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Gene of containing 26S rRNA sequence from restoring line of cotton

A DNA sequence and sequence technology, applied in the field of new genes, can solve problems such as no research reports on the cloned cotton fertility restoration gene, etc.

Inactive Publication Date: 2007-12-26
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, there is no research report on cloning the fertility restoration gene of cotton

Method used

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  • Gene of containing 26S rRNA sequence from restoring line of cotton
  • Gene of containing 26S rRNA sequence from restoring line of cotton

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Obtaining Gene Segments from Cotton Restorer Lines by Suppression Subtractive Hybridization

[0021] 1 Cultivation and selection of cotton materials for inhibiting subtractive hybridization: The cotton variety is the Y18R×P30A hybrid F1 generation self-crossing F2 generation material cultivated by Guo Sandui's research group at the Institute of Biotechnology, Chinese Academy of Agricultural Sciences. The fertility of the F1 generation selfed materials was judged according to the morphology of the stamens after sowing, and then the young buds of the fertile and sterile materials that had budded for 3 to 5 days were picked respectively. The bract leaves were cut off with a scalpel, and the young buds were quick-frozen in liquid nitrogen and stored at -70°C.

[0022] 2 Preparation of RNA: The improved thermal boric acid method was used to extract the total RNA of the upland cotton variety Y18R×P30A hybrid F1 self-bred F2 material, which was 3 to 5 days old buds, and was us...

Embodiment 2

[0026] Acquisition of Full-length cDNA Sequence of Gene GH18Rorf392 from Cotton Restorer Line

[0027]1 Obtaining cDNA from the 5' end of the cotton restorer line GH18Rorf392 gene When using the SSH fragment and the GeneRacerTMRNA Oligo sequence to form a primer pair for 5' RACE of cotton fertility restoration related genes, it was amplified from the total RNA of fertile buds of cotton F2 generation to A specific band of 1500bp (Figure 3). Cut the film containing the band with a razor blade and transfer the film to S.N.A.P. TM In the column, centrifuge through the column to obtain the fragment. The fragment was gently mixed with the pCR4-TOPO vector, incubated at room temperature for connection, transformed into Escherichia coli DH5α-T1 competent cells, and screened with 50 μg / mL kana. The obtained transformants were identified by double enzyme digestion with Pst I and Not I, and the 1500 bp band was indeed inserted (Fig. 4). After sequencing, the 5' end cDNA sequence of GH...

Embodiment 3

[0031] PCR by designing primers, recovering amplified fragments, reamplification, electrophoresis recovery, ligation and transformation to obtain complete full-length gene sequences

[0032] 1 Design primers for PCR: In Example 2, the vectors containing the 5' and 3' cDNA sequences of the GH18Rorf392 gene were obtained by RACE at the 5' and 3' ends respectively, in order to obtain the vector containing the complete full-length cDNA sequence of the GH18Rorf392 gene , so design a pair of primers: 5'-end forward splicing and 5'-end reverse splicing, where the first half sequence of the 5'-end reverse splicing primer is the sequence unique to the end of the 5' insert of pCR4-TOPO-GH18Rorf392, 5' The second half of the reverse splicing primer is the sequence unique to the front end of the pCR4-TOPO-GH18Rorf392 3' insert. At the same time, another pair of primers were designed: forward splicing at the 3' end and reverse splicing at the 3' end. The second half sequence of the splici...

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Abstract

This invention relates to a novel Gossypium hirsutum gene containing 26S rRNA sequence. The gene (GH18Rorf392) is obtained by: cloning EST sequence of cotton restorer through suppression subtractive hybridization method, designing primers according to the EST sequence, and performing 5'- and 3'-RACE to obtain full-length cDNA sequence. The gene comprises 26S rRNA sequence and 3'-end and a novel sequence at 5'-end. The gene is derived from Gossypium hirsutum restorer Y18R, and codes a protein of 392 amino acids. The gene can be used for studying fertility of cotton.

Description

technical field [0001] The present invention relates to a novel gene containing 26S ribosomal RNA (rRNA) sequence from a cotton restorer line. After obtaining the EST sequence from the cotton restorer line by suppressing subtractive hybridization, primers were designed with the EST sequence and then 5' and 3' RACE were performed to obtain its full-length cDNA sequence. The 3' end of the gene contains 26S rRNA sequence, and the 5' end is a completely new sequence. Since the gene is derived from the restorer line Y18R of upland cotton (Gossypium hirsutum) and encodes a protein of 392 amino acids, it was named GH18Rorf392. The gene can be used in the research of cotton fertility-related functions. Background technique [0002] Cotton has very obvious heterosis, and using cotton heterosis has become an effective way to improve yield, quality and disease resistance. Due to the status of cotton cytoplasmic male sterility (CMS) in the utilization of hybrid vigor, genetic breeder...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415
Inventor 郭三堆吴巧雯宋洋张锐
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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