Application of rice OsNAC42 gene in regulating cadmium accumulation amount in grains, sgRNA, recombinant vector and their applications

Abstract

The present invention relates to the field of genetic engineering technology, in particular to the application of rice OsNAC42 gene in regulating the cadmium accumulation amount in grains, sgRNA, recombinant vectors and their applications. The present invention measures the cadmium content in grains of CRISPR mutant library rice planted on Cd-polluted farmland, and screens a mutant osnac42 with significantly lower cadmium content in grains than that of the wild-type Nipponbare nip. Further, a new mutant osnac42 with a loss-of-function of this gene is created by CRISPR / Cas9 gene editing technology and planted together with the wild-type Nipponbare nip in moderately and lightly Cd-polluted farmland, and it is found that the cadmium content in grains of the osnac42 mutant is significantly lower than that of the wild-type, but the agronomic traits related to yield are basically not affected. The OsNAC42 gene can not only be used to improve the regulatory network of rice under Cd stress, but also provides a new idea for the breeding of rice varieties with low Cd accumulation.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly to the application of rice OsNAC42 gene in regulating the cadmium accumulation amount in grains, sgRNA, recombinant vectors and their applications. Background Art

[0002] Cd is a non-essential element for the human body. It often exists in the form of compounds in nature and generally has a very low content. However, due to the development of modern industry, the Cd pollution in the atmosphere and water bodies has become increasingly serious, resulting in the accumulation of Cd content in farmland soil. The Cd pollution of cultivated land poses a major challenge to the safe production of crops.

[0003] Compared with other crops, rice is prone to absorb Cd elements from the soil during growth, and finally accumulates in the grains after being transported through the above-ground parts. After consuming rice containing Cd, most of the Cd will be absorbed and accumulated in the body to produce toxicity, endangering human health, which poses a huge threat to food security. Therefore, achieving the safe production of rice plays a crucial role in food security in China and even the world. Among them, the breeding of rice varieties with low Cd accumulation is the most economical and feasible method to solve the Cd pollution of rice. Finding genes that can control the low Cd accumulation in grains without affecting the yield and clarifying their functions have important theoretical and practical significance. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides the application of rice OsNAC42 gene in regulating the cadmium accumulation amount in grains, sgRNA, recombinant vectors and their applications. The present invention determines that knocking out the rice OsNAC42 gene can not only reduce the Cd accumulation in grains, but also has basically no effect on agronomic traits such as yield, providing an opportunity for the breeding of rice varieties with low Cd accumulation, and providing gene resources and technical support for cultivating highly Cd-tolerant plants / crops.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of rice OsNAC42 gene in regulating the cadmium accumulation amount in grains, and the amino acid sequence of the protein encoded by the OsNAC42 gene is as shown in SEQ ID NO.1.

[0007] Preferably, the regulation of the cadmium accumulation amount in grains includes: reducing the cadmium accumulation amount in grains by negatively regulating the expression of the OsNAC42 gene.

[0008] Preferably, the nucleotide sequence of the rice OsNAC42 gene is as shown in SEQ ID NO.2.

[0009] The present invention also provides an sgRNA targeting the rice OsNAC42 gene, and the sgRNA includes sgRNA-S and sgRNA-A; the nucleotide sequence of sgRNA-S is as shown in SEQ ID No.3; the nucleotide sequence of sgRNA-A is as shown in SEQ ID No.4.

[0010] The present invention also provides a recombinant vector for knocking out the rice OsNAC42 gene, and the recombinant vector includes the sgRNA described in the above technical solution and a basic vector.

[0011] Preferably, the basic vector includes the VK005-01 vector.

[0012] The present invention also provides the application of the sgRNA or recombinant vector described in the above technical solution in reducing the cadmium accumulation amount in rice grains.

[0013] The present invention also provides the application of the sgRNA or recombinant vector described in the above technical solution in cultivating cadmium-tolerant rice.

[0014] Preferably, the traits of the cadmium-tolerant rice include: the cadmium content in grains is lower than that of the wild type, and other agronomic traits are equivalent to those of the wild type.

