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Plant pollen tube growth gene and application

A technology of pollen tubes and genes, applied in the field of genetic engineering, can solve problems such as unclear biological functions, and achieve the effect of solving reproductive problems

Active Publication Date: 2021-01-19
YUNNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are more than 10 genes encoding multi-copper oxidase in rice, but their biological functions are very unclear. From them, gametophytic male sterility-related genes were found and gametophytic male sterility was created, which was used to reproduce sporophytic sterility and reduce Transgenic elegant etc. are of great significance

Method used

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  • Plant pollen tube growth gene and application
  • Plant pollen tube growth gene and application
  • Plant pollen tube growth gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, the selection of the gene OsPTD1 related to the male gametophyte function:

[0030]In order to find some genes related to gametophyte development, we analyzed the BAR rice gene expression database (http: / / bar.utoronto.ca / efprice / cgi-bin / efpWeb.cgi), and found that many genes expressed in the middle and late flowers, After RT-qPCR verification, a group of genes that may be related to the development of male gametophytes were selected. Among them, the rice Loc_Os05g40740 gene is specifically highly expressed in late flowers, and its Arabidopsis homologous gene is specifically expressed in pollen. The Loc_Os05g40740 gene is initially listed as a gene related to male gametophyte development, named OsPTD1, and its amino acid sequence is shown in SEQ ID NO.1 As shown, the nucleotide sequence is shown in SEQ ID NO.2, and the genome sequence is shown in SEQ ID NO.3.

[0031] 1. RT-qPCR analysis

[0032] The temporal and spatial expression pattern of the gene was...

Embodiment 2

[0037] Embodiment 2, cloning and structure analysis of OsPTD1 gene

[0038] In order to obtain the gene structure of OsPTD1, the RACE kit of Baobio was used for analysis: the reverse transcription product of rice anther total RNA was used as a template, and the general primers provided by the kit and the 5'RACE primers and 3'RACE primers designed by ourselves were used for analysis. Three rounds of PCR amplification ( figure 2 ), the last round of product was recovered and connected to the pMD18-T vector, and the 3'UTR, 5'UTR and CDS of the OsPTD1 gene cDNA were finally determined after bacterial liquid PCR detection and sample delivery sequencing: the 5'UTR of the gene has 201bp, 3'UTR 261bp, CDS full length 1671bp, cDNA full length 2338bp. The 5'RACE-specific primers are respectively shown in SEQ ID NO.15-17, the 3'RACE-specific primers are respectively shown in SEQ ID NO.18-20, and the full-length amplification primers are shown in SEQ ID NO.21-22.

Embodiment 3

[0039] Example 3. Preparation of Male Gametophyte Sterility by Inhibiting OsPTD1 Gene Expression

[0040] In order to determine whether the OsPTD1 gene regulates pollen fertility, whether it can be used to prepare and identify male gametophytic sterile materials, the POsPTD1 promoter was used to drive the interference fragment of the OsPTD1 gene, and the male gametophytic sterile creation and identification were carried out:

[0041] 1. Vector transformation: In order to facilitate the loading of relevant target genes or DNA fragments, in order to accurately and directly identify transgenic positive strains and male gametophyte sterility, the modified gene OsMYB76R of rice anthocyanin synthesis-related genes was used to replace the GUS gene, and the The multiple cloning site of the vector pCAMBIA1301 has been transformed, and the specific operation is as follows:

[0042] (1) OsMYB76 gene modification and replacement of GUS: OsMYB76 gene is an essential gene for anthocyanin sy...

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Abstract

The invention discloses a rice pollen tube growth related gene OsPTD1 (Pollen Tube Device) and application. Protein encoded by the gene is an amino acid sequence shown as SEQ ID NO.1, or protein whichhas the same function as OsPTD1 protein and is obtained by substituting, deleting or adding at least one amino acid; and a nucleotide sequence of the protein composed of the amino acid sequence shownas SEQ ID NO.1 is shown as SEQ ID NO.2. The OsPTD1 gene encodes ascorbic acid oxidase is an essential gene for normal growth of a pollen tube, abnormal functions of the gene can cause early rupture of the pollen tube, insemination cannot be completed, and gametophyte male sterility is generated; and the gene can be used for preparing gametophyte male sterility, and plays an important role in preventing transgenosis from drifting along with pollen, bred sporophyte male genic male sterility and the like.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a rice pollen tube growth-related gene OsPTD1 and its application. Abnormal function of the gene can produce gametophyte male sterility, so it can be used to change pollen fertility, control transgene drift, prepare male gametophyte sterility and reproduce Sporophytic male sterility, etc. Background technique [0002] Plant male sterility plays an important role in the study of pollen development and the utilization of heterosis. Male sterility includes sporophytic sterility and gametophytic sterility. [0003] The sporophytic sterility is controlled by the genotype of the plant itself, and all the pollen in the anthers are sterile. In the field, the anthers usually show abnormal shape, color, and size, and are not fertile, so they are easy to find and identify; Female gametes are passed from generation to generation, and can also be passed from generation to generation through...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/46
CPCC07K14/415C12N15/8218C12N15/8289
Inventor 张毅陈云杜双林
Owner YUNNAN UNIV
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