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Method for preparing transgene using rice calmodulin binding protein kinase gene and its use

A protein kinase gene and binding protein technology, which is applied to the application field of calmodulin-binding protein kinase gene in transgenic rice, can solve problems such as difficulty, increase thousand-grain weight, increase farmer's labor intensity, etc., so as to prolong the growth period and increase the yield. Effect

Inactive Publication Date: 2005-07-20
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But this increases the labor intensity of farmers and increases the burden on farmers
At the same time, these studies have ignored the way to increase the yield by prolonging the growth cycle of crops, especially rice, and increasing the thousand-grain weight.
More importantly, it is difficult to transfer the calmodulin-binding protein kinase gene into monocotyledonous plants like rice and express it efficiently and show good traits

Method used

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  • Method for preparing transgene using rice calmodulin binding protein kinase gene and its use
  • Method for preparing transgene using rice calmodulin binding protein kinase gene and its use
  • Method for preparing transgene using rice calmodulin binding protein kinase gene and its use

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Construction of expression vector containing calmodulin-binding protein kinase gene

[0025] according to figure 1 with figure 2 As shown, two primers were used in this experiment, and their nucleic acid sequences were RF5 (5'-cgggatccatgggtctctgccatggcaag-3') and RF3 (5'cgggatcctcaggtttttgggatggtacgc-3'). Using RF5 / RF3 as the primer combination, using the pET32a+ vector cloned with the calmodulin-binding protein kinase gene as the template DNA, by PCR (94 degrees Celsius for 53 minutes, 94 degrees Celsius for 1 minute, 65 degrees Celsius for 50 seconds, 72 degrees Celsius for 1 minute30s, 30 cycles, 72 degrees Celsius for 10 minutes ) to synthesize and obtain the full-length OsCBK (Oryza sativa calcium / calmodulin-bindingprotein) of the full-length rice calmodulin-binding protein kinase gene. The intermediate vector plasmid pAHC17 was digested with the restriction endonuclease BamHI at 37 degrees Celsius to obtain pAHC17 / c , and at the same time clone the...

Embodiment 2

[0026] Example 2: Transformation and screening of plants containing calmodulin-binding protein kinase gene

[0027] 1. Preparation of Competent Agrobacterium

[0028] Put Agrobacterium EHA105 on the YM plate, culture at 26-28°C for 48 hours, pick a single colony and inoculate it into 40ml of YM liquid medium, culture it in suspension at 250rpm for about 12-16 hours, and then transfer the bacterial solution to the sterilized medium on the ultra-clean bench. In a sterile 50ml centrifuge tube, centrifuge at 4°C, 8000rpm for 8 minutes, discard the supernatant, resuspend Agrobacterium with 100mM NaCl (pre-cooled at 4°C), centrifuge at 4°C, 8000rpm for 8 minutes, discard the supernatant, and add the original bacteria 1 / 50 volume (800 μl) of 20 mM CaCl2 to resuspend the bacteria, and aliquot into 100 μl / tube. (The competent Agrobacterium at this time can be directly used for transformation), or placed in liquid nitrogen for 10 seconds, and stored in a -20°C or -80°C ice box for futu...

Embodiment 3

[0033] Example 3: Nucleic acid analysis of transformed plants containing calmodulin-binding protein kinase gene

[0034] 1. Extraction of total DNA from transformed plants for PCR

[0035] according to image 3 As shown, scrub the leaves with 70% ethanol, weigh 100mg, add 600μl extraction buffer (0.2MNaCl, 25mM EDTA, 0.5% SDS, pH 7.5), grind quickly at room temperature, and vortex mix in a 1.5ml Eppendorf tube for 5- Centrifuge at 12000 rpm for 20 seconds at room temperature for 25 minutes. Add an equal volume of isopropanol to the supernatant, and mix by inverting up and down. Precipitation overnight at -20°C, at room temperature, 12000 rpm, 15 minutes. Discard the supernatant, put it upside down on absorbent paper, and then add 200 μl of 70% ethanol to soak and wash the DNA precipitate, at room temperature, 12000 rpm, for 10 minutes. Discard the ethanol, invert it on a paper towel and add 100 μl TE (pH 7.5) to dissolve the precipitate after it dries. The DNA concentrati...

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Abstract

The present invention discloses method of preparing transgene calmodulin conjugated protein kinase gene and its application. The preparation process includes the following steps: A. restricting cloned calmodulin conjugated protein kinase gene with BamHI; B. connecting the gene in the step A to the intermediate vector pAHC17 to form pAHC17-R19(+); C. double restricting pAHC17-R19(+) with HindIII and EcoRI, and connecting to similarly restricted expression vector pCAMB1A1300 or pCAMB1A1301, to set calmodulin conjugated protein kinase gene between the ubiquitin promoter and NOS tail to form convertible or expressible vector; D. transferring the prepared vector into agrobacterium EHA105 and introducing mature embryo introduced rice callus; and E. screening positive plant. The calmodulin conjugated protein kinase gene is used in rice to produce transgenic plant with later florescence, longer growth period, and increased yield.

Description

technical field [0001] The invention belongs to the field of transgenic plants. Specifically, the invention relates to a method for preparing a transgene by using a calmodulin-binding protein kinase gene. The invention also relates to an application of a calmodulin-binding protein kinase gene in transgenic rice . Background technique [0002] During plant development, Ca 2+ It plays a very important role by interacting with its target protein. First, the calcium signal conducts signal transduction through the calcium target protein, and the calcium target protein initiates gene expression and cell life activities by binding to its target protein molecule, thus forming a calcium signal. A complex system of transduction. Intensively studied Ca in plants 2+ There are two main target proteins, calcium-binding protein kinases (CDPKs) and calmodulin (CaM). Biochemical and molecular biology analyzes have been performed on various CDPKs, and their roles in...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/54C12N15/63C12N15/82
CPCY02A40/146
Inventor 吕应堂梁述平王盈张蕾杨万年
Owner WUHAN UNIV
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