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GAT vector for mediating and regulating male fertility of plant recessive genic male sterile line and application of GAT vector

A male fertility and nuclear male technology, applied in the field of agricultural biology, can solve the problems of multi-gene transformation that cannot be screened to obtain all the transformation events of the target traits, transgene silencing, gene transfer, etc.

Pending Publication Date: 2021-11-09
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to problems such as partial gene transfer, inability of each transgenic element to function simultaneously, and transgene silencing often occur in the process of multigene transformation, transformation events that cannot be screened to obtain target traits often occur in multigene transformation.

Method used

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  • GAT vector for mediating and regulating male fertility of plant recessive genic male sterile line and application of GAT vector
  • GAT vector for mediating and regulating male fertility of plant recessive genic male sterile line and application of GAT vector
  • GAT vector for mediating and regulating male fertility of plant recessive genic male sterile line and application of GAT vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Embodiment 1 GAT carrier construction and verification

[0099] 1. Construction of GAT vector

[0100] The GAT vector adopts the strategy of segmented construction with the expression cassette as the unit and unit splicing. First in the transition vector pC1300 ( Figure 8 ) and pUC57-Simple ( Figure 9 ) to construct an expression cassette and verify it by digestion and sequencing, and then splicing the expression cassette into the final vector. The specific steps of vector construction are as follows:

[0101] 1. Synthesize the MCS pC0307 fragment (SEQ ID NO.1), connect pC1300 with Sac II and Pme I to obtain pC0307 ( Figure 5 ).

[0102] 2. Synthesize the MCS pC0308 fragment (SEQ ID NO.2), connect pC1300 with Sac II and Sph I to obtain pC0308 ( Image 6 ).

[0103] 3. Synthesize the MCS pC0309 fragment (SEQ ID NO.3), connect pC1300 with Sac II and Pme I to obtain pC0309 ( Figure 7 ).

[0104] 4. Synthesize the DNA fragment NSPT-Construct V1.81-Marker 1, cut ...

Embodiment 2

[0130] Embodiment 2 Agrobacterium-mediated genetic transformation of rice with GAT carrier

[0131] 1. Transformation of Agrobacterium with GAT vector and verification

[0132] Agrobacterium EHA105 stored at -80°C was streaked on a YEP plate containing rifampicin (25 μg / ml) + streptomycin (50 μg / ml), and cultured at 28°C. Pick a single colony and inoculate it in 5 ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 220 rpm and shake at 28° C. for 12-16 hr. Take 2ml of the bacterial liquid and transfer it to 100ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 28°C with shaking at 220rpm until OD600=0.5. Pre-cool on ice for 10 min, centrifuge at 5000 rpm for 10 min at 4°C (refrigerated centrifuge is pre-cooled to 4°C). Wash twice with sterile deionized water (10 ml each time), wash once with 10% sterile glycerol, centrifuge at 5000 rpm for 10 min at 4° C., and resuspend the bacteria in 3 ml of 10% sterile glycerol. Take...

Embodiment 3

[0140] Example 3 Molecular Identification of GAT T0 Transformation Materials

[0141] At the five-leaf stage, the transgenic plant leaves obtained in Example 2 were extracted with the CTAB method to extract the total DNA of the genome, and the extraction method was as follows:

[0142] Take 2-4cm leaves and put them into a mortar, add 800-900ul of 1.5% CTAB solution, grind, then transfer the ground liquid into a 1.5ml centrifuge tube, put it on ice or in a low-temperature refrigerator for use; sample Water bath at 65°C for 30 minutes, during which time it was inverted and mixed several times; in the fume hood, use a glass pipette to add chloroform and isoamyl alcohol solution (chloroform: isoamyl alcohol = 24:1, that is, 500ml chloroform is added to 22ml isoamyl alcohol, Gently mix) 650ul, shake on the shaker for 30min or shake by hand for about 10min after mixing, at this time, obvious stratification can be seen; centrifuge the shaken sample at 8000-10000rpm for 8min; suck th...

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Abstract

The invention provides a GAT vector for mediating and regulating male fertility of a plant recessive genic male sterile line and application of the GAT vector. The GAT vector comprises five functional element expression cassettes: a plant male fertility restoration gene element expression cassette used for restoring male fertility of recessive genic male sterility mutants, a plant pollen abortion gene element expression cassette used for removing pollen containing GAT and keeping a heterozygous state of a GAT maintainer line, a chemical herbicide positive selection expression cassette used for gene transformation and GAT maintainer line impurity removal and purification, a chemical herbicide negative selection expression cassette used for removing herbicide-sensitive GAT maintainer line pollen and seed escape and removing impurities and purifying a GAT sterile line; and a seed screening element expression cassette used for mechanically sorting seeds. The GAT vector disclosed by the invention can be used for crossbreeding and hybrid seed production of plant recessive genic male sterility materials, so that new plant varieties with high quality, high yield, wide applicability and high resistance and seeds of the new plant varieties are obtained, and the GAT vector has huge economic value.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and specifically relates to a GAT carrier for mediating and regulating the male fertility of plant recessive nuclear male sterile mutants and an application thereof. Background technique [0002] Heterosis is a phenomenon in which offspring of hybrids surpass their parents in one or more traits, and it generally exists in the biological world. The utilization of crop heterosis is an important means to increase agricultural production, and the core of the industrialization of heterosis utilization lies in the male sterility of the female parent. [0003] Taking rice as an example, China is the most successful country in the world in the industrialization of hybrid rice, and it is in a leading position in the field of hybrid rice breeding research. At present, the breeding methods of hybrid rice in my country are mainly divided into "three-line method" and "two-line method", each has its ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/66A01H5/10A01H6/46A01H1/02A01H1/04
CPCC12N15/8289A01H1/02A01H1/04A01H5/10A01H6/46C12N15/66C12N15/82
Inventor 龙湍李新鹏安保光曾翔吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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