Application of CaAUX22 gene in regulating and controlling fruit shape of pepper or tomato

Abstract

The invention belongs to the field of gene engineering, and discloses an application of a CaAUX22 gene in regulating and controlling the fruit shape of pepper or tomato. The CaAUX22 gene involved in the invention plays a key role in regulating and controlling the fruit shape of capsicum or tomato, and varieties with short and small fruits can be cultivated in capsicum by silencing the CaAUX22 gene, so that new possibility is provided for variety improvement of capsicum; the CaAUX22 gene is subjected to heterologous overexpression in tomatoes, so that tomato fruits can be lengthened, and a new thought is provided for variety improvement of the tomatoes.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and particularly relates to a gene for the fruit shape of pepper fruits CaAUX22 and its application in regulating the fruit shape of pepper or tomato Background Art

[0002] Pepper ( Capsicum annum L. ) is one of the most important solanaceous vegetables, and its vitamin C content ranks first among various vegetables. At present, the cultivation of pepper mainly focuses on the United States, Mexico, China and other places. Among them, China has the largest planting area of pepper. For the supply and demand relationship of pepper fruits, China is contrary to other countries in the world, showing a trend of oversupply. Therefore, it is necessary to breed new varieties of pepper fruits

[0003] Fruit shape is an important external quality trait of pepper fruits, and it is also an important factor affecting commercial value and an important index for consumers to evaluate. Therefore, the selection of fruit shape plays an important role in the breeding of new pepper varieties

[0004] Developing new varieties with excellent fruit shape is an effective means to improve the quality and production efficiency of pepper products. Therefore, the research on genes related to pepper fruit shape has become a hot spot in pepper molecular breeding at home and abroad. There are multiple genes controlling the fruit shape of pepper, but their modes of action are complex, with both dominant and recessive effects, and there are also interactions among genes. Therefore, the excavation and application of genes related to pepper fruit shape have become difficult points Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the above-mentioned deficiencies and defects in the background art, and provide an CaAUX22 application of a gene in regulating the fruit shape of pepper or tomato To solve the above technical problem, the technical solution proposed by the present invention is as follows CaAUX22 Application of a gene in regulating the fruit shape of pepper. Preferably, the nucleotide sequence of the C aAUX22 gene is as shown in SEQ ID NO.1

[0006] The above application ， Preferably, the CaAUX22 amino acid sequence of the gene is as shown in SEQ ID NO.2

[0007] The above application ， Preferably, silence the CaAUX2 2 gene in pepper to cultivate a pepper variety with short fruits

[0008] Based on a general inventive concept, the present invention also provides CaAUX22Application of gene in regulating tomato fruit shape, wherein the CaAUX22 nucleotide sequence of the gene is shown as SEQ ID NO.1.

[0009] The above application ， Preferably, the CaAUX22 amino acid sequence of the gene is shown as SEQ ID NO.2.

[0010] The above application ， Preferably, heterologous overexpression of the CaAUX22 gene in tomatoes causes the tomato fruits to become longer, providing a basis for cultivating long-fruit tomatoes.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows: The CaAUX22 gene involved in the present invention plays a key role in regulating pepper fruit shape or tomato fruit shape. By silencing the CaAUX22 gene, varieties with short fruits can be cultivated in peppers, which provides new possibilities for pepper variety improvement (such as varieties with higher spiciness and richer taste, etc.); heterologous overexpression of the CaAUX22 gene in tomatoes can cause the tomato fruits to become longer, which also provides a new idea for tomato variety improvement.

[0012] The CaAUX22 gene and its application of the present application provide new gene resources and methods for improving the fruit shapes of peppers and tomatoes, and have important theoretical and application values. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for description in the embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0014] Figure 1 is the single-plant phenotype of the parent and F1; Figure 2 For the identification of CaAUX22 silencing lines in Zunla materials: among them, (A) is the phenotype diagram of the CaAUX22 silenced plant mediated by TRV; (B) is the pollen phenotype diagram of the CaAUX22 silenced plant mediated by TRV; (C) is the CaAUX22 relative expression level; (D) is the phenotype diagram of the CaAUX22 silenced pepper fruit.

