 0.75μL and ddHO2 O 6.5 μL.

[0138] (2) PCR amplification procedure: pre-denaturation at 95°C for 2 min; denaturation at 94°C for 20 s, annealing at 58°C for 30 s, extension at 72°C for 15 s, 38 cycles; extension at 72°C for another 10 min; storage at 8°C.

[0139] Finally, the PCR products were sequenced, the sequencing peaks were analyzed, and the mutations were counted.

[0140] The results are as follows Figure 7 As shown, compared with the DNA sequence of the wild-type WT, the MORC6a site in the morc6abc#1 mutant has a 1 bp insertion, the MORC6b site has a 1 bp insertion, and the Morc6c site has a 1 bp insertion; the MORC6a site in the morc6abc#2 mutant has a 5 bp deletion, the MORC6b site has a 1 bp insertion, and the Morc6c site has a 1 bp insertion.

[0141] Example 6

[0142] Detection of overexpression folds in MORC6b overexpressing plants

[0143] The overexpression multiples of the positive transgenic plants screened were detected. Using the cDNA of Nipponbare and positive plants with similar plant growth as templates, qRT-PCR was performed using qGAPDH-F (as shown in SEQ ID No.29) and qGAPDH-R (as shown in SEQ ID No.30), qMORC6b-F (as shown in SEQ ID No.31) and qMORC6b-R (as shown in SEQ ID No.32) as primer pairs. The results are shown in Figure 2. Figure 8 As shown, the overexpression folds of MORC6b-OE#1 and MORC6b-OE#2 strains were approximately 7000-9000 times.

[0144] Example 7

[0145] Determination of Cd content in roots and aerial parts of MORC6a, MORC6b and MORC6c knockout transgenic plants

[0146] Stably inherited 4-day-old MORC6a, MORC6b, and MORC6c knockout transgenic plants and wild-type rice were treated with 2 μM CdCl 2 After 10 days of treatment, the roots and aerial parts were dried separately and the Cd content in the seedling stage was determined.

[0147] The digestion process is as follows:

[0148] 1) Weigh the roots or aerial parts into a digestion tube, add 2 mL of concentrated nitric acid and place in a fume hood overnight;

[0149] 2) Adjust the temperature of the digestion furnace to 100℃, shake it slightly, and wait until the tissue shape disappears and becomes liquid. Then raise the temperature to about 120℃ and heat for 2 hours until it is completely liquid;

[0150] 3) Heat up to 140℃ again to drain the acid, leaving about 1mL of liquid in the digestion tube;

[0151] 4) After cooling, adjust the volume, dilute and measure.

[0152] The results are as follows Fig. 9 and Fig.10 As shown in Figure 2, compared with the wild type, the Cd content in the roots of the morc6 mutant was significantly decreased ( Fig. 9 ), and the Cd content in the aboveground part also decreased significantly ( Fig.10 ), which indicates that the morc6 mutant has important significance and value in breeding rice germplasm with low Cd absorption.

[0153] Example 8

[0154] Determination of Cd content in roots and aerial parts of MORC6b-OE transgenic plants

[0155] Stably inherited 4-day-old MORC6b-OE transgenic plants and wild-type rice were treated with 2 μM CdCl 2 After 10 days of treatment, the roots and aerial parts were dried separately and the Cd content in the seedling stage was determined.

[0156] The digestion process is as follows:

[0157] 1) Weigh the roots or aerial parts into a digestion tube, add 2 mL of concentrated nitric acid and place in a fume hood overnight;

[0158] 2) Adjust the temperature of the digestion furnace to 100℃, shake it slightly, and wait until the tissue shape disappears and becomes liquid. Then raise the temperature to about 120℃ and heat for 2 hours until it is completely liquid;

[0159] 3) Heat up to 140℃ again to drain the acid, leaving about 1mL of liquid in the digestion tube;

[0160] 4) After cooling, adjust the volume, dilute and measure.

[0161] The results are as follows Fig.11 and Fig.12 As shown in Figure 2, compared with the wild type, the Cd content in the roots of MORC6b-OE transgenic plants was significantly increased ( Fig.11 ), and the Cd content in the aboveground part also increased significantly ( Fig.12 ).

