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Molecular engineering of a floral inducer for crop improvement

A plant and seed technology, applied in the direction of genetic engineering, the use of vectors to introduce foreign genetic material, plant peptides, etc., to achieve the effect of increasing fruit and/or seed production and improving flowering ability

Inactive Publication Date: 2012-11-21
UNIVERSITY OF WARWICK
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Evidence gathered for: FT proteins in Arabidopsis 7,8 SFT with its tomato 9 , Hd3a of rice 10 And the Cm-FTL1 / 2 of the hoist 11 The ortholog serves as an element of the non-cell-autonomous flowering signal that is transported to the SAM via the phloem, however, the role of FT mRNA activity in long-range florigen signaling remains unknown 12

Method used

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  • Molecular engineering of a floral inducer for crop improvement
  • Molecular engineering of a floral inducer for crop improvement
  • Molecular engineering of a floral inducer for crop improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0117] Cloning of the FT gene

[0118] Coding FT G172A 、FT R173A and FT R174A Arabidopsis FT derivatives by using the corresponding primer pair A and D ( figure 2 ) and PCR with the plasmid PVX / FT carrying the wild-type FT gene as a template for amplification 12 . The PCR reaction system contained: 5 μL of 10× reaction buffer (Promega), 0.2 mM dNTPs (dATP, dGTP, dCTP and dTTP), 15 pmole of each primer, 1.25 units of pfu DNA polymerase (Promega) and 50 ng of template DNA. The resulting PCR product was purified with the QIAGEN Quick PCR Purification Kit, followed by double digestion with C / al and Eagl, and then cloned into the C / al / Eagl site of a PVX-based gene expression vector 12 .

[0119] All other FT derivatives were obtained by overlap extension PCR ( figure 2 ). Design 2 mutagenic primers B and C for each sample involved ( figure 2 ), which contain specific mutations and are partially complementary to each other. Perform two separate PCR reactions (5 μL of 10...

Embodiment 2

[0121] Modification of FT gene

[0122] In order to modify the 5' (that is, any nucleotide in the first 100 nucleotides) or 3' (that is, any nucleotide in the last 100 nucleotides) end of the FT gene, chemically synthesized specific primers ( 30-110 nucleotides), the specific primer has a substitution for a codon selected for an amino acid with three nucleotides encoding alanine. Of course, using this methodology, other amino acid substitutions can be utilized and the resulting mutants tested in the same manner as described below. As described in Example 1, mutagenic primers were used in PCRs and optionally primers A or D ( figure 2 A) in order to introduce specific modifications into the FT gene.

[0123] To introduce nucleotide changes into the middle portion of the FT gene (i.e., nucleotides 101-429), two partially complementary primers B and C ( figure 2 ). Nucleotide changes were introduced into both primers to substitute codons for selecting amino acids for mutatio...

Embodiment 3

[0125] Expression of Modified Genes in Plants

[0126] RNA transcripts from each recombinant PVX vector are generated by in vitro run-off transcription 12. Typical in vitro full-length transcription reaction (50 μL containing 5 μL of 10× buffer (Biolabs), 40 units of RNasin (Promega), 2 mM each of ATP, CTP, UTP, and GTP, 0.5 mM m 7 GpppG (Biolabs), 2.5 μg of Spel-linearised carrier DNA and 200 units of T7 RNA polymerase) were incubated at 37° C. for 1 hr. The RNA transcripts were further treated with 1 unit of RNase-free DNase at 37°C for 30 min, and then mechanically inoculated at the 5-6 leaf stage of the plants. Young short-day (SD) tobacco Maryland Mammoth (MM) plants were grown in an insect-free greenhouse at 25°C with continuous light providing a long-day (LD, 16-hr) photoperiod. 12 Viral ectopic overexpression of wild-type and modified FT genes was analyzed at the RNA and protein levels as previously described, and viral delivery of wild-type and modified FT genes wa...

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Abstract

A plant comprising a modified FT polynucleotide expressing a modified polypeptide exhibits altered flowering time, floral numbers and / or increased seed production. Different mutant sequences conferring different phenotypes are disclosed.

Description

technical field [0001] The present invention relates to plants having altered flowering time, number of flowers and / or seed yield, and to methods of producing such plants. Background technique [0002] In photoperiodic flowering plants, flowering time is an important developmental switch that can be influenced by environmental signals sensed by leaves. The flowering locus T (Flowering Locus T, FT) gene is an important element that regulates the flowering signaling pathway. When a plant is induced to flower, a phloem-moving signal called "florigen" is produced, and the florigen is transported through the phloem transport flow to the shoot apical meristem (SAM), where the florigen induces flower development 1，2 . In Arabidopsis, mobile florigens have been shown to be encoded by the flowering locus T (ET) gene. FT is specifically in phloem cells but not in SAM 3 transcribed into mRNA. However, its protein product is functional at the shoot apex 4-6 . Arabidopsis FT is a ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/82
CPCC07K14/415C12N15/827C12N15/8261Y02A40/146
Inventor 洪益国S·杰克逊李春阳
Owner UNIVERSITY OF WARWICK
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