Application of a protein or a gene encoding the protein negatively regulating the length of cucumber stem to increase the length of cucumber stem

Abstract

This invention provides the application of a protein negatively regulating the cucumber pedicel length trait, or the gene encoding said protein, in the growth of cucumber pedicels, belonging to the field of gene editing technology. In this invention, the protein CsIAA16 and its encoding gene are closely related to the pedicel length of cucumbers and the pedicel length of female cucumber flowers. This invention uses the CRISPR / Cas9 system combined with Agrobacterium-mediated genetic transformation technology to perform targeted genome editing on wild-type cucumbers, successfully constructing two homozygous mutant lines. Compared with the wild type, the female flower pedicels of the two knockout mutants are significantly longer. This invention utilizes the protein CsIAA16, or the gene encoding said protein CsIAA16, which negatively regulates the cucumber pedicel length trait. CsaV3_1 G0 1520 CsaV3_1 G0 152 It can lengthen the cucumber stem, which helps in the development of cucumber varieties suitable for mechanized harvesting.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of gene editing, and particularly relates to application of a protein negatively regulating a cucumber peduncle length trait or a coding gene of the protein in increasing a cucumber peduncle. BACKGROUND

[0002] Cucumber is a common vegetable crop. At present, cucumber mechanized harvesting technology is developing rapidly. The length of cucumber peduncle is related to mechanized harvesting. A proper long cucumber peduncle is conducive to mechanized harvesting. At present, there are few studies on the regulation of cucumber peduncle length. SUMMARY

[0003] The application aims to provide application of a protein negatively regulating a cucumber peduncle length trait or a coding gene of the protein in increasing a cucumber peduncle, which is helpful for cultivating a cucumber variety suitable for mechanized picking.

[0004] The application provides a protein CsIAA16 related to a cucumber peduncle length trait or a coding gene of the protein CsIAA16 CsaV3_1G045520 in regulating a cucumber peduncle length and / or a cucumber female flower peduncle length;

[0005] The amino acid sequence of the protein CsIAA16 is shown as SEQ ID NO. 1.

[0006] The application further provides a protein CsIAA16 negatively regulating a cucumber peduncle length trait or a coding gene of the protein CsIAA16 CsaV3_1G045520 in increasing a cucumber peduncle and / or a cucumber female flower peduncle;

[0007] The amino acid sequence of the protein CsIAA16 is shown as SEQ ID NO. 1.

[0008] Preferably, the nucleotide sequence of the coding gene CsaV3_1G045520 is shown as SEQ ID NO. 2.

[0009] Preferably, the negative regulation comprises knockout.

[0010] The application further provides an sgRNA combination targeting the coding gene CsaV3_1G045520 , wherein the sgRNA combination comprises sgRNA1 and sgRNA2; the nucleotide sequence of the sgRNA1 is shown as SEQ ID NO. 3; the nucleotide sequence of the sgRNA2 is shown as SEQ ID NO. 4; and the nucleotide sequence of the coding gene CsaV3_1G045520 is shown as SEQ ID NO. 2.

[0011] The application also provides a CRISPR / Cas9 gene editing system comprising the sgRNA combination and the Cas9 protein described in the above scheme.

[0012] The application also provides application of the sgRNA combination described in the above scheme or the CRISPR / Cas9 gene editing system in at least one of the following: 1) increasing the length of cucumber peduncle; 2) increasing the length of cucumber female flower peduncle; 3) editing a coding gene CsaV3 1G045520 ; and 4) cultivating a cucumber variety suitable for mechanical picking.

[0013] The application also provides a method for increasing the length of cucumber peduncle and / or cucumber female flower peduncle, comprising the following steps: causing loss of function of protein CsIAA16 by performing gene editing on a coding region of a coding gene CsaV3_1G045520 , so as to increase the length of cucumber peduncle and / or cucumber female flower peduncle; the amino acid sequence of the protein CsIAA16 is shown as SEQ ID NO. 1; and the nucleotide sequence of the coding gene CsaV3_1G045520 is shown as SEQ ID NO. 2.

