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Application of Rapeseed bnbhlh60 Gene in Improving Rapeseed Yield

A technology of bnbhlh60 and rapeseed, applied in the field of plant genetic engineering, can solve the problems of increasing grain weight, unknown function and the like, and achieves the effects of increasing grain weight, increasing grain length and increasing the number of seed cells

Active Publication Date: 2019-06-21
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] In this study, by cloning some differentially expressed genes in the main QTL region of grain weight, it was found that overexpression of one of the genes, BnbHLH60, in Arabidopsis can significantly increase grain weight. This gene belongs to the bHLH transcription factor family, and its function is currently unknown.

Method used

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  • Application of Rapeseed bnbhlh60 Gene in Improving Rapeseed Yield
  • Application of Rapeseed bnbhlh60 Gene in Improving Rapeseed Yield
  • Application of Rapeseed bnbhlh60 Gene in Improving Rapeseed Yield

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning of the Coding Region of Rapeseed BnbHLH60 Gene:

[0027] Using the first strand of cDNA of Zhongshuang No. 11 as a template, the BnbHLH60 gene was amplified by PCR. BnbHLH60 forward primer: 5'-ACGCGTCGACATGGATCTGACCGGAGCCTTCG-3', reverse primer: 5'-GGGGTACCTCCACAGCTCCATTTTGACCTGG-3', and finally obtained the sequence containing the SEQ ID The nucleotide sequence shown in NO:1, the nucleotide sequence encodes the amino acid sequence shown in SEQ ID NO:2.

[0028] In the above scheme, in order to realize the connection with the vector, a SalI restriction site (5'-GTCGAC-3') and a KpnI restriction site (5'-GGTACC -3') and the corresponding protective bases.

Embodiment 2

[0030] Construction of overexpression vector

[0031] The cloned PCR product of rapeseed BnbHLH60 and the plasmid expression vector pD1301S (Huang et al., 2014) were subjected to double enzyme digestion, and the reaction system was as follows:

[0032]

[0033] Reaction at 37°C for >12 hours.

[0034] 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.

[0035] Ligation and transformation of the target gene and the expression plasmid vector, the specific steps are as follows:

[0036] a. Configure the ligation reaction system (10 μl)

[0037] 10×T4DNA ligase Buffer 1μl

[0038] T4DNA ligase 1μl

[0039] DNA fragments (the molar number of DNA fragments is controlled at 3-10 times of carrier DNA)

[0040] wxya 2 0 to 10 μl

[0041] b. 16°C incubator reaction > 12h;

[0042] c. Add the whole amount to 100 μl DH5α compe...

Embodiment 3

[0048] Transformation of Agrobacterium tumefaciens GV3101

[0049] Plasmid extraction was carried out using a plasmid extraction kit according to the instructions. Double enzyme digestion was used to detect whether the target fragment was successfully connected to the expression vector ( figure 1 ).

[0050] The ice-thawing method transforms Agrobacterium, and the method steps are as follows:

[0051] (1) Add 2 μg of the purified plasmid to 100 μl of competent Agrobacterium GV3101, shake and mix gently;

[0052] (2) Place it on ice for 10 minutes, and immediately place it in liquid nitrogen for 5 minutes;

[0053] (3) 28°C water bath for 5 minutes;

[0054] (4) Add 800 μl of LB medium, shake and culture on a shaker at 28° C. at 200 rpm / min for 4-5 hours;

[0055] (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; ...

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Abstract

The invention discloses application of oilseed rape BnbHLH60 gene to oilseed rape yield improvement. The nucleotide sequence of the oilseed rape BnbHLH60 gene is shown as SEQ ID NO:1. Through the overexpression of the gene in Arabidopsis, the conditions that the grain length, the grain width and the grain weight of the transgenic lines are obviously increased, and the increase of the grain length is greater than the grain width are discovered; in addition, the effect is mainly achieved through cell number addition; in addition, the influence on the number of seeds per silique is not great; the guarantee is provided for the final yield improvement, so that good application prospects are realized in high-yield breeding of crops of oilseed rape and the like.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and more specifically relates to the application of rapeseed BnbHLH60 gene in improving rapeseed yield. 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 yield. A...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H6/20
CPCC12N15/8261
Inventor 王汉中师家勤李娜詹杰鹏刘贵华王新发
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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