LOSS-OF-FUNCTION GENE OF DOMINANT GENE EIFISO4E-S RESISTANT TO TVBMV AND USES THEREOF

Abstract

The disclosure provides a loss-of-function gene of eIFiso4E-S. The loss-of-function gene is resistant to tobacco vein banding mosaic virus (TVBMV) and represented by the sequence of SEQ ID NO: 1.

Claims

1. A loss-of-function gene of a dominant gene eIFiso4E-S, the loss-of-function gene being resistant to tobacco vein banding mosaic virus (TVBMV) and the dominant gene eIFiso4E-S being represented by a sequence of SEQ ID NO: 1.

2. A polypeptide being coded by the loss-of-function gene of claim 1, and the polypeptide represented by a sequence of SEQ ID NO: 2.

3. A method of preparing a tobacco variety comprising eifiso4e-s.sup.KO and being resistant to TVBMV, the method comprising knocking out the dominant gene eIFiso4E-S (eifiso4e-s.sup.KO) of a target tobacco through chromosome segment substitution, gene introduction, gene editing, gene silencing, or physical and chemical mutagenesis.

4. The method of claim 3, wherein chromosome segment substitution comprises introducing a chromosome segment comprising eifiso4e-s.sup.KO into the target tobacco through hybrid breeding and protoplast fusion, thereby obtaining the tobacco variety resistant to TVBMV.

5. The method of claim 3, wherein gene introduction comprises introducing an exogenous gene eifiso4e-s.sup.KO into the target tobacco, thereby obtaining the tobacco variety resistant to TVBMV.

6. The method of claim 3, wherein gene editing comprises inserting, deleting, or modifying a specific nucleotide base of the dominant gene eIFiso4E-S in the target tobacco, thereby destroying the function of the sequence shown in SEQ ID NO: 1 and obtaining the tobacco variety resistant to TVBMV.

7. The method of claim 3, wherein physical and chemical mutagenesis comprises using a physical and chemical mutagen to destroy the function of the sequence shown in SEQ ID NO: 1 of the target tobacco, thereby obtaining the tobacco variety resistant to TVBMV.

8. The method of claim 3, wherein the target tobacco is a tobacco carrying a recessive resistance gene va or an eIF4E1 knockout (eif4E1.sup.KO) tobacco.

9. The method of claim 3, wherein the tobacco variety comprises a tobacco plant, a seed, and a vegetative propagule thereof.

10. A Knockout kit comprising the loss-of-function gene of the dominant gene eIFiso4E-S of claim 1.

Description

DETAILED DESCRIPTION

[0035] Unless otherwise specified in embodiments, methods refer to the conventional methods, and test materials are available from the commercial provider.

[0036] Tobacco materials comprises Nicotiana tabacum cv Yunyan87 (also known as Yunyan 87 which has a genotype of eIFiso4E-S/eIF4E1 that is susceptible to a TVBMV infection), Honghua Dajinyuan (which has a genotype of eIFiso4E-S/eIF4E1 that is susceptible to the TVBMV infection). 2-1398 (which has a genotype of eIFiso4E-S/va that is resistant to PVY infection). The tobacco materials and TVBMV are provided by Yunnan Academy of Tobacco Agriculture.

[0037] Total RNA was extracted from tobacco leaves using TRIzol reagent (Invitrogen; Carlsbad, Calif.) according to the manufacturer's instruction. Plasmid DNA purification kit, agarose gel DNA extraction kit, and DNA fragment purification kit were purchased from QIAGEN. Escherichia coli DH5a, Restriction endonucleases, DNA Marker, PrimeSTAR GXL DNA Polymerase, T4 DNA polymerase, T4 DNA ligase, and spectinomycin were purchased from Takara Bio Inc. and Roche Biotech Company. TRIzol plus RNA extraction kit was purchased from Invitrogen Corporation. The Escherichia coli DH5α, Agrobacterium tumefaciens stains EHA105 and C58C1 were stored for later use. pMD18-T vector was purchased from Takara Bio Inc.

