Sweet potato root and leaf development-related protein IbbZIP11 and its encoding gene and application

Abstract

The present invention discloses a sweet potato root tuber and leaf development-related protein IbbZIP11 and its encoding gene and application, belonging to the field of biotechnology. The present invention discloses an IbbZIP11 protein or a substance that regulates the expression of the IbbZIP11 protein encoding gene or a substance that regulates the activity or content of the IbbZIP11 protein in regulating plant leaf and / or root development and / or plant yield and in plant breeding; the IbbZIP11 protein is a protein whose amino acid sequence is SEQ ID No.1. The present invention can improve the photosynthetic rate of sweet potatoes, enlarge the root tubers, and increase the yield by knocking out the IbbZIP11 gene through gene editing technology or silencing the IbbZIP11 gene after transcription by dsRNA interference, and is a gene with great application value to the sweet potato industry.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a sweet potato root tuber and leaf development-related protein IbbZIP11 and a coding gene and application thereof. Background Art

[0002] Sweet potato (Ipomoea batatas (L.) Lam), as a homologous or allohexaploid plant, is an important root crop used as food, feed, industrial raw material and new energy source, and is widely grown in more than 100 countries and regions in the world.

[0003] As the main economic crop in the world, sweet potato is drought-resistant, barren-resistant and high-yielding in production. Therefore, it is widely planted around the world. Sweet potato in my country is mainly planted in the southwest and east China, accounting for 50% of the total value of sweet potato production in my country. In actual production, high-yield varieties often have the characteristics of strong "source", large "sink" and smooth "flow". As one of the main root crops in the world, the root system of sweet potato is mainly divided into three categories, namely, fibrous roots responsible for absorbing nutrients and water, non-storage roots formed by lignification of the mid-column, namely firewood roots, and tubers formed by the expansion of cambium cells to accumulate various nutrients. In actual production, the main harvested part is the tubers of sweet potato. The tuber yield of sweet potato (mostly calculated by fresh potato yield in production, and others include dried potato yield, starch yield, commercial potato yield and biological yield, etc.) is the most important economic trait, which is mainly determined by the yield per plant and density. The tuber yield of sweet potato per plant is mainly affected by the number of tubers per plant and the weight of tubers. In addition, sweet potato is a typical C3 crop in dicotyledonous plants. The efficiency of light energy utilization of plant populations can affect the accumulation of photosynthetic substances and is also a key factor in determining sweet potato yield. Leaves are the main site for photosynthesis in plants, which can convert light energy into organic nutrients. After distribution, part of the photosynthetic products are transported to the roots for accumulation. High-yield varieties often have a higher harvest index HI, and the ability of photosynthetic products to be transported from functional leaves to tubers is also stronger. Therefore, it is of great significance to explore the genes related to the development of sweet potato tubers and leaves through modern biotechnology and explore their genetic mechanisms that affect the development of sweet potato for breeding high-yield sweet potato varieties. Summary of the invention

[0004] The technical problem to be solved by the present invention is: how to regulate the development of sweet potato leaves and roots and / or the yield of sweet potato.

[0005] In order to solve the above technical problems, in a first aspect, the present invention provides an application, which may be A1) or A2),

[0006] A1), use of IbbZIP11 protein or a substance that regulates the expression of the gene encoding the IbbZIP11 protein or a substance that regulates the activity or content of the IbbZIP11 protein in regulating plant leaf and / or root development and / or plant yield;

[0007] A2), use of IbbZIP11 protein or a substance that regulates the expression of a gene encoding the IbbZIP11 protein or a substance that regulates the activity or content of the IbbZIP11 protein in plant breeding;

[0008] The IbbZIP11 protein may be the following P1) or P2) or P3):

[0009] P1), a protein having an amino acid sequence of SEQ ID No. 1;

[0010] P2), a protein obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in P1), which has more than 80% identity with the protein shown in P1) and has the function of regulating plant leaf and / or root development and / or plant yield;

[0011] P3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of P1) or P2).

[0012] The goals of plant breeding include developing high-yielding plants.

[0013] The substance regulating the expression of the IbbZIP11 protein encoding gene may be a substance that inhibits, reduces or downregulates the expression of the gene. The regulating plant leaf and / or root development and / or plant yield may be to improve or increase or upregulate plant leaf and / or root development and / or plant yield.

[0014] Furthermore, in the above application, the protein is derived from sweet potato.

[0015] The above proteins can be artificially synthesized, or their encoding genes can be synthesized first and then expressed biologically.

[0016] Among them, SEQ ID No. 1 consists of 167 amino acid residues.

[0017] Herein, the protein tag refers to a polypeptide or protein that is fused and expressed with a target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0018] Herein, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences for calculation, the value of the identity (%) can then be obtained.

[0019] Herein, the 80% or greater identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0020] Further, in the above application, the protein described in P2) includes protein IbbZIP11 / C46-1, protein IbbZIP11 / C46-2, IbbZIP11 / C46-3, protein IbbZIP11 / C46-4, protein IbbZIP11 / C46-5, protein IbbZIP11 / C80-1, protein IbbZIP11 / C80-2, IbbZIP11 / C80-3, protein IbbZIP11 / C80-4 and protein IbbZIP11 / C80-5,

[0021] The protein IbbZIP11 / C46-1 is a protein encoded by the IbbZIP11 / C46-1 gene, and the coding sequence of the IbbZIP11 / C46-1 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0022] The protein IbbZIP11 / C46-2 is a protein encoded by the IbbZIP11 / C46-2 gene, and the coding sequence of the IbbZIP11 / C46-2 gene is a DNA molecule obtained by deleting a total of 2 nucleotides at positions 131-132 of sequence 2 in the sequence list, replacing the nucleotide G at position 132 with A, and keeping the other nucleotides of sequence 2 unchanged;

[0023] The protein IbbZIP11 / C46-3 is a protein encoded by the IbbZIP11 / C46-3 gene, and the coding sequence of the IbbZIP11 / C46-3 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 3 nucleotides at positions 131-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged;

[0024] The protein IbbZIP11 / C46-4 is a protein encoded by the IbbZIP11 / C46-4 gene, and the coding sequence of the IbbZIP11 / C46-4 gene is a DNA molecule obtained by deleting a total of 5 nucleotides at positions 128-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0025] The protein IbbZIP11 / C46-5 is a protein encoded by the IbbZIP11 / C46-5 gene, and the coding sequence of the IbbZIP11 / C46-5 gene is a DNA molecule obtained by deleting a total of 6 nucleotides at positions 127-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0026] The protein IbbZIP11 / C80-1 is a protein encoded by the IbbZIP11 / C80-1 gene, and the coding sequence of the IbbZIP11 / C80-1 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged;

[0027] The protein IbbZIP11 / C80-2 is a protein encoded by the IbbZIP11 / C80-2 gene, and the coding sequence of the IbbZIP11 / C80-2 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting 13 nucleotides in total at positions 122-134 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged;

[0028] The protein IbbZIP11 / C80-3 is a protein encoded by the IbbZIP11 / C80-3 gene, and the coding sequence of the IbbZIP11 / C80-3 gene is a DNA molecule obtained by inserting nucleotide G at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged;

[0029] The protein IbbZIP11 / C80-4 is a protein encoded by the IbbZIP11 / C80-4 gene, and the coding sequence of the IbbZIP11 / C80-4 gene is a DNA molecule obtained by deleting a total of 4 nucleotides at positions 58-61 of sequence 2 in the sequence list, and deleting a total of 3 nucleotides at positions 126-128 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0030] The protein IbbZIP11 / C80-5 is a protein encoded by the IbbZIP11 / C80-5 gene, and the coding sequence of the IbbZIP11 / C80-5 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 134 of sequence 2 in the sequence list and keeping other nucleotides of sequence 2 unchanged.

[0031] Furthermore, in the above application, the substance that regulates the expression of the gene encoding the IbbZIP11 protein or the substance that regulates the activity or content of the IbbZIP11 protein may be a biological material, and the biological material may be any one of the following B1) to B10):

[0032] B1), an RNA molecule that inhibits, reduces or down-regulates the expression of the gene encoding the above-mentioned IbbZIP11 protein, or an RNA molecule that inhibits, reduces or down-regulates the activity or content of the above-mentioned IbbZIP11 protein;

[0033] B2), expressing the coding gene of the RNA molecule described in B1);

[0034] B3), an expression cassette containing the coding gene described in B2);

[0035] B4), a recombinant vector containing the coding gene described in B2), or a recombinant vector containing the expression cassette described in B3);

[0036] B5), a recombinant microorganism containing the coding gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

[0037] B6), a transgenic plant cell line containing the coding gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4);

[0038] B7), transgenic plant tissue containing the coding gene described in B2), or transgenic plant tissue containing the expression cassette described in B3), or transgenic plant tissue containing the recombinant vector described in B4);

[0039] B8), a transgenic plant organ containing the coding gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4);

[0040] B9), a nucleic acid molecule encoding IbbZIP11 protein;

[0041] B10), expression cassettes, recombinant vectors, recombinant microorganisms, transgenic plant cell lines, transgenic plant tissues, and transgenic plant organs containing the nucleic acid molecule described in B9).

[0042] Further, in the above application, the RNA molecule described in B1) may be an RNA transcribed from a DNA molecule as shown in formula (I): SEQ reverse-X-SEQ forward (I);

[0043] The SEQ forward sequence is the 244th to 501st position of sequence 2 in the sequence list; the SEQ reverse sequence is reverse complementary to the SEQ forward sequence; the X is an interval sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to either the SEQ forward or the SEQ reverse.

[0044] Further, in the above application, the sequence of one strand of the double-stranded RNA molecule may be a sequence obtained by transcribing a DNA fragment having a nucleotide sequence from positions 244 to 501 of sequence 2 in the sequence list;

[0045] B2) The coding gene may be as shown in formula (I): SEQ reverse-X-SEQ forward (I);

[0046] The SEQ forward sequence is the 244th to 501st position of sequence 2 in the sequence list; the SEQ reverse sequence is reverse complementary to the SEQ forward sequence; the X is an interval sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to either the SEQ forward or the SEQ reverse.

