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Cotton fiber and pollen specific expression promoter and application

A pollen-specific, cotton fiber technology, applied in the field of genetic engineering, can solve problems such as limited promoters

Inactive Publication Date: 2013-11-06
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the reported cotton fiber-specific promoters are very limited

Method used

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  • Cotton fiber and pollen specific expression promoter and application
  • Cotton fiber and pollen specific expression promoter and application
  • Cotton fiber and pollen specific expression promoter and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] [Example 1] Extraction of Cotton RNA

[0047] Select about 3g of fresh cotton material (root, hypocotyl, cotyledon, stem, stamen, leaf, pistil, ovules on the day of flowering (including protruding fiber cells), ovules 4 days after flowering (including protruding fiber cells), flowering fibers 6 days after anthesis, fibers 10 days after anthesis, fibers 18 days after anthesis, ovules 6 days after anthesis, ovules 10 days after anthesis, or ovules 18 days after anthesis). Quickly grind into a fine powder in liquid nitrogen, put it into a 50mL centrifuge tube, add 15mL of 65°C preheated RNA extraction solution (2% (W / V) CTAB, 2% (W / V) PVP, 100mmol / L Tris-HCl (pH8.0), 0.5g / L spermidine (Spermidine), 2.0mol / L NaCl, 2% mercaptoethanol (V / V, add before use), invert and mix well. 65℃ water bath for 3min, during which time mix well for 2 ~ 3 times. Chloroform:isoamyl alcohol (24:1) extracted 2 times (10,000r / min, room temperature, 5min). Take the supernatant, add 1 / 4 volume of ...

Embodiment 2

[0048] [Example 2] Synthesis of the first strand of cDNA

[0049] About 10 μg of total RNA was added to a DEPC-treated amplification tube, and the RNA was denatured in a water bath at 65° C. for 10 minutes, and then immediately ice-bathed for 3 minutes. Then add 2μL 10×RNA Reaction Buffer and 4μL 25mmol / L MgCl to the amplification tube 2 , 2 μL of 10 mmol / L dNTPs, 5 U of AMV RTase, 0.5 μL of RNase inhibitor (20 U) and 1 μL of 2.5 μmol / L Oligo-dT3' linker were added to DEPC-treated water to a final volume of 20 μL. The reverse transcription reaction program is: 30°C, 10min; 50°C, 45min; 95°C, 5min; 5°C, 5min. At the end of the program, the product was frozen at -20°C.

Embodiment 3

[0050] [Example 3] Expression patterns of GhFBP7 gene in different tissues and organs of cotton and fiber ovules in different developmental stages

[0051] Quantitative Real-time PCR was used to analyze the expression level of GhFBP7, and the first-strand cDNA of total RNA from different organs and tissues was synthesized with cDNA first-strand synthesis kit (MBI) (samples included cotton seedling roots, hypocotyls, cotyledons, true Leaves, stamens and pistils, as well as seeds and fibers from the day of flowering (0DPA) to 18 days after flowering (18DPA), were operated according to the instructions of the kit. PCR was performed using a real-time quantitative PCR kit (Bio-Rad), including 10 μL MIX buffer (including PCR buffer, DNA polymerase, dNTPs, and MgCl) in a 20 μL reaction system. 2 ), 1 μL (5 μmol / L) each of the 5’-end and 3’-end primers. The cycle parameters are 94°C pre-denaturation for 3 minutes; 94°C, 30sec, 55°C, 30sec, 72°C, 30sec, and the preset cycle number is ...

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Abstract

The invention provides cotton fiber and pollen specific expression promoter and an application. By the utilization of a genetic engineering technology, FPP1 promoters of GhFBP7 genes for cotton fiber and pollen specific expressions are successively separated. The FPP1 prompter contains 1283bp segment. The invention also verifies that the FPP1 promoter sequence has cotton pollen and fiber expression specificity. The promoter can guide specific expressions of cotton fiber and pollen in reporter gene and provides possibility and effective approaches to genetic engineering of raising cotton yield and quality.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a promoter for specific expression of cotton fiber and pollen and its application. Background technique [0002] my country is a big country of cotton production and consumption, and cotton production occupies an important position in my country's national economy. The use of traditional breeding methods has achieved great success in improving cotton varieties, but in the past 20 years, the output of cotton varieties in the world has reached a plateau. Therefore, it is difficult to greatly increase cotton yield by using existing genetic resources and traditional breeding methods. The genetic engineering method has the advantages of easy stable offspring and short breeding cycle, which can break the genetic barrier between species and realize the directional transfer of good target genes. Using genetic engineering to improve cotton yield and fiber quality is an effec...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/84C12N1/21A01H5/00
Inventor 罗明肖忠意裴炎肖月华
Owner SOUTHWEST UNIVERSITY
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