阅读说明：本技术 黄瓜CsHEC2蛋白及其编码基因在降低果实刺瘤密度中的应用 (Application of cucumber CsHEC2 protein and coding gene thereof in reducing fruit thorn tumor density ) 是由 张小兰 王中一 周朝阳 于 2021-03-23 设计创作，主要内容包括：本发明涉及植物基因工程技术领域,具体涉及黄瓜CsHEC2蛋白及其编码基因在降低果实刺瘤密度中的应用。本发明首次发现,通过抑制CsHEC2蛋白的编码基因的表达,可以显著降低黄瓜果实表面的刺瘤密度,而不影响商品期果实的瓜长和瓜横径,可在改良黄瓜品种中得到应用,有望加快不同刺瘤密度市场需求的黄瓜新品质培育。(The invention relates to the technical field of plant genetic engineering, in particular to application of a cucumber CsHEC2 protein and a coding gene thereof in reducing fruit thorn tumor density. The invention discovers for the first time that the thorn tumor density on the surface of the cucumber fruit can be obviously reduced by inhibiting the expression of the coding gene of the CsHEC2 protein without influencing the cucumber length and the cucumber transverse diameter of the fruit in the commodity period, the thorn tumor density can be applied to the improvement of cucumber varieties, and the new quality cultivation of cucumbers required by markets with different thorn tumor densities is expected to be accelerated.)

The CsHEC2 protein, its coding gene, its inhibitor, or the biological material containing its coding gene or inhibitor can be used to regulate the density of the thorns in cucumber fruit.

Application of CsHEC2 protein, or coding gene thereof, or inhibitor thereof, or biological material containing the coding gene or the inhibitor thereof in breeding cucumbers with lower fruit thorn tumor density.

3. The use according to claim 1 or 2, wherein the density of the spines in cucumber fruits is reduced by inhibiting the expression of the gene coding for CsHEC2 protein.

4. The use according to any one of claims 1 to 3, wherein the CsHEC2 protein has the amino acid sequence of any one of SEQ ID NO:

1) an amino acid sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

5. The use according to any one of claims 1 to 3, wherein the gene encoding CsHEC2 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.1, or,

(2) the coding nucleotide sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1; or, (3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.1 under strict conditions.

6. The method for constructing the transgenic cucumber with lower fruit thorn tumor density is characterized by inhibiting the expression of CsHEC2 protein in the cucumber.

7. The method as claimed in claim 6, wherein the coding gene of CsHEC2 protein is edited in cucumber by CRISPR/Cas9 technique to inhibit the expression of the coding gene.

8. The method according to claim 7, wherein the target sequences edited by the CRISPR/Cas9 vector are shown as SEQ ID No.3 and SEQ ID No. 4.

9. The method according to claim 8, wherein the sequence of the sgRNA of the CRISPR/Cas9 vector is shown as SEQ ID No.5 and SEQ ID No. 6.

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of a cucumber CsHEC2 protein and a coding gene thereof in reducing fruit thorn tumor density.

Background

Cucumber (Cucumis sativus L.), a cucurbitaceous plant, is one of the most important vegetable crops in the world (Huang et al, 2009). The cucumber fruit is an edible organ with important economic value, and can be eaten fresh or processed into pickle for eating. The surface of the cucumber fruit is usually covered with macroscopic fruit nodules and fruit spines that are born on its tip, which together constitute the wart trait (Yang et al, 2014). The wart character of fruits is an important quality character affecting the appearance and market value of cucumber fruits. And wart density is one of the important standards for the market classification of fresh cucumber, and directly influences the purchasing desire of consumers. For example, the chinese cucumber fruits mostly exhibit high density wart characteristics, while the european and american cucumber fruits have no or few warts. Therefore, analyzing the regulation mechanism of wart formation has important significance for cultivating cucumbers with different market ideal external qualities.