[0047] That is, the SEQ forward direction is a DNA molecule whose nucleotide sequence is shown as SEQ ID No.3, and the SEQ reverse direction is a DNA molecule whose nucleotide sequence is shown as SEQ ID No.4.

[0048] Furthermore, in the above application, the RNA molecule described in B1) may be a gRNA targeting the above-mentioned IbbZIP11 protein encoding gene.

[0049] Further, in the above application, the nucleotide sequence of the target sequence of the gRNA is SEQ ID No. 8, positions 43-61 and / or the nucleotide sequence of the target sequence of the gRNA is SEQ ID No. 8, positions 117-135;

[0050] B2) The coding gene may be a DNA molecule that expresses gRNA targeting the above-mentioned IbbZIP11 protein coding gene.

[0051] Furthermore, the nucleotide sequence of the target sequence is 5′- CAGAACCCAGGTTCTGAAG -3′ (SEQ ID No. 5) and / or, the nucleotide sequence of the target sequence is different from that of 5′- AGACCTCCTCGCAGATTCA -3' (SEQ ID No. 6) reverse complement.

[0052] Furthermore, in the above application, the DNA molecule described in B9) may be a DNA molecule as shown in any one of the following b1) to b3):

[0053] b1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2;

[0054] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.7.

[0055] b3) The nucleotide sequence of the coding chain is the DNA molecule shown in SEQ ID No.8.

[0056] The inhibition, reduction or down-regulation of the expression of the IbbZIP11 protein encoding gene can be achieved by gene knockout or gene silencing.

[0057] The gene knockout refers to the phenomenon of inactivating a specific target gene through homologous recombination. Gene knockout is the inactivation of a specific target gene by changing the DNA sequence.

[0058] The gene silencing refers to the phenomenon of making the gene non-expressed or low-expressed without damaging the original DNA. Gene silencing is based on the premise of not changing the DNA sequence, making the gene non-expressed or low-expressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0059] In one embodiment of the present invention, the recombinant vector can be specifically recombinant plasmids pCAMBIA1300-IbbZIP11, pCAMBIA1300-SI-X-IbbZIP11 and pHSE401-IbbZIP11. The construction process of the recombinant plasmid pCAMBIA1300-IbbZIP11 is as follows: (1) the vector pCAMBIA1300 is double-digested with restriction endonucleases Kpn I and Sal I to obtain a vector backbone; (2) the recombinant plasmid pCAMBIA1300-IbbZIP11 obtained by replacing the fragment between the restriction endonuclease Kpn I and Sal I recognition sequences of pCAMBIA1300 with the DNA molecule shown in sequence 2 in the sequence table is obtained, and the recombinant plasmid pCAMBIA1300-IbbZIP11 expresses the protein IbbZIP11 shown in sequence 1 in the sequence table.

[0060] The construction process of the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 is as follows: (1) the vector pCAMBIA1300-35SI-X is double-digested with restriction endonucleases BamH I and Sal I to obtain a vector backbone; (2) the DNA molecule shown in sequence 3 in the sequence list is used to replace the fragment between the restriction endonuclease BamH I and Sal I recognition sequences of pCAMBIA1300-35SI-X to obtain the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11-up; (3) the vector pCAMBIA1300-35SI-X-IbbZIP11-up is double-digested with restriction endonucleases Kpn I and Xba I to obtain a vector backbone; (4) the DNA molecule shown in sequence 4 in the sequence list is used to replace the restriction endonuclease Kpn I and Xba I of pCAMBIA1300-35SI-X-IbbZIP11-up. The recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 obtained by the fragment between the I recognition sequences, the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 can interfere with the protein IbbZIP11 shown in sequence 1 in the sequence table.

[0061] The construction process of the recombinant plasmid pHSE401-IbbZIP11 is as follows: (1) Log in to the website http: / / www.crisprscan.org / ?page=sequence, copy the exon sequence of the target gene to the corresponding sequence frame, and search for high-scoring targets. Log in to the website http: / / www.rgenome.net / cas-offinder / , copy the 20-nt in front of the PAM of the high-scoring target to the corresponding sequence frame, and evaluate the off-target situation; (2) Design primers DT1-BsF and DT1-F0 according to the DNA molecule shown in sequence 5 in the sequence table, and design primers DT2-R0 and DT2-BsR according to the DNA molecule shown in sequence 6 in the sequence table; (3) Use pCBC-DT1T2 diluted 100 times as a template for four-primer PCR amplification; (4) Purify and recover the PCR product, establish an enzyme cutting-ligation system, and the recombinant plasmid pHSE401-IbbZIP11 can edit the DNA molecule shown in sequence 2 in the sequence table in vivo.

[0062] The primer pair for amplifying the full length or any fragment of the coding gene of the protein IbbZIP11 related to the development of sweet potato tubers and leaves also falls within the protection scope of the present invention.

[0063] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0064] In the present invention, the recombinant microorganism in B5) or B10) may specifically be yeast, bacteria, algae or fungi. The bacteria may be gram-positive bacteria or gram-negative bacteria. The gram-negative bacteria may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens may specifically be Agrobacterium tumefaciens EHA105.

[0065] In one embodiment of the present invention, the recombinant microorganism may specifically be EHA105 / pCAMBIA1300-IbbZIP11, EHA105 / pCAMBIA1300-35SI-X-IbbZIP11 and EHA105 / pHSE401-IbbZIP11. EHA105 / pCAMBIA1300-IbbZIP11 is the recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-IbbZIP11 into Agrobacterium tumefaciens EHA105, EHA105 / pCAMBIA1300-35SI-X-IbbZIP11 is the recombinant Agrobacterium obtained by transforming the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 into Agrobacterium tumefaciens EHA105, and EHA105 / pHSE401-IbbZIP11 is the recombinant Agrobacterium obtained by transforming the recombinant plasmid pHSE401-IbbZIP11 into Agrobacterium tumefaciens EHA105.

[0066] In the present invention, the plant tissue described in B7) or B10) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

[0067] In the present invention, the transgenic plant organ described in B8) or B10) can be the root, stem, leaf, flower, fruit and seed of the transgenic plant.

[0068] In the present invention, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ may or may not include propagation materials.

[0069] Furthermore, in the above application, the plant may be any one of the following 1)-5): 1) dicotyledonous plants; 2) tubulose plants; 3) Convolvulaceae plants; 4) Ipomoea plants; 5) sweet potato.

[0070] To solve the above technical problems, in a second aspect, the present invention provides a method for regulating plant leaf and / or root development and / or plant yield, the method comprising regulating the expression of the IbbZIP11 protein encoding gene or regulating the activity or content of the IbbZIP11 protein to regulate plant leaf and / or root development and / or plant yield.

[0071] Further, the above method may include M1) and / or M2) and / or M3):

[0072] M1), comprising introducing the coding gene shown in the formula (I) in the above application into the recipient plant, inhibiting or reducing or down-regulating the expression of the IbbZIP11 protein coding gene or the activity or content of the IbbZIP11 protein, to obtain a target plant with higher plant leaf and / or root development and / or plant yield than the recipient plant;

[0073] M2), comprising knocking out the IbbZIP11 protein encoding gene in the recipient plant by introducing the encoding gene of the gRNA molecule and the encoding gene of the Cas protein into the recipient plant, thereby obtaining a target plant with higher plant leaf and / or root development and / or plant yield than the recipient plant;

[0074] M3), introducing the coding gene described in the above application B9) into the recipient plant to promote or increase the expression of the IbbZIP11 protein coding gene or the activity or content of the IbbZIP11 protein, thereby obtaining a target plant with lower plant leaf and / or root development and / or plant yield than the recipient plant.

[0075] In the present invention, the Cas protein may be a Cas9 protein.

[0076] Furthermore, the gene encoding the Cas9 protein is located on the backbone vector pHSE401 (hyg).

[0077] Furthermore, the gene encoding the Cas9 protein is a DNA molecule whose nucleotide sequence is shown in SEQ ID No.9.

[0078] Furthermore, in the above method, M2) knocking out the IbbZIP11 protein encoding gene in the recipient plant can be achieved by the following mutation;

[0079] 1) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C46-1 gene, wherein the coding sequence of the IbbZIP11 / C46-1 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 132 of sequence 2 in the sequence list and keeping the other nucleotides of sequence 2 unchanged;

[0080] 2) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C46-2 gene, wherein the coding sequence of the IbbZIP11 / C46-2 gene is a DNA molecule obtained by deleting a total of 2 nucleotides at positions 131-132 of sequence 2 in the sequence list, replacing the nucleotide G at position 132 with A, and keeping the other nucleotides of sequence 2 unchanged;

[0081] 3) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C46-3 gene, wherein the coding sequence of the IbbZIP11 / C46-3 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 3 nucleotides at positions 131-133 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged;

[0082] 4) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C46-4 gene, wherein the coding sequence of the IbbZIP11 / C46-4 gene is a DNA molecule obtained by deleting a total of 5 nucleotides at positions 128-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0083] 5) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C46-5 gene, wherein the coding sequence of the IbbZIP11 / C46-5 gene is a DNA molecule obtained by deleting a total of 6 nucleotides at positions 127-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0084] 6) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C80-1 gene, wherein the coding sequence of the IbbZIP11 / C80-1 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged;

[0085] 7) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C80-2 gene, wherein the coding sequence of the IbbZIP11 / C80-2 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting 13 nucleotides in total at positions 122-134 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged;

[0086] 8) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C80-3 gene, wherein the coding sequence of the IbbZIP11 / C80-3 gene is a DNA molecule obtained by inserting nucleotide G at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged;

[0087] 9) mutating the IbbZIP11 gene shown in sequence 2 in the sequence list into the IbbZIP11 / C80-4 gene, wherein the coding sequence of the IbbZIP11 / C80-4 gene is a DNA molecule obtained by deleting a total of 4 nucleotides at positions 58-61 of sequence 2 in the sequence list, and deleting a total of 3 nucleotides at positions 126-128 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged;

[0088] 10) The IbbZIP11 gene shown in sequence 2 in the sequence list is mutated into the IbbZIP11 / C80-5 gene, wherein the coding sequence of the IbbZIP11 / C80-5 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 134 of sequence 2 in the sequence list and keeping the other nucleotides of sequence 2 unchanged.