[0015] Figure 3Effect of heterologous overexpression of CaAUX22 on tomato fruit length: where (A) is CaAUX22 Phenotype diagram of heterologous overexpression tomato plants; (B) is CaAUX22 Phenotype diagram of heterologous overexpression tomato fruits; (C) is CaAUX22 Relative expression level in tomato overexpression plants; (D) is CaAUX22 Change in fruit length of heterologous overexpression tomatoes. Specific implementation mode

[0016] To facilitate the understanding of the present invention, the following will describe the present invention more comprehensively and meticulously in conjunction with the accompanying drawings of the specification and preferred embodiments, but the protection scope of the present invention is not limited to the following specific embodiments.

[0017] Unless otherwise defined, all professional terms used hereinafter have the same meaning as commonly understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the protection scope of the present invention.

[0018] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0019] Example 1: I. Chili pepper CaAUX22 Gene screening 1. Construction of the population Select chili pepper wild type LY0 and long fruit mutant LY143 as parents. After hybridization, an F1 population is obtained. As Figure 1 shown, after self-crossing, an F2 segregation population is obtained to complete the construction of the F1 and F2 populations.

[0020] 2. Phenotypic identification of fruit length Select three chili pepper fruits at the four-branch position at the mature green stage for each individual plant. Take pictures with a digital camera and measure the length of the chili pepper fruits with a vernier caliper to complete the preliminary identification of the phenotypic traits of the chili pepper fruit length in the above F1 and F2 populations. Among them, fruits with a measured length of 10 cm or more are long fruits, and fruits less than 10 cm are short fruits.

[0021] 3. BSA-seq analysis of fruit length genes According to the results of fruit length trait investigation, 25 long-fruit and 25 short-fruit plants were selected from the F2 population. Equal amounts of young leaves of individuals were mixed to construct two extreme pools. The DNA samples of the mixed pools were subjected to whole-genome resequencing, and the two parents were also resequenced. After sequencing, the relevant sequences and variation information were obtained by aligning to the reference genome. Based on the SNP-index statistical method of bulked segregant analysis, by comparing the differences in SNP allele frequencies in the two mixed pools (long-fruit pool and short-fruit pool), the genomic regions associated with the target trait were located; a threshold was set, and by calculating the difference in allele frequencies at the same SNP locus in the long-fruit pool and short-fruit pool, Δ(SNP-Index) was obtained, and the SNP locus region significantly associated with the target trait could be intuitively obtained. At the same time, the varBScore algorithm that can fully reduce the background noise in gene mapping and identification was used to identify the candidate regions of candidate genes again.

[0022] 4. Mapping of Fruit Length Genes As Figure 2 shown, after obtaining the target region significantly associated with the fruit length trait, we combined the variation information to develop KASP markers within the target region to further narrow down and precisely define the gene region. Through the joint analysis of the genotypes and phenotypes of the entire F2 population of plants using KASP markers, the candidate gene was finally mapped to an interval of approximately 573 kb on chromosome 3.

[0023] 5. Screening of Genes for Chili Fruit Length Traits In the mapping analysis of chili fruit length genes, we mapped the genes regulating chili fruit length mutants to an interval of 573 kb, covering 40 genes from Caz03g02700 to Caz03g03090. According to the variant-annotated genes in the BSA mixed pool resequencing, we further analyzed the expression and functional annotation of the annotated genes with variants by means of the transcriptomes of the parents' fruits at different stages, and found that only Aux22 was correlated with CaRLK. Subsequently, the expression characteristics of Aux22 and CaRLK in different tissues of wild-type and mutant were observed, and it was found that only Aux22 showed significantly higher expression in the roots, stems, leaves, flowers and fruits of the mutant than in the wild-type, and its expression in fruits was significantly the highest. Based on the above steps, the relevant gene Aux22 was initially selected as a candidate gene controlling the fruit length trait and named CaAUX22, Its nucleotide sequence is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2.