Existing studies have shown that the regulation of fruit spines and fruit tumors involves a complex network comprising multiple transcription factors and endogenous plant hormones (Che and Zhang, 2019). Several cucumber thorn mutants have been identified and cloned, such as the completely hairless mutants glaberous 3(csgl3) and trichomless-less (tril), and the dilute spike variant fw pins 1(fs1) (Pan et al, 2015; Cui et al, 2016; Wang et al, 2016; Zhang et al, 2016). Interestingly, these several mutants were allelic and resulted from different forms of mutation in the same gene (Csa6G 514870). The gene encodes a leucine zipper of class IV homologous structure (HD-ZIP IV) transcription factor, which plays an important role in the initiation and development of fruit thorn (Du et al, 2020). In addition, the map clone also finds that the HD-ZIP I transcription factor CsGL1 plays an important role in the development of fruit thorns, but the csgll mutant distributes a large number of visually invisible development-retarded fruit thorns in fruits, indicating that the gene does not regulate the initiation process of the fruit thorns (Li et al, 2015; Zhao et al, 2015). In addition, genetic studies show that the fruit tumor character has dominant effect on the fruit tumor-free character, and the fruit tumor character is controlled by a single nuclear dominant gene (CsTu), which encodes a C2H2 zinc finger transcription factor and can regulate the development of fruit tumors by promoting cytokinin synthesis. Caocheng discovered in 2001 that the surfaces of ordinary hairy cucumber fruits are covered with thorns, but some have tumor, and some have no tumor. Importantly, genetic analysis found that the fruit thorn gene CsGL1 has a stealth epitope for the fruit tumor gene CsTu, since CsTu gene is not expressed in the CsGL1 mutant without fruit thorn and without fruit tumor, while fruit thorn was present after CsTu gene mutation (Wang et a1., 2007; Zhang et al, 2010; Yang et al, 2014). The results of the above studies also indicate that the presence of fruit pricks is a prerequisite for fruit neoplasia.

In addition, phytohormones such as gibberellin, auxin and Cytokinin (CTK) have been shown to regulate the development of cucumber fruit spines and fruit tumors. The gibberellin biosynthesis gene CsGA20ox1, which has a negative regulatory effect on the development of fruit thorns, may be a potential downstream gene of the CsGL1 gene (Li et al, 2015). Transcriptome and hormone measurements indicate that CsTu is involved in CTK biosynthesis by indirectly promoting the expression of two CTK Hydroxylase (CHL) like genes, thereby stimulating cell division, ultimately leading to the initiation of fruit tumors (Yang et al, 2014).

Although spines have important significance in cucumber breeding and production, the genetic and regulatory mechanisms of their formation are still quite limited. Moreover, the related genes for regulating the development of cucumber thorn tumors influence other organs of the overground part of cucumber plants besides fruit thorn tumors, which often causes potential side effects. Therefore, the gene for specifically regulating and controlling cucumber fruit thorn tumor is discovered to have direct production guidance value for improving cucumber quality.

Reference documents:

cao Cheng xing, Zhang Song, Guo hong Yun (2001) cucumber stem and leaf hairless trait and fruit tumor thorn trait, gardening journal 28: 565-566

Che G，Zhang x(2019)Molecular basis of cucumber fruit domestication.Curr Opin Plant Biol 47：38-46

Cui JY，Miao H，Ding LH，Wehner TC，Liu PN，WangY，Zhang SP，Gu xF(2016)A New Glabrous Gene(csgl3)Identified in Trichome Development in Cucumber(Cucumis sativus L.).PLoS One 11：e0148422

Du H，Wang G，PanJ，ChenY，Xiao T， Zhang L，Zhang K，Wen H，Xiong L，YuY，He H，Pan J，Cai R(2020)The HD-ZIP IV transcription factor Tril regulates fruit spine density through gene dosage effects in cucumber.J Exp Bot 71：6297-6310

Gremski K，Ditta G，Yanofsky MF(2007)The HECATE genes regulatefemale reproductive tract development in Arabidopsis thaliana.Development 134：3593-3601