[0014] Preferably, the gene editing is performed by using the CRISPR / Cas9 gene editing system described in the above scheme.

[0015] Preferably, the gene editing comprises: transforming cucumber under mediation of Agrobacterium by using the CRISPR / Cas9 gene editing system, and screening to obtain a gene editing homozygous transgenic line.

[0016] The application provides application of a protein CsIAA16 or a coding gene CsaV3_1G045520 of the protein CsIAA16 that are negatively regulated in relation to the length of cucumber peduncle and / or the length of cucumber female flower peduncle in increasing the length of cucumber peduncle and / or the length of cucumber female flower peduncle. In the application, the protein CsIAA16 and its coding gene are closely related to the length of cucumber peduncle and the length of cucumber female flower peduncle. It has been verified that, by using the CRISPR / Cas9 system in combination with the Agrobacterium-mediated genetic transformation technology to perform targeted genome editing on wild-type cucumber, two homozygous mutant lines are successfully constructed. Phenotype observation finds that, compared with the wild type, the female flower peduncle of the two knockout mutants is significantly longer; and t test is performed on the field investigation data, and the length of the female flower peduncle of the two knockout mutants is significantly longer than the average length of the wild type (P<0.01). By negatively regulating the protein CsIAA16 or the coding gene CsaV3_1G045520 of the protein CsIAA16 that are related to the length of cucumber peduncle, the length of cucumber peduncle can be increased, which is helpful for cultivating a cucumber variety suitable for mechanical picking. BRIEF DESCRIPTION OF DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the accompanying drawings needed in the embodiments will be briefly introduced as follows. Obviously, the accompanying drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without any creative effort.

[0018] Figure 1 For CsIAA16 The mutant strain gene structure schematic diagram and the phenotype comparison diagram of the female and male flower peduncle of the knockout mutant; wherein A is CsIAA16 The gene editing target point, gene structure and coding protein sequence schematic diagram, wherein the wild type (CU2) CsIAA16 The gene structure and the positions of the two CRISPR / Cas9 editing target points (target point 1 and target point 2) (annotated by blue vertical lines). The solid black cabinet shape represents the CDS region, the green color represents the PAM sequence, and the red dashed line represents the base deletion at the corresponding target point; B and C are the wild type CU2 and CsIAA16 The phenotype comparison of the female and male flower peduncle of the knockout mutant (actual object diagram), wherein B is the female flower and female flower peduncle phenotype, from top to bottom, the wild type CU2, Csiaa16-1-ff and Csiaa16-2-ff, C is the male flower and male flower peduncle phenotype, from top to bottom, the wild type CU2, Csiaa16-1-ff and Csiaa16-2-ff, the scale is 2 cm; Figure 1 B and C shown in the above are the day of flowering; D and E are the wild type CU2 and CsIAA16 The analysis of the length difference of the female and male flower peduncle of the knockout mutant (bar chart), wherein the horizontal coordinate is the strain (WT, Csiaa16-1-ff, Csiaa16-2-ff), and the vertical coordinate is the average length of the peduncle (unit: mm); the error line represents the standard error (SEM), and different lowercase letters represent that the difference between the strains reaches a very significant level (P P <0.01, t test), and the same letter represents no significant difference (P P >0.05);

[0019] Figure 2 For CsIAA16 The phenotype comparison diagram of the cucumber peduncle shortening and cucumber length of the overexpression strain; wherein A is the phenotype comparison of the female flower peduncle and ovary of the wild type (CU2) and the overexpression strain (OE) on the day of flowering: the scale is 2 cm; B is the female flower peduncle length (FPL) and ovary length statistics of the wild type (CU2) and the overexpression strain (OE) on the day of flowering: “ ” represents P<0.01, and ''ns'' represents no significant difference (P>0.05), n≥6; C is the length of cucumber stem and the length of cucumber of CU2 and OE after 25 days of pollination, scale=4cm; D is the length of cucumber stem and the length of cucumber of CU2 and OE after 25 days of pollination, ''t'' P <0.01, and ''ns'' represents no significant difference (P>0.05), n≥6. DETAILED DESCRIPTION

