Encoding sequence for Late Embryogenesis Abundant protein of Jatropha curcas and application in plants

A technology of jatropha curcas and leprosy, applied in the fields of molecular biology and genetic engineering

Inactive Publication Date: 2010-08-11
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Before the present invention was published, there was no disclosure or report of the Jatropha curcas LEA protein sequence and its nucleic acid sequence mentioned in this patent application

Method used

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  • Encoding sequence for Late Embryogenesis Abundant protein of Jatropha curcas and application in plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1 Cloning of the LEA protein gene of Jatropha curcas

[0094] 1. Organization separation (isolation)

[0095] The seeds of Jatropha curcas come from the Panzhihua area of ​​Sichuan Province. The seeds of Jatropha curcas are collected and stored in the laboratory.

[0096] 2. RNA isolation (RNA isolation)

[0097] Peel off the seed coat of Jatropha curcas seeds, take out the seed kernels, grind them in a mortar, add liquid nitrogen, grind them into powder, take 100mg and transfer to a 1.5mL EP tube to extract total RNA (two-step lysis method) . Use formaldehyde denaturing gel electrophoresis to identify the quality of total RNA, and then measure the RNA content on a spectrophotometer.

[0098] 3. Cloning of Full-length cDNA

[0099] According to the amino acid conservative sequence of some plant LEAs, design merging primers, using the principle of homologous gene cloning, SMARTTM RACE cDNA amplification method (Clonetech kit) for full-length cDNA cloning in four stages:

...

Embodiment 2

[0109] Example 2 Sequence information and homology analysis of Jatropha curcas LEA protein gene

[0110] The length of the full-length cDNA of the new Jatropha curcas LEA protein is 1070 bp, and the detailed sequence is shown in SEQ ID NO. 1, in which the open reading frame is located at the 59-820 nucleotides. According to the full-length cDNA, the amino acid sequence of the LEA protein of Jatropha curcas was deduced, with a total of 254 amino acid residues, a molecular weight of 26454.62, and a pI of 4.64. See SEQ ID NO.2. for the detailed sequence.

[0111] The full-length cDNA sequence of the Jatropha curcas LEA protein and its encoded protein were used BLAST program to perform nucleotide summation in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR databases The protein homology search revealed that it has 67% homology with the Arabidopsis LEA protein (NM_113148) at the nucleotide level (see Table 2); at the amino acid ...

Embodiment 3

[0112] Example 3 Prediction of the structure and type of Jatropha curcas LEA protein

[0113] 1. The structural domain analysis of Jatropha curcas LEA protein

[0114] The amino acid sequence of the LEA protein of Jatropha curcas was searched for the domain in the NCBI database (website: http: / / www.ncbi.nlm.nih.gov / Blast.cgi), and the results are as attached figure 1 Shown.

[0115] (1) In the amino acid sequence, there are the following domain regions: the framed region (ie SEQ ID. 1 in the sequence listing is from the amino-terminal (N-terminal) amino acid residues 14-72, 129-190, 193-254 Amino acid residues). The framed area constitutes the SMP mature protein functional module (Seed maturation protein, SMP).

[0116] (2) Functional analysis There is a functional module of seed mature protein in the amino acid sequence of the gene, so it can be predicted that it does have corresponding functions.

[0117] 2. Classification prediction of Jatropha curcas LEA protein

[0118] The amino...

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Abstract

The invention belongs to the technical fields of molecular biology and genetic engineering, relates to a new Late Embryogenesis Abundant protein ('LEA protein' for short) which is expressed in the Jatropha curcas, and an encoding sequence and application in improving drought tolerance of plants, and relates to fusion gene structures containing the genes, new recombined expression vectors carrying the fusion gene structures, and transformation plant cells, and transgenic plants of the genes generated by the transformation cells and offsprings thereof, which comprise plant seeds and plant tissues. By introducing the genes into plant hosts, drought stress resistance enhanced transgenic plants (crops) can be obtained. The genes have important theoretical and practical significances for culturing new breeds of drought resistance enhanced crops, and can be applied to breeding and identifying plant resistance breeds needed by farming and animal husbandry and ecological environment management.

Description

Technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering. Specifically, the present invention relates to a LEA protein (late embryogenesis abundant protein, LEA) expressed in Jatropha curcas and its nucleic acid sequence. The invention also relates to the preparation method and use of the protein and nucleic acid sequence. Background technique [0002] Drought stress is one of the main limiting factors for plants, especially cash crops. Among the various drought response mechanisms of plants, one of the strategies is to reduce the dehydration of cells by accumulating a series of proteins in plant cells during the lack of water (Sun Liping, Li Dequan. Research progress in molecular biology of LEA protein, Biotechnology Bulletin, 2003, 6: 5-13). Although there have been reports in the literature that a large number of stress-related genes have been isolated from plants (Thomashow et al., Plant cold acclimation: Freezing to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/63C12N1/19C12N5/10C12P21/02C12N15/82C12Q1/68C12N15/11
Inventor 林娟周明琦金元杰
Owner FUDAN UNIV
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