Hu B，Li D，Liu x，Qi J，Gao D，Zhao S，Huang S，Sun J，Yang L(2017)Engineering Non-transgenic Gynoecious Cucumber Using an Improved Transformation Protocol and Optimized CRISPR/Cas9 System.Mol Plant 10：1575-1578

Huang S，Li R，Zhang Z，Li L，Gu X，Fan W，Lucas WJ，Wang X，Xie B，NiP，RenY，Zhu H，Li J，Lin K，Jin W，Feiz，Li G，staub J，Kilian A，van der Vossen EA，WuY，Guo J，He J，Jia z，RenY，Tian G， LuY，Ruan J，Qian W，Wang M，Huang Q，Li B，Xuanz，Cao J，Asan，Wuz，Zhang J，Cai Q，BaiY，Zhao B，HanY，LiY，Li X，Wang S，Shi Q，Liu S，Cho WK，Kim JY，XuY，Heller-Uszynska K，Miao H，Cheng Z，Zhang S，Wu J，YahgY，Kang H，Li M，Liang H，Ren X，ShiZ，Wen M，Jian M，Yang H，Zhang G，Yang Z，Chen R，Liu S，Li J，Ma L，Liu H，Zhou Y，Zhao J，Fang X，Li G， Fang L，LiY，Liu D，Zheng H，ZhangY，Qin N，Li Z，Yang G，，Yang S，Bolund L，Kristiansen K，Zheng H，Li S，Zhang X，Yang H，Wang J，Sun R，Zhang B，Jiang S，Wang J，Du Y，Li S(2009)The genome of the cucumber，Cucumis sativus L.Nat Genet 41：1275-1281

Li Q，Cao C，Zhang C，Zheng S，Wang Z，Wang L，Ren Z(2015)The identification of Cucumis sativus Glabrous 1(CsGL1) requiredfor theformation oftrichomes uncovers a novel function for the homeodomain-leucine zipper I gene.J Exp Bot 66：2515-2526

Pan Y，Bo K，Chengz，Weng Y(2015)The loss-of-function GLABROUS 3mutation in cucumber is due to LTR-retrotransposon insertion in a class IV HD-ZIP transcriptionfactor gene CsGL3 that is epistatic over CsGL1.BMC Plant Biol15：302

Schuster C，Gaiuochet C，Lohmann JU(2015)Arabidopsis HECATE genesfunction in phytohormone control during gynoecium development.Development 142：3343-3350

Schuster C，Gaillochet C，Medzihradszky A，Busch W，Daum G，，Krebs M，Kehle A，Lohmann JU(2014)A regulatoryframeworkfor shoot stem cellcontrol integrating metabolic，transcriptional，and phytohormone signals.Dev Cell28∶438-449

Wang G，Qin Z，Zhou x，Zhao ZY(2007)Genetic analysis and SSR markers of tuberculatetrait in Cucumis sativus.Chin BullBot 168-172

Wang YL，Nie JT，Chen HM，Guo CL，Pan J，He HL，Pan JS，Cai R(2016)Identification and mapping of Tril，a homeodomain-leucine zipper gene involved in multicellulartrichome initiation in Cucumis sativus.TheorAppl Genet 129：305-316

Xing HL，Dong L，Wang ZP，Zhang HY，Han CY，Liu B，Wang xC，Chen QJ(2014)A CRISPR/Cas9 toolkitfor multiplex genome editing in plants.BMC Plant Biol14∶327

Yan S，Ning K，Wang Z，Liu X，Zhong Y，Ding L，Zi H，Cheng Z，Li X，Shan H，Lv Q，Luo L，Liu R，Yan L，Zhou Z，Lucas WJ，Zhang x(2020)CsIVPfunctions in vasculature development and downy mildew resistance in cucumber.PLoS Biol18：e3000671

Yang x，Zhang W，He H，Nie J，Bie B，Zhao J，Ren G，LiY，Zhang D，Pan J，Cai R(2014)Tuberculatefruit gene Tu encodes a C2 H2 zinc finger protein that is required for the warty rfuitphenotype in cucumber(Cucumis sativus L.).Plant J 78∶1034-1046