[0020] The application provides a protein CsIAA16 related to the length of cucumber stem and a coding gene of the protein CsIAA16 CsaV3_1G045520 The application also provides an application of the protein CsIAA16 in regulating the length of cucumber stem and / or the length of female flower stem of cucumber, and the amino acid sequence of the protein CsIAA16 is shown in SEQ ID NO. 1.

[0021] The application also provides a protein CsIAA16 related to the length of cucumber stem and a coding gene of the protein CsIAA16 CsaV3_1G045520 The application also provides an application of the protein CsIAA16 in regulating the length of cucumber stem and / or the length of female flower stem of cucumber, and the amino acid sequence of the protein CsIAA16 is shown in SEQ ID NO. 1.

[0022] In the application, the nucleotide sequence of the coding gene CsaV3_1G045520 is shown in SEQ ID NO. 2.

[0023] In the application, the amino acid sequence shown in SEQ ID NO. 1 is specifically as follows: MGSLLGFEETELRLGLPGGDGRNDGDAVKKRGFTETVDLKLNIVTDSNQGNKTTDKNVVSSFVNKDLPKPPPAKAQVVGWPPVKSLRRNIVGPPKKNKLEETEKNAVISGGGCSVGAAAFIKVSMDGAPYLRKVDLKMYGSYQELSDALRKMFASFTAGQCGAQKMRDYMNERKLIDVSNGSDYVPTYEDKDGDWMLVGDVPWDMFVDSCKRLRIMKGSEAIGLAPRALEKCKNRN .

[0024] In the application, the nucleotide sequence shown in SEQ ID NO. 2 is specifically as follows:

[0025] ​

[0026] In this invention, the coding gene CsaV3_1G045520 The CDS nucleotide sequence is shown in SEQ ID NO.7, specifically as follows:

[0027] .

[0028] As one implementation method, the negative regulation includes knockout.

[0029] This invention also provides a targeted gene encoding method. CsaV3_1G045520 The sgRNA combination, wherein the sgRNA combination includes sgRNA1 and sgRNA2;

[0030] The nucleotide sequence of the sgRNA1 is shown as SEQ ID NO. 3; the nucleotide sequence of the sgRNA2 is shown as SEQ ID NO. 4; the nucleotide sequence of the coding gene CsaV3_1G045520 is shown as SEQ ID NO. 2.

[0031] In the present application, the sgRNA combination can target the coding region of the coding gene CsaV3 1G045520 based on CRISPR / Cas9 gene editing, and can knockout the coding gene CsaV3_1G045520 together with the Cas9 protein.

[0032] In the present application, the nucleotide sequence shown as SEQ ID NO. 3 is specifically AAGAGACAGAGCTCCGTCT.

[0033] In the present application, the nucleotide sequence shown as SEQ ID NO. 4 is specifically GATCTTCCGAAGCCTCCTC.

[0034] In the present application, the target points recognized by the sgRNA1 and the sgRNA2 are both located in the first exon region of the coding gene CsaV3 1G045520 .

[0035] In the present application, the target point recognized by the sgRNA1 is the DNA molecule shown as 296-314 from the 5' end of SEQ ID NO. 2, i.e. the DNA molecule shown as 23-41 from the ATG of SEQ ID NO. 2.

[0036] In the present application, the target point recognized by the sgRNA2 is the DNA molecule shown as 469-487 from the 5' end of SEQ ID NO. 2, i.e. the DNA molecule shown as 196-214 from the ATG of SEQ ID NO. 2.

[0037] The present application also provides a CRISPR / Cas9 gene editing system comprising the sgRNA combination and the Cas9 protein described in the above scheme.