[0015] Preferably, the other agronomic traits include one or more of plant height, grain size, tiller number, and panicle shape.

[0016] Beneficial effects:

[0017] The present invention provides the application of the rice OsNAC42 gene in regulating the cadmium accumulation amount in grains, and the amino acid sequence of the protein encoded by the OsNAC42 gene is as shown in SEQ ID NO.1. By measuring the cadmium content in grains of the CRISPR mutant library rice planted on moderately and lightly Cd-polluted farmland, the present invention screened a mutant osnac42 with a significantly lower cadmium content in grains than that of the wild type Nipponbare (Oryza sativa). Further, a mutant osnac42 with a loss-of-function of the gene OsNAC42 (LOC_Os09g32040) was created by the CRISPR / Cas9 gene editing technology and planted together with the wild type Nipponbare on moderately and lightly Cd-polluted farmland. It was found that the cadmium content in grains of the OsNAC42 mutant was significantly lower than that of the wild type, but the agronomic traits related to yield were basically not affected. The OsNAC42 gene can not only be used to improve the regulatory network of rice under Cd stress, but also provides a new idea for the breeding of rice varieties with low Cd accumulation. Description of the drawings

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for use in the embodiments.

[0019] Figure 1 It is the determination result of the cadmium content in the grains of wild-type rice nip, mutant materials osnac42-1 and osnac42-2;

[0020] Figure 2 It is the comparison diagram of the appearance and tiller number of wild-type rice nip, mutant materials osnac42-1 and osnac42-2 at the mature stage;

[0021] Figure 3 It is the comparison diagram of the plant height of wild-type rice nip, mutant materials osnac42-1 and osnac42-2 at the mature stage;

[0022] Figure 4 It is the comparison diagram of the yield of wild-type rice nip, mutant materials osnac42-1 and osnac42-2 at the mature stage;

[0023] Figure 5 It is the comparison diagram of the 1000-grain weight of wild-type rice nip, mutant materials osnac42-1 and osnac42-2 at the mature stage;

[0024] Figure 6 It is the comparison diagram of the panicle shape of wild-type rice nip, mutant materials osnac42-1 and osnac42-2 at the mature stage;

[0025] Figure 7 It is the comparison diagram of the grain size of wild-type rice nip, mutant materials osnac42-1 and osnac42-2 at the mature stage. Specific embodiments

[0026] The present invention provides the application of rice OsNAC42 gene in regulating the cadmium accumulation amount in grains. The amino acid sequence of the protein encoded by the OsNAC42 gene is as shown in SEQ ID NO.1, specifically as follows:

[0027] MGMNRWGRSTDVYKRRIATGRTYIADVDVYKSHSHRSARTGDKWSRMDRKYNGSRASRTTGGYWKATGKDRSICNGGGGGTASGRAVGSKKTVYHHGRARGRSDWVMHYTADAAARDRAYAYKHKSGAGKNGYGARDWDDDDHHHDAAAAATTSGRAATTHADGGDDVAINDDIIADSTHVTSVIHRVHGWSDDGGKSDVADATTSGSAMANTCAIDGMISDDTVMSGSASVSHDYHGCGVHRVGVADSTTVSSAVVTCTVRDIGMINDNASDSTVHHDCHAAGDGSRATNIANSTMVSNDTGSNITSITTMNGTNAVTSRSMSVGSYDRDGDDINDDDIGSTRSNIADDGVDSYSDAMGSDRTGVVANHYVGASGINGHTTAHNVANVRNHMKDNHVVSHSSDATIIHTVNNRSSNASSWNGASADSVSSAMAANIGNRTRISSRSARVSSTINTRRRGGGIISMVVAIMWTSNGSAVKSKGWKST。

[0028]

[0029] In the present invention, the regulation of cadmium accumulation in grains preferably includes: reducing the cadmium accumulation in grains by negatively regulating the expression of the OsNAC42 gene.

[0030] The present invention has determined that the grain Cd content of mutants with loss-of-function of the OsNAC42 gene is significantly decreased compared with that of wild-type plants, but the agronomic traits such as yield are equivalent to those of wild-type plants, indicating that the OsNAC42 gene is a key gene that can not only control low Cd accumulation in grains but also has no effect on yield; the OsNAC42 gene can provide gene resources and technical support for the breeding of rice varieties with low Cd accumulation.