Example 1

[0038] Analysis of eIFiso4E Gene Sequence of Common Tobacco.

[0039] Pepper eIFiso4E gene (Genebank: DQ022080) was used as a reference and two homologous sequences of eIFiso4E gene in common tobacco (N. tabacum) were found in Genbank database by Blastn. The nucleotide sequences of eIFiso4E gene in Nicotiana sylvestris (N. sylvestris) and Nicotiana tomentosiformis (N. tomentosiformis) respectively have a high coincidence rate with that of common tobacco. The two nucleotide sequences were named eIFiso4E-S and eIFiso4E-T, respectively.

[0040] Specific primers for synthesizing the dominant gene eIFiso4E-S:

TABLE-US-00001 isoS-F: (SEQ ID NO: 3) (5′-ATGGCCACTGAAGCACCGATAGAG-3′) isoS-R: (SEQ ID NO: 4) (5′-TCACACAGTATATCGACTCT-3′)

Example 2

[0041] Cloning and Sequence Analysis of eIFiso4E Gene of Common Tobacco.

[0042] (1) Total RNA extraction from tobacco: Fresh young leaves were collected from common tobacco Honghua Dajinyuan and total RNA was extracted from the fresh young leaves using TRIzol reagent (Invitrogen). Total RNA was reverse transcribed into cDNA by RT-PCR using Oligo dT-Adapter as a primer.

[0043] (2) Cloning of eIFiso4E-S gene: cDNA was used as a template and eIFiso4E-S gene was amplified by PCR using the specific primers isoS-F and isoS-R. Total reaction volume for PCR was 50 μL, including 4.0 μL of 100 ng/μL cDNA, 10.0 μL of 5×PCR buffer, 4 μL of dNTPs (2.5 mmol/L each), 2.0 μL of the specific primer isoS-F, 2.0 μL of the specific primer isoS-R, 1 μL of PrimeSTAR GXL DNA Polymerase, and 27 μL of ddH.sub.2O. All reagents were purchased from Takara. PCR conditions: 98° C. for 2 min, then 35 cycles of 98° C. for 10 s, 60° C. for 15 s, 68° C. for 1 min, followed by 68° C. for 10 min.

[0044] (3) Recovery and purification of PCR products: PCR products were electrophoresed on a 1.5% agarose gel with 1×TAE as the electrophoresis buffer. Bromophenol blue dye migrated at 120 V for 60 min. The gel was then transferred into a gel imaging system for recording the dye bands. The small portion containing DNA fragment of interest was cut out from the gel under UV light. The DNA fragment of interest was recovered with a DNA fragment purification kit (purchased from QIAGEN).

[0045] (4) Cloning and sequencing of PCR products: The recovered DNA fragment of interest was introduced into a cloning vector. 30 positive clones were screened, sequenced by Thermo Fisher Scientific Inc. (Guangzhou), and aligned to the eIFiso4E-S gene sequence. The nucleotide sequences showing more than 99% sequence identity were identified as the DNA sequence of the eIFiso4E-S gene. The eIFiso4E-S gene sequence was shown in SEQ ID NO: 1.

Example 3

[0046] Polypeptide Sequence Encoded by eIFiso4E-S Gene.

[0047] The nucleotide sequence of eIFiso4E-S gene was translated to an amino acid sequence by a Molecular Evolutionary Genetics Analysis software MEGA. The amino acid sequence, also called a polypeptide sequence, was shown in SEQ ID NO: 2.

Example 4

[0048] Construction of eIFiso4E-S Targeting Vector.

[0049] An eIFiso4E-S targeting vector was constructed using a plasmid pRGEB31 which was disclosed in the reference “Xie, K. and Y. Yang (2013). “RNA-guided gene editing in plants using a CRISPR-Cas system.” Mol Plant 6(6)”. In accordance with the principle of CRISPR/Cas9 technology, a target site was designed in the first exon of eIFiso4E-S gene. A PAM site was found on the sequence of the first exon (The PAM site is NGG or CCN which takes the form of

TABLE-US-00002 5′-NNNNNNNNNNNNNNNNNNNNNGG -3′ or 5′-CCNNNNNNNNNNNNNNNNNNN-3′).