[0038] The present application also provides the application of the sgRNA combination or the CRISPR / Cas9 gene editing system described in the above scheme in at least one of the following: 1) increasing the cucumber stalk; 2) increasing the female flower stalk of cucumber; 3) editing the coding gene CsaV3 1G045520 ; 4) cultivating a cucumber variety suitable for mechanical picking.

[0039] The application further provides a method for increasing the length of cucumber peduncle and / or the length of cucumber female flower peduncle, comprising the following steps: increasing the length of cucumber peduncle and / or the length of cucumber female flower peduncle by gene editing on the coding region of a coding gene CsaV3_1G045520 , so as to cause the loss of function of protein CsIAA16; the amino acid sequence of the protein CsIAA16 is shown as SEQ ID NO. 1; the nucleotide sequence of the coding gene CsaV3_1G045520 is shown as SEQ ID NO. 2.

[0040] As an implementation form, the gene editing is performed by using the CRISPR / Cas9 gene editing system described in the above scheme.

[0041] The application further provides that the gene editing comprises: transforming cucumber under the mediation of Agrobacterium by using the CRISPR / Cas9 gene editing system, and screening to obtain a gene editing homozygous transgenic line.

[0042] In order to further illustrate the application, the application of the protein or the coding gene of the protein related to the length of cucumber peduncle provided by the application in increasing the length of cucumber peduncle is described in detail below in combination with the drawings and examples, but they should not be understood as limiting the protection scope of the application.

[0043] Example 1

[0044] The 19 bp single-direction RNA (sgRNA1 and sgRNA2, the nucleotide sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4, respectively) targets for CsIAA16 (the coding gene of CsIAA16, the nucleotide sequence is shown as SEQ ID NO. 2) are designed by using the CRISPR-GE online tool (http: / / skl.scau.edu.cn / ), and the specificity thereof is verified by BLAST comparison with the cucumber genome database. The target fragment of CsaV3_1G045520 is successfully obtained by PCR amplification with pCBC-DT1T2 plasmid as a template and inserting the homologous arms of the plasmid at both ends of the target sequence. CsIAA16 Subsequently, the purified PCR product is inserted into the linearized pBSE402 vector by Bsa I restriction endonuclease, and finally the recombinant plasmid pBSE402-IAA16 is constructed. After sequencing verification, the plasmid is introduced into Agrobacterium EHA105 strain for genetic transformation of cucumber.

[0045] The genetic transformation of cucumber is performed as follows: the seeds of CU2 variety cucumber are sown in sterile MS medium after sterilization treatment, and are placed in a 28°C dark environment for 24 h. Then the distal third of the cotyledon is cut, the hypocotyl is removed, and a U-shaped incision is made at the proximal end of the cotyledon. A surgical knife is used to make an additional slit at the U-shaped incision to separate the cotyledon to obtain a suitable explant. The pre-activated Agrobacterium strain is resuspended in inoculation liquid, and the infection concentration is adjusted to an optical density (OD) of 0.2 at a wavelength of 600 nm.

[0046] To promote wound formation and improve infection efficiency, the cotyledon explant is placed in a 100-watt ultrasonic processor for 30 s of ultrasonic treatment. Then the explant is placed in a 20 mL syringe, and an appropriate amount of Agrobacterium suspension is sucked to remove air bubbles and excess liquid. The syringe plunger is pushed to the 10 mL mark, and the needle is sealed with a rubber plug. Vacuum is formed by pulling the plunger back to the 20 mL mark and holding for 90 s, and then gently shaking to ensure uniform pressure distribution on the surface of the explant. Repeating the vacuum treatment step can further improve the infection efficiency.