[0031] The present invention provides an opportunity for the breeding of rice varieties with low Cd accumulation, and provides gene resources and technical support for the cultivation of highly Cd-tolerant plants / crops.

[0032] The present invention also provides an sgRNA targeting the rice OsNAC42 gene, and the sgRNA includes sgRNA-S and sgRNA-A;

[0033] The nucleotide sequence of the sgRNA-S is shown in SEQ ID No.3, specifically: 5’-GGATGGAGAACCCGCCGCTCCGG-3’;

[0034] The nucleotide sequence of the sgRNA-A is shown in SEQ ID No.4, specifically: 5’-CCGGAGCGGCGGGTTCTCCATCC-3’.

[0035] The sgRNA provided by the present invention can specifically knockout the OsNAC42 gene, and the transgenic rice prepared by using the sgRNA can reduce the Cd accumulation in grains and has no effect on yield.

[0036] The present invention also provides a recombinant vector for knocking out the rice OsNAC42 gene, and the recombinant vector includes the sgRNA described in the above technical solution and a basic vector.

[0037] In the present invention, the basic vector preferably includes the VK005-01 vector.

[0038] The present invention has no special requirements for the construction method of the recombinant vector, and the construction method well-known to those skilled in the art can be adopted.

[0039] The present invention also provides the application of the sgRNA or recombinant vector described in the above technical solution in reducing the cadmium accumulation in rice grains. The transgenic rice prepared by using the sgRNA provided by the present invention is planted in soil with a Cd content of 0.76 mg / kg and a pH value of 4.65. Compared with the wild type, it can not only reduce the Cd accumulation in grains, but also has no effect on the yield of rice.

[0040] The present invention also provides the application of the sgRNA or recombinant vector described in the above technical solution in cultivating cadmium-tolerant rice.

[0041] In the present invention, the traits of the cadmium-tolerant rice preferably include: the cadmium content in grains is lower than that of the wild type, and other agronomic traits are equivalent to those of the wild type; the other agronomic traits preferably include one or more of plant height, grain size, tiller number, and panicle shape. The transgenic rice prepared by using the sgRNA provided by the present invention can reduce the Cd accumulation in grains and has no effect on agronomic traits such as yield, plant height, grain size, tiller number, and panicle shape.

[0042] To further illustrate the present invention, the application of the rice OsNAC42 gene in regulating the cadmium accumulation in grains, the sgRNA, the recombinant vector, and their applications provided by the present invention will be described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] A recombinant vector for knocking out the rice OsNAC42 gene, which is constructed by the following method:

[0045] Synthesize the sgRNA containing the BspQ I restriction site, and the sgRNA containing the BspQ I restriction site is composed of sgRNA-S1 and sgRNA-A1;

[0046] The nucleotide sequence of the sgRNA-S1 is as shown in SEQ ID No.5, specifically: 5’- GCTCTTC GCGTAGCGGAACGCGTCCTC-3’;

[0047] The nucleotide sequence of the sgRNA-A1 is as shown in SEQ ID No.6, specifically: 5’- GAAGAGC GAGGACGCGTTCCGCTACGC-3’;

[0048] Among them, the underlined nucleotide sequence is the BspQ I restriction site sequence;

[0049] Dilute the synthesized sgRNA-S1 and sgRNA-A1 to 10 μM respectively. After mixing them according to the volume ratio of sgRNA-S1:sgRNA-A1:ddH2O = 5:5:15, incubate them in a metal bath at 95 °C for 3 min, and then let them cool naturally at room temperature for 10 min to obtain Oligo dimers for standby.