[0050] Synthesis and annealing of gDNA oligo: the target site of gRNA was used as a template. Two target sites were determined for the first exon of eIFiso4E-S gene. The specific primers were as follows:

TABLE-US-00003 gRNA1F (SEQ ID NO: 5) 5′-GGCAcgcctctatcggtgatcag-3′; gRNA1R (SEQ ID NO: 6) 5′-AAACctgaagcaccgatagaggcg-3′; gRNA2F (SEQ ID NO: 7) 5′-GGCActagagaggagatggacattc-3′; gRNA2R (SEQ ID NO: 8) 5′-AAACgaatgtccatctcctctctag-3′.

[0051] Primer annealing: a set of complementary DNA oligo was synthesized and annealed to form a dsDNA.

[0052] An annealing system was as follows:

TABLE-US-00004 F-primer: 20 ul R-Primer: 20 ul 10X Annealing buffer:  5 ul H.sub.2O:  5 ul

[0053] The annealing conditions: 95° C. for 5 min, 90° C. for 1 mim, 80° C. for 1 mim, 70° C. for 1 mim, 60° C. for 1 mim, 50° C. for 1 mim, 40° C. for 1 mim, 30° C. for 1 mim, 20° C. for 1 mim, and 10° C. for 1 mim.

[0054] A Restriction Digestion System of pRGEB31 Plasmid:

TABLE-US-00005 Plasmid: 5 ul 10X buffer: 5 ul BsaI: 2 ul H.sub.2O: 37 ul 

[0055] The digested products of large fragments were recovered:

[0056] A System for Connecting the Recovered Products and the dsDNA.

TABLE-US-00006 Recovered products 3 ul dsDNA 10 ul  T4 DNA buffer 2 ul T4 DNA Ligase 1 ul H.sub.2O 4 ul

[0057] The connected product was transformed into Escherichia coli and the colonies were screened using colony PCR for positive colonies. The forward primer for PCR: OsU3 5′F 5′-aaggaatctttaaacatacgaacag-3′ (SEQ ID NO: 9). The reverse primer for PCR was the primer gRNA1R or gRNA2R. The positive colonies were incubated in a shaking incubator and sequenced to analysis whether gRNA was correct. The primer for sequencing was OsU3 5′F.

Example 5

[0058] Plant Transformation and Detection of eIFiso4E-S Targeting Vector.

[0059] The recombinant plasmid pRGEB31 was transformed into Agrobacterium GV3101 by electroporation. Calluses were induced from the wild-type tobacco Yunyan 87 and the tobacco 2-1398 comprising a homozygous va gene locus, followed by Agrobacterium-mediated plant transformation. The induced calluses were infected with Agrobacterium GV3101 and screened for hygromycin B resistant calluses. The resistant calluses differentiated and regenerated a whole transgenic tobacco.

[0060] Detection of eIFiso4E-S Gene Mutation in Transgenic Tobacco.

[0061] Specific primers were designed upstream and downstream of the target site in accordance with eIFiso4E-S gene sequence. The specific primers were as follows:

TABLE-US-00007 EditestF: (SEQ ID NO: 10) 5′ -caattccattacgcctctccgttcgct -3′; EditestR: (SEQ ID NO: 11) 5′-ggaacaaaatccgaatttatcaataact-3′.

[0062] The genomic DNA of the transgenic tobacco was used as a template, and the eIFiso4E-T gene was amplified by PCR using the specific primers EditestF and EditestR. The PCR products were sequenced by Thermo Fisher Scientific (Guangzhou) using the primer EditestF.

Example 6

[0063] Recombination of eIFiso4E-S.sup.KO and Va Leads to Resistance to TVBMV.

