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Screening method of transgenic rice

A technology of transgenic rice and a screening method, which is applied in the field of efficient and safe genetic transformation and screening of transgenic rice, can solve the problems that the phosphomannose isomerase gene cannot be used as a marker gene of transgenic tobacco, cannot use antibiotics, etc., and achieves rapid identification and screening. The effect of improving efficiency and reducing conversion costs

Pending Publication Date: 2020-11-24
HUAZHONG AGRI UNIV
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Problems solved by technology

However, the above marker genes have some limitations
Some plant species are naturally resistant to antibiotics, resulting in the genetic transformation of these plants cannot be selected using antibiotics, such as orchids are naturally resistant to hygromycin and kanamycin
Some plants are not sensitive to some non-metabolites, such as tobacco is not sensitive to mannose, phosphomannose isomerase gene cannot be used as a marker gene of transgenic tobacco
At the same time, herbicide and antibiotic resistance traits may also be horizontally transferred to wild-type related species through transgene escape channels such as pollen dispersal and seed dispersal, which may have a negative impact on the natural environment, animals, plants and human beings on the earth.

Method used

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Experimental program
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Effect test

Embodiment 1

[0036] Construction of Overexpression Vector of Phosphite Dehydrogenase Gene (PhiQ)

[0037]According to the nucleotide sequence of the PhiQ gene, PCR primers were designed and synthesized to amplify the PtxQ gene. The PCR system and amplification conditions are shown in Table 1.

[0038] The primer sequences are as follows:

[0039] PhiQ-F: 5'-tgcaggtcgactctagagATGAAGCCGAAGGTGGTCCT-3',

[0040] PhiQ-R: 5'-tcgagctcggtacccgggTTAGGCGGCCTTCACGCCTGGGT-3';

[0041] Table 1 PCR reaction system and amplification conditions

[0042]

[0043] The obtained PCR product was subjected to agarose gel electrophoresis, and the PCR product of PhiQ gene was recovered using the gel recovery kit provided by Beijing Kangwei Century Biotechnology Co., Ltd. The pCAMBIA1300-35S plasmid was digested with BamH I, subjected to agarose gel electrophoresis, and then the linearized pCAMBIA1300-35S plasmid was recovered using the gel extraction kit provided by Beijing Kangwei Century Biotechnology Co....

Embodiment 2

[0045] rice genetic transformation

[0046] (1) Test materials The wild-type (ie non-transgenic) rice variety Zhonghua 11 (also known as ZH11, from the Institute of Crop Science, Chinese Academy of Agricultural Sciences).

[0047] (2) Rice transgenic method

[0048] 1) Preparation of rice mature embryo callus

[0049] Remove the husks from the 11 seeds of wild-type rice Zhonghua, pick plump and smooth seeds in a beaker, disinfect them with 75% ethanol solution for 1 min in an ultra-clean workbench, pour off the ethanol, and then use 20% (v / v) NaClO The solution (approximately 1-1.2% available chlorine) was disinfected for 10 minutes. Pour off the NaClO solution, wash 5-6 times with sterile water, and finally soak in sterile water for 30 minutes, pour off the sterile water, and dry the seeds on absorbent filter paper. After drying, the seeds were transferred to the induction rice medium (Table 6) with tweezers, and placed in a dark incubator at 28°C for 28-30 days. Then ope...

Embodiment 3

[0086] Comparison of Survival Rates of Wild-type and Transgenic Rice Callus Under Phosphite Treatment

[0087] Select the wild-type ZH11 and transgenic PhiQ rice calli with basically the same growth status, size and color, and put them on the rice subculture medium with five phosphite treatment concentrations of 1-5mM, and at the same time, use the normal phosphate concentration of 2.9mM as the experiment As a control, culture was carried out in a dark incubator at 28°C. After 14 days of culture, the phenotypes of wild-type ZH11 and transgenic PhiQ rice callus were observed and compared, and the survival rates of wild-type ZH11 and transgenic PhiQ rice callus were compared.

[0088] Depend on figure 1 It can be seen that on the rice subculture medium containing phosphite as the only phosphorus source, the growth of wild-type ZH11 rice callus was significantly inhibited, and the survival rate was significantly reduced compared with normal phosphate treatment conditions; while ...

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Abstract

The invention relates to the technical field of plant genetic engineering, and discloses a screening method of transgenic rice. The invention establishes a high-efficiency transgenic rice screening and identifying method using a phosphite dehydrogenase gene as a selection marker and phosphite as a screening agent. The method is used for screening transgenic rice calluses, the efficiency is higherthan that of existing hygromycin and glufosinate-ammonium screening, and the method has the advantages of being safer and more environmentally friendly. Meanwhile, a method for screening transgenic rice positive seedlings under the conditions of water culture and foliar spraying of phosphite is established, it is found that transgenic rice seedlings can be screened under the conditions of water culture and foliar spraying of phosphite, and an efficient screening identification method for transgenic rice in the seedling stage is established.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to an efficient and safe screening method for genetic transformation of transgenic rice. Background technique [0002] During plant genetic transformation, only a small fraction of cells are able to acquire the target gene during the transformation process. Therefore, in order to efficiently screen these transformed cells that have acquired the target gene, the use of efficient marker genes is very necessary. These genes can generally encode proteins, giving transformed cells a selective advantage over untransformed cells. At the same time, transgenic plant selection systems must allow selective growth and differentiation of successfully transformed cells and inhibit the growth of unsuccessfully transformed cells. Therefore, in the process of plant genetic transformation, the selection of marker genes is very critical, which can help researchers identify successfu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895A01H1/04A01H4/00A01G31/00A01G7/06A01G7/00
CPCA01G7/00A01G7/06A01G31/00A01H1/04A01H4/008C12Q1/6895C12Q2600/13
Inventor 王创吴高兵刘诗琦袁丽丽刘同同
Owner HUAZHONG AGRI UNIV
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