Zhang H，Wang L，Zheng S，Liu Z，Wu x，Gao Z，Cao C，Li Q，Ren Z(2016)Afragment substitution in the promoter of CsHDZIV11/CsGL3 is responsible for fruit spine density in cucumber(Cucumis sativus L.).TheorAppl Genet 129：1289-1301

Zhang W，He H，Guan Y，Du H，Yuan L，Li Z，Yao D，Pan J，Cai R(2010)Identification and mapping of molecular markers linked to the tuberculate fruit gene in the cucumber(Cucumis sativus L.).Theor Appl Genet 120：645-654

Zhao JL，Pan JS，Guan Y，Zhang WW，Bie BB，Wang YL，He HL，Lian HL，Cai R(2015)Micro-trichome as a class I homeodomain-leucine zipper gene regulates multicellular trichome development in Cucumis sativus.J Integr Plant Bio1 57：925-935

Disclosure of Invention

The invention aims to provide application of cucumber CsHEC2 protein and coding gene thereof in reducing fruit thorn tumor density.

Specifically, the invention firstly provides the CsHEC2 protein, or a coding gene thereof, or an inhibitor thereof, or a biological material containing the coding gene or the inhibitor thereof for regulating the density of the spines of cucumber fruits.

The invention also provides application of the CsHEC2 protein, or an encoding gene thereof, or an inhibitor thereof, or a biological material containing the encoding gene or the inhibitor thereof in breeding cucumbers with lower fruit thorn tumor density.

Preferably, the thorn tumor density of the cucumber fruits is reduced by inhibiting the expression of a gene coding the CsHEC2 protein.

Preferably, the CsHEC2 protein has an amino acid sequence of any one of the following:

1) an amino acid sequence shown as SEQ ID NO. 2; or the like, or, alternatively,

2) the amino acid sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more amino acid residues in the amino acid sequence shown in SEQ ID NO. 2.

Preferably, the encoding gene of the CsHEC2 protein has any one of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.1 (obtained by cloning from a cucumber variety Sinomenium), or,

(2) the coding nucleotide sequence of the protein with the same function is obtained by replacing, deleting or inserting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 1; or the like, or, alternatively,

(3) a nucleotide sequence which can be hybridized with the nucleotide sequence shown in SEQ ID NO.1 under strict conditions.

The invention also provides a method for constructing the transgenic cucumber with lower fruit thorn tumor density, which can inhibit the expression of CsHEC2 protein in the cucumber.

Preferably, the encoding gene of CsHEC2 protein is edited in cucumber by CRISPR/Cas9 technology, and the expression of the encoding gene is inhibited.

More preferably, the target sequences edited by the CRISPR/Cas9 vector are shown in SEQ ID NO.3 and SEQ ID NO. 4.

The CsHEC2 gene in the knockout plant forms a stop codon in advance and cannot be translated into complete protein, so that the CsHEC2 gene loses functions.

More preferably, the sequence of the sgRNA of the CRISPR/Cas9 vector is shown in SEQ ID No.5 and SEQ ID No. 6.

Based on the scheme, the invention has the following beneficial effects:

the invention discovers for the first time that the thorn tumor density on the surface of the cucumber fruit can be obviously reduced by inhibiting the expression of the coding gene of the CsHEC2 protein without influencing the cucumber length and the cucumber transverse diameter of the fruit in the commodity period, the thorn tumor density can be applied to the improvement of cucumber varieties, and the new quality cultivation of cucumbers required by markets with different thorn tumor densities is expected to be accelerated.

Drawings

FIG. 1 shows the results of Sanger sequencing chromatogram alignment of wild type WT, Cschec 2#1 and Cschec 2#2 editing plant target one in example 3.

FIG. 2 shows the results of Sanger sequencing chromatogram alignment of wild type WT, Cschec 2#1 and Cschec 2#2 editing plant target two in example 3.

