A kind of cotton phytosulfokine precursor gene ghpskp and its preparation method and application

A gene and cotton fiber technology, applied in the direction of botanical equipment and methods, biochemical equipment and methods, applications, etc., to achieve the effect of high efficiency, high accuracy, promotion of differentiation and elongation

Inactive Publication Date: 2015-09-30
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Sauter et al. found that PSK signal affects root length by affecting the size of root tip primary layer cells (Sauter M(2009)PSK-α promotes root growth in Arabidopsis.New phytologist), and in subsequent studies, St¨ 1 hrwohldt found that the same phenomenon exists in the development of hypocotyl cells and ro

Method used

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  • A kind of cotton phytosulfokine precursor gene ghpskp and its preparation method and application
  • A kind of cotton phytosulfokine precursor gene ghpskp and its preparation method and application
  • A kind of cotton phytosulfokine precursor gene ghpskp and its preparation method and application

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Experimental program
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Embodiment 1

[0040] Isolation and Cloning of GhPSKP Gene and Its Expression Characteristics in Different Tissues of Cotton

[0041] In the preliminary work of the applicant's State Key Laboratory of Crop Genetic Improvement, an EST (abbreviation: see SEQ ID NO: 1) that is highly homologous to other plant PSKP genes was isolated from the expression profiles of cotton fiber development at different stages, Bioinformatics Analysis showed that the EST contained a complete ORF (see SEQ ID NO: 2), and it was predicted by bioinformatics that this ORF could translate into a PSKP precursor polypeptide (SEQ ID NO: 3), so it was named GhPSKP. The gene is highly expressed at various stages of fiber elongation, and 10DPA fiber total RNA is extracted from upland cotton variety YZ1 (the extraction method is based on Zhu et al. An improved simple protocol for isolation of high quality RNA from Gossy pium spp. suitable for cDNA library construction. Acta Agronomica Sinica.2005, 31.1657-1659.), using revers...

Embodiment 2

[0044] Construction and transformation of overexpression vector and RNAi

[0045] In order to verify the function of the GhPSKP gene in cotton, the applicant constructed an overexpression and RNAi vector to transform cotton.

[0046] According to the ORF region of GhPSKP cDNA obtained in Example 1 (see SEQ.ID.No.2), overexpression primers were designed, and the restriction sites of BamHI and SalI and protective bases were added to both ends of the primers respectively, and the primers were respectively Name the primers GhPSKPoef and GhPSKPoer, and then use the cDNA of the GhPSKP gene as a template for PCR amplification, and the obtained PCR product is connected to pCAMBIA2300S digested with the same enzyme (the precursor of the pCAMBIA2300S vector was obtained from the Australian CAMBIA experiment Center for the Application of Molecular Biology to International Agriculture donated pCAMBIA2300 to construct an overexpression vector p35s::GhPSKP. The overexpression vector p35s::G...

Embodiment 3

[0093] Correlation Analysis of GhPSKP Transgenic Lines

[0094]1. Molecular identification of GhPSKP transgenic lines

[0095] GhPSKP gene overexpression transgenic line obtained 5 families, RNAi transgenic line obtained 5 families. Genomic DNA was extracted from transgenic lines, and analyzed by Southern blot to identify the transgenic effect and copy number. For DNA extraction and Southern experiments, refer to J. Sambrook et al., translated by Huang Peitang et al., Molecular Cloning Experiment Guide (Third Edition), Science Press, 2002 edition. The results see ( Figure 6 ).

[0096] 2. Expression analysis of GhPSKP transgenic lines

[0097] The total RNA of transgenic strains was extracted by single plant, each strain had at least three biological repetitions, and expression analysis was carried out by Realtime PCR and Northern blot. The Realtime-PCR method was the same as in Example 2, and the probe used for Northern hybridization was the GhPSKP gene The full-length ...

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Abstract

The invention discloses a cotton phytosulfokine precursor (PSKP) gene GhPSKP and its preparation method and use. Through the preparation method, an EST which is highly homologous with a PSKP gene of other plants is separated from expression profiles of different cotton fiber growth periods, and bioinformatics analysis proves that the EST contains a complete ORF and is named as GhPSKP; the gene GhPSKP can be expressed highly in all fiber elongation periods, 10 DPA fiber total RNA is extracted from an upland cotton variety YZ1, the total RNA is reversely-transcribed by a reverse transcriptase Superscript III so that cDNA is obtained, and a full-length cDNA of the gene GhPSKP is obtained by amplification adopting primers of GhPSKPF and GhPSKPR; and the PCR product obtained by amplification is introduced into a pGEM-T carrier, positive clone is screened and is subjected to sequencing so that the full-length cDNA of the gene GhPSKP is obtained. An experiment proves that the gene GhPSKP can be preferentially expressed in the cotton fiber fast-elongation stage. Through excess expression of the gene GhPSKP based on a transgenosis method, fiber elongation is effectively promoted. The gene GhPSKP can effectively promote cotton fiber elongation and improve cotton fiber quality.

Description

technical field [0001] The invention belongs to the technical field of cotton genetic engineering. It specifically relates to a cotton Phytosulfokine (PSK) precursor gene GhPSKP, and also relates to a preparation method of the cotton Phytosulfokine (PSK) precursor gene GhPSKP, and also relates to a use of the cotton Phytosulfokine (PSK) precursor gene GhPSKP. The applicant isolated and cloned the gene GhPSK Precursor (GhPSKP) encoding the precursor polypeptide of the substance from the cotton fiber cDNA library, and further confirmed that the gene was predominantly expressed during the rapid elongation stage of cotton fibers. Overexpression of this gene in cotton by transgenic means can effectively promote fiber elongation. The invention can effectively promote the elongation of the cotton fiber and improve the quality of the cotton fiber. Background technique [0002] Cotton is an important economic crop worldwide and the most important textile fiber crop. It is the main ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/10C07K14/415C12N15/63C12N15/84A01H5/00
Inventor 张献龙韩杰谭家福涂礼莉
Owner HUAZHONG AGRI UNIV
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