Temperature sensing male fertile gene and use thereof

A technology of male fertility and temperature-sensitive sterile lines, applied in the field of genetic engineering, can solve the problem of unclear molecular basis of Annong S-1 temperature-sensitive sterile lines, inability to better select rice, and isolate and clone temperature-sensitive sterile Gene tms5 and other issues to achieve the effect of improving selection efficiency

Active Publication Date: 2008-12-31
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

At present, there is no report on the isolation and cloning of the temperature-sensitive sterile gene tms5 in rice. The molecular basis of Annong S-1 temperature-sensitive sterility is still unclear. good rice breeding

Method used

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  • Temperature sensing male fertile gene and use thereof
  • Temperature sensing male fertile gene and use thereof
  • Temperature sensing male fertile gene and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Fine mapping of tms5 (screening of polymorphic molecular markers and linkage mapping of tms5 gene)

[0034] According to the public rice genome sequence database (http: / / www.ncbi.nlm.nih.gov), multiple molecular markers have been developed in this region, as shown in Table 1:

[0035] Table 1 Molecular markers of polymorphisms closely linked or co-segregated with tms5

[0036]

[0037] AN1, PL1 and NAC1 in Table 1 are insertion / deletion (InDel) markers; RA1 is a single nucleotide polymorphism (SNP) marker. The detection method of SNP marker is as follows: in the first PCR, use the primers "RA1F" and "RA1R" to amplify for 33 cycles, then take 0.5 μl of the PCR product as the template for the second PCR, prepare two reactions for each sample, and use Primers "RA1F" and "RASR" are specific reactions for thermosensitive sterile genotypes, and primers "RA1F" and "RANR" are specific reactions for wild genotypes. Amplify for 8-10 cycles, use 6% polyacrylamide Ge...

Embodiment 2

[0039] Cloning and sequence analysis of embodiment 2tms5

[0040] According to the genome DNA sequence of rice (japonica rice) released by GenBank and the analysis of RiceGAAS software (http: / / ricegaas.dna.affrc.go.jp), there are only 2 predicted genes in the 8.4kb region of the location. These 2 genes They are pectate lyase (Pectate lyase, abbreviated as PL) and nuclease Z (RNase Z, abbreviated as RZ). The pectin lyase gene encodes a protein of 446 amino acids, and the nuclease Z gene encodes a protein of 302 amino acids.

[0041] Using primers PLf (5'-CTCCTCTCGATGCACTCATG-3') and PLr (5'-GATAGCATGACATGGCATTG-3') and RZf (5'-CACGTCGTCAACAACGACTAC-3') and RZr (5'-CCTGTTAACCGGTIGAGGTG-3') from the normal variety Annong N and thermosensitive male sterile line Annong S-1 were amplified by PCR technique to amplify the complete genome sequence of PL and RZ (including promoter and coding region), and cloned into plasmid vector pUC18. After sequencing analysis, it was found that th...

Embodiment 3

[0042] Example 3 Genetic transformation identifies the function of the target gene

[0043] Using primers RZf (5'-GTTCGGTGTGTGAAGGGGTG-3') and RZr (5'-CCTGTTAACCGGTTGAGGTG-3') the 4kb RZ gene fragment (comprising promoter region, coding region, and its downstream region sequence) amplified from Annong N ) was cloned into the binary transformation vector pCAMBIA1300. The expression vector was introduced into common soil Agrobacterium tumefaciens strain EHA105 by electric shock method. T 0 transgenic plants. Through molecular detection, transformants containing exogenous genes were obtained, and the pollen fertility of these transformants was normal under high temperature conditions (28-35°C), see Figure 5 and Figure 6 .

[0044] Harvest T 0 Generation seeds, planted T 1 Under high temperature conditions (28-35°C), the pollen fertility of the T1 generation segregated, that is, the pollen of the plants containing the transgene was fertile, while the pollen of the plants ...

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Abstract

The invention discloses a thermo-sensitive male fertility gene and an application thereof and also discloses a genetic marker of the thermo-sensitive male fertility gene, and provides a protein coded by the thermo-sensitive male fertility gene and a carrier containing the thermo-sensitive male fertility gene and a recombinant microorganism. The invention also discloses the application of the thermo-sensitive male fertility gene in cultivating a thermo-sensitive male sterile line and an application of the genetic marker in rice breeding. The thermo-sensitive fertility gene TSSNR is acquired through the map-based cloning technique of Annong S-1 thermo-sensitive sterility gene locus tms5, functions of the gene are validated through transgene experiments, and the transgene technique of RNAi or antisense RNA or the over-expression dominant negative principle is provided, functions of the TSSRZ gene in normal rice varieties are incapable, so as to cultivate the thermo-sensitive male sterile line, and the application value is significant.

Description

technical field [0001] The invention relates to a temperature-sensitive male fertility gene and its application, belonging to the field of genetic engineering. Background technique [0002] Plants produce hybrids by crossing between different varieties. Hybrids have advantages in growth potential, adaptability, yield, etc., which are called heterosis. The use of heterosis can improve the yield, quality and resistance of crops. The way to utilize heterosis in rice breeding is mainly through hybrid rice breeding, and the methods of hybrid rice breeding mainly include two-line method and three-line method. The three-line method requires sterile lines, maintainer lines and restorer lines, while the two-line method only requires sterile lines and restorer lines. The sterile line in the two-line method has the characteristics of fertility conversion, and its male sterility or fertility is controlled by the external photoperiod and temperature conditions, and the sterile line ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/63C07K14/415C12N1/21C12N5/10A01H1/00C12Q1/68
Inventor 庄楚雄周海姜大刚李静唐佩君卢森吴平刘勤坚朱丽雅刘耀光梅曼彤
Owner SOUTH CHINA AGRI UNIV
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