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Cloning method and application of lily bulbil formation regulation gene LlWOX11

A technology of lily pearl buds and genes, which is applied in the field of plant genetic engineering and can solve problems such as unreported functional research.

Active Publication Date: 2021-09-14
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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  • Cloning method and application of lily bulbil formation regulation gene LlWOX11
  • Cloning method and application of lily bulbil formation regulation gene LlWOX11
  • Cloning method and application of lily bulbil formation regulation gene LlWOX11

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Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0045] 1. RNA extraction and cDNA synthesis

[0046] RNA extraction: Extract the leaf axil tissue of Juandan containing bulbils, 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. Ⅱ 1st 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 gently blow and mix with a pipette. Incubate at 42°C for 2 minutes.

[0049]

[0050] (2) Add directly to the previous reaction tube ⅡSuperMix plus, use a pipette to mix gently by pipetting. Carry out the following reactions in a PCR instrument: ...

Embodiment 2

[0064] Embodiment 2 LlWOX11 gene expression analysis

[0065] 1. RNA extraction and cDNA synthesis

[0066] RNA extraction: Take the axil tissue of the bulbils in Lilac and different tissues (shoot tips, root tips, scales, young leaves, mature leaves, stems, petals, stigmas, anthers and ovaries), and use RNAprep from Tiangen Biochemical Technology (Beijing) Co., Ltd. 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. Ⅱ 1st Strand cDNASynthesis SuperMix for qPCR (gDNA digester plus) reverse transcription kit for cDNA synthesis.

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

[0069] Fluorescent quantitative primers were designed using Primer 6.0, qLlWOX11-F / R, the sequence of the forward primer was: 5′-TCGCTGCGTCCAATCCTCGG-3′, and the sequence of the reverse primer was: 5′-CTGCCTGTTTCTCATCAATCCC-3′....

Embodiment 3

[0072] Example 3 LlWOX11 subcellular localization

[0073] 1. Construction of pCAMBIA2300-LlWOX11 vector

[0074] According to the CDS sequence of the LlWOX11 gene, adapter primers were designed, and the full-length sequence of the gene was amplified by PCR (removing the stop codon). : 5'-CCATGGTACTAGTGTCGACTCTAGACTACGAAGGCCTCGAAACCAA-3'. The PCR product was recovered and used for later use. The pCAMBIA2300 vector was digested with XmaI and XbaI, and 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 is 1:5. Gently blow and mix with a pipette gun, then recombine in a PCR machine at 50°C for 15-20min, transfer the recombined product into Escherichia coli competent transformation, and send the monoclonal to be sequenced after being tested positive.

[0076] 2. Agrobacterium transformation

[0077] The positive plasmid was ...

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Abstract

The invention discloses a cloning method and application of a lily bulbil formation regulation gene LlWOX11. The nucleotide sequence of the lily bulbil formation regulation gene LlWOX11 is as shown in SEQ ID NO. 1. The WOX11 gene has the main function of participating in development of lateral roots or adventitious roots in model plants of arabidopsis thaliana and rice. In lilium brownii, the bulbil induction rate can be remarkably reduced by inducing LlWOX11 gene silencing through a VIGS technology; and after the LlWOX11 is over-expressed, the induction rate of the bulbil is increased, which indicates that the LlWOX11 plays an important regulation function in the formation of the bulbil of the lily, and the LlWOX11 gene can effectively and manually intervene in the formation of the bulbil of the lily and has a wide application prospect in the aspect of improving the propagation efficiency of the 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 bulbil formation regulatory gene L1WOX11. Background technique [0002] Lily (Lilium) is a world-renowned bulbous flower. Its flowers are large, colorful, and fragrant. It has high economic value 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 values. [0003] Lilium lancifolium, also known as Yixing lily, is native to my country. It has strong adaptability and a wide distribution range. 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 as many as hundreds of bulbils. Therefore, bulbil reproduction has become an important reproductive strategy for...

Claims

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Application Information

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Patent Type & Authority Applications(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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