[0050] Digest the VK005-01 vector with BspQ I restriction endonuclease at 50 °C in a water bath for 2 h. After the digestion, use a gel extraction kit (SteadyPure DNA Gel Extraction Kit purchased from Aikerui Biotech, product number AG21006) for extraction. The specific extraction operation is as follows: Add 3 volumes of buffer MB and transfer all of it to the centrifugal column. After centrifuging at 12,000 rpm for 1 min, discard the filtrate. Add 700 μL of Washing buffer solution, centrifuge at 12,000 rpm for 1 min, discard the filtrate, and centrifuge again at 12,000 rpm for 2 min to remove the residual liquid. Place the centrifugal column in a new 1.5 mL centrifuge tube, add 30 μL of Elution buffer preheated at 65 °C, let it stand at room temperature for 1 min, and then centrifuge at 12,000 rpm for 1 min to obtain the digested product of VK005-01.

[0051] Use T4 ligase for vector ligation. After mixing according to the volume ratio of VK005-01 digested product:Oligo dimer:T4 ligase:T4 Buffer = 1:7:1:1, let it stand at room temperature for 4 h to obtain the recombinant vector.

[0052] Application Example 1

[0053] Transform the recombinant vector constructed in Example 1 into DH5α competent cells and use a kanamycin-resistant medium for positive monoclonal screening to obtain a positive clone bacterial solution. The kanamycin-resistant medium consists of the following components at the following concentrations: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, kanamycin 0.5 g / L, and agar powder 20 g / L.

[0054] Extract the recombinant plasmid from the obtained positive clone bacterial solution using a plasmid extraction kit (PlasmidExtraction Mini Kit purchased from TOROVID, product number PDE-100).

[0055] Transfer the extracted recombinant plasmid into Agrobacterium tumefaciens GV3101, and then infect the callus of Oryza sativa Nipponbare (Oryza sativa, denoted as nip) to screen and obtain positive transgenic mutants osnac42-1 and osnac42-2. The specific method is as follows:

[0056] The Agrobacterium tumefaciens GV3101 into which the recombinant plasmid has been transferred is inoculated onto YEB solid medium (purchased from Solarbio, catalog number LA7580), and monoclonal colonies are obtained by culturing at 28 °C; the monoclonal colonies are picked from the YEB solid medium and inoculated into 20 mL of YEB liquid medium containing 50 mg / L kanamycin antibiotic and 50 mg / L rifampicin, and cultured with shaking at 28 °C until the late logarithmic growth phase to obtain a bacterial solution; then, 0.5 mL is taken from the bacterial solution and transferred to 50 mL of YEB medium containing 50 mg / L kanamycin antibiotic and 50 mg / L rifampicin, and cultured with shaking at 28 °C until the OD 600 is 0.5; after centrifuging the cultured Agrobacterium tumefaciens at 4000 g for 10 min, the precipitate is resuspended with an equal volume of AAM-AS medium to obtain an AAM-AS resuspension; the AAM-AS medium is based on AAM medium (Solarbio; LA8580) and only contains 100 μmol / L acetosyringone; the pH of the AAM-AS medium is 5.2;

[0057] Take nip seeds 14 days after flowering, disinfect them with 70% ethanol for 1 min, and rinse them 3 times with sterile water; then disinfect them by shaking with a sodium hypochlorite solution containing 2% available chlorine for 60 min, and rinse them 5 times with sterile water, and then isolate the immature embryos under sterile conditions and inoculate them on N6D2S2 medium, and culture them in the dark at 28 °C for 4 days to induce callus; the N6D2S2 medium is based on N6 medium (purchased from Haibo Biotech, catalog number HBZ0601-2), and also only contains casein hydrolysate 500 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, phytagel 2.5 g / L, hygromycin 50 mg / L and cefotaxime 300 mg / L, and the pH value of the N6D2S2 medium is 5.8;

[0058] The callus is immersed in the AAM-AS resuspension for 20 min, dried with sterile filter paper and then transferred to N6D2C medium, and cultured in the dark at 25 °C for 3 days to obtain the infected callus; the N6D2C medium is based on N6 medium, and also only contains casein hydrolysate 500 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, phytagel 2.5 g / L, glucose 10 g / L and acetosyringone 100 μmol / L, and the pH value of the N6D2C medium is 5.2;

[0059] Wash the infected callus 5 times with sterile water containing 300 mg / L cephalosporin, blot dry with sterile filter paper, and then transfer it to the N6D2S1 medium for one round of screening. The N6D2S1 medium is based on the N6 medium and further contains only casein hydrolysate 500 g / L, sucrose 30 g / L, 2,4-D 2 mg / L, phytagel 2.5 g / L, hygromycin 25 mg / L, and cephalosporin 600 mg / L. The pH value of the N6D2S1 medium is 5.8.