[0064] The eIFiso4E-S targeting vector in Example 4 was transformed into the tobacco 2-1398, and the resulting tobacco was named tobacco eifiso4e-s.sup.KO/va. Genomic DNA was extracted from TO generation seedlings, amplified by PCR, sequenced, and screened for tobacco 2-1398g1-1C which comprises eIFiso4E-S.sup.KO. Referring to SEQ ID NO: 1, a base A was added or deleted at position 11 in the sequence of eIFiso4E-S.sup.KO compared with eIFiso4E. The tobacco 2-1398g1-1C were selfed, producing Ti generation seeds. Ti generation seedlings were nursed in a plot by conventional methods until each seedling has 4-5 leaves. Genomic DNA was extracted from the leaves, and screened for the individuals which are homozygous/biallelic for eIFiso4E-S gene and wild-type for eIFiso4E-T gene. Diseased leaves infected with TVBMV were squeezed into juice, diluted 40 times, and inoculated on the individuals. The va tobacco 2-1398 was used as a control group. The disease was investigated on the 14.sup.th, 21.sup.st, and 28.sup.th after inoculation. Referring to Table 1, the control group (va tobacco 2-1398) had an incidence rate of 100% on 14.sup.th after inoculation. The eifiso4e-s.sup.KO/va tobacco had an incidence rate of 0, which shows a strong resistance to TVBMV.

TABLE-US-00008 TABLE 1 Resistance of tobacco eifiso4e-s.sup.KO/va against TVBMV Incidence rate, % Materials Genotype Number 14 dpi 21 dpi 28 dpi 35 dpi 2-1398g1-1C eifiso4e-s.sup.KO/va 24 0 0 0 0 2-1398 va 32 100 100 100 100

[0065] On the 32.sup.nd day after inoculation, the leaves from each individual were collected for total RNA extraction. Oligo T primers were used in reverse transcription to obtain cDNA. PCR primers were designed according to the VPg sequence of TVBMV. PCR was carried out using an annealing temperature of 60° C. The amplified product was 765 bp in size.

TABLE-US-00009 TVBMVVPg_F: (SEQ ID NO: 12) 5′ -AACTCAAGAGTCGTTGGAACA-3′; TVBMVVPgR: (SEQ ID NO: 13) 5′ -CAAGCAAGCATATACACTTAGC-3′.

[0066] No target bands were amplified in the 12 samples which are collected from the tobacco 2-1398g1-1C inoculated with TVBMV, but target bands were amplified in the tobacco 2-1398 inoculated with TVBMV. The results further confirmed that the tobacco 2-1398g1-1C is resistant to TVBMV, while the control 2-1398 is sensitive to TVBMV.

Example 7

[0067] Breeding Application of Allele eifiso4e-s.sup.KO of eIFiso4E-S

[0068] An allele of the gene eIFiso4E-S having the sequence of SEQ ID NO: 1 was named eifiso4e-s.sup.KO. A chromosome fragment containing the allele eifiso4e-s.sup.KO was transferred into a target tobacco by conventional breeding methods. The target tobacco may be va or eif4E1.sup.KO tobacco. Germplasm resources comprising eifiso4e-s.sup.KO were screened from a tobacco population by a functional or linked molecular marker of gene eifiso4e-s.sup.KO, or by artificial inoculation of TVBMV. The germplasm resources comprise wild species of tobacco, hybrid and cultivated species of wild and cultivated species. The chromosome fragment comprising the gene eifiso4e-s.sup.KO was screened from the germplasm resources and introduced into the target tobacco through conventional hybrid breeding, protoplast fusion, or chromosome segment substitution, thereby obtaining a non-transgenic tobacco with increased in TVBMV resistance. The non-transgenic tobacco was bred into a commercial variety through hybridization, backcrossing, etc., thereby improving the resistance of tobacco against TVBMV.

Example 8

[0069] Breeding Application of Gene-Edited eIFiso4E-S Gene.

[0070] The gene eIFiso4E-S in the target tobacco was modified using biotechnology such as gene editing, losing the biological function of interacting with TVBMV VPg. The resulting tobacco can resist TVBMV. The target tobacco may be va or eif4E1.sup.KO tobacco.

[0071] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.