Cloning and application of gene OsAP65 controlling development of paddy rice pollen tube

A technology of pollen tube development and genetics, which is applied in the fields of application, genetic engineering, and plant genetic improvement, and can solve the problems of lagging research on the function of aspartic acid protease genes and unclear biological functions, etc.

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are at least 96 AP members in rice (Chen, et al. 2009. Aspartic proteases gene family in rice: Gene structure and expression, predicted protein features and phylogenetic relation. Gene, 442: 108-118), about aspartic proteases in rice The research on gene function of S5 is a major regulator of the reproductive barrier and compatibility of indica-japonica hybrids in rice.Proc Natl Acad Sci U S A, 105: 11436-11441) and OsCDR1 (Prasad, et al. 2009. Overexpression of rice (Oryza sativa L.) OsCDR1 leads to constitutive activation of defense responses in rice and Arabidopsis. Mol Plant Microbe Interact, 22: 1635-1644) known In addition to the function, the structure and expression of oryzasin1 and OsAsp1 were analyzed, the specific biological function is still unclear (Asakura, et al. 1995.Rice aspartic proteinase, oryzasin, expressed during seed ripening and germination, has a gene organization distinct from those of animal and microbial aspartic proteinases. Eur J Biochem, 232:77-83; Bi, et al. 2005. The rice nucellin gene ortholog OsAsp1 encodes an active aspartic protease without aplant-specific insert and is strongly expressed in early embryo. Plant Cell Phy siol, 46:87-98)

Method used

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  • Cloning and application of gene OsAP65 controlling development of paddy rice pollen tube
  • Cloning and application of gene OsAP65 controlling development of paddy rice pollen tube
  • Cloning and application of gene OsAP65 controlling development of paddy rice pollen tube

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Obtaining and phenotypic identification of OsAP65 mutants

[0049] The rice T-DNA insertion mutants used in this example were constructed from the vector pGA2715 by Pohang University of Science and Technology in South Korea (for the construction method of the mutant library, see the paper: Jeong, et al. 2002. T-DNA insertion mutagenesis for activation tagging in rice. Plant Physiol, 130: 1636-1644). The screening and identification method of mutants is: the mutant rice seeds obtained from the above-mentioned rice T-DNA insertion mutant flanking sequence database (RiceGE: http: / / signal.salk.edu / cgi-bin / RiceGE) are soaked according to conventional methods 1. Sow in the seedling field after germination, and transplant to the field after 20 days (the planting density is 5 inches × 8 inches, and the planting site is the experimental field of Huazhong Agricultural University in Shizishan District, Hongshan District, Wuhan City, Hubei Province, China), and carry out...

Embodiment 2

[0055] Embodiment 2: Mutant phenotype observation

[0056] 1. Reciprocal cross experiment

[0057] Since the segregation ratio of wild-type and T-DNA insertion heterozygous plants in the self-pollination progeny of OsAP65 heterozygous plants is about 1:1, which deviates from the Mendelian ratio of 1:2:1 (homozygous for T-DNA insertion: T -DNA insertion heterozygous: wild type). So we preliminarily believe that this mutant is a gametocyte development-deficient mutant. In order to determine whether the mutant affects the development of male gametes or the development of female gametes, or whether the development of both male and female gametes is affected, we conducted reciprocal cross experiments. Using the OsAP65 heterozygous plant as the female parent and the pollen of the wild-type plant (Minghui 63, or MH63, a rice variety widely grown in China) as the male parent, the segregation ratios of the offspring were 1.43:1 (67:47 ). When the OsAP65 heterozygous plant is used a...

Embodiment 3

[0064] Embodiment 3: complementary experiment

[0065] 1. Construction of Complementary Vectors

[0066] The construction method of the complementary vector OsAP65-pU2301-FLAG is as follows: primers are designed to amplify the coding region sequence of OsAP65 (starting from ATG, excluding TAA). Using primers 65CDS-KpnI-F2 (5'-GGGGTACCATGGCGAGTCGCCGGTCGCGTCGGC-3') and 65OE-R2 (5'-GAAGATCT TAGCGGTTGGAGCTCTTGCTCAG-3') to amplify the OsAP65CDS sequence, the PCR product was subjected to agarose electrophoresis, and the target was recovered from the agarose gel Fragment, the target fragment was connected with the pGEM-T vector (purchased from Promega (Beijing) Biotechnology Co., Ltd. as an agent, that is, Promega Company of the United States), and the connected product was connected by electrotransformation (the electrotransformer was a product of Eppendorf Company, this The voltage used in the invention is 1800V, and the specific operation refers to the instruction manual of the i...

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Abstract

The invention belongs to the technical field of plant genetic engineering and particularly relates to the cloning, functional verification and application of an aspartic protease gene OsAP65 controlling the development of a paddy rice pollen tube. The nucleotide sequence of the aspartic protease gene OsAP65 is shown as the sequence table SEQ ID NO: 1; the coding region of the aspartic protease gene OsAP65 is positioned from the 129th base to 2024th base of the gene segment; the sequence of the protein of the aspartic protease gene OsAP65 is shown as the sequence table SEQ ID NO: 2. A research result shows that the cloned aspartic protease gene OsAP65 participates in the development process of the paddy rice pollen tube and has a significant value for theoretical research such as the molecular mechanism in the development of the paddy rice pollen tube.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering. It specifically relates to the cloning, functional verification and application of an aspartic acid protease gene OsAP65 controlling rice pollen tube development. Background technique [0002] The life cycle of higher plants consists of alternate generations of gametophytes and sporophytes, and the development of gametophytes and the process of fertilization are key steps to achieve the alternation of generations. As one of the participants in the sexual reproduction of higher plants, the male gametophyte's occurrence and development are crucial to the process of plant generation alternation. Pollen germination and pollen tube growth are important steps in the development of the male gametophyte. Germinated pollen tubes show a polar growth pattern, elongating only to one end. The organelles in normal growing pollen tubes show a typical regional distribution. The top growth are...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/57C12N9/50C12N15/10C12N15/84A01H5/00
Inventor 张启发黄建燕赵晓波安贞焕
Owner HUAZHONG AGRI UNIV
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