Corn pullulanase

A technology of pullulanase and nucleic acid fragments, applied in the directions of enzymes, hydrolase, glycosylase, etc., can solve the problems such as the undocumented corn pullulanase gene

Inactive Publication Date: 2000-06-21
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although pullulanase genes from other plants have been described (US Patent No. 5514576; Nakamura Y. et al. (1996) Plants (2):

Method used

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  • Corn pullulanase

Examples

Experimental program
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Effect test

Embodiment 1

[0060] Construction of maize cDNA library, isolation and sequencing of cDNA clones

[0061] A cDNA library representing mRNA was prepared from the endosperm of maize (Zea mays) LE392 20 days after pollination, following the product instructions (Stratagene Cloning SystemLa jolla, CA) at Uni-ZAP TM cDNA libraries were prepared in XR vectors. According to the protocol provided by Stratagene put Uni-ZAP TM The XR library was converted into a plasmid library. In transformation, the cDNA insert was obtained in the plasmid vector pBluescript. cDNA library normalization essentially followed the protocol disclosed in US Patent No. 5,482,845. cDNA inserts randomly picked from bacterial clones containing the recombinant pBluescript plasmid were amplified by polymerase chain reaction using primers specific for vector sequences flanking the inserted maize cDNA sequence. The sequence of the amplified insert DNA was determined in a dye-primer sequencing reaction according to the protoco...

Embodiment 2

[0063] Identification and characterization of cDNA clones

[0064]By running BLAST (Basic Local Aligment Search Tool; Altschul; S.F. et al. (1990) J. Molecular Biology, 215: 403-410; also check www.ncbi.nlm.nih.gov / BLAST / ) to find and GenBank database obtained Sequence similarity was used to identify the cDNA encoding maize pullulanase. The BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI) was used to analyze the cDNA sequences obtained in the examples for similarity to those containing published DNA sequences obtained in the GenBank database. DNA sequences were translated in all reading frames and compared with all published proteins obtained in the GenBank database using the BLASTX algorithm provided by NCBI (Gish.W and States.D.J. (1993) Nature Genetics, 3:266-272) sequence is similar.

[0065] The BLASTN search using the clone cen3n.pk0028.d2 shows that this nucleotide sequence is consistent with the nucleotide encoding r...

Embodiment 3

[0070] Expression of Chimeric Genes in Plant Cells

[0071] A chimeric gene can be constructed that includes a positively positioned maize pullulanase. The maize 27KD zein promoter located at the 5' end of the maize pullulanase fragment and the 10KD zein promoter located at the 3' end of the maize pullulanase fragment. A maize pullulanase fragment of the gene can be generated by polymerase chain reaction (PCR) using appropriate oligonucleotide primers from a cDNA clone comprising the maize pullulanase. When inserted into the digested vector pML103 as follows, a cloning site (NcoI or SmaI) can be incorporated into the oligonucleotide to provide the correct positioning of the DNA fragment. Amplification was performed in 100 μl of standard PCR mix consisting of 0.4 mM of each oligonucleotide and 0.3 pM of target DNA prepared in 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 0.001% w / v gelatin, 200mM dGTP, 200mM dATP, 200mM dTTP, 200mMdCTP and 0.025 units of Amplitaq ...

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Abstract

This invention relates to isolated nucleic acid fragments encoding all or a substantial portion of a corn pullulanase. The invention also relates to the construction of chimeric genes encoding all or a portion of a corn pullulanase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of corn pullulanase in a transformed host cell.

Description

field of invention [0001] The invention belongs to the field of plant molecular biology. More specifically, the present invention relates to nucleic acid fragments encoding enzymes involved in starch biosynthesis in corn plants and seeds. Background of the invention [0002] Corn starch is an important component of food, feed and industrial products. Broadly speaking, it consists of 2 types of glucan polymers: a few polymers with relatively long chains - amylose, and a shorter chain but highly branched molecule called amylopectin. Their synthesis is dependent on complex interactions between multiple enzymes (Smith, A. et al., (1995) Plant Physiology, 107: 673-677; Preiss, J. (1988) Plant Biochemistry 14: 181-253). Important among them are ADP-glucose pyrophosphorylase, which catalyzes the production of ADP-glucose; a series of starch synthases that use ADP-glucose as a substrate to form polymers using α-1,4 linkages; and several starches Branching e...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/29C12N15/56C12N15/82
CPCC12Y302/01041C12N15/8245C12N9/2457
Inventor K·E·布罗伊
Owner EI DU PONT DE NEMOURS & CO
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