Preparation method of citrus dominant function mutant

A mutant and functional technology, applied in the field of biotechnology research and development, can solve the problems of difficult plant materials and long citrus childhood, and achieve the effect of improving the infection efficiency

Active Publication Date: 2020-11-06
HUNAN INST OF GARDENING
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

However, citrus has a long childhood period, and it generally takes at least 5 years to grow from seeds to seed plants and bear fruit. Therefore, it is not easy for citrus to quickly obtain plant materials that are homozygous for the insertion of related genes through methods such as self-crossing and backcrossing. Using the method of large-scale construction of citrus T-DNA insertion mutation population to discover the important reasons for the identification of functional genes of citrus agronomic traits

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  • Preparation method of citrus dominant function mutant
  • Preparation method of citrus dominant function mutant
  • Preparation method of citrus dominant function mutant

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preparation example Construction

[0043] The invention provides a method for preparing a citrus dominant functional mutant, comprising the following steps:

[0044] Step 1, using the first pair of primers shown in SEQ ID NO: 1 and 2, using the pCAMBIA1300 plasmid as a template, PCR amplification obtains the vector backbone containing the transfer DNA domain, and then the first strong promoter gene, reporter Both the gene and the second strong promoter gene are connected between the left and right boundaries of the transfer DNA domain of the vector backbone, and the destination vector containing the target sequence in the transfer DNA domain is constructed, wherein the first strong promoter is located in the reporter gene upstream of , the second strongest promoter is located downstream of the reporter gene. The vector backbone of the present invention contains a transfer DNA domain, and the distance between the right border and the second strong promoter is suitable, a distance of more than ten base pairs, so ...

Embodiment 1

[0074] 1.1 Obtaining carrier elements

[0075] a. Use primers Right298XS-F (SEQ ID NO: 1: 5'-GCGTCGACTCTAGAGGTAAACCTAAGAGAAAAGAGCG-3') and Left6582HS-R (SEQ ID NO: 2: 5'-GCGTCGACAAGCTTTTGTTTACACCCACAATATATCCTGC-3') to amplify plasmid pCAMBIA1300 to obtain vector element pVC ( 6286bp) PCR product. (The pCAMBIA1300 plasmid comes from the laboratory of Hunan Horticultural Research Institute; PCR reaction system (30 μL): 3 μL of 10×PCR buffer, 1 μL of dNTPs (each 2.5 mmol / L), 0.5 μL of forward primer (10 μmol / L), reverse Primer (10 μmol / L) 0.5 μL, Pyrobest DNA polymerase (5U / μL) 1 μL, template (0.2 μg / L) 1 μL, sterile deionized water 23 μL; amplification conditions: 95°C for 5min; 95°C for 20s, 55°C ℃20s, 72℃6min, 25 cycles; 72℃10min.)

[0076] b. with primers NOSp2462H-F (SEQ ID NO: 5'-CCAAGCTTGCCAATATCCTGTCAAACACTG-3') and NOSp2837S-R (SEQ ID NO: 6: 5'-GCGTCGACGCGAAACGATCCAGATCC-3') amplified plasmid pBI121 to obtain vector element NOSp ( 377bp) PCR product.

[0077] c. Usin...

Embodiment 2

[0113] 1. Preparation of dominant functional mutants of rock sugar orange and red navel orange

[0114] ① Seeds of rock sugar orange or navel orange, which were stripped of the outer testa and treated with 1% NaClO for 10 min, were sown on MS salt medium (pH 5.7) and cultured in the dark for about 20 days, and then cultured in the light (12 h light / 12 h dark) for 10 days. The growth temperature was maintained at 26-28°C. At this time, the thickness of the stem of the seedling plant reaches 1mm-3mm, and the plant height is greater than 5cm.

[0115] ② Pick a single colony of Agrobacterium (the strain contains pVC-NOSpGUSter-35 plasmid or pBI121 plasmid) in LB liquid medium (containing 50 μg / mL Rif and 50 μg / mL kanapenicillin), in a shaker at 28 ° C, Cultivate overnight at 180rpm; then transfer at 1:50 and continue to culture to OD 600 1.2-1.8; then centrifuge at 5000rpm for 5min, discard the supernatant, resuspend with the transformation medium, and adjust to the OD of the ba...

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Abstract

The invention discloses a method for preparing a citrus dominant functional mutant. The method comprises the following steps: 1, taking a vector pCAMBIA1300 as a template by utilizing a first primer pair, amplifying to obtain a vector framework containing a transfer DNA structural domain, connecting a first strong promoter gene, a reporter gene and a second strong promoter gene into the left and right boundaries of the transfer DNA structural domain of the carrier framework, and constructing to obtain a destination vector of which the transfer DNA structural domain contains a target sequence;2, integrating the target sequence onto citrus seedling genome by agrobacterium tumefaciens mediated transformation, and screening to obtain a positive transformant; 3, culturing the positive transformant into a plant, planting in soil or grafting onto stock for preservation, thereby obtaining the citrus dominant functional mutant. According to the method disclosed by the invention, available citrus genetic materials at modern times can be created, and the problem that directly available citrus mutants cannot be rapidly created by utilizing a T-DNA insertion method at present can be solved.

Description

technical field [0001] The invention belongs to the technical field of biotechnology research and development, and relates to a method for preparing a citrus dominant function mutant. Background technique [0002] After the T-DNA sequence is inserted into the plant genome, it can destroy the transcription of the gene at the insertion site. The plants with the T-DNA sequence inserted can obtain the homozygous line of the T-DNA insertion of the gene in their progeny through methods such as self-crossing and backcrossing, that is, a mutant with the loss of the gene function can be obtained. Using the obtained T-DNA insertion homozygous mutants and wild-type plant materials to perform phenotypic detection under different growth conditions has become an important means to discover and identify functional genes in Arabidopsis, rice and other plants. However, citrus has a long childhood period, and it generally takes at least 5 years to grow from seeds to seed plants and bear frui...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/65A01H5/00A01H6/78
CPCC12N15/8205C12N15/8261
Inventor 孔佑涵李卫东李先信吴娟娟苑平张平
Owner HUNAN INST OF GARDENING
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