[0089] In one embodiment of the present invention, the IbbZIP11 genes on the six chromosomes of the recipient sweet potato (hexaploid) mutate into the IbbZIP11 / C46-1 gene, the IbbZIP11 / C46-2 gene, the IbbZIP11 / C46-3 gene, the IbbZIP11 / C46-4 gene, and the IbbZIP11 / C46-5 gene, respectively, and the IbbZIP11 gene on one chromosome is mutated, to obtain plants with higher leaf and / or root development and / or plant yield than the recipient sweet potato.

[0090] In one embodiment of the present invention, the IbbZIP11 genes on the six chromosomes of the recipient sweet potato (hexaploid) are mutated into the IbbZIP11 / C80-1 gene, the IbbZIP11 / C80-2 gene, the IbbZIP11 / C80-3 gene, the IbbZIP11 / C80-4 gene, and the IbbZIP11 / C80-5 gene, respectively, and the IbbZIP11 gene on one chromosome is mutated, to obtain plants with higher leaf and / or root development and / or plant yield than the recipient sweet potato.

[0091] Furthermore, in the above method, the method includes introducing the coding gene described in B9) in the above application into the recipient plant, promoting or increasing the expression of the IbbZIP11 protein coding gene or the activity or content of the IbbZIP11 protein, and obtaining the target plant with lower plant leaf and / or root development and / or plant yield than the recipient plant.

[0092] Furthermore, in the above method, the DNA molecule described in B9) is a DNA molecule as shown in any one of the following b1) to b3):

[0093] b1), the coding sequence of the coding strand is the DNA molecule shown in SEQ ID No.2;

[0094] b2) The nucleotide sequence of the coding strand is the DNA molecule shown in SEQ ID No.7.

[0095] b3) The nucleotide sequence of the coding chain is the DNA molecule shown in SEQ ID No.8.

[0096] Furthermore, in the above method, the plant is any one of the following 1)-5): 1) dicotyledonous plants; 2) tubulose plants; 3) Convolvulaceae plants; 4) Ipomoea plants; 5) sweet potato.

[0097] In the above method, the coding gene is introduced into the recipient plant, specifically by transforming plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and cultivating the transformed plant tissues into plants. The transformed cells, tissues or plants are understood to include not only the final product of the transformation process, but also the materials obtained by asexual reproduction thereof and transgenic progeny.

[0098] In the above method, the plant leaf and root development indicators include photosynthetic rate, stomatal conductance, transpiration rate, chlorophyll content in leaves, number of tubers per plant, fresh weight of tubers per plant and dry matter rate of tubers.

[0099] In order to solve the above technical problems, in a third aspect, the present invention also provides the protein in the above application or the biomaterial in the above application.

[0100] The beneficial technical effects achieved by the present invention are as follows:

[0101] 1. Experiments have shown that the root and leaf development-related protein IbbZIP11 protein and its encoding gene provided by the present invention can change the root and leaf development of plants: when the IbbZIP11 gene is overexpressed in sweet potatoes, the leaves turn yellow, the photosynthetic rate decreases, the root expansion is hindered, and there is almost no yield; when the expression of the IbbZIP11 gene is interfered with and edited in sweet potatoes, the leaf color returns to normal, the photosynthetic rate is restored, the expansion is more developed and the yield is increased.

[0102] 2. The present invention provides a method for preparing high-yield sweet potatoes, and the method provided by the present invention can obtain sweet potatoes with higher yields.

[0103] 3. The present invention can improve the photosynthetic rate of sweet potatoes, enlarge the root tubers, and increase the yield by knocking out the IbbZIP11 gene through gene editing technology or interfering with the expression of the IbbZIP11 gene through dsRNA, indicating that the IbbZIP11 gene has great application value for the development of the sweet potato industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 PCR analysis of sweet potato transgenic plants.

[0105] Figure 2 This is the physical map of the DT1T2-PCR amplification product containing dual gene editing targets.

[0106] Figure 3To identify the leaf and root morphology of sweet potato transgenic plants.

[0107] Figure 4 These are the statistical results of physiological indicators of sweet potato transgenic plants.

[0108] Figure 5 Yield determination results of sweet potato transgenic plants; A is fresh weight (kg), B is the number of tubers per plant, and C is dry matter rate.

[0109] Figure 6 This is a map of the vector pCAMBIA1300-GFP. DETAILED DESCRIPTION

[0110] The sweet potato salt-tolerant mutant ND98 was preserved for this experiment and is recorded in the following literature: He Shaozhen. In vitro screening of sweet potato salt-tolerant mutants and cloning of salt-tolerant candidate genes. Doctoral dissertation of China Agricultural University, 2008; the public can obtain it from the Sweet Potato Genetics and Breeding Laboratory of China Agricultural University. The obtained materials can only be used to repeat this experiment and cannot be used for other purposes.

[0111] Cloning vector T-Vector pMD TM 19 (Simple) was purchased from Baoli Biotechnology (Dalian) Co., Ltd. with a product catalog number of 3271 and is referred to as pMD19-T in the following examples.

[0112] The vector pCAMBIA1300-GFP was kindly provided by Professor Jin Weiwei from the College of Agronomy, China Agricultural University, and is described in the following literature: Luo HS, Meng DX, Liu HB, Xie MJ, Yin CF, Liu F, Dong ZB, Jin WW. Ectopic Expression of the Transcriptional Regulator silky3 Causes Pleiotropic Meristem and Sex Determination Defects in Maize Inflorescences. Plant Cell. 2020, 32: 3750-3773; the map of the vector pCAMBIA1300-GFP is shown in Figure 6 In the following examples, it is referred to as pCAMBIA1300.

[0113] pCAMBIA1300-35SI-X was purchased from Wuhan Transduction Laboratory Co., Ltd. and was modified based on pCAMBIA1300. 35S promoter + Intro + NOS terminator were added to the MCS, and the forward and reverse sequences were inserted respectively to form a hairpin structure.

[0114] Vectors pHSE401 (hyg) and pCBC-DT1T2 were kindly provided by Professor Qijun Chen from the College of Life Sciences, China Agricultural University, and are described in the following literature: Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ. ACRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014, 14(1): 327. The public can obtain them from the applicant, and the obtained materials can only be used to repeat this experiment and cannot be used for other purposes.

[0115] PrimeScript TM 1st Strand cDNA Synthesis Kit is a product of Takara Biotech (Dalian) Co., Ltd., with the product catalog number being 6110A.

[0116] The following examples were processed using SPSS statistical software, and the experimental results were expressed as mean ± standard deviation, using T test, ns means no significant difference, P < 0.05 (*) means significant difference, P < 0.01 (**) means extremely significant difference. The quantitative tests in the following examples were repeated three times unless otherwise specified, and the results were averaged.

[0117] Example 1. Acquisition of IbbZIP11 gene

[0118] The steps for obtaining the IbbZIP11 gene are as follows:

[0119] 1. Obtaining the template

[0120] Total RNA from young leaves of sweet potato salt-tolerant mutant ND98 was extracted using a plant total RNA extraction kit and the total RNA was purified by PrimeScript TM 1st Strand cDNA Synthesis Kit was used to reverse transcribe the first-strand cDNA.

[0121] 2. According to the nucleotide sequence of the EST sequence shown in Sequence 7 in the sequence table, primers IbbZIP11-orf-F and IbbZIP11-orf-R were designed and artificially synthesized. The nucleotide sequences of IbbZIP11-orf-F and IbbZIP11-orf-R are as follows:

[0122] IbbZIP11-orf-F: 5′-ATGGCTTCCTTCTAGTGGGACAT-3′;

[0123] IbbZIP11-orf-R: 5′-TCAGTATTGCAGCATGTCAGCAG-3′.

[0124] 3. After completing step 2, using the cDNA obtained in step 1 as a template, and using the IbbZIP11-orf-F and IbbZIP11-orf-R synthesized in step 2 as primers, a PCR amplification product of about 500 bp was amplified by homologous cloning, and the above PCR amplification product was connected to the cloning vector pMD19-T to obtain recombinant plasmid 1. The recombinant plasmid 1 was sequenced to obtain the nucleotide sequence of the protein IbbZIP11.

[0125] The results show that the nucleotide sequence of the PCR amplification product obtained in step 3 is shown in sequence 2 in the sequence table. The gene shown in the sequence is named IbbZIP11 gene, the protein encoded by it is named IbbZIP11 protein or protein IbbZIP11, and the amino acid sequence is shown in sequence 1 in the sequence table.

[0126] Example 2: Construction of recombinant plasmid

[0127] A. Construction of recombinant plasmid pCAMBIA1300-IbbZIP11

[0128] 1. Artificially synthesize a double-stranded DNA molecule shown in sequence 2 of the sequence table. Use the double-stranded DNA molecule as a template and pCAMBIA1300-IbbZIP11-F (Kpn I) and pCAMBIA1300-IbbZIP11-R (Sal I) as primers for PCR amplification to obtain a double-stranded DNA molecule containing restriction endonuclease Kpn I at the N-terminus and restriction endonuclease Sal I at the C-terminus, and recover a fragment 1 of about 500 bp.

[0129] Among them, the nucleotide sequence of pCAMBIA1300-IbbZIP11-F (Kpn I) is as follows:

[0130] 5′-TACGAATTCGAGCTC GGTACC ATGGCTTCCTCTAGTGGGACAT-3′ (underlined is the recognition sequence of restriction endonuclease Kpn I);

[0131] The nucleotide sequence of pCAMBIA1300-IbbZIP11-R (Sal I) is as follows:

[0132] 5′-CTTGCATGCCTGCAG GTCGAC GTATTGCAGCATGTCAGCAG-3′ (the underline is the recognition sequence of restriction endonuclease Sal I).

