STSCI PROTEIN FOR CHANGING SELF-INCOMPATIBILITY OF DIPLOID POTATO MATERIALS

Abstract

Provided is a StSCI protein for changing the self-incompatibility of diploid potato materials, wherein the amino acid sequence of the StSCI protein includes or consists of the following sequence: 1) the amino acid sequence represented by SEQ ID NO: 1; or 2) a functional homologous sequence having at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 1; or 3) a protein in which one or more (e.g., 1-10) amino acids are added, deleted, or replaced in the amino acid sequence represented by SEQ ID NO: 1 and has the activity of inhibiting self-incompatibility. The advantage of the application is that the StSCI protein may inhibit the cytotoxicity of multiple types of S-RNase, which is hereditary and fundamentally overcomes the defect of self-incompatibility of diploid potatoes, thereby facilitating to realize the cultivation of a high-generation homozygous inbred line of diploid potatoes.

Claims

1. A StSCI protein for changing the self-incompatibility of diploid potato materials, wherein the amino acid sequence of the StSCI protein comprises or consists of the following sequence: 1) the amino acid sequence represented by SEQ ID NO: 1; or 2) a functional homologous sequence having at least 95% sequence identity with the amino acid sequence represented by SEQ ID NO: 1; or 3) a protein in which one or more (e.g., 1-10) amino acids are added, deleted, or replaced in the amino acid sequence represented by SEQ ID NO: 1 and has the activity of inhibiting self-incompatibility.

2. A nucleotide sequence encoding the StSCI protein of claim 1, wherein the nucleotide sequence encoding the StSCI protein comprises or consists of the following sequence: 1) the nucleotide sequence represented by SEQ ID NO: 2; or 2) a complementary sequence, a degenerate sequence, or a homologous sequence of the nucleotide sequence represented by SEQ ID NO: 2; wherein the homologous sequence is a polynucleotide sequence having at least 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, and encoding a protein with the activity of inhibiting self-incompatibility, or a corresponding cDNA molecule thereof; or 3) a nucleotide sequence hybridizing with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encoding a protein with the activity of inhibiting self-incompatibility.

3. A promoter for regulating the nucleotide sequence according to claim 2, wherein the nucleotide sequence of the promoter comprises or consists of the following sequence: 1) the nucleotide sequence represented by SEQ ID NO: 3; or 2) a functional homologous sequence having at least 90% sequence identity with the nucleotide sequence represented by SEQ ID NO: 3; or 3) a polynucleotide hybridizing to the nucleotide sequence represented by SEQ ID NO: 3 under stringent conditions or a complementary sequence thereof.

4. A PCR primer for amplifying the nucleotide sequence according to claim 2.

5. The PCR primer according to claim 4, wherein the primer is an upstream primer and/or a downstream primer, the upstream primer comprises the sequence of 5′-ATGGACTATTTCCTATTGCTACCAGAAGG-3′ (SEQ ID NO: 4); and the downstream primer comprises the sequence of 5′-TCATTCTGGTCTAAATTCAATTCCTTGAA-3′ (SEQ ID NO: 5).

6. A vector comprising 1) a nucleotide sequence represented by SEQ ID NO: 2, or 2) a complementary sequence, a degenerate sequence, or a homologous sequence of the nucleotide sequence represented by SEQ ID NO: 2, or 3) a nucleotide sequence hybridizing with the nucleotide sequence of SEQ ID NO: 2 under stringent conditions and encoding a protein with the activity of inhibiting self-incompatibility, and the promoter according to claim 3 or a heterologous promoter.

7. A microorganism or plant cell into which the nucleotide sequence of claim 2 has been introduced.

8. A microorganism or plant cell into which the vector according to claim 6 has been introduced.

9. A method for expressing a target gene in a plant, wherein the vector according to claim 6 is transferred into a plant body to obtain the expression of the target gene.

10. A method for expressing a target gene in a plant, wherein the microorganism according to claim 7 is transferred into a plant body to obtain the expression of the target gene.

11. A method for expressing a target gene in a plant, wherein the microorganism according to claim 8 is transferred into a plant body to obtain the expression of the target gene.

12. A method for producing a self-compatible diploid potato, comprising that allowing the diploid potato plant to express the protein according to claim 1.

13. A method for producing a self-compatible diploid potato, comprising that transferring the nucleotide sequence according to claim 2 into a diploid potato, so that the StSCI protein is expressed in the diploid potato.

14. A method for producing a self-compatible diploid potato, comprising that transferring the vector according to claim 6 into a diploid potato, so that the StSCI protein is expressed in the diploid potato.

15. A method for producing a self-compatible diploid potato, comprising that transferring the microorganism or plant cell according to claim 7 into a diploid potato, so that the StSCI protein is expressed in the diploid potato.

16. A method for producing a self-compatible diploid potato, comprising that transferring the microorganism or plant cell according to claim 8 into a diploid potato, so that the StSCI protein is expressed in the diploid potato.