FIG. 3 shows the genotypes of wild-type WT, Cschec 2#1 and Cschec 2#2 mutants in example 3.

FIG. 4 is a phenotype map of cucumber fruits showing wild type WT, Cschec 2#1 and Cschec 2#2 mutants in example 3. Wherein, A is the fruit on the day of flowering; b is a partial enlarged view of the fruit on the day of flowering; c is a local electron microscope scanning image of the fruit on the day of flowering; graph D shows fruits 10 days after flowering.

FIG. 5 is a data sheet showing cucumber fruits of wild type WT, Cschec 2#1 and Cschec 2#2 mutants in example 3. Wherein A is 40mm of fruit at day of flowering²Counting the density of the spines in the region; b, the fruit length of the fruit 10 days after the flower; and C, the fruit thickness of the fruit 10 days after the flower blossoming.

FIG. 6 is an electron micrograph of cucumber leaves showing wild-type WT, Cshec2#1 and Cshec2#2 mutants in example 3.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The CsHEC2 gene is short for Csa2G285890 gene. The PKSE402G vector was offered by the national institute of agricultural sciences, Huangsanwen and Yangli teachers (Hu et al, 2017.Engineering Non-transgenic Gynoecius Cucumber Using an Improved Transformation Protocol and Optimized CRISPR/Cas9 System. mol Plant10: 1575-. The pCBC-DT1T2 template plasmid was gifted by the aged army teacher of the university of agriculture of China (Xing et al, 2014.A CRISPR/Cas9 toolkit for multiplex genome editing in plants BMC Plant Biol 14: 327). Agrobacterium EHA105 competent cells were purchased from Shanghai Diego Biotechnology Ltd. BsaI endonuclease and T4 ligase were purchased from New England Biolabs (New England Biolabs).

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or instruments used are conventional products available from regular distributors, not indicated by the manufacturer.

Example 1 cloning of CsHEC2 Gene

HECs transcription factors play an important role in the development of female reproductive organs and the control of meristems in Arabidopsis thaliana, but their function in cucumber is not yet known (Gremski et al, 2007; Schuster et al, 2014; Schuster et al, 2015). Importantly, the recent research results of the inventors show that the bHLH transcription factor CsIVP/HEC3 gene specifically expressed by vascular tissues regulates the leaf shape and plant height by directly activating the expression of vascular development regulatory factors CsYAB5, CsBP and CsAUX4 (Yan et al, 2020), so that the function of the CsHECs family gene is being systematically researched. The CsHEC2 is found to have strong expression in the spine tumor tissue shown by cucumber fruits by fluorescence quantification and in situ hybridization technology, so that the CsHEC2 gene is cloned and functionally analyzed.

1. Obtaining of test materials

The Xintai Mici cucumber variety is offered by Zhang Zhenxian professor laboratory of China university of agriculture. Planting in an illumination incubator at the seedling stage, taking a tender growing point when the cucumber seedling grows to the second true leaf and unfolding, quickly putting into liquid nitrogen for freezing, and storing in a refrigerator at-80 ℃ for later use.

2. Extraction of RNA

Total RNA was extracted from the sample using Promega Kit (Eastep Super isolation Kit, Promega).

3. Obtaining of cDNA

The RNA extracted in the step 2 is used as a template, and a Kit (FastKing gDNA dispensing RT SuperMix Kit, Tiangen Biotech) of Tiangen Biotech is adopted for reverse transcription to synthesize cDNA.

4. Amplification of target Gene

And 3, carrying out PCR amplification by using the cDNA obtained in the step 3 as a template and primers CsHEC2-F and CsHEC2-R to obtain the CsHEC2 gene full length with the PCR product length of 648 bp. The primer sequences are as follows:

CsHEC2-F:5’-ATGGACGATATCGACATCCTCA-3’(SEQ ID NO.7)

CsHEC2-R:5’-TCAAGACTGCATTTGCAAAGAA-3’(SEQ ID NO.8)

TABLE 1 amplification System for the full-Length sequence of the coding region of the target Gene CsHEC2

PCR amplification reaction procedure: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 15s, extension at 72 ℃ for 40s, and 34 cycles; final extension at 72 ℃ for 5 min.