[0133] 2. Double-digest the vector pCAMBIA1300 with restriction endonucleases Kpn I and Sal I, and recover the vector backbone 1 of about 11,000 bp.

[0134] 3. Connect fragment 1 to vector backbone 1 to obtain recombinant plasmid pCAMBIA1300-IbbZIP11.

[0135] According to the sequencing results, the structure of the recombinant plasmid pCAMBIA1300-IbbZIP11 is described as follows: the small fragment between the restriction endonuclease Kpn I and Sal I recognition sequences of the vector pCAMBIA1300 is replaced with the DNA molecule shown in sequence 2 in the sequence table, and the other nucleotide sequences of the pCAMBIA1300 vector are kept unchanged to obtain the recombinant plasmid. The recombinant plasmid pCAMBIA1300-IbbZIP11 expresses the IbbZIP11 protein shown in sequence 1 in the sequence table.

[0136] The recombinant plasmid pCAMBIA1300-IbbZIP11 has an expression cassette A, the nucleotide sequence of which is shown in Sequence 7 in the sequence listing, wherein positions 1 to 847 of Sequence 7 in the sequence listing from the 5′ end are the CaMV35S promoter, positions 854 to 1354 are the coding gene for the IbbZIP11 protein, and positions 1361 to 2080 are the coding gene for the GFP protein, wherein the CaMV35S promoter and the coding gene for the GFP protein are both sequences on the backbone vector.

[0137] B. Construction of recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11

[0138] 1. Using the above-mentioned recombinant plasmid pCAMBIA1300-IbbZIP11 as a template, pCAMBIA1300-35SI-X-IbbZIP11-up-F (BamH I) and pCAMBIA1300-35SI-X-IbbZIP11-up-R (Sal I) as primers for PCR amplification to obtain a double-stranded DNA molecule containing restriction endonuclease BamH I at the N-terminus and restriction endonuclease Sal I at the C-terminus, and recover a fragment of about 300 bp 2.

[0139] Among them, the nucleotide sequence of pCAMBIA1300-35SI-X-IbbZIP11-up-F (BamH I) is as follows:

[0140] 5′-ACGGGGGACTCTAGT GGATCC CTCAATGTTGAGGCTGAAAACTC-3′ (the underline is the recognition sequence of restriction endonuclease BamH I);

[0141] The nucleotide sequence of pCAMBIA1300-35SI-X-IbbZIP11-up-R (Sal I) is as follows:

[0142] 5′-AATTACCCTCTACTA GTCGAC GTATTGCAGCATGTCAGCAGC-3′ (the underline is the recognition sequence of restriction endonuclease Sal I).

[0143] 2. Double-digest the vector pCAMBIA1300-35SI-X with restriction endonucleases BamH I and Sal I, and recover the vector backbone 2 of about 10,000 bp.

[0144] 3. Connect fragment 2 to vector backbone 2 to obtain the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11-up.

[0145] 4. Using the above-mentioned recombinant plasmid pCAMBIA1300-IbbZIP11 as a template, PCR amplification was performed with pCAMBIA1300-35SI-X-IbbZIP11-down-F (Xba I) and pCAMBIA1300-35SI-X-IbbZIP11-down-R (Kpn I) as primers to obtain a double-stranded DNA molecule containing restriction endonuclease Xba I at the N-terminus and restriction endonuclease Kpn I at the C-terminus, and a fragment of about 300 bp was recovered. 3.

[0146] Among them, the nucleotide sequence of pCAMBIA1300-35SI-X-IbbZIP11-down-F (Xba I) is as follows:

[0147] 5′-TCAATTGTTAGGATT TCTAGA GTATTGCAGCATGTCAGCAGC-3′ (the underline is the recognition sequence of restriction endonuclease Xba I);

[0148] The nucleotide sequence of pCAMBIA1300-35SI-X-IbbZIP11-down-R(Kpn I) is as follows:

[0149] 5′-TTCGAGCTCAGATCT GGTACC CTCAATGTTGAGGCTGAAAACTC-3′ (the underline is the recognition sequence of restriction endonuclease Kpn I).

[0150] 5. Double-digest the vector pCAMBIA1300-35SI-X-IbbZIP11-up with restriction endonucleases Kpn I and Xba I to recover the vector backbone 3 of about 10,000 bp.

[0151] 6. Connect fragment 3 to vector backbone 3 to obtain recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11.

[0152] According to the sequencing results, the structure of the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 is described as follows: the small fragment between the restriction endonuclease BamH I and Sal I recognition sequences of the vector pCAMBIA1300-35SI-X is replaced with the DNA molecule shown in sequence 3 in the sequence table, and the small fragment between the restriction endonuclease Kpn I and Xba I recognition sequences is replaced with the DNA molecule shown in sequence 4 in the sequence table. The recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 can interfere with the expression of the IbbZIP11 protein shown in sequence 1 in the sequence table.

[0153] The nucleotide sequence of SEQ ID NO: 3 is identical to that of SEQ ID NO: 244-501.

[0154] C. Construction of recombinant plasmid pHSE401-2gR-IbbZIP11

[0155] 1. Log in to http: / / www.crisprscan.org / ?page=sequence, copy sequence 2 in the sequence list to the corresponding sequence box, and search for high-scoring targets. Log in to http: / / www.rgenome.net / cas-offinder / , copy the 20-nt before the PAM of the high-scoring target to the corresponding sequence box, and evaluate the off-target situation.

[0156] 2. Using 100-fold diluted pCBC-DT1T2 (kindly donated by Professor Qijun Chen from the College of Life Sciences, China Agricultural University) as a template, design primer DT1-BsF according to the DNA molecule shown in sequence 5 in the sequence table: 5′-ATATATGGTCTCGATTG CAGAACCCAG GTTCTGAAG GTT-3′ and DT1-F0:5′-TG CAGAACCCAGGTTCTGAAG GTTTTAGAGCTAGAAATAGC-3′ (the underline is sequence 5 in the sequence list, which is the target sequence), and primer DT2-R0: 5′-AAC was designed based on the DNA molecule shown in sequence 6 in the sequence list. AGACCTCCTCGCAGATTCA CAATCTCTTAGTCGACTCTAC-3′ and DT2-BsR: 5′-ATTATTGGTCTCGAAAC AGACCTCCTCGCAGATTCA CAA-3′ (the underline is sequence 6 in the sequence list, i.e., the reverse complementary sequence of the second target sequence), and four-primer PCR amplification was performed. DT1-BsF and DT2-BsR are normal primer concentrations; DT1-F0 and DT2-R0 are diluted 20 times. The DT1T2-PCR amplification product contains the sgRNA expression cassette for the gene editing dual targets, and its physical map is as follows Figure 2 shown.

[0157] Sequence 5 is identical to the 43rd to 61st nucleotide sequence of Sequence 8 in the sequence listing, and Sequence 6 is identical to the 117th to 135th nucleotide sequence of Sequence 8 in the sequence listing.

[0158] 3. Purify and recover the PCR product, establish the enzyme digestion-ligation system to obtain the recombinant plasmid pHSE401-IbbZIP11. The ligation system is: PCR fragment 2μl, pHSE401 (hyg) 2μl, 10x NEB T4 Buffer 1.5μl, 10x BSA 1.5μl, Bsa I (NEB) 1μl, T4 Ligase (NEB) 1μl, ddH2O 6μl, total volume 15μl; ligation reaction conditions are: 37℃ 5h, 50℃ 5min, 80℃ 10min.

[0159] The recombinant plasmid pHSE401-IbbZIP11 can edit the DNA molecule shown in sequence 8 in the sequence table in vivo.

[0160] Among them, the pHSE401 (hyg) vector contains the coding gene of the Cas9 protein, and the coding gene of the Cas9 protein is a DNA molecule with a nucleotide sequence shown in SEQ ID No.9.

[0161] Example 3. Obtaining recombinant Agrobacterium and regeneration of sweet potato transgenic plants

[0162] A. Regeneration of transgenic sweet potato plants overexpressing IbbZIP11

[0163] 1. The recombinant plasmid pCAMBIA1300-IbbZIP11 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105 / pCAMBIA1300-IbbZIP11.

[0164] 2. Peel off the stem apex meristem of Li Zixiang about 0.5 mm long, place it on an embryonic callus induction solid medium (MS solid medium containing 2.0 mg / L 2,4-D and 3.0% sucrose), and cultivate it at 27±1°C for 8 weeks to obtain embryonic callus. Then, place the embryonic callus in an embryonic callus induction liquid medium (MS liquid medium containing 2.0 mg / L 2,4-D and 3.0% sucrose), and culture it on a horizontal shaker with alternating light and dark for 3 days (specific conditions are: 100 r / min; 27°C; the cycle of alternating light and dark culture is: light time is 13 h, dark time is 11 h; light intensity is 500 lx), and obtain embryonic cell masses with a diameter of 0.7-1.3 mm.

[0165] 3. After completing step 2, the embryonic cell mass was transformed with EHA105 / pCAMBIA1300-IbbZIP11 using the Agrobacterium-mediated method, and then placed on a co-culture medium (MS solid medium containing 30 mg / L AS and 2.0 mg / L 2,4-D) and cultured in the dark at 28°C for 3 days.

[0166] 4. After completing step 3, wash the embryonic cell mass twice with MS liquid culture medium containing 900 mg / L cefotaxime sodium (CS) and 2.0 mg / L 2,4-D, and then place it on a selective culture medium (solid MS culture medium containing 2.0 mg / L 2,4-D, 300 mg / L CS and 5 mg / L hygromycin (hyg)), and culture it in the dark at 27±1°C for 6-8 weeks (the selective culture medium needs to be replaced once a week).

[0167] 5. After completing step 4, place the embryonic cell mass on somatic embryo induction medium (MS solid medium containing 1.0 mg / L ABA) and culture at 27±1°C with alternating light and dark (the cycle of alternating light and dark culture is: light time is 13 h, dark time is 11 h; light intensity is 3000 lx) for 2-4 weeks to obtain resistant callus.

