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Application of vector containing CYP704B2 gene

A CYP704B2, gene technology, applied in the field of genes and their encoded proteins, can solve the problems of unclear molecular regulation mechanism and network of pollen outer wall components

Active Publication Date: 2011-11-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the molecular regulatory network for the development of rice tapetum, especially the composition of the pollen exine and its molecular regulatory mechanism and network are still unclear.

Method used

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  • Application of vector containing CYP704B2 gene
  • Application of vector containing CYP704B2 gene
  • Application of vector containing CYP704B2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Obtaining and Morphological Observation of CYP704B2 Mutant Plants

[0045] The mutant was mutagenized with japonica rice 9522 (Wild type / WT) seeds by 60Coγ-rays at a dose of 280Gy. A male sterile mutant in the mutagenized F2 generation was backcrossed for three generations, and a stable genetic mutant CYP704B2 regulated by a recessive single gene was obtained. The CYP704B2 mutant was backcrossed with japonica rice 9522, all the F1 generations were fertile, and segregation occurred in the self-crossed F2 generation, of which 78 were normal plants and 23 were mutant plants (χ2=0.161), indicating that the phenotype of the male sterile mutant was composed of Caused by a single gene mutation.

[0046] Morphological observation of CYP704B2 mutant plants. Such as figure 1 , the phenotype comparison between wild type and mutant CYP704B2 shows that: the wild type rice spikes droop after fruiting (left), while the CYP704B2 mutant spikelets are erect without fruiting (right), a...

Embodiment 2

[0048] Schematic diagram of determination of monomer components of anther cutin of CYP704B2 mutant

[0049] Step 1, the extraction of anther cutin monomer. Select about 4 mg of freeze-dried anthers from the wild-type and mutants at the bispore stage, use 700 μl of chloroform to extract the waxy components on the surface of the anthers, then add 700 μl of chloroform to treat at 50°C for 30 minutes, shake slightly overnight on a shaker, and repeat this step About 4 to 6 times to ensure that all lipids have been extracted. The defatted anthers dried with a silica gel desiccator were further used to analyze the cutin monomer components on the surface of the anthers. Dried defatted anthers were treated with 1 ml of methanol solution containing 1 N hydrochloric acid at 80°C for 2 hours. This transesterification reaction can depolymerize the defatted anthers and release the easily extracted anthers, which can be used for GC and GC-MS analysis. After adding 2ml of saturated NaCl aqu...

Embodiment 3

[0052] Location and cloning of CYP704B2 gene

[0053] Step 1, positioning the population, crossing CYP704B2 with the indica rice line Longtefu B, selfing to obtain the F2 generation, and selecting male sterile plants among them as the positioning population;

[0054] Step two, rice DNA extraction. The improved CTAB method is used for extraction, including the following steps: take 0.1-0.2 grams (about half a piece) of leaves and put them in a small mortar, add an appropriate amount of liquid nitrogen, grind them to powder immediately, put them into a 2ml centrifuge tube, add 700ul 100 Put the 1.5xCTAB solution preheated at ℃ in a centrifuge tube, mix carefully and put it in a 56℃ water bath, take out the centrifuge tube after 20 minutes, add an equal volume of chloroform / isoamyl alcohol, mix vigorously, centrifuge at 13000rpm for 10 minutes, and take the supernatant In a new tube, add 900ul of absolute ethanol and mix well, then put it at -20°C for more than half an hour. Th...

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Abstract

Application of a CYP704B2 gene and a coding protein thereof belongs to the technical field of gene engineering. The invention relates to a gene which codes the following protein (a) or (b): (a) a protein consisting of an amino acid sequence shown as SEQ ID NO:2; (b) a protein derived from (a) by substitution, deletion or adding one or a plurality of amino acids on the amino acid sequence of (a), and with aliphatic acid hydroxylase activity. The invention also relates to a plasmid, comprising a gene shown as SEQ ID NO:1. The invention also relates to a host cell, comprising a gene shown as SEQ ID NO:1. The invention also relates to a polypeptide, an amino acid sequence of which is shown as SEQ ID NO:2. The CYP704B2 gene of the invention is an anther specific expressed gene, which can regulate paddy rice anther epidermal layer and pollen extine cutin monomer component synthesis, and deletion of which can specifically lead to paddy rice male sterility.

Description

[0001] The patent application of the present invention is the Chinese invention patent application number 200910309083.1, the application date is October 30, 2009, the applicant is "Shanghai Jiaotong University", and the invention name is "CYP704B2 gene and its encoded protein". technical field [0002] The invention relates to a gene in the technical field of genetic engineering and the encoded protein thereof, in particular to the application of a carrier with CYP704B2 gene (cytochrome P450 gene). Background technique [0003] Rice is one of the most important food crops in my country. More than 60% of the population in China takes rice as the staple food. It is the largest rice producer and consumer in the world. The annual sown area of ​​rice in my country is 30 million hectares, accounting for 20% of the world; the output is 185 million tons, accounting for nearly 1 / 3 of the world, and the output per unit area is 6.35 tons / ha, which is 65% higher than the global average ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A01H1/06A01H5/00C12N15/84
Inventor 张大兵李晖梁婉琪袁政
Owner SHANGHAI JIAO TONG UNIV
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