[0060] After two weeks, transfer it to the N6D2S2 medium for the second round of screening, with one round every two weeks. Take out the vigorously growing resistant callus after 3 rounds of screening and transfer it to the pre-differentiation medium. In the differentiation incubator, culture it for 7 days under the conditions of a 12-hour light cycle, 28 °C during the day, and 25 °C at night. Then transfer it to the differentiation medium and culture it in the differentiation incubator until regenerated seedlings are produced under the conditions of a 12-hour light cycle, 28 °C during the day, and 25 °C at night. The pre-differentiation or differentiation medium is based on the MS medium and further contains only casein hydrolysate 300 g / L, hygromycin 50 mg / L, 6-BA 3 mg / L, 6-KT 3 mg / L, 6-ZT 0.2 mg / L, and phytagel 2.5 g / L. The pH of the pre-differentiation medium is 5.8.

[0061] Transfer the regenerated seedlings to the rooting and strong-seedling medium and carry out rooting and strong-seedling cultivation under the conditions of a 12-hour light cycle, 28 °C during the day, and 25 °C at night. The composition of the rooting and strong-seedling medium is: based on the 1 / 2MS medium, add sucrose 10 g / L.

[0062] When the small seedlings grow to 10 cm, open the sealing film of the container for 3 days, and then transfer the small seedlings to the artificial climate chamber for cultivation to obtain the plants for standby.

[0063] Take the leaves of the plants, grind them into powder with liquid nitrogen, add 600 μL of TPS DNA extraction solution, mix well, incubate in a water bath at 65 °C for 15 min, and invert and mix 3 times during this period. Centrifuge at 12000 rpm at room temperature for 10 min. The TPS DNA extraction solution consists of the following components: 1M Tris-HCl, 0.5M EDTA, and 5M KCl.

[0064] Then, pipette 300 μL of the supernatant and add an equal volume of isopropanol, mix well, let stand for 10 min, centrifuge at 12000 rpm at room temperature for 10 min, and discard the supernatant. Add 500 μL of 70% ethanol to wash the precipitate, centrifuge at 12000 rpm at room temperature for 5 min, discard the supernatant, and dry the precipitate in the ultra-clean bench. Add 50 μL of ddH2O to dissolve the DNA to obtain the plant gDNA.

[0065] Genomic PCR amplification was performed using the hygromycin sequence primers Hyg-F and Hyg-R with 2×Rapid Taq MasterMix (Vazyme, P222-01) to obtain PCR products. The nucleotide sequence of Hyg-F is as shown in SEQ ID NO.7: 5’-TTTCTTTGCCCTCGGACGAGT-3’; the nucleotide sequence of Hyg-R is as shown in SEQ ID NO.8: 5’-ATGAAAAAGCCTGAACTCACC-3’.

[0066] PCR amplification system: 10 μL of 2×Rapid Taq MasterMix, 0.5 μL of Hyg-F (10 μM), 0.5 μL of Hyg-R (10 μM), 1 μL of sample DNA, and 8 μL of ddH2O.

[0067] PCR amplification reaction program: Pre-denaturation at 95 °C for 3 min; denaturation at 95 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 15 s, repeated for 32 cycles; final extension at 72 °C for 3 min.

[0068] The PCR products were detected for bands by agarose gel electrophoresis. If the target band was present, it was initially identified as a positive transgenic mutant, and mutants osnac42-1 and osnac42-2 were screened.