[0168] 6. After completing step 5, place the resistant callus on MS solid culture medium and culture at 27±1°C with alternating light and dark (light time is 13h, dark time is 11h; light intensity is 3000lx) for 4-8 weeks to obtain 51 transgenic sweet potato plants, which are named O1 to O51 in sequence.

[0169] 7. Extract the genomic DNA of the young leaves of the transgenic sweet potato plants obtained in step 6, and use the genomic DNA as a template, 35S-F: 5′-TCCTTCGCAAGACCCTTCCTC-3′ and IbbZIP11-orf-R: 5′-TCAGTATTGCAGCATGTCAGCAG-3′ as primers to perform PCR amplification to obtain PCR amplification products; if the PCR amplification product contains a band of about 500 bp, the corresponding transgenic sweet potato plants are transgenic sweet potato positive plants. Replace the genomic DNA of the young leaves of the transgenic sweet potato plants with an equal volume of water, and perform PCR amplification as a negative control. Replace the genomic DNA of the young leaves of the transgenic sweet potato plants with the genomic DNA of the young leaves of the wild-type sweet potato variety Lizixiang, and perform PCR amplification as a control. Replace the genomic DNA of the young leaves of the transgenic sweet potato plants with the recombinant plasmid pCAMBIA1300-IbbZIP11, and perform PCR amplification as a positive control.

[0170] Some experimental results can be found in Figure 1 A in (WT is the genomic DNA of young leaves of sweet potato salt-tolerant strain Lizixiang, P is the positive control, W is the negative control, O2, O3, O4, O5, O6, O7, O8, O9, O10, O20, O21, O22, O23 and O24 are all transgenic sweet potato plants). The results showed that O2, O4, O5, O7, O8, O9, O10, O20, O22, O23 and O24 were all transgenic sweet potato positive plants.

[0171] Asexual propagation was used to propagate transgenic sweet potato positive plants, and the plants propagated from one transgenic seedling were regarded as a strain.

[0172] 8. Extract RNA from young leaves of sweet potato transgenic positive plants determined in step 7, and use RNA from young leaves of wild-type sweet potato varieties to replace RNA from young leaves of sweet potato transgenic positive plants as a control, and perform qRT-PCR amplification to detect the transcription level of IbbZIP11 gene in different transgenic lines, using the sweet potato specific actin gene (β-actin) as the internal reference gene. The primer sequences used are as follows:

[0173] qIbactin-F:5'-AGCAGCATGAAGATTAAGGTTGTAGCAC-3';

[0174] qIbactin-R:5'-TGGAAAATTAGAAGCACTTCCTGTGAAC-3';

[0175] qIbbZIP11-F:5'-GGGACATGCTCAGGGGTCATCTA-3';

[0176] qIbbZIP11-R:5'-ATTCTCTTCCTTTTCCTTGATCC-3'

[0177] Some experimental results can be found in Figure 1 The results showed that after the IbbZIP11 gene was introduced into the sweet potato variety Lizixiang, the expression level of the IbbZIP11 gene in nine transgenic lines (O2, O4, O5, O7, O8, O9, O10, O20, O22, O23 and O24) was significantly increased. Therefore, the three lines with higher expression levels, O4, O7 and O9, were selected for subsequent experiments.

[0178] B. Regeneration of sweet potato transgenic positive plants expressing IbbZIP11

[0179] 1. The recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105 / pCAMBIA1300-35SI-X-IbbZIP11.

[0180] 2. Follow the methods 2 to 6 in step A, replace EHA105 / pCAMBIA1300-IbbZIP11 with EHA105 / pCAMBIA1300-35SI-X-IbbZIP11, and keep other steps unchanged to obtain 63 sweet potato plants to be interfered with, which are named R1 to R63 in sequence.

[0181] 3. Extract the genomic DNA of the young leaves of the plants to be interfered with obtained in step 2, and use the genomic DNA as a template, 35SI-XF: 5′-GACGCACAATCCCACTATCC-3′ and 35SI-XR: 5′-GATAATCATCGCAAGACCGG-3′ as primers to perform PCR amplification to obtain PCR amplification products; if the PCR amplification product contains a band of about 900 bp, the corresponding sweet potato transgenic plants are sweet potato transgenic positive plants. Replace the genomic DNA of the young leaves of the sweet potato transgenic plants with an equal volume of water, and perform PCR amplification as a negative control. Replace the genomic DNA of the young leaves of the sweet potato transgenic plants with the genomic DNA of the young leaves of the wild-type plants of the sweet potato variety Lizixiang, and perform PCR amplification as a control. Replace the genomic DNA of the young leaves of the sweet potato transgenic plants with the recombinant plasmid pCAMBIA1300-35SI-X-IbbZIP11, and perform PCR amplification as a positive control.

[0182] Some experimental results can be found in Figure 1 B in (WT is the genomic DNA of young leaves of the salt-tolerant sweet potato strain Lizixiang, P is the positive control, W is the negative control, and R1-R21 are all transgenic sweet potato plants). The results showed that except for R1, R3, R4, and R5, the rest were transgenic sweet potato positive plants.

[0183] Asexual propagation was used to propagate transgenic sweet potato positive plants, and the plants propagated from one transgenic seedling were regarded as a strain.

[0184] The expression level of IbbZIP11 gene in different transgenic lines was detected by qRT-PCR according to the method in step A. Some experimental results are shown in Figure 1 The results showed that after pCAMBIA1300-35SI-X-IbbZIP11 was introduced into the sweet potato variety Lizixiang, the expression level of the IbbZIP11 gene in 10 transgenic lines (R3, R13, R14, R20, R21, R23, R24, R26 and R27) was significantly reduced. Therefore, two lines with lower expression levels, R20 and R23, were selected for subsequent experiments.

[0185] C. Regeneration of transgenic sweet potato plants with gene-edited IbbZIP11

[0186] 1. The recombinant plasmid pHSE401-IbbZIP11 was transformed into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium A, which was named EHA105 / pHSE401-IbbZIP11.

[0187] 2. Follow the methods 2 to 6 in step A, replace EHA105 / pCAMBIA1300-IbbZIP11 with EHA105 / pHSE401-IbbZIP11, and keep other steps unchanged to obtain 80 sweet potato plants to be interfered with, which are named C1 to C80 in sequence.

[0188] 3. Extract the genomic DNA (SEQ ID No. 8) of the young leaves of the intended interference plant obtained in step 2, and use the genomic DNA as a template, and use U626-IDF: 5′-TGTCCCAGGATTAGAATGATTAGGC-3′ and U629-IDR: 5′-AGCCCTCTTCTTTCGATCCATCAAC-3′ as primers to perform PCR amplification to obtain PCR amplification products; if the PCR amplification product contains a band of about 750 bp, the corresponding sweet potato intended transgenic plant is a sweet potato transgenic positive plant. Replace the genomic DNA of the young leaves of the sweet potato intended transgenic plant with an equal volume of water, and perform PCR amplification as a negative control. Replace the genomic DNA of the young leaves of the sweet potato intended transgenic plant with the genomic DNA of the young leaves of the sweet potato intended transgenic plant, and perform PCR amplification as a control. Replace the genomic DNA of the young leaves of the sweet potato intended transgenic plant with the genomic DNA of the young leaves of the sweet potato wild-type plant of the sweet potato variety Lizixiang, and perform PCR amplification as a control. Replace the genomic DNA of the young leaves of the sweet potato intended transgenic plant with the recombinant plasmid pHSE401-IbbZIP11, and perform PCR amplification as a positive control.

[0189] Some experimental results can be found in Figure 1 D in (WT is the genomic DNA of young leaves of the salt-tolerant sweet potato line Lizixiang, P is the positive control, W is the negative control, and C36-C56 are all transgenic sweet potato plants). The results showed that except C38, all of them were transgenic sweet potato positive plants.

[0190] The genomic DNA of the sweet potato transgenic positive plants identified above was used as a template, and HiTOM-bZIP-F and HiTOM-bZIP-R were used as primers for PCR amplification. The PCR product was sent to the laboratory of Professor Wang Kejian at the China National Rice Research Institute for sequencing. The sequencing results are shown in Figure 1 The results showed that base deletion or increase occurred at two target positions of C8, C11, C17, C22, C28, C32, C35, C40, C46, ​​C51, C55, C58, C62, C65, C68, C72 and C80 to varying degrees. Two strains, C46 and C80, were selected for subsequent experiments. Deep sequencing revealed that five copies were detected at the target position of IbbZIP11 in both strains, C46 and C80.

[0191] Among them, the nucleotide sequence of HiTOM-bZIP-F is as follows:

[0192] 5′- GGAGTGAGTACGGTGTGC ATGGCTTCCTCTAGTGGGACA-3′;

[0193] The nucleotide sequence of HiTOM-bZIP-R is as follows:

[0194] 5′- GAGTTGGATGCTGGATGGAGATGAGCCACCTGGAACACC-3′.

[0195] The transgenic sweet potato positive plants C46 and C80 were propagated asexually, and the plants propagated from one transgenic seedling were regarded as a line.

[0196] The mutant line C46 is a quintallolic mutant plant. Compared with the wild-type sweet potato chestnut fragrance, the IbbZIP11 gene is:

[0197] The IbbZIP11 gene in one chromosome mutates into the IbbZIP11 / C46-1 gene, which is a DNA molecule obtained by deleting a total of 1 nucleotide at position 132 of sequence 2 in the sequence list and keeping other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C46-1 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 132 of sequence 2 in the sequence list and keeping other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C46-1 composed of 47 amino acids;

[0198] The IbbZIP11 gene in another chromosome mutated into the IbbZIP11 / C46-2 gene, which is a DNA molecule obtained by deleting a total of 2 nucleotides at positions 131-132 of sequence 2 in the sequence list, replacing the nucleotide G at position 132 with A, and keeping the other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C46-2 gene is a DNA molecule obtained by deleting a total of 2 nucleotides at positions 131-132 of sequence 2 in the sequence list, replacing the nucleotide G at position 132 with A, and keeping the other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C46-2 composed of 47 amino acids;

[0199] The IbbZIP11 gene in another chromosome mutated into the IbbZIP11 / C46-3 gene, which is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 3 nucleotides at positions 131-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C46-3 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 3 nucleotides at positions 131-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C46-3 composed of 38 amino acids;

[0200] The IbbZIP11 gene in another chromosome mutated into the IbbZIP11 / C46-4 gene, which is a DNA molecule obtained by deleting a total of 5 nucleotides at positions 128-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C46-4 gene is a DNA molecule obtained by deleting a total of 5 nucleotides at positions 128-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C46-4 composed of 44 amino acids;

[0201] The IbbZIP11 gene in another chromosome mutated into the IbbZIP11 / C46-5 gene. The IbbZIP11 / C46-5 gene is a DNA molecule obtained by deleting a total of 6 nucleotides at positions 127-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C46-5 gene is a DNA molecule obtained by deleting a total of 6 nucleotides at positions 127-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C46-5 composed of 166 amino acids.