[0162] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A gene for regulating the heavy metal content in the root and aerial parts of rice, characterized in that: The gene includes any one or more of MORC6a, MORC6b and MORC6c, and the nucleotide sequences of the MORC6a, MORC6b and MORC6c genes are shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.

2. The gene according to claim 1, characterized in that The heavy metals include cadmium.

3. Use of the gene according to claim 1 or 2 in regulating the heavy metal content in rice roots and aerial parts or in cultivating rice germplasm related to cadmium content.

4. Application of knocking down or knocking out MORC6a, MORC6b and MORC6c genes in reducing the cadmium content in rice roots and aboveground parts or in cultivating rice germplasm with low cadmium content.

5. A method for reducing the cadmium content in the root and aboveground part of rice or cultivating rice germplasm with low cadmium content, characterized in that: The following steps are involved: Knock down the expression of the MORC6a, MORC6b and MORC6c genes according to claim 1 or 2 in rice or knock out the MORC6a, MORC6b and MORC6c genes.

6. The method according to claim 5, characterized in that The knockdown or knockout method includes gene editing; the gene editing method includes CRISPR / Cas9 gene editing method.

7. The method according to claim 6, characterized in that When the CRISPR / Cas9 gene editing method is used for the knockdown or knockout, the nucleotide sequences of the gene editing target are shown as SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6, respectively.

8. A method for constructing a vector for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on CRISPR / Cas9 gene editing method, characterized in that: The following steps are involved: (1) Using the pYLsgRNA-OsU3 vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; Using the mixture of the first-round PCR amplification products as a template, a second-round PCR amplification was performed using the third pair of primers; The product of the second round of PCR amplification was used as a template, and the third round of PCR amplification was performed using the fourth pair of primers to obtain a U3 promoter-driven sgRNA expression cassette; the first pair of primers were UF and MORC6a-OsU3T1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.8; the second pair of primers were MORC6a-gRT1 and gR-R, and the nucleotide sequences were shown in SEQ ID No.9 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GGL and Pgs-GG2, and the nucleotide sequences were shown in SEQ ID No.11 and SEQ ID No.12; (2) Using the pYLsgRNA-OsU6c vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; Using the mixture of the first-round PCR amplification products as a template, a second-round PCR amplification was performed using the third pair of primers; The product of the second round of PCR amplification was used as a template, and the third round of PCR amplification was performed using the fourth pair of primers to obtain the U6c promoter-driven sgRNA expression cassette; the first pair of primers were UF and MORC6b-OsU6cT1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.13; the second pair of primers were MORC6b-gRT2 and gR-R, and the nucleotide sequences were shown in SEQ ID No.14 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GG2 and Pgs-GG3, and the nucleotide sequences were shown in SEQ ID No.15 and SEQ ID No.16; (3) Using the pYLsgRNA-OsU6a vector as a template, the first pair of primers and the second pair of primers were used for the first round of PCR amplification; Using the mixture of the first-round PCR amplification products as a template, a second-round PCR amplification was performed using the third pair of primers; The product of the second round of PCR amplification was used as a template, and the third round of PCR amplification was performed using the fourth pair of primers to obtain the U6a promoter-driven sgRNA expression cassette; the first pair of primers were UF and Morc6c-OsU6aT1, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.17; the second pair of primers were Morc6c-gRT3 and gR-R, and the nucleotide sequences were shown in SEQ ID No.18 and SEQ ID No.10; the third pair of primers were UF and gR-R, and the nucleotide sequences were shown in SEQ ID No.7 and SEQ ID No.10; the fourth pair of primers were Pps-GG3 and Pgs-GGR, and the nucleotide sequences were shown in SEQ ID No.19 and SEQ ID No.20; (4) The U3 promoter-driven sgRNA expression cassette, the U6c promoter-driven sgRNA expression cassette, and the U6a promoter-driven sgRNA expression cassette were digested with BsaⅠ and then ligated to the pYLCRISPR / Cas9Pubi-H vector by T4 to obtain a binary vector for simultaneously knocking out the three genes MORC6a, MORC6b, and MORC6c.

9. The vector constructed by the construction method of claim 8 for knocking down or knocking out MORC6a, MORC6b and MORC6c genes based on the CRISPR / Cas9 gene editing method.

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