[0024] SEQ ID NO:1 ATGGCAACTGAAATGGAAATTACTGAGCTAAGATTGGGCCTTCCAGGGGGGATAATGGAGAAAAATGAGAAGAAAAGGGTGTATTCTGAAATGACTACTAGTGATCGGAACAGCAGCAGCAACGTTAACGTTAACGTTAAGTGTCAGAACAAGAATGAAGTTGTGGGGTGGCCACCAGTGTGTGCTTACAGGAGAAAGAACAGCTTTAATGGACGTGAAGGATCGAATAAAATGTACGTGAAAGTTAGCATGGATGGTGCTCCATTTCTGAGGAAAGTTGATTTGAGTACTCATAAGGGTTATGATGAACTTGTTATGGCTCTTGAAAAGCTCTTTGATTGCTATGGAATTGGAGAAGCATTGGAGGATGCAGACAAGTCAGAGTTCGTACCAATATATGAAGACAAAGATGGAGATTGGATGCTTGTTGGCGATGTTCCATGGCAAATGTTTAGTGAGTCATGCAAAAGGCTAAGGATCATGAAGAGATCAGAGGCAAAAGTGATAGGCCTTGGAGCCAGGGACTTTCTCAAGGGGATGTCTGAAAAATAA SEQ ID NO:2 MATEMEITELRLGLPGGIMEKNEKKRVYSEMTTSDRNSSSNVNVNVKCQNKNEVVGWPPVCAYRRKNSFNGREGSNKMYVKVSMDGAPFLRKVDLSTHKGYDELVMALEKLFDCYGIGEALEDADKSEFVPIYEDKDGDWMLVGDVPWQMFSESCKRLRIMKRSEAKVIGLGARDFLKGMSEK II. VIGS verification CaAUX22 Regulating the fruit length of LY143 To investigate CaAUX22 whether it has an impact on the fruit shape of pepper, the function was further verified by VIGS gene silencing CaAUX22 On the Solanaceae online website https: / / solgenomics.net / , select the tool "VIGS Tool", and use CaAUX22The full-length coding region was placed on the website to obtain an assessment of the silencing region. According to the assessment results and the mutation situation of this gene in the two parents, a silencing fragment of 200 - 500 bp was set. The silencing vector used was pTRV2, and the restriction endonucleases used were EcoRI and BamHI. Using wild-type LY143 as a template, the selected silencing region fragment was amplified and the target fragment was recovered by gel extraction. Subsequently, it was ligated to the digested pTRV2 vector. The recombinant vector was also transformed into Escherichia coli competent DH5α and plated on a plate containing 50 mg / ml kanamycin. After monoclonal colonies grew out, colony PCR identification was carried out and sequencing was confirmed. The primers used were the universal primers RNA2F / R on the vector.

[0025] The correctly sequenced recombinant vector was transformed into the Agrobacterium tumefaciens strain GV3101 of Tsingke Biological. The steps of Agrobacterium transformation were as follows: a. The Agrobacterium competent cells stored at -80 °C were placed at room temperature. After partial melting, they were inserted on ice. b. The correctly sequenced plasmid was added and gently mixed. Then it was left standing on ice for 5 min, in liquid nitrogen for 5 min, in a 37 °C water bath for 5 min, and in an ice bath for 5 min in sequence. c. 700 μl of sterile LB medium without antibiotics was added to the centrifuge tube. After mixing, it was resuscitated in a shaker at 28 °C and 200 rpm for 2 hours. d. The resuscitated bacterial solution was centrifuged, and 100 μl was taken and spread on a plate containing 50 mg / ml kanamycin and 50 mg / ml rifampicin. Monoclonal colonies were subjected to colony PCR identification.