[0202] The mutant line C80 is a quintallolic mutant plant. Compared with the wild-type sweet potato chestnut fragrance, the IbbZIP11 gene is:

[0203] The IbbZIP11 gene in one chromosome mutates into the IbbZIP11 / C80-1 gene, the IbbZIP11 / C80-1 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C80-1 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C80-1 composed of 38 amino acids;

[0204] The IbbZIP11 gene in one chromosome mutates into the IbbZIP11 / C80-2 gene, the IbbZIP11 / C80-2 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting 13 nucleotides in total at positions 122-134 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C80-2 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting 13 nucleotides in total at positions 122-134 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C80-2 composed of 38 amino acids;

[0205] The IbbZIP11 gene in one chromosome mutates into the IbbZIP11 / C80-3 gene, the IbbZIP11 / C80-3 gene is a DNA molecule obtained by inserting nucleotide G at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C80-3 gene is a DNA molecule obtained by inserting nucleotide G at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C80-3 composed of 38 amino acids;

[0206] The IbbZIP11 gene in one chromosome mutates into the IbbZIP11 / C80-4 gene, the IbbZIP11 / C80-4 gene deletes a total of 4 nucleotides at positions 58-61 of sequence 2 in the sequence list, and deletes a total of 3 nucleotides at positions 126-128 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged to obtain a DNA molecule, resulting in premature termination of translation; the coding sequence of the IbbZIP11 / C80-4 gene is a DNA molecule obtained by deleting a total of 4 nucleotides at positions 58-61 of sequence 2 in the sequence list, and deleting a total of 3 nucleotides at positions 126-128 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C80-4 composed of 25 amino acids;

[0207] The IbbZIP11 gene in a chromosome mutates into the IbbZIP11 / C80-5 gene. The IbbZIP11 / C80-5 gene deletes a total of 1 nucleotide at position 134 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged to obtain a DNA molecule, resulting in premature termination of translation. The coding sequence of the IbbZIP11 / C80-5 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 134 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged, encoding a protein IbbZIP11 / C80-5 composed of 47 amino acids.

[0208] Example 4: Morphological Identification

[0209] 1. Leaf morphology identification of transgenic plants

[0210] The sweet potato plants are wild-type plants (WT), plants of O4, plants of O7, plants of O9, plants of R20, plants of R23, plants of C46, ​​and plants of C80 of the sweet potato variety Lizixiang.

[0211] The experimental results are shown in Figure 3 , the above sweet potato plants planted in the field were observed, and the leaf morphology of the transgenic strains was significantly different from that of the wild-type plants. The leaves of the overexpression strains turned yellow and the leaves were narrow and irregular at the edges; the leaves of the interference and gene-edited strains turned green and the leaves were normal heart-shaped. Therefore, overexpression of the IbbZIP11 gene in sweet potatoes can lead to poor leaf development, while interference and editing of the expression of the IbbZIP11 gene promote leaf development.

[0212] 2. Root morphology identification of transgenic plants

[0213] The sweet potato plants are wild-type plants (WT), plants of O4, plants of O7, plants of O9, plants of R20, plants of R23, plants of C46, ​​and plants of C80 of the sweet potato variety Lizixiang.

[0214] The experimental results are shown in Figure 3 , the above sweet potato plants planted in the field were observed, and the root expansion of the transgenic strains was significantly different from that of the wild-type plants. The root expansion of the overexpression strains was blocked, and there was no expanded tuber; the root expansion of the interference and gene editing strains was more developed. Therefore, overexpression of the IbbZIP11 gene in sweet potato can inhibit the development of sweet potato tubers, while interference and editing of the expression of the IbbZIP11 gene promote the development of sweet potato tubers.

[0215] 3. Determination of physiological and biochemical indicators

[0216] (1) Determination of photosynthetic indexes and chlorophyll content

[0217] Photosynthesis is the process by which plants use light energy to assimilate CO2 and H2O into organic matter and release O2. It is the main way for plants to produce organic matter. Photosynthesis is mainly carried out in the mesophyll cells of leaves. The organic matter produced in the chloroplasts is continuously transported to other parts, including flowers, stems, roots, etc. for consumption and storage. Therefore, the yield traits as a sink often have a certain relationship with the photosynthetic performance as a source. The photosynthetic rate, transpiration rate, stomatal conductance, etc. of plants can be used as a physiological indicator to measure yield.

[0218] The chlorophyll content of sweet potato leaves was detected according to the method of reference (Zhao Haibo, Lin Qi, Sun Xusheng, et al. Effects of nitrogen and phosphorus application on photosynthetic characteristics and yield of Jimai 22 after flowering [J]. Journal of Wheat Crops, 2009, 029(004): 663-667). The sweet potato plants were wild-type plants (WT), O4 plants, O7 plants, O9 plants, R20 plants, R23 plants, C46 plants, and C80 plants of the sweet potato variety Lizixiang grown in the field for 100 days. 14 different third expanded leaves were measured for each strain, and the results were averaged.

[0219] The experimental results are shown in Figure 4 A. The results showed that the chlorophyll content of O4, O7 and O9 plants was significantly lower than that of the wild-type plants (WT) (46%-48%, n=14), and the chlorophyll content of R20, R23, C46 and C80 plants was higher than that of the wild-type plants (WT) (2%-7%, n=14).

[0220] The photosynthetic rate, transpiration rate and stomatal conductance of sweet potato leaves were detected according to the method of reference (Zhao Haibo, Lin Qi, Sun Xusheng, et al. Effects of nitrogen and phosphorus application on photosynthetic characteristics and yield of Jimai 22 after flowering [J]. Journal of Wheat Crops, 2009, 029(004): 663-667). The sweet potato plants were wild-type plants (WT), O4 plants, O7 plants, O9 plants, R20 plants, R23 plants, C46 plants and C80 plants of the sweet potato variety Lizixiang grown in the field for 100 days. Six different third expanded leaves were measured for each strain, and the results were averaged.

[0221] The experimental results are shown in Figure 4The results showed that compared with the wild-type plants (WT), the photosynthetic rate of the plants overexpressing the O4, O7 and O9 lines was significantly reduced (47%-54%, n=6), the transpiration rate was significantly reduced (22%-34%, n=6), and the stomatal conductance was significantly reduced (33%-65%, n=6). The photosynthetic rate of the plants of the interference lines R20 and R23 increased (2%-5%, n=6), the transpiration rate increased (26%-36%, n=6), and the stomatal conductance had no significant difference. The photosynthetic rate of the plants of C46 and C80 was slightly increased (5%-6%, n=6), the transpiration rate increased (20%-28%, n=6), and the stomatal conductance had no significant difference.

[0222] (2) Yield measurement

[0223] The number of tubers per plant, fresh weight of tubers per plant, and dry weight of tubers per plant of the wild-type, overexpression, interference, and gene-edited sweet potato varieties of Lizixiang grown in the field for 100 days were counted. Three biological replicates were set for each strain, and the results were averaged. Wash the tubers, cut them into long and thin strips, weigh about 100 g and put them in a kraft paper bag, dry them in an oven at 80°C until constant weight, and calculate the percentage of dry weight to fresh weight as the dry matter ratio.

[0224] The experimental results are shown in Figure 5 The results showed that the overexpression strains had difficulty in forming tubers, did not produce tubers, and had no yield; the number of tubers in the interference strains was significantly increased compared with the wild type (146%-153%, n=3), and the fresh weight was significantly increased (162%-180%, n=3); the number of tubers in the gene-edited strains was increased compared with the wild type (92%-153%, n=3), and the fresh weight was significantly increased (179%-185%, n=3).

