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New application of OsBBTI4 (oryza sativa Bowman-Birk trypsin inhibitor 4) gene

A rice and genetic technology, applied in the direction of protease inhibitors, application, genetic engineering, etc., can solve the problems of inferring the role of OsBBTI4 gene, etc., and achieve the effect of reducing cadmium accumulation and promoting efflux

Inactive Publication Date: 2016-04-06
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, even if the cadmium tolerance of the OsBBTI4 gene is known, the role of the OsBBTI4 gene on cadmium accumulation in rice cannot be deduced because of two mechanisms

Method used

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  • New application of OsBBTI4 (oryza sativa Bowman-Birk trypsin inhibitor 4) gene
  • New application of OsBBTI4 (oryza sativa Bowman-Birk trypsin inhibitor 4) gene
  • New application of OsBBTI4 (oryza sativa Bowman-Birk trypsin inhibitor 4) gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1: Expression of the OsBBTI4 gene in Saccharomyces cerevisiae reduces the accumulation of cadmium in yeast

[0065] According to conventional methods, the recombinant expression vector OsBBTI4-pYES2 of Saccharomyces cerevisiae was constructed, such as figure 1 shown.

[0066] Cultivate Saccharomyces cerevisiae strain Δycf1 (purchased from Euroscarf, European Yeast Research Center, http: / / web.uni-frankfurt.de / fb15 / mikro / euroscarf / , strain number Y04069), use pYES2 plasmid and recombinant OsBBTI4-pYES2 to the above yeast to convert. Since the OsBBTI gene is placed under the regulation of the yeast galactose-induced promoter PGAL1 (see figure 1 shown), OsBBTI4-pYES2 was transformed into Saccharomyces cerevisiae and grown on the selective growth synthetic medium (Selective Growth Synthetic Medium, SDmedium) supplemented with galactose, which could induce the heterologous overexpression of OsBBTI4 in yeast.

[0067] The method used for yeast transformation is the ...

Embodiment 2

[0080] Example 2: Construction of OsBBTI4 Gene Transgenic Overexpression Vector and RNAi Vector and Rice Genetic Transformation

[0081] Take 100ng of rice genomic DNA as a template, use primers OsBBTI4OEF: 5'-CGGGATCCATGAGCAACACCACCATGGC-3' and OsBBTI4OER: 5'-CGGGATCCCTAGTTCTCCGCTCGGGGTT-3' (SEQ ID NO.3 and SEQ ID NO.4) to amplify and construct OsBBTI4 gene transgene overexpression vector Fragment; primers OsBBTI4RIF: 5'-GGGGTACCACTAGTATGAGCAACACCACCATGGC-3' and OsBBTI4RIR: 5'-CGGGATCCGAGCTCCTAGTTCTCCGCTCGGGGTT-3' (SEQ ID NO.5 and SEQ ID NO.6) were used to amplify the fragment for constructing the OsBBTI4 gene transgenic RNAi vector. The rice transgene overexpression vector used in the present invention is pCU1301 [ChenR, ZhaoX, ShaoZ, WeiZ, WangY, ZhuL, ZhaoJ, SunM, HeR, HeG (2007) RiceUDP-glucosepyrophosphorylase1isessentialforpollencallosedepositionanditscosuppressionresultsinanewtypeofthermosensitivegenicmalesterility. On this basis, the rice transgenic overexpression vec...

Embodiment 3

[0094] Embodiment 3: Determination of heavy metal cadmium content in transgenic rice seeds by atomic absorption spectrometry

[0095] Seeds of the above-mentioned homozygous strains were sown into 9 cm diameter petri dishes to germinate at 37°C, and after 7 days, the seedlings were transferred to nutrient solution (control) and nutrient solution supplemented with 0.01 mM Cd for cultivation and growth. The composition of the nutrient solution is: mother solution 1: 91.4gNH 4 NO 3 , 32.4gMgSO 4 ·7H 2 O, add water to make up to 1L; mother liquor 2: 88.6gCaCl 2 , add water to 1L; mother liquor 3: 40.3gNaH 2 PO 4 , 71.4gK 2 SO 4 , add water to 1L; mother liquor 4: 0.943gH 3 BO 4 , 1.5gMnCl 2 4H 2 O, 0.074g (NH 4 ) 6 Mo 7 o 24 4H 2 O, 0.031gCuSO 4 ·5H 2 O, 0.035gZnSO 4 ·7H 2 O, add water to make up to 1L; mother liquor 5: 6.9gFeSO 4 ·7H 2 O, 9.3gNa 2 EDTA·2H 2 O, add water to make up to 0.5L. When in use, add 5mL of No. 1-5 mother liquor for every 4L of nutr...

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Abstract

The invention relates to the field of engineering bacterium saccharomyces cerevisiae and plant genetic engineering, and provides application of an OsBBTI4 (oryza sativa Bowman-Birk trypsin inhibitor 4) gene to reduction of heavy metal cadmium accumulation in oryza sativa. The cDNA (complementary desoxyribonucleic acid) overall length sequence of the gene is shown in SEQ ID NO.1, and the encoded amino acid sequence of the gene is shown in SEQ ID NO.2. OsBBTI4 gene encoded protein OsBBTI4 is related to heavy metal cadmium accumulation of oryza sativa. By means of transgenosis overexpression or RNAi(ribonucleic acid interfere) of the OsBBTI4 gene in oryza sativa, the expression quantity of the OsBBTI4 gene in transgenic oryza sativa strains is regulated and controlled, cadmium content of transgenic oryza sativa seeds can be affected, and a low-cadmium-enrichment transgenic oryza sativa strain is obtained. The OsBBTI4 gene can be applied to genetic engineering breeding of engineering bacteria and oryza sativa for changing in-vivo cadmium content, and genetic engineering breeding of other plants for in-vivo cadmium enrichment.

Description

technical field [0001] The invention belongs to the field of biological genetic engineering, in particular to rice trypsin inhibitor OsBBTI4 ( O ryza s ativa B owman- B irk t rypsin i nhibitor4) and its encoding gene and its application in regulating cadmium accumulation in rice. Background technique [0002] Heavy metal cadmium is a non-essential toxic element for organisms. With the development of modern society, environmental pollution is becoming more and more serious, and more and more toxic active cadmium ions are released from the earth's crust into water, atmosphere and soil. These cadmium ions enter the biosphere and enter the human body through the food chain, seriously endangering human health. [0003] Rice is a crop rich in heavy metal cadmium. In April 2014, the Ministry of Land and Resources and the Ministry of Environmental Protection released data showing that the heavy metal pollution of my country's land is very serious. Calculated according to the...

Claims

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Application Information

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IPC IPC(8): C12N15/15C07K14/81C12N15/82A01H5/00
CPCC07K14/811C12N15/8218C12N15/8243
Inventor 张美孙雯王晶郭艳
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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