Method for cultivating phosphor highly active paddy rice utilizing inverted citrate synthetase gene

A technology for citrate synthase and rice, which is applied in the fields of genetic engineering and rice molecular breeding, and can solve the problems of not finding an effective way and the like

Inactive Publication Date: 2004-06-02
HUAZHONG AGRI UNIV
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Problems solved by technology

Although plant genetic engineering has achieved great success in improving crop resistance, insect resistance, stress resistance, herbicide resistance, quality, hybrid seed production technology, etc., it has not yet improved the efficient use of soil phosphorus by crops. find an effective way

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  • Method for cultivating phosphor highly active paddy rice utilizing inverted citrate synthetase gene

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Embodiment 1

[0022] Embodiment 1: Cloning of citrate synthase gene coding sequence

[0023] a. According to Lynda et al. (Lynda, Gilles F. Molgat and Harry W. Duckworth. Cloning, sequencing, and expression of the gene for NADH-sensitive citrate synthase of Pseudomonas aeruginosa. J Bacteriol, 1989, 171(10): 5542-5550) Primer P1 (5'-GC GGA TCC ATGGCT GAC AAA AAA GCG-3') and P2(5'-CG GGA TCC TCA GCC GCG ATC CTT GAG-3').

[0024] A BamHI endonuclease recognition site is designed in the primers to facilitate gene modification;

[0025] b. clone the citrate synthase gene coding sequence from the genome of Pseudomonas (numbering is Pa 1.112, this bacterial strain is purchased by Institute of Microbiology, Chinese Academy of Sciences) with the method of PCR cloning (part a of embodiment 1 has disclosed this nucleotide sequence of the gene);

[0026] c. Digested with BamHI, connected to the vector pBluescript-SK, named pBS-CS;

[0027] d. Use a KpnI restriction site at the 5' end of the cod...

Embodiment 2

[0028] Embodiment 2: Construction of recombinant gene and expression vector

[0029] a. Carry out double digestion of plasmid pBS-CS1 with SmaI and XbaI enzymes, and recover the digestion products;

[0030] b. At the same time, carry out double digestion with SmaI and XbaI enzymes on the vector pRTL2-NS containing double 35S promoters and 3' end regulatory sequences for enhanced expression, and recover the digestion products;

[0031] c. The two digested products were ligated with T4 DNA ligase, and the recombinant plasmid was named pRTL2-Cs;

[0032] d. Recombinant plasmid pRTL2-CS was digested with HindIII, and the digested product was recovered; at the same time, the binary Ti plasmid vector pCAMBIA1301 was digested with HindIII, and dephosphorylated, and the digested product was recovered;

[0033] e. The two digested products were ligated with T4 DNA ligase, and the recombined binary Ti plasmid vector was named p1301-CS;

[0034] f. Introducing p1301-CS into Agrobacteri...

Embodiment 3

[0035] Embodiment 3: Determination of available phosphorus content in phosphorus-deficient soil for potted plants

[0036] Use molybdenum-antimony resistance method (Huazhong Agricultural University Teaching Experiment Book "Soil Chemistry Experiment Guide", 1993, Huazhong Agricultural University Textbook Section) to measure the content of available phosphorus in the phosphorus-deficient soil for potting to be 2.25mg / kg. In order to increase the content of total phosphorus in the soil, we add phosphate rock powder to the soil at a ratio of 1 / 1000. It has been determined that the content of available phosphorus in the phosphate rock powder is 11.76mg / kg.

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Abstract

The present invention belongs to the field of genetic engineering and new plant variety breeding technology, and features that Citrate synthase from bacteria is introduced into rice acceptor, and by means of report gene, PCR detection and molecular hybridization to identify transgenic plant, examine the soil null phosphate absorbing case and agricultural character of filial plant, select transgenic rice plant line with obviously increased stooling, increased yield and low phosphate tolerance, and further breed high efficiency phosphate rice variety and create new resource for breeding.

Description

Technical field: [0001] The invention belongs to the technical fields of genetic engineering and rice molecular breeding. Specifically, it involves the application of transgenic methods to introduce exogenous citrate synthase gene (CS) into rice receptors, increase the synthesis and secretion of citric acid, enhance the tolerance of rice to low phosphorus, and cultivate a new variety of rice with high phosphorus efficiency or A method of creating new resources. Background technique: [0002] The lack of available phosphorus in soil has affected the growth and yield of crops to varying degrees in the world. For a long time, research on plant nutrition and fertilization has mostly used measures such as soil improvement and fertilization to improve soil fertility, which not only increases the cost of agriculture, but also increases the pollution of the environment, especially water bodies, due to the extensive use of chemical fertilizers. Conventi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H1/00A01H5/00C12N15/09C12N15/52
Inventor 张启发周泽民林拥军贺立源徐才国
Owner HUAZHONG AGRI UNIV
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