[0047] The Agrobacterium-infected explants are evenly inoculated in co-culture medium, and the surface is covered with a filter paper layer. Then it is placed in a 23°C dark environment for 3 days. After co-culture is completed, it is transferred to the growth induction medium, and continues to be cultured under light for 3-4 weeks to promote the differentiation of adventitious buds. GFP-positive bud bodies are selected by fluorescence microscopy, and are transferred to rooting medium. After about 3 weeks, the transgenic positive seedlings with well-developed root systems are removed from the medium, washed thoroughly to remove residual medium, and then transplanted into sterile soil for culture in a light incubator. When the seedlings grow to more than 15 cm, they are transplanted to a greenhouse for planting until they have the ability to pollinate and bear fruit.

[0048] The above CRISPR / Cas9 system is combined with Agrobacterium-mediated genetic transformation technology to perform targeted genome editing on wild-type cucumber. Two homozygous mutant lines are successfully constructed. In the two lines, CsIAA16 the CDS region target 1 is deleted by 6 bp and 26 bp respectively, and the target 2 is deleted by 1 bp, as shown in Figure 1 A of the reference example. The two lines are named Csiaa16-1-ff (the nucleotide sequence shown in SEQ ID NO. 2 from 1 to 221 is replaced by the nucleotide shown in SEQ ID NO. 5) and Csiaa16-2-ff (the nucleotide sequence shown in SEQ ID NO. 2 from 1 to 221 is replaced by the nucleotide shown in SEQ ID NO. 6), both of which result in a frameshift mutation and premature termination of translation. The CsIAA16 protein is prematurely terminated in the two lines, and the CsIAA16 function is lost.

[0049] In this invention, the nucleotide represented by SEQ ID NO.5 is specifically:

[0050] atgggaagcttacttgggtttgaagagacagagcttggtctgcccggcggggacggaagaaacgatggcgacgccgtgaaaaaaagaggtttcactgaaactgtgga tttgaagctgaatattgtgactgattccaaccaaggcaacaaaacgaccgacaaaaatgtcgtttcgagcttcgtcaataaagatcttccgaagcctctccggccaa.

[0051] In this invention, the nucleotide represented by SEQ ID NO.6 is specifically:

[0052] atgggaagcttacttgggtctgcccggcggggacggaagaaacgatggcgacgccgtgaaaaaaagaggtttcactgaaactgtggatttgaagctg aatattgtgactgattccaaccaaggcaacaaaacgaccgacaaaaatgtcgtttcgagcttcgtcaataaagatcttccgaagcctctccggccaa.

[0053] Phenotypic observation revealed that, compared to the wild type (WT, CU2), the female flower stalks of both knockout mutants were significantly longer. The average length of the female flower stalk in Csiaa 16-1-ff was 28.13 mm, and the average length of the female flower stalk in Csiaa 16-2-ff was 26.26 mm (Table 1). A t-test of the field survey data showed that the female flower stalk lengths of both Csiaa 16-1-ff and Csiaa 16-2-ff were significantly longer than the average length of 21.12 mm for the wild type CU2. P <0.01). Overall, the female pedicel growth was more pronounced in the Csiaa 16-2-ff line than in the Csiaa 16-1-ff line. No significant differences were observed between the wild-type and mutant lines in male pedicel development, etc. (C and E). Compared to the wild-type, the pedicel of the mutant line was significantly longer, while no significant differences were observed in fruit length between the wild-type and mutant lines; therefore, knockout mutation does not affect the fruit. See Table 2 and... Figure 1 .

[0054] Table 1. Average length of female flower stalks on the day of flowering in different groups (unit: mm)

[0055]

[0056] Table 2: Length of fruit peduncle, fruit and average length of fruit / fruit peduncle (unit: cm) of different groups at 5-25 days

[0057]

[0058] Example 2

[0059] This example constructed CsIAA16 The overexpression vector of the gene was constructed, and a cucumber genetic transformation system mediated by Agrobacterium was used. After PCR molecular identification and breeding of offspring, three genetically stable CsIAA16 overexpression transgenic lines OE-CsIAA16-1, OE-CsIAA16-2 and OE-CsIAA16-3 were obtained.

