High-temperature-resistant cabbage type rape glyoxalase gene and protein as well as applications thereof

A kind of Brassica napus, glyoxalase technology, applied in the field of mutation and genetic engineering, to achieve the effect of expanding heat resistance

Active Publication Date: 2015-08-05
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

In recent years, studies have found that the GLYI gene is a gene family containing multiple genes [5] , not only participate in the process of plant growth and development, but also play an important role in the response of plants to biotic stress, heavy metals, high salinity, drought and other abiotic stresses, and to alleviate the damage caused by them [5][6][7] , but in higher plants, there are few reports about whether the GLYI gene has the function of heat tolerance

Method used

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  • High-temperature-resistant cabbage type rape glyoxalase gene and protein as well as applications thereof
  • High-temperature-resistant cabbage type rape glyoxalase gene and protein as well as applications thereof
  • High-temperature-resistant cabbage type rape glyoxalase gene and protein as well as applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] 1. Embodiment 1: Screening of heat-resistant Brassica napus

[0059] First, we screened mature seeds of Brassica napus through different high-temperature treatments to obtain germplasm resources of Brassica napus with different heat resistance, such as figure 1 It was shown that there was a significant difference in the germination rate of the seeds of heat-tolerant Brassica napus 3382 and heat-sensitive Brassica napus 2682 after high temperature treatment at 65°C.

[0060] In order to ensure that the seeds have a germination rate of more than half after treatment, the mature seeds of Brassica napus heat-resistant germplasm resource 3382 were treated as follows:

[0061] Control: 37°C (2h) → standing for 2h (3382CK), the germination rate is 100%;

[0062] Treatment: 37°C (2h) → standing for 2h → 65°C (2h) (3382TR), the germination rate is over 80%;

[0063] Subsequent analysis of protein extraction and two-dimensional electrophoresis is expected to find related prote...

Embodiment 2

[0064] 2. Example 2: Extraction, two-dimensional electrophoresis and mass spectrometry analysis of seed protein

[0065] 1) Extraction of seed protein

[0066] The seeds of 3382 seeds before and after high temperature treatment were extracted by TCA-acetone method for two-dimensional electrophoresis analysis (Ganlu, Li Dianrong, Zang Xin, etc., 2010, the establishment of a protein two-dimensional electrophoresis system in Brassica napus, Acta Crops, 36 (4) :612-619), the specific steps are as follows:

[0067] ① Liquid nitrogen sample 1g;

[0068] ②In 10ml 10%TCA-0.07%DTT(g / v)-acetone solvent, vortex, vibrate vigorously for 15s, place at -20°C for 1h, shake once every 15min during the period;

[0069] ③ Centrifuge at 15000×g (number of rotations) for 30 minutes at 4°C, and remove the supernatant;

[0070] ④Add 15mL 0.07% DTT(g / v)-acetone, precipitate at -20°C for 1 hour, shake twice every 1 hour;

[0071] ⑤ Centrifuge at 15000×g for 30min at 4°C, remove the supernatant, th...

Embodiment 3

[0079] 3. Example 3: Cloning of the BnGLYI coding region

[0080] The RNA of the seeds before and after 3382 heat treatment was extracted by the method of LiCl, and the reverse transcription kit FSK100 of Toyobo was used to reverse-transcribe the cDNA. According to the sequence (Y13239) of the Brassica napus GLYI gene, primers were designed to amplify the full length of the gene (Table 1 ).

[0081] Table 1 is used to amplify the primers of BnGLYI

[0082]

[0083]

[0084]Note: Veena, Reddy VS, Sopory SK, 1999, Glyoxalase I from Brassica juncea: molecular cloning, regulation and its over-expression confer tolerance in transgenic tobacco under stress. Plant J 17: 385–395.

[0085] Using the inverted cDNA as a template, use KOD-PLUS DNA polymerase (product of TOYOBO, Japan, purchased from Beijing Dingguo Biotechnology Co., Ltd.), and perform PCR amplification with the following system:

[0086]

[0087] Make up to 50 μL with ultra-pure water, mix and centrifuge, add ...

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Abstract

The invention discloses a high-temperature-resistant cabbage type rape glyoxalase gene and protein as well as applications thereof, and relates to the technical field of mutation and genetic engineering. The nucleotide sequence of the high-temperature-resistant cabbage type rape glyoxalase gene namely a BnGLYI gene is shown by SEQ ID NO1; and the amino acid sequence of the protein of the high-temperature-resistant cabbage type rape glyoxalase gene namely the BnGLYI gene is shown by SEQ ID NO2. The invention lays an experimental foundation for boosting the resource development of good genes of cabbage type rapes, and improving the heat resistance and cold resistance of the cabbage type rapes; heat-resistant and cold-resistant gene resources are further expanded; the BnGLYI gene is converted into yeast cells to ensure that the heat resistance and cold resistance of the yeast cells are significantly improved, so that the heat resistance and cold resistance of other microorganisms and plants are expected to be further improved.

Description

technical field [0001] The invention relates to the technical field of mutation and genetic engineering; in particular to a high-temperature-resistant Brassica napus glyoxalase gene and protein and applications thereof; in particular to a high-temperature-resistant Brassica napus glyoxalase gene (Glyoxalase I, BnGLYI) The nucleotide sequence relates to the amino acid sequence of the high-temperature-resistant BnGLYI protein, the recombinant strain expressed by the BnGLYI gene in Pichia pastoris, relates to using the gene to construct an expression yeast expression vector, and also relates to a method for transforming yeast using the above gene sequence. Background technique [0002] Seeds provide us with most of our food and important sources of animals and industrial raw materials. High-quality seeds are of great importance to society and economy [1] . Many biotic and abiotic factors affect seed viability, germination and storability, for example, high ambient temperature ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N9/88C12N15/81C12N15/82
Inventor 伍晓明闫贵欣高桂珍陈碧云许鲲李锋李俊乔江伟
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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