Iris hollandica gene ihhd12 and expression protein and application thereof

Abstract

This invention discloses a Dutch iris IhHD12 Genes, their expressed proteins, and applications belong to the field of plant genetic engineering technology. The Dutch iris mentioned above... IhHD2 The gene, whose nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the expressed protein are shown in SEQ ID NO.2. Yeast activity experiments showed that this transcription factor possesses transcriptional activation activity. Functional identification based on transgenic rice plants revealed that the transformation of Dutch iris... IhHD12 The rice plants from the genetic line exhibit a phenotype of reduced plant height. This characteristic can provide some reference for breeding new dwarf Dutch iris varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to a species of Dutch iris. IhHD12 Genes, their expressed proteins, and applications. Background Technology

[0002] Dutch Iris ( Iris × hollandica Dutch irises (Iris truncata) are bulbous flowering plants belonging to the genus Iris in the family Iridaceae. They are the most widely cultivated and used type of bulbous iris, characterized by their upright plant shape, unique flower shape, and rich flower colors. Currently, they are mainly used for cut flower production. In addition, their natural flowering period is similar to that of tulips, and they bloom uniformly, so they can also be used in flower fields and flower gardens. However, due to the slow progress of Dutch iris breeding, there are currently few varieties circulating internationally, and most of them are large-bulb varieties, which can reach a height of 70-90 cm. Their excessive height makes them difficult to fully meet the requirements of ground cover cultivation. Although the technical difficulties of hybridization between small-bulb varieties have been solved (ZL202311015040.9), with the aim of obtaining more dwarf Dutch iris varieties through this method, the limited variety resources of this group restrict the space for genetic improvement through hybridization. In addition to conventional hybridization breeding, molecular breeding technology has become an effective means to accelerate the breeding process due to its advantages such as overcoming interspecific isolation barriers, strong predictability, high selection efficiency, and the ability to achieve recombination and aggregation of superior genes. Understanding the genes that control target traits is a prerequisite for using molecular biology methods to breed new varieties. However, so far, there have been no reports on genes that regulate the height of Dutch irises. Summary of the Invention

[0003] The purpose of this invention is to provide a gene, its expressed protein, and its application for dwarfing Dutch irises. This invention provides a selectable candidate gene for regulating the height of Dutch irises. In the future, by overexpressing this gene in Dutch irises, new varieties with reduced plant height can be obtained, which can then be used for flower fields and flower displays, showing promising application prospects.

[0004] The present invention achieves the above objectives through the following technical solutions: A type of Dutch iris IhHD12 The gene, whose nucleotide sequence is shown in SEQ ID NO.1.

[0005] The Dutch iris IhHD12 The gene expresses a protein whose amino acid sequence is shown in SEQ ID NO.2.

[0006] Used for cloning. IhHD12 The primer pair for the gene, and the primer sequences of the primer pair are as follows: IhHD12-F: TCGACTCGGTGCAAAAGTT; IhHD12-R:TATGGTGTGTCCGAGTTGCAT.

[0007] Contains the Dutch iris IhHD12 The vector or host bacterium of the gene.

[0008] This invention also provides Dutch iris IhHD12 Application of genes in suppressing rice plant height.

[0009] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a novel gene derived from Dutch iris. IhHD12 The gene is 606 bp in length and encodes 201 amino acid residues. Yeast activation experiments showed that this protein has transcriptional activation activity. Overexpression of this gene in rice resulted in a phenotype of reduced plant height. Therefore, Dutch iris... IhHD12 The gene and its encoded protein can be used for the genetic improvement of Dutch iris plant height. Attached Figure Description

[0010] Figure 1 A schematic diagram of the structure of IhHD12 (A) and a diagram of transcriptional activity analysis (B); Figure 2 for IhHD12 Gene overexpression phenotype identification diagram A: Plant height phenotype of wild-type (WT) and transgenic lines (#1-#3); B: Plant height statistics of wild-type and transgenic lines; Significance marker ** indicates P<0.01. Detailed Implementation

[0011] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0012] Plant materials: The Dutch irises used for gene cloning were obtained from the Iris Germplasm Resource Bank of the Institute of Botany, Chinese Academy of Sciences, Jiangsu Province. The rice callus tissue used for transgenic research was preserved in a tissue culture room with a photoperiod of 12 h light / 12 h dark and a culture temperature of 28℃.