[0060] The results are shown in Tables 3, 4 and Figure 2 The results show that: CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIAA16 CsIA The length of fruit peduncle of the gene overexpression plant was significantly shorter than that of the wild type, and the length of fruit was not significantly different.

[0061] Table 3: Length of female flower peduncle and length of ovary (unit: cm) on the day of flowering

[0062]

[0063] Table 4: Length of fruit at 25 days and length of fruit peduncle (unit: cm)

[0064]

[0065] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, but not all the embodiments. Other embodiments can be obtained according to the present embodiments without creativity, which are within the scope of the present application.

Claims

1. The application of knocking out the gene encoding the protein CsIAA16, which is associated with the trait of cucumber pedicel length, in the growth of cucumber pedicels and / or cucumber female flower pedicels; the amino acid sequence of the protein CsIAA16 is shown in SEQ ID NO.

1.

2. The application of a combination of sgRNAs targeting the gene CsaV3_1G045520 or a CRISPR / Cas9 gene editing system in at least one of the following: 1) To lengthen the cucumber stem; 2) Increase the length of the female flower stalk of cucumber; 3) Edit the coding gene CsaV3_1G045520; 4) Develop cucumber varieties suitable for mechanized harvesting; The nucleotide sequence of the encoding gene CsaV3_1G045520 is shown in SEQ ID NO.2; the CDS nucleotide sequence of the encoding gene CsaV3_1G045520 is shown in SEQ ID NO.

7. The sgRNA combination includes sgRNA1 and sgRNA2; the nucleotide sequence of the target gene of sgRNA1 is shown in SEQ ID NO. 3; the nucleotide sequence of the target gene of sgRNA2 is shown in SEQ ID NO. 4; The CRISPR / Cas9 gene editing system includes the aforementioned sgRNA combination and Cas9 protein.

3. A method for increasing the pedicel of cucumber and / or the pedicel of female cucumber flowers, characterized in that, Includes the following steps: By editing the coding region of the gene CsaV3_1G045520, the function of the protein CsIAA16 is lost, thereby increasing the length of cucumber pedicels and / or cucumber female flower pedicels. The amino acid sequence of the protein CsIAA16 is shown in SEQ ID NO.1; The nucleotide sequence of the encoding gene CsaV3_1G045520 is shown in SEQ ID NO.2; The CDS nucleotide sequence of the gene encoding CsaV3_1G045520 is shown in SEQ ID NO.

7.

4. The method according to claim 3, characterized in that, The gene editing was performed using a CRISPR / Cas9 gene editing system; the CRISPR / Cas9 gene editing system includes an sgRNA combination and a Cas9 protein; the sgRNA combination includes sgRNA1 and sgRNA2; the nucleotide sequence of the target gene of sgRNA1 is shown in SEQ ID NO.3; the nucleotide sequence of the target gene of sgRNA2 is shown in SEQ ID NO.

4.

5. The method according to claim 4, characterized in that, The gene editing includes: transforming cucumbers with the CRISPR / Cas9 gene editing system mediated by Agrobacterium tumefaciens, and screening to obtain gene-edited homozygous transgenic lines.

6. A method for increasing the pedicel of cucumber and / or the pedicel of female cucumber flowers, characterized in that, The steps include: replacing protein CsIAA16 with one of the following sequences: MGSLLGFEETELGLPGGDGRNDGDAVKKRGFTETVDLKLNIVTDSNQGNKTTDKNWVSSFVNKDLPKPLRPSMNSHLSLFFIIIISKKRLMTVNKEKFSASKVHNFYILFSHIFLFSRKQLDE; or MGSLLGSARRGRKKRWRRREKKRFH; the amino acid sequence of protein CsIAA16 is shown in SEQ ID NO.

1.

7. A transgenic cucumber strain, characterized in that, The gene encoding protein CsIAA16 in the transgenic cucumber line was knocked out; the amino acid sequence of protein CsIAA16 is shown in SEQ ID NO.1.

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