5. Detection and recovery of PCR products

After the amplification reaction is finished, electrophoresis detection is carried out by using 1% agarose gel, and the expected target band is cut off rapidly under an ultraviolet lamp. Gel recovery is carried out by adopting a gel recovery kit, and sequencing is carried out on gel recovery products.

Example 2 CRISPR/Cas9 vector construction of CsHEC2 Gene

1. Target sequence design of sgRNA sequence

Target sequences are searched and designed on the CsHEC2 gene, and the length of the target sequences is 19 bp.

The target point-nucleotide sequence is as follows:

5’-AGTACATGAGGAGTGCAAT-3’(SEQ ID NO.3)

the reverse complementary sequence is: 5'-ATTGCACTCCTCATGTACT-3' (SEQ ID NO.5)

The target dinucleotide sequence is:

5’-GGCGATAAAGGCGCCGAAG-3’(SEQ ID NO.4)

the reverse complement sequence is: 5'-CTTCGGCGCCTTTATCGCC-3' (SEQ ID NO. 6).

2. Primer design and amplification constructed by CRISPR/Cas9 vector

The following four partially overlapping primers were synthesized based on the target sequences selected in step 1 above. Four-primer PCR amplification was performed using 100-fold diluted pCBC-DT1T2 plasmid as a template. -BsF/-BsR is normal primer concentration; 20-fold dilution of-F0/-R0. The PCR system and the amplification procedure are listed in the following Experimental scheme. The four primer sequences are as follows (SEQ ID NO.9-12 from top to bottom):

CsHEC2-DT1-BsF:

5’-ATATATGGTCTCGATTGAGTACATGAGGAGTGCAATGTT-3’CsHEC2-DT1-F0:

5’-TGAGTACATGAGGAGTGCAATGTTTTAGAGCTAGAAATAGC-3’

CsHEC2-DT2-R0:

5’-AACCTTCGGCGCCTTTATCGCCCAATCTCTTAGTCGACTCTAC-3’

CsHEC2-DT2-BsR:

5’-ATTATTGGTCTCGAAACCTTCGGCGCCTTTATCGCCCAA-3’

3. construction of enzyme digestion-ligation System of vector

And (3) purifying and recovering the PCR amplification product, and establishing an enzyme digestion-connection system to obtain a PKSE402G-CRISPR/Cas9-CsHEC2 vector.

TABLE 2 enzyme digestion-ligation system constructed by CRISPR/Cas9 vector

The reaction procedure was as follows: incubation at 37 ℃ for 2min, 16 ℃ for 5min, cycle repeated 50 times, and incubation at 80 ℃ for 5min (heat inactivation of the enzyme).

4. Large intestine competent transformation and colony sequencing

The vector ligated in step 3 above was transformed into E.coli competent DH 5. alpha. and the resulting bacterial suspension was plated on a medium plate containing kanamycin sulfate (50mg/L) for selection. The obtained single colony is subjected to colony PCR identification by using primers U626-F + U629-R of 726bp, and colonies which accord with an expected target band are selected and subjected to vector sequencing by using the primers U626-F and U629-F. The primer sequences are as follows (SEQ ID NO.13-15 from top to bottom):

U626-F:5’-TGTCCCAGGATTAGAATGATTAGGC-3’

U629-F:5’-TTAATCCAAACTACTGCAGCCTGAC-3’

U629-R:5’-AGCCCTCTTCTTTCGATCCATCAAC-3’。

example 3 application of CsHEC2 gene in reduction of fruit thorn tumor density by using CRISPR/Cas9 system

1. Recombinant vector transformation agrobacterium tumefaciens EHA105

The PKSE402G-CRISPR/Cas9-CsHEC2 vector bacterial liquid with correct sequencing obtained in the example 2 is subjected to plasmid extraction by a plasmid extraction kit. The obtained recombinant plasmid PKSE402G-CRISPR/Cas9-CsHEC2 is extracted to carry out transformation on agrobacterium-infected EHA105 by a heat shock transformation method, so as to obtain recombinant agrobacterium EHA105(PKSE401-CRISPR/Cas9-CsHEC 2). The specific transformation procedure was performed according to the Agrobacterium competent transformation instruction of Shanghai Dingwei Biotechnology Ltd.

