A rice gene related to low nitrogen stress and nitrogen utilization ospimt2 Cloning and application of

A rice and genetic technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., to achieve the effect of improving nitrogen utilization rate, broad application and market prospects, and low lipid peroxidation level

Inactive Publication Date: 2019-09-27
福建省农业科学院水稻研究所
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no literature report in this area, but it is worthy of further exploration as a NUE-related gene

Method used

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  • A rice gene related to low nitrogen stress and nitrogen utilization <i>ospimt2</i> Cloning and application of
  • A rice gene related to low nitrogen stress and nitrogen utilization <i>ospimt2</i> Cloning and application of
  • A rice gene related to low nitrogen stress and nitrogen utilization <i>ospimt2</i> Cloning and application of

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment one, OsPIMT2 Cloning of the full-length CDS of the gene.

[0018] Through the NCBI database, the PIMT gene coding sequence (coding sequence, CDS) sequence of Arabidopsis and wheat was searched to obtain highly homologous expressed sequence tags (expressed sequence tags, ESTs), and the cDNA sequence was spliced ​​through the sequence. Primers were designed according to its conserved sequence and the full-length cDNA was cloned using Smarter RACE technology. The 5'RACE and 3'RACE primers were as follows:

[0019] MT2R5: 5' CCCTCTGCTACGAACAATGCTCTGTCAAT 3'

[0020] MT2R3: 5'GCAGCGGTTACTTGACAGC 3'.

[0021] First, extract RNA from leaves and roots of japonica rice Nipponbare, reverse transcribe mRNA through SMARTer™ RACE cDNA Amplification Kit, and prepare cDNA for 5'RACE and 3'RACE containing specific linker sequences respectively. The reverse transcription process is provided according to the kit manual. Dilute the 5' and 3' RACE cDNA templates 50 times as ...

Embodiment 2

[0022] Embodiment two, OsPIMT2 Cloning of full-length genome sequences.

[0023] Through the NCBI database, using the existing full-length CDS to compare with the rice genome, it was determined that OsPIMT2 The open reading frame and its promoter region were predicted by bioinformatics software, and the coverage was designed OsPIMT2 The primer sequences of the promoter region, the exon region, the intron region and the terminator region, the primer sequences are as follows:

[0024] MT2g-F: 5'GTACAATTCAGTTTTTGTTATTGTTTTATATT 3'

[0025] MT1g-R: 5'TATATTTAATAATAAATCAAATGATATGAAAA 3'.

[0026] Using the improved CTAB method to extract rice leaf genomic DNA as a template for gene cloning, the amplified product was connected to the pJET1.2 cloning vector and then sent for sequencing. The results showed that OsPIMT2 The gene contains 3 introns and 4 exons.

Embodiment 3

[0027] Embodiment three, OsPIMT2 Effects of transgenic plants on the adaptability of rice to low nitrogen stress.

[0028] (one) OsPIMT2 Construction of transgenic vectors.

[0029] Design primers containing enzyme cleavage sites and use Nipponbare cDNA as a template to carry out OsPIMT2 Amplification of the coding region. The primer sequences are as follows:

[0030]PIMT2-CDS-F: 5'GCCGACATGTGCCTCGCCGCCGCCAT 3'

[0031] PIMT2-CDS-R: 5'CAAGTAGACTGAGCGTCGAGAT 3'.

[0032] Pass this snippet through Pci with Bst Obtained after EII double digestion and connection with plant overexpression vector pCAMBIA1301 OsPIMT2 Overexpression vector MT2-OE.

[0033] By Blast comparison, select OsPIMT2 The specific region was used as the target sequence, and primers with enzyme cleavage sites were designed, which were connected to the RNAi plant interference vector pTCK303 after one PCR and two enzyme digestions. The vector construction method was carried out by referring to the lite...

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Abstract

The invention relates to the technical field of plant molecular biology and gene engineering, and in particular relates to a rice protein (rice OsPIMT2) related to low nitrogen stress resistance as well as cloning, functional verification and application of a coding gene thereof. Full-length cDNA is cloned out by using a Smarter RACE technology to further obtain a full-length genomic sequence of OsPIMT2. Then, an over-expression and dry interference expression vector of an OsPIMT2 gene is constructed, and the agrobacterium mediated transformation of keng rice (Nipponbare) and the analysis of low nitrogen stress of a transgenic plant show that an over-expression transgenic plant of the OsPIMT2 gene, compared with a wild plant, has a lower lipid preoxidation level and a higher biomass (such as dry weight, plant height and the like). Therefore, the expression of the gene can enable the rice to use limited nitrogen to obtain and generate a higher biomass, thereby providing a certain theory and a certain basis for culturing a rice variety with a high nitrogen utilization ratio in the future.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a rice gene related to low nitrogen stress response and nitrogen utilization ( OsPIMT2 ) Isolation and cloning, functional verification and application. Background technique [0002] Nitrogen is an essential macroelement for plant growth and development. The growth of crops and food production require sufficient nitrogen, and the effects of nitrogen deficiency on growth and yield are very common. Nitrogen deficiency can lead to drastic changes in plant growth and physiology, such as growth of lateral roots, yellowing of leaves, decrease in yield, and increase of reactive oxygen species (ROS) in vivo. Therefore, nitrogen fertilizers are widely used worldwide to meet the nitrogen requirements of crops. However, although excessive use of nitrogen fertilizers will temporarily increase crop yields, the accompanying economic and environmental problems cannot be ign...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11A01H5/00A01H6/46
Inventor 张建福谢华安魏毅东何炜许惠滨连玲蔡秋华朱永生谢鸿光罗曦郑燕梅吴方喜蒋家焕林强魏林艳
Owner 福建省农业科学院水稻研究所
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