[0225] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art. Sequence Listing <110> China Agricultural University <120> Sweet potato root and leaf development-related protein IbbZIP11 and its encoding gene and application <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 167 <212> PRT <213> Sweet potato (Ipomoea batatas) <400> 1 Met Ala Ser Ser Ser Gly Thr Cys Ser Gly Ser Ser Met Met Gln Asn 1 5 10 15 Pro Gly Ser Glu Gly Asp Leu Gln Gln Leu Met Asp Gln Arg Lys Arg 20 25 30 Lys Arg Met Ile Ser Asn Arg Glu Ser Ala Arg Arg Ser Arg Met Arg 35 40 45 Lys Gln Lys His Leu Asp Asp Leu Val Phe Gln Val Ala His Leu Arg 50 55 60 Lys Glu Asn Ser Gln Ile Ile Thr Ser Met Ser Ile Thr Thr Gln His 65 70 75 80 Tyr Leu Asn Val Glu Ala Glu Asn Ser Ile Leu Arg Ala Gln Met Ala 85 90 95 Glu Leu Ser His Arg Leu Gln Ser Leu Asn Glu Ile Met Ser Phe Leu 100 105 110 Asn Gly Asn Asn Asn Leu Gly Phe Glu Ile Glu Asp Met Gln Gln Gln 115 120 125 Gln Pro Tyr Leu Tyr Glu Thr Leu Asp Asp Gly Phe Phe Met Asn Asn 130 135 140 Ser Cys Trp Asn Phe Val Cys Val Asn Lys Pro Ile Met Ala Thr Ala 145 150 155 160 Ala Ala Asp Met Leu Gln Tyr 165 <210> 2 <211> 504 <212> DNA <213> Sweet potato (Ipomoea batatas) <400> 2 atggcttcct ctagtgggac atgctcaggg tcatctatga tgcagaaccc aggttctgaa 60 ggggatcttc aacaattgat ggatcagagg aaaaggaaga gaatgatatc taaccgtgaa 120 tctgcgagga ggtctaggat gaggaaacag aagcatctgg atgatctggt gttccaggtg 180 gctcatctga ggaaagagaa cagtcagatc atcaccagca tgagcatcac cacccagcat 240 tatctcaatg ttgaggctga aaactccatc ctcagagctc aaatggctga gcttagccac 300 aggctgcagt ccctgaatga gattatgagt ttcttgaatg gaaacaataa tttgggtttt 360 gagattgagg atatgcagca acagcagcct tacctttatg aaactctgga tgatgggttc 420 tttatgaaca acagttgttg gaattttgtg tgtgtcaaca agcccatcat ggctactgca 480 gctgctgaca tgctgcaata ctga 504 <210> 3 <211> 258 <212> DNA <213> Sweet potato (Ipomoea batatas) <400> 3 ctcaatgttg aggctgaaaa ctccatcctc agagctcaaa tggctgagct tagccacagg 60 ctgcagtccc tgaatgagat tatgagtttc ttgaatggaa acaataattt gggttttgag 120 attgaggata tgcagcaaca gcagccttac ctttatgaaa ctctggatga tgggttcttt 180 atgaacaaca gttgttggaa ttttgtgtgt gtcaacaagc ccatcatggc tactgcagct 240 gctgacatgc tgcaatac 258 <210> 4 <211> 258 <212> DNA <213> Artificial Sequence <400> 4 gtattgcagc atgtcagcag ctgcagtagc catgatgggc ttgttgacac acacaaaatt 60 ccaacaactg ttgttcataa agaacccatc atccagagtt tcataaaggt aaggctgctg 120 ttgctgcata tcctcaatct caaaacccaa attattgttt ccattcaaga aactcataat 180 ctcattcagg gactgcagcc tgtggctaag ctcagccatt tgagctctga ggatggagtt 240 ttcagcctca acattgag 258 <210> 5 <211> 19 <212> DNA <213> Sweet potato (Ipomoea batatas) <400> 5 cagaacccag gttctgaag 19 <210> 6 <211> 19 <212> DNA <213> Sweet potato (Ipomoea batatas) <400> 6 tgaatctgcg aggaggtct 19 <210> 7 <211> 2080 <212> DNA <213> Artificial Sequence <400> 7 agattagcct tttcaatttc agaaagaatg ctaacccaca gatggttaga gaggcttacg 60 cagcaggtct catcaagacg atctacccga gcaataatct ccaggaaatc aaataccttc 120 ccaagaaggt taaagatgca gtcaaaagat tcaggactaa ctgcatcaag aacacagaga 180 aagatatatt tctcaagatc agaagtacta ttccagtatg gacgattcaa ggcttgcttc 240 acaaaccaag gcaagtaata gagattggag tctctaaaaa ggtagttccc actgaatcaa 300 aggccatgga gtcaaagatt caaatagagg acctaacaga actcgccgta aagactggcg 360 aacagttcat acagagtctc ttacgactca atgacaagaa gaaaatcttc gtcaacatgg 420 tggagcacga cacacttgtc tactccaaaa atatcaaaga tacagtctca gaaccaaa 480 gggcaattga gacttttcaa caaagggtaa tatccggaaa cctcctcgga ttccattgcc 540 cagctatctg tcactttatt gtgaagatag tggaaagga aggtggctcc tacaaatgcc 600 660 atggaccccc acccacgagg agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa 720 780 cttcgcaaga cccttcctct atataaggaa gttcatttca tttggagaga acacggggga 840 cgagctcggt accatggctt cctctagtgg gacatgctca gggtcatcta tgatgcagaa 900 cccaggttct gaaggggatc ttcaacaatt gatggatcag agaaaagga agaatgat 960 atctaaccgt gaatctgcga ggaggtctag gatgagaaa cagaagcatc tggatgatct 1020 ggtgttccag gtggctcatc tgaggaaga gaacgtcag atcatcacca gcatgagcat 1080 caccacccag cattatctca atgttgaggc tgaaaactcc atcctcagag ctcaaatggc 1140 tgagcttagc cacaggctgc agtccctga tgagattatg agtttcttga atggaaacaa taatttgggt tttgagattg aggatatgca gcaacagcag ccttaccttt atgaaactct ggatgatggg ttctttatga acaacagttg ttggaatttt gtgtgtgtca acaagcccat catggctact gcagctgctg acatgctgca atacgtcgac atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa 1440 gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta 1560 cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta 1680. caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa 1740. gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacaa 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860. 1860 gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac 1920 ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca cccagtccgc 1980 cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc 2040 cgccgggatc actctcggca tggacgagct gtacaagtaa 2080 <210> 8 <211> 504 <212> DNA <213> Sweet potato (Ipomoea batatas) <400> 8 atggcttcct ctagtgggac atgctcaggg tcatctatga tgcagaaccc aggttctgaa 60 ggggatcttc aacaattgat ggatcagagg aaaaggaaga gaatgatatc taaccgtgaa 120 tctgcgagga ggtctaggat gaggaaacag aagcatctgg atgatctggt gttccaggtg 180 gctcatctga ggaaagagaa cagtcagatc atcaccagca tgagcatcac cacccagcat 240 tatctcaatg ttgaggctga aaactccatc ctcagagctc aaatggctga gcttagccac 300 aggctgcagt ccctgaatga gattatgagt ttcttgaatg gaaacaataa tttgggtttt 360 gagattgagg atatgcagca acagcagcct tacctttatg aaactctgga tgatgggttc 420 tttatgaaca acagttgttg gaattttgtg tgtgtcaaca agcccatcat ggctactgca 480 gctgctgaca tgctgcaata ctga 504 <210> 9 <211> 4101 <212> DNA <213> Artificial Sequence <400> 9 gacaagaagt actcgatcgg cctcgatatt gggactaact ctgttggctg ggccgtgatc 60 accgacgagt acaaggtgcc ctcaaagaag ttcaaggtcc tgggcaacac cgatcggcat 120 tccatcaaga agaatctcat tggcgctctc ctgttcgaca gcggcgagac ggctgaggct 180 acgcggctca agcgcaccgc ccgcaggcgg tacacgcgca ggaagaatcg catctgctac 240 ctgcaggaga ttttctccaa cgagatggcg aaggttgacg attctttctt ccacaggctg 300 gaggagtcat tcctcgtgga ggaggataag aagcacgagc ggcatccaat cttcggcaac 360 attgtcgacg aggttgccta ccacgagaag taccctacga tctaccatct gcggaagaag 420 ctcgtggact ccacagataa ggcggacctc cgcctgatct acctcgctct ggcccacatg 480 attaagttca ggggccattt cctgatcgag ggggatctca acccggacaa tagcgatgtt 540 gacaagctgt tcatccagct cgtgcagacg tacaaccagc tcttcgagga gaaccccatt 600 aatgcgtcag gcgtcgacgc gaaggctatc ctgtccgcta ggctctcgaa gtctcggcgc 660 ctcgagaacc tgatcgccca gctgccgggc gagaagaaga acggcctgtt cgggaatctc 720 attgcgctca gcctggggct cacgcccaac ttcaagtcga atttcgatct cgctgaggac 780 gccaagctgc agctctccaa ggacacatac gacgatgacc tggataacct cctggcccag 840 atcggcgatc agtacgcgga cctgttcctc gctgccaaga atctgtcgga cgccatcctc 900 ctgtctgata ttctcagggt gaacaccgag attacgaagg ctccgctctc agcctccatg 960 atcaagcgct acgacgagca ccatcaggat ctgaccctcc tgaaggcgct ggtcaggcag 1020 cagctccccg agaagtacaa ggagatcttc ttcgatcagt cgaagaacgg ctacgctggg 1080 tacattgacg gcggggcctc tcaggaggag ttctacaagt tcatcaagcc gattctggag 1140 aagatggacg gcacggagga gctgctggtg aagctcaatc gcgaggacct cctgaggaag 1200 cagcggacat tcgataacgg cagcatccca caccagattc atctcgggga gctgcacgct 1260 atcctgagga ggcaggagga cttctaccct ttcctcaagg ataaccgcga gaagatcgag 1320 aagattctga ctttcaggat cccgtactac gtcggcccac tcgctaggg caactcccgc 1380 ttcgcttgga tgacccgcaa gtcagaggag acgatcacgc cgtggaactt cgaggaggtg 1440 gtcgacaagg gcgctagcgc tcagtcgttc atcgagagga tgacgaattt cgacaagaac 1500 ctgccaaatg agaaggtgct ccctaagcac tcgctcctgt acgagtactt cacagtctac 1560 aacgagctga ctaaggtgaa gtatgtgacc gagggcatga ggaagccggc tttcctgtct 1620 ggggagcaga agaaggccat cgtggacctc ctgttcaaga ccaaccggaa ggtcacggtt 1680 aagcagctca aggagacta cttcaagaag attgagtgct tcgattcggt cgagatctct 1740 ggcgttgagg accgcttcaa cgcctccctg gggacctacc acgatctcct gaagatcatt 1800 aaggataagg acttcctgga caacgaggag aatgaggata tcctcgagga cattgtgctg 1860 acactcactc tgttcgagga ccgggagatg atcgaggac gcctgaagac ttacgcccat 1920 ctcttcgatg acaaggtcat gaagcagctc aagaggagga ggtacaccgg ctgggggagg 1980 ctgagcagga agctcatcaa cggcattcgg gacaagcagt ccgggaagac gatcctcgac ttcctgaaga gcgatggctt cgcgaaccgc aatttcatgc agctgattca cgatgacagc ctcacattca aggaggatat ccagaaggct caggtgagcg gccagggga ctcgctgcac gagcatatcg cgaacctcgc tggctcgcca gctatcaaga aggggattct gcagaccgtg aaggttgtgg acgagctggt gaaggtcatg ggcaggcaca agcctgagaa catcgtcatt gagatggccc gggagatca gaccacgcag aagggccaga agaactcacg cgagaggatg aagaggatcg aggagggcat taaggagctg gggtcccaga tcctcaagga gcacccggtg 2400. gagaacacgc agctgcaga tgagaagctc tacctgtact acctccaga tggccgcgat atgtatgtgg accaggagct ggatattaac aggctcagcg attack cgatcatatc gttccacagt cattcctgaa ggatgactcc attgacaaca aggtcctcac caggtcggac aagaaccggg gcaagtctga taatgttcct tcagaggagg tcgttaagaa gatgaagaac tactggcgcc agctcctga tgccaagctg atcacgcagc ggaagttcga taacctcaca aaggctgaga ggggcgggct ctctgagctg gacaaggcgg gcttcatcaa gaggcagctg 2760 gtcgagacac ggcagatcac taagcacgtt gcgcagattc tcgactcacg gatgaacact 2820 aagtacgatg agaatgacaa gctgatccgc gaggtgaagg tcatcaccct gaagtcaaag 2880 ctcgtctccg acttcaggaa ggatttccag ttctacaagg ttcggggagat caacaattac 2940 caccatgccc atgacgcgta cctgaacgcg gtggtcggca cagctctgat caagagtac 3000 ccaaagctcg agagcgagtt cgtgtacggg gactacaagg tttacgatgt gaggaagatg 3060 atcgccaagt cggagcag gattggcaag gctaccgcca agtacttctt ctactctaac 3120 attatgaatt tcttcaagac agagatcact ctggccaatg gcgagatccg gaagcgcccc 3180 ctcatcgaga cgaacggga gacgggggag atcgtgtggg aaagggcag ggatttcgcg 3240 accgtcagga aggttctctc catgccacaa gtgaatatcg tcaagaagac agaggtccag 3300 actggcgggt tctctaaagga gtcaattctg cctaagcgga acagcgacaa gctcatcgcc 3360 cgcaagaagg actgggatcc gaagtac ggcgggttcg acagccccac tgtggcctac 3420 tcggtcctgg ttgtggcgaa ggttgagaag ggcaagtcca agaagctcaa gagcgtgaag 3480 gagctgctgg ggatcacgat tatggagcgc tccagcttcg agaagaaccc gatcgatttc 3540 ctggaggcga agggctacaa ggaggtgaag aaggacctga tcattaagct ccccaagtac 3600 tcactcttcg agctggagaa cggcaggaag cggatgctgg cttccgctgg cgagctgcag 3660 aaggggaacg agctggctct gccgtccaag tatgtgaact tcctctacct ggcctcccac 3720 tacgagaagc tcaagggcag ccccgaggac aacgagcaga agcagctgtt cgtcgagcag 3780 cacaagcatt acctcgacga gatcattgag cagatttccg agttctccaa gcgcgtgatc 3840 ctggccgacg cgaatctgga taaggtcctc tccgcgtaca acaagcaccg cgacaagcca 3900 atcagggagc aggctgagaa tatcattcat ctcttcaccc tgacgaacct cggcgcccct 3960 gctgctttca agtacttcga cacaactatc gatcgcaaga ggtacacaag cactaaggag 4020 gtcctggacg cgaccctcat ccaccagtcg attaccggcc tctacgagac gcgcatcgac 4080 ctgtctcagc tcgggggcga c 4101

