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Method for adjusting starch composition of root crops

A plant and plant cell technology, applied in the field of biotechnology or botany, can solve the problems of difficulty in meeting the urgent needs of the starch processing industry, and time-consuming

Active Publication Date: 2010-03-10
江苏三黍生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Relying on traditional breeding to change the quality of starch takes a long time, requires a lot of manpower and material resources, and is difficult to meet the urgent needs of the starch processing industry

Method used

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  • Method for adjusting starch composition of root crops
  • Method for adjusting starch composition of root crops
  • Method for adjusting starch composition of root crops

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Embodiment 1, vector construction

[0077] According to the published GBSSI gene sequence (NCBI accession number: X74160), the appropriate region in GBSSI (that is, the 1371-1570th position in SEQ ID NO: 1) was obtained, through an artificially synthesized intron (SEQ ID NO: 2) Forward and reverse connection as a spacer sequence, inserted into CaMV 35S (conventional constitutive promoter) or P54 (cassava vascular bundle specific expression promoter, Genbank accession number: AY217353, using the 1080bp sequence upstream of the start codon ( That is, in the EcoRI and XbaI sites of the pCAMBIA1300 binary vector driven by the -1~-1080)) promoter, the recombinant vectors pC-35S-GBSSI-RNAi and pC-P54-GBSSI-RNAi ( figure 1 ), used to transform cassava to prepare transgenic plants.

[0078] The intron sequence used was (SEQ ID NO: 2):

[0079] 5-GTACTATAGTATTTTGGTACCTTTACAATCGGTTTTTTACCTTTTCTTCTTTATTTATTAAATTTATAG-3.

Embodiment 2

[0080] Embodiment 2, preparation and plant regeneration of transgenic cassava and sweet potato

[0081] Cassava brittle calli were transformed with Agrobacterium containing the two recombinant binary vectors of pC-35S-GBSSI-RNAi and pC-P54-GBSSI-RNAi, respectively, and transgenic plants were obtained through regeneration through embryogenesis and organogenesis. The specific method is as follows:

[0082] 1. Use a sterilized toothpick to pick a single clone of Agrobacterium containing the recombinant binary vector from the plate, place it on the resistant YEB medium, and culture it overnight at 28°C and 240rpm with shaking.

[0083] 2. Take 25μl of bacterial liquid, add it to 50ml of fresh YEB medium, and cultivate for 12-20 hours to OD 600 0.5-1.0.

[0084] 3. Centrifuge the bacterial solution at 6000 rpm at 4°C for 10 min, suspend and wash with 50 ml liquid MS medium (PH 5.3), and centrifuge again. Resuspend the bacterial liquid in liquid MS medium containing 200 μM acetos...

Embodiment 3

[0097] Embodiment 3, transgenic plant detection

[0098] RT-PCR was used to detect the expression of GBSSI gene in transgenic cassava from the molecular level. Total RNA was extracted from the leaves of wild-type and transgenic cassava seedlings using TIANGEN's TRIZOL (DP421) reagent, with oligo (dT) as the first-strand primer, and M-MLV reverse transcriptase (TIANGENER104-03) reversed Record it as cDNA, perform double PCR amplification with the respective specific primers of GBSSI and c15 gene, and obtain the result.

[0099] see results figure 2 , indicating that the expression of the GBSSI gene in the eight transgenic cassava lines was greatly disturbed.

[0100] Similarly, RT-PCR can be used to detect the expression of the GBSSI gene in the transgenic sweet potato at the molecular level, and it was found that the expression of the GBSSI gene in the transgenic plants was greatly disturbed.

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Abstract

The invention relates to a method and a substance for adjusting starch composition of root crops. The invention discloses polynucleotide constructs which is expressed by specific interference granulebound starch synthase I (GBSSI) and can be processed after being introduced into plant., and a brand-new way for changing the starch quality of the plant by interrupting the expression of GBSSI genesbased on micro-molecular RNA interface technology to achieve the aim of adjusting the starch composition in the plant.

Description

technical field [0001] The invention belongs to the field of biotechnology or botany, and in particular relates to a substance and a method for regulating starch composition of tuber plants. Background technique [0002] Starch is the storage material of higher plants, algae and some microbial cells. It is a colorless, odorless white powder that is widely found in various plants, especially seeds (such as rice, wheat, corn) and roots (such as cassava). , sweet potato, potato) are rich in content and can be used as raw materials for industrial production of starch, such as corn, sweet potato, potato, wild acorn, kudzu root and other starchy substances. In addition to food, starch is used industrially to make dextrin, maltose, and glucose. It is also used to prepare printing paste, sizing textiles, gluing paper, and pressing pharmaceutical tablets. [0003] Starch is a polysaccharide formed by the condensation of many glucose molecules, which has two kinds of straight chain a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/82A01H1/00A01H5/00C12N15/113
Inventor 张鹏赵姗姗
Owner 江苏三黍生物科技有限公司
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