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Application of oilseed rape alpha-6 microtubulin gene in increasing yield of oilseed rape

A technology of tubulin and rapeseed, applied in the field of plant genetic engineering, can solve the problem of increasing grain weight and achieve the effect of increasing grain weight

Active Publication Date: 2021-06-04
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The applicant found that (rape α-6 tubulin gene, the present invention or called BnaTUA6 gene) can significantly increase grain weight by overexpression in Arabidopsis, and the function of this gene regulating seed weight has not yet been reported

Method used

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  • Application of oilseed rape alpha-6 microtubulin gene in increasing yield of oilseed rape
  • Application of oilseed rape alpha-6 microtubulin gene in increasing yield of oilseed rape
  • Application of oilseed rape alpha-6 microtubulin gene in increasing yield of oilseed rape

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Cloning of the Coding Region of Rapeseed BnTUA6 Gene:

[0034] Using the first strand of cDNA of Brassica napus L. as a template to amplify BnTUA6 gene by PCR, BnTUA6 forward primer: 5'-ACGGGGGACTGGTACATGAGGGAGTGCATCTCGATC-3', reverse primer: 5'-GCTCACCATGGGATCGTACTCCTCGCCTTCGTCATC -3', finally obtained comprising the nucleotide sequence shown in SEQ ID NO:1, encoding the protein shown in SEQ ID NO:2.

[0035] In the above scheme, in order to realize the connection with the vector, a Kpn I restriction site (5'-GGTACC-3') and a BamH I restriction site (5' -GGATCC-3') and the corresponding protected bases.

Embodiment 2

[0037] Construction of overexpression vector

[0038] Both the PCR product of the cloned rapeseed BnTUA6 and the plasmid expression vector pCAMBIA2300S (that is, the 35S promoter added at its multiple cloning site on the basis of the pCAMBIA2300 vector backbone) were subjected to double enzyme digestion, and the reaction system was as follows:

[0039]

[0040] Reaction at 37°C for >1 hour.

[0041] Then, the double-digested plasmid and the PCR product were purified and recovered using the kit according to the instructions, and the concentration of the recovered result was detected by 1% agarose gel.

[0042] Ligation and transformation of the target gene and the expression plasmid vector, the specific steps are as follows:

[0043] a. Configure the ligation reaction system (10 μl)

[0044] 10×T4 DNA ligase Buffer 1μl

[0045] T4 DNA ligase 1μl

[0046] DNA fragments (the molar number of DNA fragments is controlled at 3-10 times of carrier DNA)

[0047] wxya 2 0 to 10 μ...

Embodiment 3

[0055] Transformation of Agrobacterium tumefaciens GV3101

[0056] Plasmid extraction was carried out using a plasmid extraction kit according to the instructions. Use double enzyme digestion to detect whether the target fragment is successfully connected to the expression vector.

[0057] The ice-thawing method transforms Agrobacterium, and the method steps are as follows:

[0058] (1) Add 2 μg of the purified plasmid to 100 μl of competent Agrobacterium GV3101, shake and mix gently;

[0059] (2) Place it on ice for 5 minutes, and immediately place it in liquid nitrogen for 5 minutes;

[0060] (3) 37°C water bath for 5 minutes;

[0061] (4) Add 800 μl of LB medium, shake and culture on a shaker at 28° C. at 200 rpm / min for 1 h;

[0062] (5) Centrifuge to remove most of the supernatant, precipitate, gently absorb and mix with a gun, remove about 100 μl of the bacterial solution, and apply it on an LB plate containing 50 mg / L of Rif, Gen and Kan;

[0063] (6) After culturi...

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Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to application of an oilseed rape alpha-6 tubulin gene in increasing the yield of oilseed rape. The nucleotide sequence of the oilseed rape BnaTUA6 gene is shown as SEQ ID NO:1, and the encoding protein of the oilseed rape BnaTUA6 gene is shown as SEQ ID NO:2. By conducting overexpression on the gene in arabidopsis thaliana, it is found that the grain weight and seed size of a transgenic line are remarkably increased, the number of grains per silique is not remarkably changed, and a guarantee is provided for increasing the final yield. Therefore, the oilseed rape alpha-6 tubulin gene has a good application prospect in high-yield breeding of crops such as oilseed rape.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and more specifically relates to the application of rapeseed α-6 tubulin gene in improving the yield of rapeseed. Background technique [0002] Seed size is an important adaptive trait in plant life history. Seed dispersal, germination, seedling settlement, and population distribution patterns are all related to seed weight (size) (Zhu et al., 2012; Zhao et al., 2013). Among plant traits, seed size is at the center and is a central feature of a plant's life history. As a special selection method, domestication plays an important role in the cultivation of many crops, and many genes controlling important agronomic traits are mutated. Seed weight (size) is one of the target traits for crop domestication and artificial breeding (Harlan et al., 1973). [0003] Grain weight, as one of the three factors constituting the yield of rapeseed, plays an important role in the formation of the final...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/10A01H6/20
CPCC07K14/415C12N15/8261
Inventor 王汉中师家勤梁华兵王新发詹杰鹏刘贵华
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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