Claims

1. Use of a substance that reduces the expression of a gene encoding an IbbZIP11 protein in promoting plant leaf development and / or plant root development and / or increasing plant yield; the IbbZIP11 protein is a protein whose amino acid sequence is SEQ ID No.1; the encoding gene is a DNA molecule whose coding sequence is SEQ ID No.2; the substance that reduces the expression of the gene encoding the IbbZIP11 protein is a biological material, and the biological material is any one of the following B1) to B5): B1), an RNA molecule that inhibits or reduces the expression of the gene encoding the IbbZIP11 protein; B2), expressing the coding gene of the RNA molecule described in B1); B3), an expression cassette containing the coding gene described in B2); B4), a recombinant vector containing the coding gene described in B2) or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the coding gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); The plant is sweet potato.

2. The use according to claim 1, characterized in that: B1) The RNA molecule is an RNA transcribed from a DNA molecule as shown in formula (I): SEQ reverse-X-SEQ forward (I); The SEQ forward sequence is the 244th to 501st position of sequence 2 in the sequence list; the SEQ reverse sequence is reversely complementary to the SEQ forward sequence; the X is an interval sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse; or / and, B1) The RNA molecule is a gRNA targeting the protein-coding gene of claim 1, and the nucleotide sequence of the target sequence of the gRNA is positions 43-61 of SEQ ID No. 8 and / or the nucleotide sequence of the target sequence of the gRNA is positions 117-135 of SEQ ID No.

8.

3. The use according to claim 1, characterized in that: B2) The coding gene is as shown in formula (I): SEQ reverse-X-SEQ forward (I); The SEQ forward sequence is the 244th to 501st position of sequence 2 in the sequence list; the SEQ reverse sequence is reversely complementary to the SEQ forward sequence; the X is an interval sequence between the SEQ forward and the SEQ reverse, and the X is not complementary to the SEQ forward and the SEQ reverse; or, B2) The coding gene is a DNA molecule that expresses gRNA targeting the above-mentioned IbbZIP11 protein coding gene.

4. Use of the biological material according to any one of claims 1 to 3 in sweet potato breeding, wherein the purpose of the sweet potato breeding includes cultivating high-yield sweet potatoes.

5. A method for promoting sweet potato leaf development and / or sweet potato root development and / or increasing sweet potato yield, characterized in that: The method comprises introducing the coding gene shown in formula (I) in the application of claim 2 or 3 into the recipient sweet potato, inhibiting the expression of the IbbZIP11 protein coding gene to promote the leaf development of the recipient sweet potato and / or promote the root development of the recipient sweet potato and / or increase the yield of the recipient sweet potato.

6. A method for promoting sweet potato leaf development and / or sweet potato root development and / or increasing sweet potato yield, characterized in that: The method comprises introducing the coding gene of the gRNA molecule and the coding gene of the Cas protein in claim 2 or 3 into the recipient sweet potato to knock out the IbbZIP11 protein coding gene in the recipient sweet potato, so as to promote the leaf development of the recipient sweet potato and / or promote the root development of the recipient sweet potato and / or increase the yield of the recipient sweet potato.

7. A method for inhibiting the development of sweet potato leaves and / or inhibiting the development of sweet potato roots and / or reducing the yield of sweet potato, characterized in that: The method comprises introducing the coding gene of the application described in claim 1 into the recipient sweet potato, promoting or increasing the expression of the IbbZIP11 protein coding gene in the recipient sweet potato, so as to inhibit the leaf development of the recipient sweet potato and / or inhibit the root development of the recipient sweet potato and / or reduce the yield of the recipient sweet potato.

8. The method according to claim 6, characterized in that: The knockout of the IbbZIP11 protein encoding gene in the recipient sweet potato is to mutate the IbbZIP11 protein encoding gene shown in SEQ ID No.2 into the IbbZIP11 / C46-1 gene, the IbbZIP11 / C46-2 gene, the IbbZIP11 / C46-3 gene, the IbbZIP11 / C46-4 gene, IbbZIP11 / C46-5 gene, the IbbZIP11 / C80-1 gene, IbbZIP11 / C80-2 gene, IbbZIP11 / C80-3 gene, IbbZIP11 / C80-4 gene or IbbZIP11 / C80-5 Gene, Said IbbZIP11 The coding sequence of the / C46-1 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C46-2 gene is a DNA molecule obtained by deleting a total of 2 nucleotides at positions 131-132 of sequence 2 in the sequence list, replacing the nucleotide G at position 132 with A, and keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C46-3 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 3 nucleotides at positions 131-133 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C46-4 gene is a DNA molecule obtained by deleting a total of 5 nucleotides at positions 128-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C46-5 gene is a DNA molecule obtained by deleting a total of 6 nucleotides at positions 127-132 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C80-1 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C80-2 gene is a DNA molecule obtained by inserting nucleotide A at positions 58-59 of sequence 2 in the sequence list, deleting 13 nucleotides in total at positions 122-134 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C80-3 gene is a DNA molecule obtained by inserting nucleotide G at positions 58-59 of sequence 2 in the sequence list, deleting a total of 5 nucleotides at positions 129-133 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C80-4 gene is a DNA molecule obtained by deleting a total of 4 nucleotides at positions 58-61 of sequence 2 in the sequence list, deleting a total of 3 nucleotides at positions 126-128 of sequence 2 in the sequence list, and keeping the other nucleotides of sequence 2 unchanged; Said IbbZIP11 The coding sequence of the / C80-5 gene is a DNA molecule obtained by deleting a total of 1 nucleotide at position 134 of sequence 2 in the sequence list, while keeping the other nucleotides of sequence 2 unchanged.

9. Protein, characterized in that: The protein is the IbbZIP11 protein described in claim 1.

10. A DNA molecule encoding the protein according to claim 9.

11. The DNA molecule according to claim 10, characterized in that The nucleotide sequence of the DNA molecule is SEQ ID No.

2.

12. An expression cassette comprising the DNA molecule of claim 10 or 11.

13. A recombinant vector comprising the DNA molecule according to claim 10 or 11 or the expression cassette according to claim 12.

14. A recombinant microorganism comprising the DNA molecule according to claim 10 or 11, the expression cassette according to claim 12 or the recombinant vector according to claim 13.

15. The RNA molecule according to B1) of any one of claims 1 to 3.

16. The coding gene described in B2) of any one of claims 1 to 3.

17. The expression cassette of any one of claims 1 to 3.

18. The recombinant vector according to any one of claims 1 to 3.

19. The recombinant microorganism according to any one of claims 1 to 3.

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