17. A diploid potato plant, potato tuber, and potato leaf obtained by the method according to claim 12.

18. A method for breeding a diploid potato, comprising that utilizing a self-compatible diploid potato plant comprising the nucleotide sequence according to claim 2, to perform self-crossing or hybridize with other diploid potato plant.

19. A method for breeding a diploid potato, comprising that utilizing the diploid potato plant according to claim 17 to perform self-crossing or hybridize with other diploid potato plant.

20. A method for breeding a diploid potato, comprising that doubling a self-compatible diploid potato plant comprising the nucleotide sequence according to claim 2, then hybridizing with other tetraploid potato plant, and then reducing into a diploid potato plant.

21. A method for breeding a diploid potato, comprising that doubling the diploid potato plant according to claim 17 into a tetraploid plant, then hybridizing with other tetraploid potato plant, and then reducing into a diploid potato plant.

22. A method for breeding a tetraploid potato, comprising that doubling a self-compatible diploid potato plant comprising the nucleotide sequence according to claim 2, then hybridizing with other tetraploid potato plant.

23. A method for breeding a tetraploid potato, comprising that doubling the diploid potato plant according to claim 17 into a tetraploid plant, then hybridizing with other tetraploid potato plant.

24. A food prepared from the diploid potato plant according to claim 17.

25. A food prepared from the diploid potato plant obtained by the method for breeding a diploid potato according to claim 18.

26. A food prepared from the diploid potato plant obtained by the method for breeding a diploid potato according to claim 20.

27. A food prepared from the tetraploid potato plant obtained by the method for breeding a tetraploid according to claim 22.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0087] FIG. 1 is a plot showing the initial localization interval of the StSCI gene in Example 1.

[0088] FIG. 2 are photos showing the identification of the self-compatibility phenotype of the diploid potato materials in Example 2, wherein panel A is the diploid potato material S15-65 having complementary expression of the StSCI gene, and panel B is the diploid potato material PI 225689 having overexpression of the StSCI gene.

[0089] FIG. 3 is a diagram showing the identification of the self-compatibility phenotype of the self-compatible material RH89-039-16 after knocking out the StSCI gene.

DETAILED DESCRIPTION OF EMBODIMENTS

[0090] The technical solutions in the examples of the present application will be clearly and completely described with reference to the drawings in the examples of the present application. Obviously, the described examples are only a part of the examples of the present application, rather than all the examples. Based on the examples in this application, all of the other examples obtained by those of ordinary skill in the art without creative work shall fall within the protection scope of this application.

[0091] In the following examples, the diploid potato material RH89-039-16 is self-compatible, and the diploid potato material PI 225689 and S15-65 are self-incompatible.

[0092] Unless otherwise specified, all of the operation methods in the following examples belong to the prior art commonly used by those skilled in the art, and will not be explained repeatedly herein.

Example 1: StSCI Gene Position

[0093] The pollen of the diploid potato material RH89-039-16 is used to pollinate the originally self-incompatible diploid material PI 225689, and the seeds are harvested as the F1 generation. The self-compatibility traits of F1 generation plants are investigated, and it is found that self-compatible plants and self-incompatible plants respectively account for half of them, indicating that the self-compatibility gene in RH89-039-16 is in a heterozygous state, while RH89-039-16 shows self-compatibility traits, indicating that the gene for controlling the self-compatibility of RH89-039-16 is dominant, and this dominant gene is named as StSCI gene. The leaves of 40 self-compatible individual plants and 40 self-incompatible individual plants are respectively selected from the F1 generation, mixing and extracting the genomic DNA, then sequencing the genomic DNA of the two mixed pools.

[0094] The sequencing results are analyzed, and the difference between the SNP-index of the two pools is used to initially locate the StSCI gene in the 2.5 Mb interval at the end of chromosome 12, thereby obtaining the initial positioning interval of the StSCI gene (FIG. 1).

[0095] Genome resequencing is performed on the self-compatible diploid potato material RH89-039-16 and the self-incompatible diploid potato material PI 225689, and the molecular markers for StSCI gene positioning are developed according to the genomic sequence information of RH89-039-16 and PI 225689 in the initial positioning interval of in the StSCI gene. Since the StSCI gene is heterozygous in RH89-039-16, and is homozygous and recessive in PI 225689, the molecular markers to be linked to the StSCI gene and to be developed should also be heterozygous in RH89-039-16, and be homozygous in PI 225689. The molecular markers in the initial positioning interval are developed according to this principle, and the individual plants of F1 population are used as the positioning population to finely position the StSCI gene, finally positioning the StSCI gene in a 76 Kb interval. There are a total of 6 annotation genes in this interval. Using the sequence information of these 6 annotation genes, 6 pairs of quantitative primers are designed to respectively detect the expression levels of these 6 annotation genes, meanwhile the total plant RNA from pollen parts of RH89-039-16 and PI 225689 is extracted, and the expression levels of the annotated genes in pollen are detected by real-time fluorescent quantitative PCR (polymerase chain reaction), wherein three of them are highly expressed in pollen of RH89-039-16. Among the three genes highly expressed in pollen of RH89-039-16, the gene encoded as PGSC0003DMG400016861 is not expressed at all in pollen of PI 225689. This gene may be responsible for the self-compatibility of RH89-039-16 and the self-incompatibility of PI 225689, and it is a candidate gene of the StSCI gene.