[0026] Finally, the correct Agrobacterium monoclonal was selected and large-scale cultured and then inoculated onto the LY143 material at the 4 - 6 leaf stage. The infection solution at the time of inoculation consisted of 10 mM MgCl2, 10 mM MES, and 200 μM AS. The inoculation method was to inoculate the back of the leaf. The negative control was inoculated with the empty vector pTRV2, and the positive control was inoculated with the reporter gene pTRV2 - PDS 。

[0027] After the pepper plants were inoculated with the bacterial solution, they were placed in the dark at 16 °C for 24 h. Then the inoculated plants were transferred to an artificial climate chamber with a 16 h light / 8 h dark photoperiod and 70% relative humidity and cultured for three weeks. Three weeks after virus inoculation, we took the appearance of photobleaching in the leaves of the positive control plants as a signal of effective inoculation. Using the TRV virus detection primers to perform PCR identification and screening on the CaAUX22 newly emerged leaves to obtain positive lines, and the silencing efficiency of the positive plants was evaluated by qPCR.

[0028] Finally, phenotypic observations of relevant traits were carried out on the positive lines. The results showed that compared with the control, the silencing CaAUX22The fruit shape of the strain changed significantly, showing a phenomenon of shortening of the plant fruits, such as Figure 2 shown.

[0029] III. Overexpression of CaAUX22 gene To further understand the effect of CaAUX22 gene on the development of tomato fruit shape, a heterologous overexpression vector was constructed: specific primers containing restriction enzyme sites SacI and BamHI were designed, the overexpression vector used P1300S, and the CaAUX22 fragment obtained by amplification was identified by agarose gel electrophoresis and the recovered product was then ligated to the digested P1300S vector. After homologous recombination, the recombinant product was transformed into Escherichia coli DH5a and plated on a plate containing 50 mg / ml kanamycin. Subsequently, monoclonal colonies were selected for shaking culture, and the colonies were verified by PCR using primers 1300S-F / R and sequenced. The plasmid with correct sequencing was then transformed into Agrobacterium tumefaciens GV3101 for inoculation. During inoculation, transformed regenerated plants were obtained through processes such as Agrobacterium-mediated genetic transformation, screening culture, differentiation, and rooting of resistant buds. CaAUX22 When the regenerated plants took root and grew well, the leaves of the regenerated plants were cut, and the leaf DNA was extracted using the CTAB method. PCR detection was performed using specific primers for the screening marker gene NPTII F / R, and positive plants were identified according to the presence or absence of bands on the agarose gel electrophoresis. After obtaining positive plants, the seedlings were transferred from the medium to sterilized vermiculite for two weeks of acclimation. After the acclimation period, the plants were moved to the substrate and grown in an incubator at a day temperature of 25 °C / night temperature of 18 °C and a light of 16 h. At the T0 generation, plant lines were screened and subcultured by combining plant phenotypes and

[0030] the expression level of CaAUX22 . At the T3 generation, three overexpression lines were selected for comparison and analysis with the untransformed Micro Tom tomato material. CaAUX22 The results showed that

[0031] the lengths of fruits at the green ripe stage and red ripe stage in the overexpressing plants were significantly higher than those of the control group WT, indicating that CaAUX22 played a regulatory role in the length of tomato fruits, as CaAUX22 shown. Figure 3 In summary, the present invention provides new gene resources and methods for improving the fruit shapes of peppers and tomatoes, and has important theoretical and application values.

[0032] ​

Claims

1. CaAUX22 Application of gene in regulating pepper fruit shape ， It is characterized in that The said CaAUX22 nucleotide sequence of the gene is shown as SEQ ID NO. 1 2. The application according to claim 1 ， characterized in that the CaAUX22 amino acid sequence of the gene is as shown in SEQ ID NO.

2.

3. The application according to claim 1, wherein Silence CaAUX2 gene 2 in peppers to cultivate pepper varieties with short fruits.

4. CaAUX22 Application of a gene in regulating tomato fruit shape, characterized in that, The CaAUX22 nucleotide sequence of the gene is shown in SEQ ID NO.

1.

5. The application according to claim 1 ， Characterized in that the CaAUX22 amino acid sequence of the gene is as shown in SEQ ID NO.

2.

6. The application according to claim 4, characterized in that Heterologous overexpression in tomatoes CaAUX22 gene to cultivate long-fruit tomatoes.

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