2. Agrobacterium infection genetic transformation cucumber

The recombinant agrobacterium in the step 1 is subjected to cucumber transgenosis by utilizing an agrobacterium-mediated cucumber cotyledon transformation method, the cucumber variety is Xintai mici, and the specific genetic transformation step is described with reference to a 2017 article (Hu et al, 2017). And performing GFP fluorescence screening on the differentiated regenerated buds under a body type fluorescence microscope to obtain T0 generation positive transgenic cucumber plants. The obtained plants are domesticated and domesticated, planted in a greenhouse, and artificially pollinated to collect progeny seeds.

3. Cucumber plant edited by gene CsHEC2

And (3) breeding the T0 generation plants in the step (2) in a greenhouse of a scientific and technological garden of Chinese agriculture university to obtain T1 generation seeds, selecting GFP fluorescence-free seeds from the T1 generation seeds for seedling culture (GFP fluorescence-free shows that a CRISPR/Cas9 vector is separated to obtain a plant which does not contain a transgenic vector but is edited), and extracting true leaf genome DNA by adopting a classical CTAB method when a first true leaf of a seedling is completely unfolded. The CsHEC2 gene was cloned by PCR amplification using the primers CsHEC2-F and CsHEC2-R of example 1, and the PCR amplified gel products of different individuals of different strains were subjected to Sanger sequencing after gel electrophoresis of the obtained products and preliminary band observation by an ultraviolet lamp. The obtained gene editing plants were subjected to genotype identification by sequence alignment with the wild type CsHEC2 gene according to the company sequencing results (fig. 1, 2, 3). Sequencing chromatogram analysis of fig. 1 and fig. 2 shows that the CRISPR/Cas9 system is used to successfully realize the editing of cucumber CsHEC2 gene, and two complete mutants are obtained: both Cshec2#1 (homozygous allele, target one deletion of 2bp, target two deletion of 1bp, 86 amino acids) and Cshec2#2 (homozygous allele, target one deletion of 7bp, target two deletion of 2bp, 33 amino acids) produced premature stop codons, resulting in the production of truncated protein end products, achieving loss of function of the Cshec2 gene (fig. 3).

4. Cucumber plant phenotype observation edited by gene CsHEC2

And (3) carrying out phenotype observation and photographing on the plants edited by the T2-generation cucumber gene CsHEC2 obtained in the step (3) after greenhouse planting and growth and development. The results are shown in FIG. 4, and compared with the wild-type plant fruit, it was found that: fruits at day of flowering (a, B, C in fig. 4) and fruits at commodity stage (10 days after flowering) (D in fig. 4) from Cshec2#1 and Cshec2#2 knockout plants both showed a significant reduction in shot density, but no significant change in morphology. Statistical analysis found that the fruit demonstrated indeed a significant decrease in bur density (a in fig. 5), and importantly that the fruit length (B in fig. 5) and fruit thickness (C in fig. 5) of the commercial fruit were not statistically significantly different. In addition, we also observed leaves from plants, and found that there were no significant number and morphological changes in the spines on the front and back of the leaves from Cshec2#1 and Cshec2#2 knockout plants (fig. 6). The results show that the gene CsHEC2 specifically regulates the initiation of fruit spines, and can remarkably reduce the density of spines without influencing the fruit length and fruit thickness of fruits and the surface spines of leaves after knockout.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of agriculture in China

<120> application of cucumber CsHEC2 protein and coding gene thereof in reducing fruit thorn tumor density

