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Genetic intelligent breeding and seed production carrier for crops

A technology of crops and vectors, applied in the field of agricultural biology, can solve the problems of multi-gene transformation that cannot be screened to obtain transformation events that achieve the target traits, gene transfer, transgene silencing, 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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  • Genetic intelligent breeding and seed production carrier for crops
  • Genetic intelligent breeding and seed production carrier for crops
  • Genetic intelligent breeding and seed production carrier for crops

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Embodiment 1 GAT carrier construction and verification

[0093] 1. Construction of GAT vector

[0094] 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 6 ) and pUC57-Simple ( Figure 7 ) 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:

[0095] 1. Synthesize the MCS pC0308 fragment (SEQ ID NO.17), connect pC1300 with Sac II and Sph I to obtain pC0308 ( Figure 4 ).

[0096] 2. Synthesize the MCS pC0309 fragment (SEQ ID NO.23), connect pC1300 with Sac II and Pme I to obtain pC0309 ( Figure 5 ).

[0097] 3. Synthesize the DNA fragment NSPT-Construct V1.81-Marker 1, cut it with Kpn I+Hind III and join it into pC1300 to generate pC1300-Marker 1. The sequence of NSPT-Construct V1.81-Marker 1 consist...

Embodiment 2

[0120] Embodiment 2 Agrobacterium-mediated genetic transformation of rice with GAT carrier

[0121] 1. Transformation of Agrobacterium with GAT vector and verification

[0122] 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 bacterial liquid and transfer it to 100ml of YEP liquid medium containing the above-mentioned antibiotics, culture at 28°C and shake at 220rpm until OD 600 = 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 1 ...

Embodiment 3

[0130] Example 3 Molecular Identification of GAT T0 Transformation Materials

[0131] 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:

[0132] 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 genetic intelligent breeding and seed production carrier for crops. The seed breeding and production system contains a GAT system carrier, and the carrier is pC1300-MMCK, pC0308-MMCK or pC0309-MMCK; the nucleotide sequences of the carrier are respectively shown as SEQ ID NO.1-3. The genetic intelligent breeding and seed production carrier creatively solves the maintenance and reproduction problems of recessive genic male sterility, and can be used for crossbreeding and hybrid seed production of plant recessive genic male sterility materials, so that new varieties of plants with high quality, high yield, wide applicability and high resistance and seeds of the plants are obtained, and the genetic intelligent breeding and seed production carrier has huge economic value.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a carrier for genetic intelligent breeding and seed production of crops and its application. 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 advantages and disadvantages. The "three-li...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/02A01H1/04C12N15/82C12Q1/6895
CPCA01H1/02A01H1/04C12N15/8289C12Q1/6895C12Q2600/13C12N15/82
Inventor 李新鹏安保光龙湍曾翔吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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