[0069] Further, genomic PCR amplification was performed using the target position sequence primers OsNAC42-F and OsNAC42-R with 2×Rapid Taq MasterMix. The PCR amplification system and reaction program were the same as above, except that Hyg-F was replaced with OsNAC42-F and Hyg-R was replaced with OsNAC42-R. The nucleotide sequence of OsNAC42-F is as shown in SEQ ID NO.9: 5’-TCCGGCTGAAGAAGAACCAC-3’; the nucleotide sequence of OsNAC42-R is as shown in SEQ ID NO.10: 5’-CAAATGGAGCCCACCCTCAT-3’. The PCR products were sequenced to determine the specific mutation types of mutants osnac42-1 and osnac42-2. Among them, osnac42-1 had a single-base deletion, with a single-base G deletion at the 21st bp in the sequence shown in SEQ ID NO.2; osnac42-2 had a single-base insertion, with a single-base C insertion at the 23rd bp in the sequence shown in SEQ ID NO.2.

[0070] The wild-type rice NIP, mutant materials osnac42-1 and osnac42-2 were respectively planted in soil with a Cd content of 0.76 mg / kg and a pH value of 4.65. After the grains matured, they were threshed, washed, and dried respectively. 15 evenly grown grains were taken and weighed, 2 mL of HNO3 was added, and they were left to digest overnight. Then, they were heated and digested in a metal block at 120 °C until the liquid became clear and transparent. After cooling, deionized water was added to make the volume up to 10 mL, and the cadmium content in the grains was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES). Four parallel repeated experiments were carried out for each of the three materials. The results are shown in Table 1 and Figure 1 .

[0071] Table 1 Cadmium content in different rice grains (unit: mg / kg)

[0072] Group nip osnac42-1 osnac42-2 Replicate 1 0.914 0.810 0.762 Replicate 2 0.887 0.814 0.767 Replicate 3 0.899 0.822 0.777 Replicate 4 0.950 0.825 0.782

[0073] The wild-type rice NIP, mutant materials osnac42-1 and osnac42-2 were respectively planted in soil without cadmium pollution (Cd content less than or equal to 0.10 mg / kg) and with a pH value of 5.6. The tiller number, plant height, 100-grain weight, panicle shape, and grain size of different groups were observed and counted. Four parallel repeated experiments were carried out for each of the three materials. The results are shown in Figures 2 - 7 .

[0074] As can be seen from Table 1 and Figures 1 - 7 it can be known that the Cd content in the rice grains of mutant materials osnac42-1 and osnac42-2 is significantly lower than that of the wild-type rice variety NIP, but the agronomic traits such as yield, plant height, grain size, tiller number, and panicle shape are comparable to those of the wild-type rice variety NIP (i.e., there is no significant difference).

[0075] In summary, the OsNAC42 gene provided by the present invention is a key gene that can not only reduce the enrichment of Cd in grains but also has no effect on yield; the OsNAC42 gene can provide gene resources and technical support for the breeding of Cd-low-accumulating rice varieties.

[0076] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of rice OsNAC42 gene in regulating cadmium accumulation in grains, wherein the nucleotide sequence of the rice OsNAC42 gene is as shown in SEQ ID NO.2, and the regulation of cadmium accumulation in grains is: negatively regulating the expression of the OsNAC42 gene reduces the cadmium accumulation in rice grains.

2. Use of sgRNA or recombinant vector in reducing cadmium accumulation in rice grains; the sgRNA is sgRNA-S or sgRNA-A; the nucleotide sequence of the sgRNA-S is as shown in SEQ ID No.3; the nucleotide sequence of the sgRNA-A is as shown in SEQ ID No.4; the recombinant vector comprises the sgRNA and a basic vector.

3. The use according to claim 2, wherein, The basic vector is the VK005-01 vector.

4. Use of sgRNA or recombinant vector in cultivating cadmium-tolerant rice; the sgRNA is sgRNA-S or sgRNA-A; the nucleotide sequence of the sgRNA-S is as shown in SEQ ID No.3; the nucleotide sequence of the sgRNA-A is as shown in SEQ ID No.4; the recombinant vector comprises the sgRNA and a basic vector.

5. The use according to claim 4, wherein, The cadmium content in the grains of the cadmium-tolerant rice is lower than that of the wild type, and other agronomic traits are equivalent to those of the wild type; the other agronomic traits are one or more of plant height, grain size, tiller number, and panicle shape.

6. The use according to claim 4, wherein, The basic vector is the VK005-01 vector.

Images