[0096] A primer pair (represented by SEQ ID NO: 4 and SEQ ID NO: 5) for amplifying a region including the gene and the promoter is designed according to the annotated sequence information of the gene PGSC0003DMG400016861 in the DM genome, the genomic DNAs of RH89-039-16 and IP65C are respectively used as templates, and the PCR products are sequenced after PCR amplification. It is found that in the promoter region of this gene, RH89-039-16 has a 538 bp insert sequence as compared with PI 225689. This insert sequence is located 100 bp upstream of the initiation codon, the insert sequence includes an annotated promoter core element, and this sequence is the reason that the gene is expressed in RH89-039-16 but not in PI 225689.

[0097] The nucleotide sequence of the StSCI gene is represented by SEQ ID NO: 2.

[0098] The above inserted sequence is the promoter for regulating the StSCI gene, and its nucleotide sequence is represented by SEQ ID NO: 6.

Example 2: StSCI Gene Verification

[0099] In order to verify that gene PGSC0003DMG400016861 is the inducing gene responsible for the self-compatibility in RH89-039-16, a plant binary vector containing a 1245 bp of promoter region upstream of the initiation codon of the gene PGSC0003DMG400016861 in RH89-039-16 and a 3352 bp of full-length region of the gene is constructed by using pCAMBIA1301 as the backbone vector. With the aid of the Agrobacterium transformation system, the gene PGSC0003DMG400016861 in RH89-039-16 and its promoter are transferred into the originally self-incompatible diploid potato material S15-65 to obtain a complementary transgenic plant. Meanwhile an overexpression vector connecting the encoding region of gene PGSC0003DMG400016861 with CaMV35S as the promoter is constructed, and the same method is used to transfer it into the self-incompatible material PI 225689.

[0100] Meanwhile, a gene knockout vector containing the gene PGSC0003DMG400016861 with 4 target positions is constructed by using CRISP/Cas9 technology, and the gene PGSC0003DMG400016861 is knocked out in the self-compatible RH89-039-16 material. It is found in the results in FIG. 2 that, the complementary expression or overexpression of the gene PGSC0003DMG400016861 in the self-incompatible material S15-65 may make the self-incompatible material S15-65 and PI 225689 become self-compatible, which are respectively shown in A and B of FIG. 2. After knocking out the gene PGSC0003DMG400016861 in the self-compatible material RH89-039-16, the originally self-compatible RH89-039-16 became self-incompatible (as shown in FIG. 3), indicating that the gene PGSC0003DMG400016861 is the StSCI gene responsible for self-compatibility of RH89-039-16.

Example 3: A Method for Producing a Self-Compatible Diploid Potato

[0101] The pCAMBIA1301 vector carrying the nucleotide sequence encoding the StSCI protein (SEQ ID NO: 2) and the promoter (SEQ ID NO: 3) is transferred into a diploid potato identified as self-incompatible by using Agrobacterium LBA440. As a result, it is found that the introduction of the above pCAMBIA1301 vector allows the StSCI gene in diploid potatoes to express the StSCI protein, thereby turning the self-incompatible diploid potato into self-compatible.

Example 4: A Method for Producing a Self-Compatible Diploid Potato

[0102] The pCAMBIA1301 vector carrying the nucleotide sequence encoding the StSCI protein (SEQ ID NO: 2) and the CaMV35S promoter is transferred into a diploid potato identified as self-incompatible by using Agrobacterium LBA440. As a result, it is found that the introduction of the above pCAMBIA1301 vector allows the StSCI gene in diploid potatoes to express the StSCI protein, thereby turning the self-incompatible diploid potato into self-compatible.

Example 5: A Method for Breeding a Diploid Potato by Self-Crossing

[0103] Diploid potato materials (such as RH89-039-16) capable of expressing the StSCI gene in pollen are self-compatible. Therefore, after RH89-039-16 enters the full flowering stage, the pollen from the stamens of RH89-039-16 is picked to spread on the stigma of the pistils of RH89-039-16 for self-pollination. Since RH89-039-16 is self-compatible, after self-pollination RH89-039-16 may produce seeds, thereby obtaining diploid potato seeds of RH89-039-16.

Example 6: A Method for Breeding a Diploid Potato by Hybridization

[0104] Diploid potato materials (such as RH89-039-16) capable of expressing the StSCI gene in pollen are self-compatible, while most diploid potato materials such as PI 225689 are self-incompatible. After the above RH89-039-16 and PI 225689 materials enter the full flowering stage, the pollen from the stamens of RH89-039-16 is picked to spread on the stigma of PI 225689 for hybridizing pollination. Half of the seeds produced by using genome recombination are capable of expressing StSCI gene, and thus they are self-compatible, thereby obtaining a self-compatible diploid potato material by hybridization.

[0105] The above descriptions are only preferred examples of this application, and are not intended to limit this application. Any modification, equivalent replacement, etc. made within the spirit and principle of this application shall be covered in the protection scope of this application.