<130> KHP211111947.7

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 648

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggacgata tcgacatcct caaatccaca ctctcccaat ccgacatgtc gtccacattc 60

ttcccaaaca acaccgcccc caattgcact cctcatgtac tcccaataat cccaccccca 120

gcttatttct ccgactactc cccgccgccc ggaacctcct tattccaaac caccccaaca 180

ataatccccg aaactcctgc gcggcagagg cggagcggcg taagtggggg aggaatggcg 240

gcgatgagag agatgatatt tagaatagcg gcgatgcagc cggtggagat tgatccggag 300

gcgataaagg cgccgaagcg gcggaatgtg agaatatcga aagatccaca gagtgtggcg 360

gcgaggcacc ggcgggaaag gattagccag aaaattagga ttctgcagcg gctggtgccg 420

ggcgggacga agatggacac agcgtcgatg ctggatgagg cggtgcatta tgtgaagttt 480

ttgaaacggc aagtccaaac gctggagcag gctgggttta attataataa taataataac 540

aacaataaca attttaataa ttttgttaat tccgcgaatt taaattacgc ttctgccctt 600

ttcaaggctt gtcaaataat gcctgcttct ttgcaaatgc agtcttga 648

<210> 2

<211> 215

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Asp Asp Ile Asp Ile Leu Lys Ser Thr Leu Ser Gln Ser Asp Met

1 5 10 15

Ser Ser Thr Phe Phe Pro Asn Asn Thr Ala Pro Asn Cys Thr Pro His

20 25 30

Val Leu Pro Ile Ile Pro Pro Pro Ala Tyr Phe Ser Asp Tyr Ser Pro

35 40 45

Pro Pro Gly Thr Ser Leu Phe Gln Thr Thr Pro Thr Ile Ile Pro Glu

50 55 60

Thr Pro Ala Arg Gln Arg Arg Ser Gly Val Ser Gly Gly Gly Met Ala

65 70 75 80

Ala Met Arg Glu Met Ile Phe Arg Ile Ala Ala Met Gln Pro Val Glu

85 90 95

Ile Asp Pro Glu Ala Ile Lys Ala Pro Lys Arg Arg Asn Val Arg Ile

100 105 110

Ser Lys Asp Pro Gln Ser Val Ala Ala Arg His Arg Arg Glu Arg Ile

115 120 125

Ser Gln Lys Ile Arg Ile Leu Gln Arg Leu Val Pro Gly Gly Thr Lys

130 135 140

Met Asp Thr Ala Ser Met Leu Asp Glu Ala Val His Tyr Val Lys Phe

145 150 155 160

Leu Lys Arg Gln Val Gln Thr Leu Glu Gln Ala Gly Phe Asn Tyr Asn

165 170 175

Asn Asn Asn Asn Asn Asn Asn Asn Phe Asn Asn Phe Val Asn Ser Ala

180 185 190

Asn Leu Asn Tyr Ala Ser Ala Leu Phe Lys Ala Cys Gln Ile Met Pro

195 200 205

Ala Ser Leu Gln Met Gln Ser

210 215

<210> 3

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agtacatgag gagtgcaat 19

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ggcgataaag gcgccgaag 19

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

attgcactcc tcatgtact 19

<210> 6

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cttcggcgcc tttatcgcc 19

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggacgata tcgacatcct ca 22

<210> 8

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tcaagactgc atttgcaaag aa 22

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atatatggtc tcgattgagt acatgaggag tgcaatgtt 39

<210> 10

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tgagtacatg aggagtgcaa tgttttagag ctagaaatag c 41

<210> 11

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aaccttcggc gcctttatcg cccaatctct tagtcgactc tac 43

<210> 12

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

attattggtc tcgaaacctt cggcgccttt atcgcccaa 39

<210> 13

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgtcccagga ttagaatgat taggc 25

<210> 14

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

ttaatccaaa ctactgcagc ctgac 25

<210> 15

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

agccctcttc tttcgatcca tcaac 25