[0013] Strains and vectors: E. coli DH5α Agrobacterium EHA105 All competent yeast strains were purchased from Vidi (Shanghai) Co., Ltd. AH109 The strains, pGBKT7, and pGADT7 vectors were purchased from TaKaRa, Dalian Biotechnology Co., Ltd. (Beijing), and the pEASY-Blunt vector was purchased from TransGen Biotech Co., Ltd. (Beijing). The rice overexpression vector pCAMBIA1305 was stored in the laboratory.

[0014] Example: (1) Dutch Iris IhHD12 Gene cloning Total RNA was extracted from the young shoots of Dutch iris 'Discovery' using the TaKaRa MiniBEST Plant RNA Extraction Kit, and then reverse transcribed into cDNA. Based on comparison with the Dutch iris transcriptome database, selected cDNA was obtained. IhHD1 The full-length sequence of gene 2 was obtained, and full-length primers were designed. PCR amplification was then performed using KOD high-fidelity enzyme from TOYOBO. The amplified product was identified by agarose gel electrophoresis and then purified. The purified product was then ligated with pEASY-Blunt, and after the reaction, it was transformed into *E. coli*. DH5α Competent cells were then plated on LB agar medium containing kanamycin and incubated overnight at 37°C (inverted) for 12-16 hours. Single clones were picked and sequenced by a sequencing company (Qingke) to obtain... IhHD12 Full-length gene sequence.

[0015] clone IhHD12 The primers used are as follows: IhHD12-F: TCGACTCGGTGCAAAAGTT; IhHD12-R:TATGGTGTGTCCGAGTTGCAT.

[0016] (2) Analysis of IhHD12 transcriptional activation activity Related adapter primers were designed, and the full-length IhHD12 sequence was constructed into the pGBKT7 vector. Then, pGBKT7-IhHD12 and the empty pGBKT7 vector were co-transformed with the empty pGADT7 vector into competent yeast cells according to the Clontech yeast transformation kit instructions. AH109 The transformed yeast cells were then plated onto SD / -Trp-Leu yeast-deficient medium. After incubation at 30°C inverted for approximately 3 days, single clones were picked and dissolved in 0.9% NaCl solution, and then spotted at different concentration gradients onto SD / -Trp-Leu (two-fold deficiency), SD / -Trp-Leu-His (three-fold deficiency), and SD / -Trp-Leu-His-Ade yeast-deficient mediums, and incubated inverted at 30°C. The results showed that only yeast cells transformed with the full-length IhHD12 gene could grow on SD / -Trp-Leu-His-Ade-deficient medium, while those transformed with the empty vector could not, indicating that IhHD12 possesses transcriptional activation activity.

