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Lettuce HPT protein coded sequence

A protein and lettuce technology, applied in the field of protein coding sequences, can solve problems such as the undiscovered vitamin E, the undiscovered lettuce HPPD protein sequence and its nucleic acid sequence, etc.

Inactive Publication Date: 2011-01-12
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In the analysis of prior art documents, no technical measures have been found to utilize genetic engineering technology to increase the vitamin E content in plants of lettuce, nor have any reports related to the lettuce HPPD protein sequence and nucleic acid sequence mentioned in the present invention

Method used

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  • Lettuce HPT protein coded sequence
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Cloning of Lettuce HPT Gene

[0044] 1. RNA extraction (RNA extraction)

[0045] Lettuce leaf tissue was taken, ground in liquid nitrogen, added to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shaken thoroughly, and total RNA was extracted according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer.

[0046] 2. Cloning of Full-length cDNA

[0047] According to the conserved sequence of amino acids encoded by related genes in Arabidopsis, using the principle of homologous gene cloning, the RACE (Rapid Amplification of cDNA Ends) method (Clontech) kit) was used to carry out full-length cDNA cloning in three stages:

[0048] (1) Synthesis of first-strand cDNA:

[0049] Use the 5'-CDS primer A and SMART2 A oligo primers provided by the Clontech kit, and use the extracted total RNA as a template to synthesize 5'-RACE-Re...

Embodiment 2

[0058] Sequence information and homology analysis of HPT-related genes in lettuce

[0059] The full-length cDNA of the new lettuce HPT obtained in the present invention is 1670bp in length, and the detailed sequence is shown in SEQ ID NO.3, wherein the open reading frame is located at nucleotides 299-1483 (1185 nucleotides). According to the obtained cDNA, the amino acid sequence of lettuce HPT was deduced, with a total of 395 amino acid residues, a molecular weight of 44kD, and an isoelectric point of 9.66. See SEQ ID NO.4 for the detailed sequence.

[0060] Using the vectorNTI 9.0 software to carry out homologous comparison of the relevant amino acid sequences of HPT derived from various plants, it was found that the cloned lettuce HPT gene is similar to that of Arabidopsis thaliana, Medicago sativa, soybean (Glycine max), corn (Zea mays), green algae (O.lucimarinus) and two kinds of blue-green algae (Synechococcus, Synechocystis) encoded amino acid sequence similarities we...

Embodiment 3

[0062] Eukaryotic expression of lettuce HPT-related proteins or polypeptides in Arabidopsis cells and identification of vitamin E content in transgenic plants

[0063] 1. Containing the construction of the expression vector of target gene (lettuce HPT gene)

[0064] According to the full-length coding sequence of lettuce HPT (SEQ ID NO.3), design primers to amplify the complete coding reading frame, and introduce BamHI and SacI restriction endonuclease sites on the forward and reverse primers respectively, so as to construct the expression vector . Using the 5'-RACE-Ready cDNA obtained in Example 1 as a template, after PCR amplification, the coding region sequence of the lettuce HPT was connected to the intermediate vector pMD18-T for sequencing, and then the coding region of the correctly sequenced HPT The sequence was further cloned into the expression vector pHB, and then transformed into Agrobacterium tumefaciens (such as GV3101, used in this example was donated by the Un...

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Abstract

The invention relates to a lettuce HPT protein coded sequence in the technical field of gene engineering, which comprises that: a polypeptide nucleotide sequence with lettuce HPPD protein activity is coded; and the nucleotide sequence consists of nucleotide sequences at 299-1,483 positions in SEQ ID NO.3 with at least 70 percent of homology. A method for transforming plants to increase the contentof pant vitamin E comprises the following steps: connecting coded polypeptide purified nucleotide sequence with the lettuce HPT protein activity to a plant expression regulatory sequence to form a plant expression vector containing lettuce HPT protein gene; transforming the expression vector in the step (1) into Agrobacterium; transforming the Agrobacterium containing the expression vector into Arabidopsis by an immigration method; and screening the Arabidopsis by antibiotics to obtain transgenic cells containing the lettuce HPT protein gene, and regenerating transgenic plants and descendantscomprising plant seeds and plant tissues. In the invention, the gene is expressed in the plant, so the content of the vitamin E in the obtained transgenic plants is obviously improved.

Description

technical field [0001] The present invention relates to a protein coding sequence in the technical field of genetic engineering, specifically, the present invention relates to a lettuce HPT protein coding sequence. Background technique [0002] Since the biological function of vitamin E was discovered in 1922, long-term medical research has shown that vitamin E is not only related to the reproductive system, but also closely related to the normal metabolism of the central nervous system, digestive system, cardiovascular system and muscle system ( Traber and Sies, 1996). Vitamin E is a lipid vitamin, and its natural products are divided into eight types according to the structure, namely α, β, γ, δ-tocopherol (tocopherol) and α, β, γ, δ-tocotrienol (tocotrienol), of which α-tocopherol has the highest biological activity and is considered to be the main active ingredient of vitamin E. Vitamin E is involved in the construction and maintenance of cell membranes, and is an impo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/82A01H1/00A01H5/00A01H5/10
Inventor 唐克轩任薇薇唐岳立
Owner SHANGHAI JIAOTONG UNIV