A kind of cloning method of lily bulb formation regulation gene llwox11 and its application

A technology of gene and lily, applied in the field of cloning of lily bulb bud formation regulation gene LlWOX11, can solve problems such as unreported functional research, achieve broad application prospects, and improve the effect of lily reproduction efficiency

Active Publication Date: 2022-08-02
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few isolated genes related to plant bulbil formation, and they mainly focus on gene cloning and expression analysis, and no functional studies have been reported.

Method used

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  • A kind of cloning method of lily bulb formation regulation gene llwox11 and its application
  • A kind of cloning method of lily bulb formation regulation gene llwox11 and its application
  • A kind of cloning method of lily bulb formation regulation gene llwox11 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Cloning and bioinformatics analysis of LlWOX11 gene

[0045] 1. RNA extraction and cDNA synthesis

[0046] RNA extraction: Take the leaf axil tissue of dandelion buds, and use the RNAprepPure polysaccharide and polyphenol plant total RNA extraction kit from Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the total plant RNA. The extraction method is carried out according to the instructions.

[0047] cDNA synthesis: using Yisheng Biotechnology (Shanghai) Co., Ltd. Ⅱ1st Strand cDNASynthesis SuperMix for qPCR (gDNA digester plus) reverse transcription kit for cDNA synthesis, follow the steps below:

[0048] (1) Prepare the following mixture in an RNAse-free centrifuge tube, and mix with a pipette. Incubate at 42°C for 2 min.

[0049]

[0050] (2) Add directly to the reaction tube in the previous step ⅡSuperMix plus, mix by pipetting gently with a pipette. Perform the following reactions in a PCR machine: 25°C, 5 min; 42°C, 30 min; 85°C, 5...

Embodiment 2

[0064] Embodiment 2 LlWOX11 gene expression analysis

[0065] 1. RNA extraction and cDNA synthesis

[0066] RNA extraction: Take the leaf axil tissue containing pearl buds and different tissues (shoot tips, root tips, scales, young leaves, mature leaves, stems, petals, stigmas, anthers and ovaries), and use Tiangen Biochemical Technology (Beijing) Co., Ltd. RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit extracts plant total RNA, and the extraction method is carried out according to the instructions.

[0067] cDNA synthesis: using Yisheng Biotechnology (Shanghai) Co., Ltd. Ⅱ1st Strand cDNASynthesis SuperMix for qPCR (gDNA digester plus) reverse transcription kit for cDNA synthesis.

[0068] 2. Quantitative Real-time RT-PCR

[0069] Primer 6.0 was used to design fluorescent quantitative primers, qLlWOX11-F / R, the forward primer sequence was: 5'-TCGCTGCGTCCAATCCTCGG-3', and the reverse primer sequence was: 5'-CTGCCTGTTCTCCATCAATCCC-3'. Take lilyActin (...

Embodiment 3

[0072] Example 3 Subcellular localization of LlWOX11

[0073] 1. Construction of pCAMBIA2300-LlWOX11 vector

[0074] According to the CDS sequence of the LlWOX11 gene, the linker primers were designed, and the full-length sequence of the gene was amplified by PCR (the stop codon was removed). The upstream primer sl-LlWOX11-F was: 5′-TTGGAGAGGACAGGGTACCCGGGATGGATAGCCACACACCCAAC-3′; : 5'-CCATGGTACTAGTGTCGACTCTAGACTACGAAGGCCTCGAAAACCAA-3'. The PCR product was recovered and used for later use. After the pCAMBIA2300 vector was digested with XmaI and XbaI, it was recovered for use.

[0075] The LlWOX11 linker product was fused into the pCAMBIA2300 linear vector by homologous recombination. The molar ratio of vector to insert in the homologous recombination system was 1:5. After mixing gently with a pipette, recombine in a PCR machine at 50°C for 15-20 min. The recombined product is transferred into E. coli competent transformation, and the single clone is sent for sequencing aft...

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Abstract

The present invention discloses a cloning method and application of a lily bulb bud formation regulating gene L1WOX11, and its nucleotide sequence is shown in SEQ ID NO.1. The main function of WOX11 gene in model plants Arabidopsis and rice is to participate in the development of lateral or adventitious roots. In lily, the induction of LlWOX11 gene silencing by VIGS technology can significantly reduce the induction rate of bulblets; after overexpression of LlWOX11, the induction rate of bulblets increases, indicating that LlWOX11 plays an important regulatory function in the formation of bulblets in lily, and the LlWOX11 gene can inhibit the formation of lily bulbs. Effective manual intervention for bulb formation will have broad application prospects in improving the reproductive efficiency of lily.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a cloning method and application of a lily bulb bud formation regulation gene L1WOX11. Background technique [0002] Lilium is a world-famous bulbous flower. Its flowers are large in color, rich in fragrance, and have high economic and ornamental value. It is one of the top five fresh cut flowers in the world. In addition, lily is rich in starch, protein, vitamins, alkaloids, etc., and has important edible and medicinal value. [0003] Lilium lancifolium, also known as Yixing lily, is native to my country. It has strong adaptability and wide distribution. It is an important species in the genus Lilium with ornamental, edible and medicinal values. It is a natural triploid and cannot reproduce by seeds, but its leaf axils can produce up to hundreds of pearl buds. Therefore, bud propagation has become an important reproductive strategy. [0004] Bulbs are form...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/10C12N15/84A01H5/00A01H5/10A01H6/20
CPCC07K14/415C12N15/1096C12N15/8218C12N15/8205C12N15/8261C12Q2531/113
Inventor 杨盼盼明军何国仁徐雷锋
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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