[0017] The primer sequences for constructing the IhHD12 pGBKT7 vector are as follows (lowercase letters indicate vector homologous arms): IhHD12-BD-F: catggaggccgaattc ATGGCCGAGAGCGACCAGGA; IhHD12-BD-R: tagttatgcggccgctgcag TTATCCATTCATAGGCCAGA; (3) IhHD12 Phenotypic identification of transgenic rice Using primer pairs with adapters IhHD12 The full-length gene sequence was amplified with high fidelity. The pCAMBIA1305 vector was then digested with TaKaRa's XbaI rapid digester. Both the amplified fragments and the digested vector products were recovered and purified by agarose gel electrophoresis and then recombinantly constructed using Novizan's recombinase. The recombinant product was transformed into *E. coli*. DH5α Competent cells were plated on LB medium containing kanamycin and cultured overnight at 37°C. Single colonies were then picked and sequenced by a sequencing company (Qingke). After obtaining correctly constructed cells, they were transformed into Agrobacterium competent cells. EHA105 competent cells, then containing pCAMBIA1305-IhHD12 plasmid EHA105 Agrobacterium infection was performed on rice callus. Positive seedlings were obtained and, after DNA testing, were planted in a greenhouse, with three generations of seed harvesting until homozygous lines were achieved. Rice seeds from the T3 generation homozygous lines were then hydroponically cultured in an incubator with an 18-hour light / 6-hour dark photoperiod and a day / night temperature of 30℃ / 25℃. After 10 days of culture, seedling height was measured. The average height of the three transgenic lines was approximately 3.76 cm, 2.95 cm, and 3.86 cm, respectively, while the wild-type line was approximately 7.30 cm. The significantly lower height of the transgenic lines compared to the wild-type indicates that they originated from Dutch iris. IhHD12 Genes can serve as a new genetic resource for regulating plant height phenotype.

[0018] The primer sequences for constructing the IhHD12 pCAMBIA1305 vector are as follows (lowercase letters indicate vector homologous arms): IhHD12-GFP-F: cggagctagctctagaATGGCCGAGAGCGACCAGGA; IhHD12-GFP-R: tgctcaccatggatccTCCATTCATAGGCCAGAAAT; The primer sequences for detecting DNA in positive transgenic rice plants are as follows: IhHD12-test-F: AGGAGGTTCAGCGAGGAGCA; IhHD12-test-R: GGACTGGAGCGACTCGAAGC.

[0019] SEQ ID NO.1: ATGGCCGAGAGCGACCAGGAAGACAGCAGCTACATCTGGCTGGAGGAGCCGGGCGGGGGTGGCGGGGGCAAGCTGGGCAAGAAGAGGAGGTTCAGCGAGGAGCAGGTGAAGTCGCTGGAGCTCATGTTCGAGACGCAGGCCAAGCTCGAGCCGCGCAAGAAGCTGCAGCTGGCCAAGGAGCTCGGCCTGCACCCTCGCCAGGTCGCGATATGGTTCCAGAACAAGAGGGCGAGATGGAAGTCGAAGCAGCTCGAGAGGGAGTATGGCGCGCTCAAAGCAGACTACGATGCTCTTCTATCCAGCTTCGAGTCGCTCCAGTCCGAGAAGCAAGCTCTCGCCAAACAGTTGCACAAGTTGGCAGAGGTATTTAACAAACAAGTCCTTGAAGAAAGAGGCAAGGAGGAGGAGAAAGCTGTGCCATGGATCGAGAAATCGGACGACGAAGAGAACAGGGACGGTGGTAGATTCCGAGAGAAAGAAGAGGTGGGTCTGTGCGCAGAACCAGCTGCGGACGACGAGACCTTGGTGTCGAGCGAGCAGCACTTCTTGCTCCAGCAGCAGCCCACTTCATCACAGTGGTGGGATTTCTGGCCTATGAATGGATAA 606 bp SEQ ID NO.2: MAESDQEDSSYIWLEEPGGGGGGKLGKKRRFSEEQVKSLELMFETQAKLEPRKKLQLAKELGLHPRQVAIWFQNKRARWKSKQLEREYGALKADYDALLSSFESLQSEKQALAKQLHKLAEVFNKQVLEERGKEEEKAVPWIEKSDDEENRDGGRFREKEEVGLCAEPAADDETLVSSEQHFLLQQQPTSSQWWDFWPMNG 201 aa 。

Claims

1. A type of Dutch iris IhHD12 Genes, characterized by, The Dutch Iris IhHD12 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The Dutch iris as described in claim 1 IhHD12 The protein expressed by the gene is characterized by, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. Containing the Dutch iris as described in claim 1 IhHD12 The vector or host bacterium of the gene.

4. The Dutch Iris as described in claim 1 IhHD12 Application of genes in suppressing the height of rice seedlings.

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